Structural and Functional Characterisation of LIM kinases

Structural and functional

characterisation of

LIM kinases

Kevin Yves Mark Knut Mittelstaedt

Submitted in total fulfilment of the requirements

of the degree of Doctor of Philosophy

November 2012

St. Vincent’s Institute of Medical Research

Department of Medicine, St. Vincent’s Hospital

The University of Melbourne

iii

ABSTRACT

LIMK1 and LIMK2 constitute a family of serine/threonine protein kinases that

serve as important regulators of actin dynamics. They phosphorylate and

inactivate the actin depolymerising factor, cofilin, thereby leading to the

accumulation of actin filaments. Both LIMKs have been implicated in pathological

conditions such as tumour cell invasion and ocular hypertension, where the

regulation of actin polymerisation is affected. Several small-molecule LIMK

inhibitors have been identified, but the lack of a three-dimensional structure of the

kinase domain has hampered structure-based development of these compounds.

This thesis describes the expression and purification of the LIMK1 kinase domain

for X-ray crystallographic studies to enable rational structure-based optimisation

of LIMK inhibitors. The GST-tagged LIMK1 kinase domain was expressed in

baculovirus-infected insect cells and purified using ammonium sulphate

precipitation prior to glutathione affinity and size-exclusion chromatography.

Limited proteolysis of the kinase domain revealed improved domain boundaries,

yielding a catalytically active fragment consisting of residues 332-607. The low

solubility of the kinase domain in the absence of the GST tag was enhanced by

addition of the solubilising agent, NV10. However, crystallisation trials yielded

only amorphous precipitates, but no protein crystals. Despite the development of

improved algorithms to predict important protein features such as domain

boundaries and regions of high entropy, the purification of soluble protein and the

crystallisation thereof remain the major bottleneck in structure determination. The

recent determination of the crystal structure of the LIMK1 kinase domain in

complex with staurosporine has paved the way for co-crystallisation studies using

LIMK-specific inhibitors to determine the inhibitor:enzyme interactions and

improve the compounds’ potency and substrate specificity.

The second part of this thesis investigates the molecular mechanisms leading to

the obesity phenotype of LIMK2a knockout mice. Phenotypic characterisation

revealed a significant increase in the body mass and fat mass in male LIMK2a

knockout mice compared to male wildtype mice. The knockout mice were

hyperlipidaemic and showed signs of insulin resistance. Their adipocytes were

hypertrophic and showed impaired glucose uptake and lipolysis. The inverse

iv

correlation between LIMK2a expression and body mass was also observed in the

adipose tissue of obese ob/ob and lean wildtype mice. It was hypothesised that

altered actin organisation as a result of lack of LIMK2a may be responsible for the

enlargement of adipocytes and possible changes in the adipogenic potential of

progenitor cells. In cultured 3T3-L1 pre-adipocytes, the level of LIMK2a mRNA and

total LIMK2 protein was transiently up-regulated following stimulation with an

adipogenic differentiation cocktail. However, adipogenic differentiation of LIMK2-

and LIMK2a-deficient MEFs showed no difference in lipid accumulation or cellular

morphology when compared to wildtype MEFs. These results indicate that LIMK2

or LIMK2a are not involved in the adipogenic cell fate decision in MEF cultures.

However, it remains to be determined whether the lack of LIMK2a affects the

adipogenic potential of mesenchymal stem cells or the morphology of fully

differentiated adipocytes derived from these progenitors.

v

DECLARATION

This is to certify that

i. the thesis comprises only my original work towards the PhD except

where indicated in the Preface,

ii. due acknowledgement has been made in the text to all other material

used,

iii. the thesis is fewer than 100,000 words in length, exclusive of tables,

maps, bibliographies and appendices.

Kevin Mittelstaedt

November 2012

vi

PREFACE

The thesis comprises only my original work toward the PhD except for the

reagents and experiments as stated below.

Subcloning of the mouse and rat LIMK kinase domain was based on DNA

constructs previously generated by former members of the Cytoskeleton and

Cancer Unit at St Vincent’s Institute. The expression construct encoding His6-

tagged LIMK1 kinase domain was generated by Dr. Rong Li. Additional large-scale

amplification of selected baculoviruses was carried out by the Fermentation group

at CSIRO Molecular and Health Technologies (Parkville, Victoria). Mass

spectrometry analysis was performed by Dr. Rohan Steel. Discussion of the

structure of the LIMK1 kinase domain is based on a crystal structure by Beltrami et

al., which is deposited at the Protein Data Bank under accession number 3S95.

Phenotypic characterisation of the LIMK2a knockout mouse was carried out in

collaboration with A/Prof. Matthew Watt and Dr. Andrew Hoy (Department of

Physiology, Monash University, Victoria) who performed the following

experiments: measurement of body mass and fat mass; indirect calorimetry;

measurement of metabolites in blood, adipose tissue, muscle and liver; lipid and

glucose metabolic assays; and expression of adipogenic marker genes and lipolytic

proteins (shown in Figure 8-1, 8-2, 8-3 and 8-4). Sectioning and staining of adipose

tissue was performed by Mr. Sheng Chen in the laboratory of A/Prof. Ora Bernard.

Recombinant Flag-ROCK and LIMK2 knockout mice were kindly provided by Ms.

Alice Schofield and Lexicon Pharmaceuticals, respectively.

Kevin Mittelstaedt

November 2012

vii

ACKNOWLEDGEMENTS

First and foremost, I would like to thank my supervisor, A/Prof. Ora Bernard, for

giving me the opportunity to perform this research in her laboratory, for making

me feel welcome in Melbourne, and for her endless patience with me on this

extraordinary journey. I also wish to thank my co-supervisor, Dr. Boris Sarcevic,

for his feedback and constructive discussions at critical stages of my candidature.

I wish to express my gratitude to Drs. Rong Li, Juliana Antonipillai, Jiong Zhou and

Cristina Gamell, as well as Ms. Priscilla Soo, Ms. Alice Schofield and Mr. Sheng Chen

for creating and maintaining an enjoyable and productive working environment as

well as for technical assistance, advice and critical discussion. In particular, I would

like to acknowledge the contributions of Dr. Rong Li, who established and taught

me the procedures for bacmid generation and protein expression in insect cells.

I would like to thank Dr. David Asher for his priceless advice on how to deal with

the “fickle mind” of insect cells, and Dr. Michael Gorman for guidance in the field of

protein purification and crystallisation as well as for his encouraging words in

times when LIMK proteins were not on their best behaviour. Also, I am grateful to

Dr. John Scott for sharing with me his knowledge in in vitro phosphorylation

assays, Dr. Jon Oakhill for assistance with ion-exchange chromatography, and Dr.

Rohan Steel for mass spectrometry analysis.

I wish to acknowledge A/Prof. Matthew Watt for providing the invaluable in vivo

data that formed the basis for our stimulating collaboration. Additional thanks go

to Mr. Cletus Pinto who helped with establishing procedures for quantitative real-

time PCR and data analysis, and Mr. Tony Blick for extensive discussions about

statistics. I am also grateful to the countless staff and students on the St. Vincent’s

campus who helped me explore the multitude of promising aspects of my research

projects that did not find their way into this thesis. I extend my appreciation to the

many other people I have met on the way, who kindly offered their time, support

and friendship, and contributed to my personal and professional development.

viii

I take this opportunity to express my endless gratitude to my beautiful girlfriend,

Dr. Sih Min Tan, who has been a source of inspiration, and has supported and

motivated me in every imaginable way throughout this journey. I also wish to

thank my parents, who taught me the value of a good education and always

inspired me to be the best I can be.

Last but no least, I would like to acknowledge the financial support I have received

from The University of Melbourne (Melbourne International Research Scholarship

and Melbourne International Fee Remission Scholarship) and the St. Vincent’s

Institute Foundation (Postgraduate Student Award).

ix

TABLE OF CONTENTS

Abstract .............................................................................................................................. iii

Declaration ......................................................................................................................... v

Preface ............................................................................................................................... vi

Acknowledgements .......................................................................................................... vii

Table of contents ............................................................................................................... ix

List of abbreviations ....................................................................................................... xiv

List of figures ................................................................................................................. xviii

List of tables ................................................................................................................... xxii

List of publications ........................................................................................................ xxiii

Chapter 1 − Literature review .......................................................................................... 1

1.1 Introduction ..................................................................................................... 3

1.2 Protein kinases ................................................................................................. 4

1.3 Protein kinase inhibitors ............................................................................... 12

1.4 LIM kinases as regulators of actin dynamics ............................................... 17

1.4.1 Gene structure and expression pattern of LIMKs ........................ 17

1.4.2 Actin polymerisation and depolymerisation ................................ 23

1.4.3 LIMK substrates .............................................................................. 25

1.4.4 Regulators of LIMK activity ............................................................ 28

1.4.4.1 Rho GTPases ..................................................................... 28

1.4.4.2 Regulation of LIMK1 activity .......................................... 29

1.4.4.3 Regulation of LIMK2 activity .......................................... 30

1.4.5 LIMK knockout mice ....................................................................... 30

1.5 Regulation of actin dynamics in cell migration ........................................... 31

1.5.1 Cofilin-mediated actin dynamics in cell motility .......................... 31

1.5.2 Regulation of cofilin activity in directed cell migration .............. 32

1.5.3 The role of LIMK1 in cancer metastasis ........................................ 34

1.5.4 Inhibitors of the Rho GTPase/cofilin pathway ............................. 35

1.6 Protein structure determination .................................................................. 39

1.6.1 Protein crystallography .................................................................. 39

1.6.2 High-resolution structures of LIM kinases ................................... 43

1.7 Hypothesis and aims ...................................................................................... 43

Chapter 2 − Materials & Methods ................................................................................... 47

x

2.1 Generation of expression constructs............................................................ 49

2.1.1 Bioinformatic analysis .................................................................... 49

2.1.2 DNA constructs ............................................................................... 49

2.1.3 Polymerase chain reaction ............................................................. 50

2.1.4 Polyadenylation and cloning of His6-GST-tagged constructs ..................................................................................... 52

2.1.5 Agarose gel electrophoresis ........................................................... 52

2.1.6 Purification and preparative digestion of DNA ............................ 53

2.1.7 DNA ligation .................................................................................... 53

2.1.8 Heat-shock transformation ............................................................ 54

2.1.9 Amplification and purification of plasmid DNA ........................... 54

2.1.10 Analytic digestion of plasmid DNA .............................................. 54

2.1.11 DNA sequencing ............................................................................ 55

2.1.12 Generation of recombinant bacmid DNA .................................... 56

2.1.13 Isolation of recombinant bacmid DNA ........................................ 56

2.1.14 Analysis of recombinant bacmid DNA ........................................ 57

2.2 Insect cell culture and baculovirus production ........................................... 57

2.2.1 Cell culture materials...................................................................... 57

2.2.2 Cell lines & culture maintenance ................................................... 58

2.2.3 Transfection .................................................................................... 58

2.2.4 Analysis of recombinant protein expression ................................ 58

2.2.5 Baculovirus plaque assay ............................................................... 59

2.2.6 Baculovirus clone amplification .................................................... 59

2.3 Protein expression in insect cells ................................................................. 60

2.3.1 Micro-scale expression ................................................................... 60

2.3.2 Small/medium-scale protein expression ...................................... 60

2.4 Protein purification ....................................................................................... 61

2.4.1 Ammonium sulphate precipitation, glutathione-affinity chromatography and GST tag removal ...................................... 61

2.4.2 Ion exchange chromatography ...................................................... 62

2.4.3 Size exclusion chromatography ..................................................... 62

2.4.4 Facilitation of protein solubility .................................................... 62

2.5 Protein characterisation ................................................................................ 63

2.5.1 Determination of protein concentration ...................................... 63

2.5.2 SDS-polyacrylamide gel electrophoresis ...................................... 63

2.5.3 Western blot .................................................................................... 64

2.5.4 In vitro kinase assay ....................................................................... 65

2.5.5 Limited proteolysis ......................................................................... 65

2.5.6 In-gel digestion and mass spectrometry ....................................... 66

2.5.7 Concentrating proteins ................................................................... 66

2.6 Screening for crystallisation conditions ...................................................... 66

2.7 Protein structure analysis ............................................................................. 67

Chapter 3 − Expression, purification and crystallisation of the LIMK1 kinase

domain .............................................................................................................................. 69

3.1 Protein expression for structural studies .................................................... 71

xi

3.2 Construct design ............................................................................................ 71

3.3 Generation of expression constructs and baculoviruses ............................ 79

3.4 Optimisation of baculovirus amplification and protein expression .......... 83

3.5 Cell lysis and affinity chromatography ........................................................ 85

3.6 Proteolytic tag removal ................................................................................. 90

3.7 Limited proteolysis ........................................................................................ 97

3.8 Purification of the N-terminally truncated LIMK1 kinase domain .......... 106

3.9 Solubility of LIMK1 kinase domain ............................................................ 110

3.10 Purification of the LIMK1 kinase domain by ion-exchange and

size-exclusion chromatography ............................................................ 112

3.11 Catalytic activity of the purified LIMK1 kinase domain fragment ......... 116

3.12 Crystallisation trials .................................................................................. 118

Chapter 4 − Discussion .................................................................................................. 119

4.1 Optimisation of baculovirus production .................................................... 121

4.2 Construct design .......................................................................................... 121

4.3 Ammonium sulphate precipitation and catalytic activity ........................ 123

4.4 Protein stability ............................................................................................ 124

4.5 Protein solubility ......................................................................................... 125

4.6 Crystallisation propensity ........................................................................... 127

4.7 Structure of the LIMK1 kinase domain/staurosporine complex ............. 128

Chapter 5 − Conclusion .................................................................................................. 137

Chapter 6 − Literature review ...................................................................................... 143

6.1 Introduction ................................................................................................. 145

6.2 Overweight and obesity .............................................................................. 146

6.3 Obesity-induced adipocyte dysfunction .................................................... 146

6.4 Obesity-induced insulin resistance ............................................................ 150

6.5 Regulation of adipose tissue development and function ......................... 153

6.5.1 Adipose tissue development and composition .......................... 154

6.5.2 Adipogenic differentiation in vitro .............................................. 156

6.5.3 Lineage commitment of adipocyte progenitor cells .................. 157

6.5.4 Actin cytoskeletal regulators in adipocytes ................................ 158

6.6 Hypothesis and aims .................................................................................... 159

Chapter 7 − Materials & methods ................................................................................. 161

7.1 Animals and animal care ............................................................................. 163

xii

7.2 Genotyping of mouse tails ........................................................................... 165

7.3 Indirect calorimetry and feeding ................................................................ 166

7.4 Plasma metabolite analysis ......................................................................... 166

7.5 Tissue metabolite analysis .......................................................................... 166

7.6 Lipogenesis and lipolysis ............................................................................ 167

7.7 Insulin tolerance test ................................................................................... 167

7.8 Haematoxylin and eosin staining ............................................................... 168

7.9 Glucose uptake ............................................................................................. 168

7.10 RNA extraction, reverse transcription and quantitative real-

time PCR.................................................................................................. 168

7.11 Adipogenic differentiation of 3T3-L1 pre-adipocytes ............................ 171

7.12 Preparation of mouse embryonic fibroblasts ......................................... 171

7.13 Western blot ............................................................................................... 172

7.14 Oil Red O staining ...................................................................................... 173

7.15 siRNA-mediated knockdown of LIMK2 ................................................... 173

7.16 In vitro phosphorylation of LIMK2 using ROCK1 .................................... 174

7.17 Fractionation of adipose tissue extract.................................................... 175

7.18 Fractionation of NIH3T3 cell extract ....................................................... 175

7.19 In vitro phosphorylation and identification of potential LIMK2

substrates ............................................................................................... 176

7.20 Statistical analysis ..................................................................................... 176

Chapter 8 − Functional characterisation of LIMK2 in adipocytes ............................. 177

8.1 Phenotypic characterisation of the LIMK2a knockout mouse ................. 179

8.2 LIMK2 mRNA expression in adipose tissue from lean and obese

mice ......................................................................................................... 185

8.3 LIMK2 mRNA and protein expression profile during adipogenesis ........ 187

8.4 Knockdown of LIMK2 expression in 3T3-L1 cells..................................... 190

8.5 Protein expression levels of LIMK2, cofilin and phospho-cofilin in

MEFs ........................................................................................................ 194

8.6 Differentiation of MEFs extracted from wildtype and LIMK2a

knockout embryos ................................................................................. 196

8.7 Differentiation of LIMK2 knockout MEFs .................................................. 199

8.8 Identification of novel LIMK2 substrates .................................................. 201

xiii

Chapter 9 − Discussion .................................................................................................. 209

9.1 Obesity phenotype ....................................................................................... 211

9.2 Insulin-stimulated glucose uptake ............................................................. 213

9.3 Lipid metabolism ......................................................................................... 214

9.4 LIMK2 expression in adipose tissue ........................................................... 215

9.5 LIMK2 expression and function during adipogenesis .............................. 217

9.6 Identification of novel LIMK2 substrates .................................................. 220

Chapter 10 − Conclusion ............................................................................................... 223

Chapter 11 - Final Conclusion ....................................................................................... 229

Bibliography ................................................................................................................... 233

List of abbreviations

xiv

LIST OF ABBREVIATIONS

[ -32P]ATP Phosphorus-32-labelled adenosine triphosphate

2DG 2-deoxyglucose

AcMNPV Autographa californica multicapsid nucleopolyhedrovirus

ADF Actin-depolymerising factor

ADP Adenosine diphosphate

AMP Adenosine monophosphate

AMP-PNP Adenylyl imidodiphosphate

ANOVA Analysis of variation

ATGL Adipose triglyceride lipase

ATP Adenosine triphosphate

BDT Big dye terminator

BMI Body mass index

bp Base pair

BSA Bovine serum albumin

C/EBP CCAAT/enhancer binding protein

cAMP Cyclic adenosine monophosphate

cDNA Complementary deoxyribonucleic acid

CGI-58 Comparative gene identification-58

CoA Coenzyme A

cpm Counts per minute

dATP Deoxyadenosine triphosphate

DEPC Diethylpyrocarbonate

dH2O Deionised water

DMEM Dulbecco's modified Eagle's medium

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DNase Deoxyribonuclease

dNTP Deoxyribonucleotide

DTT Dithiothreitol


xv

ECL Enhanced chemiluminescence

EDTA Ethylene diamine tetraacetic acid

EGF Epidermal growth factor

EGFR Epidermal growth factor receptor

EGTA Ethylene glycol tetraacetic acid

F-actin Filamentous actin

FBS Fetal bovine serum

FFA Free fatty acid

G-actin Globular actin

GAP GTPase-activating protein

GAPDH Glyceraldehyde 3-phosphate dehydrogenase

GDI Guanine nucleotide dissociation factor

GEF Guanine nucleotide exchange factor

GLUT Glucose transporter

GSH Reduced glutathione

GST Glutathione S-transferase

GTP Guanosine triphosphate

GTPase Guanosine triphosphatase

HEPES Hydroxyethylpiperazineethanesulfonic acid

HET Heterozygous

HFD High-fat diet

His6 Hexahistidine

HRP Horseradish peroxidase

HSL Hormone-sensitive lipase

IBMX Isobutyl methyxanthine

IGF Insulin-like growth factor

IPTG Isopropylthio-β-galactoside

IR Insulin receptor

IRS Insulin receptor substrate

JNK c-Jun N-terminal kinase

KO Knockout

LB Luria Bertani


xvi

LIM Functional protein domain named after Lin-11, Isl-1 and Mec-3

LIMK LIM domain-containing protein kinase

LTR Long terminal repeat

MBP Maltose-binding protein

MEF Mouse embryonic fibroblast

MLC Myosin light chain

MOI Multiplicity of infection

MPD 2-methyl-2,4-pentanediol

mRNA Messenger RNA

MW Molecular weight

NBF Neutral buffered formaline

ob/ob Leptin-deficient

P1…P4 1st…4th generation of baculovirus (1st…4th amplification)

PAGE Polyacrylamide gel electrophoresis

PAK p21-activated kinase

PBS Phosphate-buffered saline

PBS-T Phosphate-buffered saline supplemented with 0.1% Tween-20

PCR Polymerase chain reaction

PDB Protein Data Bank

PDZ Functional protein domain named after PSD-95, DLG and ZO-1

PEG Polyethylene glycol

PI3K Phosphoinositide 3-kinase

PKx Protein kinase x (x = A, R)

PPAR Peroxisome proliferator-activated receptor

RER Respiratory exchange ratio

RNA Ribonucleic acid

RNase Ribonuclease

ROCK Rho-associated protein kinase

RT Room temperature

S.E.M. Standard error of the mean

SDS Sodium dodecyl sulphate

Sf9 Spodoptera frugiperda 9


xvii

Sf21 Spodoptera frugiperda 21

shRNA Short hairpin RNA

siRNA Small interfering RNA

SOC Super optimal broth

SSH Slingshot

TBE Tris/borate/EDTA

TCEP Tris(2-carboxyethyl)phosphine

TE Tris/EDTA

TESK Testis-specific protein kinase

TEV Tobacco etch virus

TGF Transforming growth factor

TNF Tumour necrosis factor

TNFR Tumour necrosis factor receptor

v/v Volume per volume

w/v Weight per volume

WCL Whole cell lysate

WT Wildtype

List of figures

xviii

LIST OF FIGURES

Figure 1-1: Phylogenetic tree of human protein kinases. ...................................... 5

Figure 1-2: Ligand-induced dimerisation of the EGF receptor. ............................ 7

Figure 1-3: Crystal structure of the catalytic subunit of PKA in complex with ATP and a peptide inhibitor........................................................... 9

Figure 1-4: Schematic diagram of interactions between protein kinase residues, ATP and peptide substrate prior to phosphoryl transfer. .................................................................................................. 11

Figure 1-5: Comparison of the structures of staurosporine and the non-hydrolysable ATP analogue, AMP-PNP, when bound to the catalytic subunit of PKA. ....................................................................... 14

Figure 1-6: Rotation of the N-lobe of the PKA catalytic subunit in complex with staurosporine. ............................................................... 16

Figure 1-7: Isoforms of LIMK1 resulting from alternative splicing. ................... 19

Figure 1-8: Isoforms of LIMK2. .............................................................................. 21

Figure 1-9: Polymerisation and depolymerisation of actin. ................................ 24

Figure 1-10: Concentration-dependent effect of cofilin on actin dynamics. ............................................................................................... 27

Figure 1-11: Localisation of cofilin activity in response to EGF stimulation. ............................................................................................ 33

Figure 1-12: Inhibitors of the Rho GTPase/cofilin pathway. .............................. 37

Figure 1-13: Crystallisation phase diagram. ......................................................... 42

Figure 1-14: Protein crystallography pipeline from gene to structure and beyond. ................................................................................................... 45

Figure 3-1: Sequence alignment of the catalytic domain of LIMK1 and other crystallised protein kinases. ...................................................... 74

Figure 3-2: Multiple sequence alignment of LIMK kinase domain constructs. ............................................................................................. 76

List of figures

xix

Figure 3-3: Secondary structure prediction for the kinase domains of human LIMK1 and rat LIMK2. .............................................................. 78

Figure 3-4: Expression of GST-LIMK kinase domain proteins. ........................... 82

Figure 3-5: Optimisation of protein expression. .................................................. 84

Figure 3-6: Expression of endogenous GST-like proteins in insect cells. ........... 87

Figure 3-7: Ammonium sulphate precipitation of LIMK1 kinase domain. ......... 89

Figure 3-8: Thrombin-mediated GST tag removal. .............................................. 92

Figure 3-9: Degradation of LIMK1 kinase domain. .............................................. 94

Figure 3-10: Titration of thrombin used for GST tag removal. ........................... 96

Figure 3-11: Multiple sequence alignment of LIMK1 kinase domain and previously crystallised kinase domains. ............................................. 98

Figure 3-12: Limited proteolysis of GST-tagged LIMK1 kinase domain using Proteinase K. ............................................................................. 100

Figure 3-13: Peptides resulting from the tryptic digestion of a LIMK1 kinase domain fragment resistant to Proteinase K. ......................... 102

Figure 3-14: Amino acid sequence of the truncated LIMK1 kinase domain designed for structure determination by x-ray crystallography. ................................................................................... 103

Figure 3-15: Purification of truncated GST-tagged and tag-free LIMK1 kinase domain. .................................................................................... 107

Figure 3-16: Stability and solubility of truncated LIMK1 kinase domain. ....... 109

Figure 3-17: On-resin cleavage of GST-hLIMK1 kinase domain in the presence and absence of NV10. ......................................................... 111

Figure 3-18: Purification of LIMK1 kinase domain by ion-exchange chromatography. ................................................................................. 113

Figure 3-19: Purification of the LIMK1 kinase domain by size-exclusion chromatography. ................................................................................. 115

Figure 3-20: In vitro phosphorylation of cofilin by purified LIMK1. ................ 117

Figure 4-1: Structure of the LIMK1 kinase domain/staurosporine complex. ............................................................................................... 130

List of figures

xx

Figure 4-2: Polar interactions between staurosporine and residues in the catalytic cleft of LIMK1 kinase domain. ...................................... 131

Figure 4-3: Active conformation of LIMK1 kinase domain in complex with staurosporine. ............................................................................. 133

Figure 4-4: Sequence alignment of LIM and TES kinases. ................................. 135

Figure 6-1: Lipid metabolism. .............................................................................. 149

Figure 6-2: Insulin-induced glucose uptake in adipocytes. ............................... 151

Figure 6-3: Adipose tissue composition. ............................................................. 155

Figure 6-4: Potential role of LIMK2a in the pathogenesis of obesity and insulin resistance. ............................................................................... 160

Figure 8-1: Obesity and adipose tissue phenotype of the LIMK2a knockout mouse. ................................................................................. 180

Figure 8-2: Metabolic characteristics of the LIMK2a knockout mouse. ........... 181

Figure 8-3: Lipid and glucose metabolism in LIMK2a knockout mice. ............ 183

Figure 8-4: Expression of adipogenic marker genes and lipolytic proteins. ............................................................................................... 184

Figure 8-5: Expression of LIMK2 isoforms in adipose tissue from obese and lean mice at mRNA level. ............................................................. 186

Figure 8-6: Expression of LIMK2 isoforms in 3T3-L1 cells undergoing adipogenic conversion. ....................................................................... 188

Figure 8-7: Expression of LIMK and cofilin in 3T3-L1 cells undergoing adipogenesis. ....................................................................................... 189

Figure 8-8: LIMK2 expression and cofilin phosphorylation after transient LIMK2 knockdown and differentiation of 3T3-L1 pre-adipocytes. .................................................................................... 191

Figure 8-9: Lipid accumulation in 3T3-L1 cells after transient LIMK2 knockdown. ......................................................................................... 193

Figure 8-10: Protein expression of LIMK1 and LIMK2 in mouse embryonic fibroblasts. ........................................................................ 195

Figure 8-11: Lipid accumulation in differentiating LIMK2a knockout mouse embryonic fibroblasts. ............................................................ 197

List of figures

xxi

Figure 8-12: Lipid staining in differentiating LIMK2a knockout mouse embryonic fibroblasts. ........................................................................ 198

Figure 8-13: Lipid accumulation in differentiating LIMK2 knockout mouse embryonic fibroblasts. ............................................................ 200

Figure 8-14: In vitro phosphorylation of cofilin by activated and non-activated LIMK2. ................................................................................. 202

Figure 8-15: In vitro phosphorylation of fractionated adipose tissue extract by activated and non-activated LIMK2. ................................ 204

Figure 8-16: In vitro phosphorylation of fractionated NIH3T3 cell extract by activated LIMK2. ............................................................................ 205

List of tables

xxii

LIST OF TABLES

Table 1-1: LIMK expression in mouse tissues. ..................................................... 22

Table 2-1: PCR primers for subcloning of LIMK kinase domain constructs. ............................................................................................. 51

Table 3-1: List of affinity-tagged kinase domain constructs for LIMK1 and LIMK2. ............................................................................................. 80

Table 3-2: Extended list of affinity-tagged kinase domain constructs for LIMK1 and LIMK2. .............................................................................. 105

Table 7-1: Nucleotide sequences of PCR primers used for genotyping. ........... 165

Table 7-2: Sequences of oligonucleotides used for real-time PCR. ................... 170

Table 7-3: Primary and secondary antibodies used for Western blotting. ...... 172

Table 8-1: Peptides identified in differentially phosphorylated protein bands. ................................................................................................... 207

List of publications

xxiii

LIST OF PUBLICATIONS

Mittelstaedt, K., Hoy, A.J., Watt, M.J. and Bernard, O. (2012). LIMK2a deficiency

induces obesity and adipose tissue dysfunction in male mice. Manuscript in

preparation.

List of publications

xxiv

1

CHAPTER 1 − LITERATURE REVIEW

Chapter 1 − Literature review

2


3

1.1 Introduction

The cytoskeleton has proven to be a powerful tool in the course of evolution.

Eukaryotic cells have acquired the ability to utilise a variety of structural and

regulatory proteins to form complex and versatile networks of filaments that could

not be accomplished by the prokaryotic homologues (Wickstead & Gull, 2011).

Among the most remarkable of processes enabled by the eukaryotic cytoskeleton

are the delicate movement, alignment and segregation of chromosomes by

microtubules during mitosis (Civelekoglu-Scholey & Scholey, 2010), and the

formation of structures providing resistance to pressure and shear, as exemplified

by the abundance of intermediate filaments in cells subjected to high mechanical

stress in vivo (Flitney et al., 2009). In addition to microtubules and intermediate

filaments, actin filaments make a significant contribution to the workload handled

by intracellular filamentous structures. Due to their high degree of dynamics and

organisation, actin networks can perform a variety of tasks in a rapid and well-

coordinated fashion. Bundles of aligned actin filaments give rise to filopodial

protrusions used for chemotaxis, whereas a branched network of filaments

supports the leading edge of migrating cells and to generate tensile force for the

support of different cell shapes (Rafelski & Theriot, 2004).

As is the case for other complex biological systems, dysregulation of critical

components can lead to altered performance and eventually disease. Considering

the ubiquitous nature and great functional importance of actin networks, it is not

surprising that dysregulation of actin dynamics has been implicated in the

increased invasiveness of cancer cells as well as in the pathogenesis of muscle- and

neuro-degenerative diseases (Nurnberg et al., 2011; Ono, 2010; Ramakers, 2002).

This thesis focuses on the LIM kinases (LIMKs), a family of protein kinases that is

involved in the regulation of actin dynamics. LIMK1 has been implicated in cancer

cell invasion and metastasis due to its pivotal role in regulating cell motility.

Therefore, it is considered a potential target for therapeutic intervention and there

are considerable efforts underway to develop LIMK1-specific small-molecule

inhibitors. Despite exhaustive characterisation of LIMK1 at a functional level,

detailed structural insights have remained elusive. High-resolution models of the


4

protein defining the structural features that are unique to LIMKs (especially those

located around the substrate-binding sites for adenosine triphosphate [ATP] and

the phosphoryl group-accepting protein) would be highly advantageous for the

development of inhibitors, as they can accelerate the drug discovery process

through enabling structure-based, rational drug design and optimisation.

1.2 Protein kinases

Protein phosphorylation is the most common type of post-translational

modification used in signal transduction and plays essential roles in virtually all

basic cellular processes. Approximately 30% of all cellular proteins are

phosphorylated on at least one residue (Cohen, 2000). The phosphoryl group is

transferred from adenosine triphosphate (ATP) to a substrate molecule in an

enzymatic reaction that is catalysed by a kinase. The covalent bond between the

substrate molecule and the phosphoryl group can be broken by phosphatases.

Phosphorylation and dephosphorylation allow intracellular signalling events to be

initiated and ‘reset’ for tight control of cellular functions.

Among the 518 known protein kinases encoded in the human genome, 478 kinases

are related in the sequence of the catalytic domain and are hence referred to as

typical protein kinases (Figure 1-1). The remaining 40 kinases that possess

biochemical kinase activity but lack sequence similarity to conventional kinases

are termed atypical protein kinases (Manning et al., 2002).


5

Figure 1-1: Phylogenetic tree of human protein kinases.

Most of the 518 human protein kinases are shown in a dendrogram based on sequence similarity between protein kinase domains. Each kinase forms the tip of a branch. Distance between two kinases is inversely related to their similarity. Seven major groups of kinases are coloured differently and are named AGC (containing protein kinase A, protein kinase G and protein kinase C families), CAMK (calcium/calmodulin-dependent protein kinase), CK1 (casein kinase 1), CMGC (containing cyclin-dependent protein kinase, mitogen-activated protein


6

kinase, glycogen synthase kinase 3 and Cdc2-like kinase families), STE (homologues of yeast Sterile 7, Sterile 11 and Sterile 20 kinases), TK (tyrosine kinase) and TKL (tyrosine kinase-like). Atypical protein kinases are not shown. Adapted from (Manning et al., 2002).

Another commonly used classification system groups the kinases according to the

phosphoryl group-accepting residue in the protein substrate. The 90 human

tyrosine kinases are divided into receptor tyrosine kinases containing a trans-

membrane domain (58), and non-receptor tyrosine kinases (32) (Robinson et al.,

2000). The receptor type kinases are unique to metazoans and mediate cell-to-cell

communication during embryonic development and adult homeostasis. Prominent

examples for receptor tyrosine kinases include the epidermal growth factor

receptor (EGFR) and the insulin receptor (IR), which are critical components in the

regulation of chemotactic movement and insulin-induced signalling processes,

respectively (Boura-Halfon & Zick, 2009; Wang et al., 2007). Receptor tyrosine

kinases are activated by ligand-induced oligomerisation, which juxtaposes the

cytoplasmic kinase domains (Figure 1-2). This juxtaposition leads to stabilisation

of a catalytically competent kinase conformation by varied mechanisms such as

allosteric activation and trans-phosphorylation (Zhang et al., 2006).


7

Figure 1-2: Ligand-induced dimerisation of the EGF receptor.

The ectodomain of the EGF receptor is composed of four subdomains (L1, CR1, L2, CR2) and is connected to the cytoplasmic kinase domain through a single-pass transmembrane domain. Upon ligand binding (EGF; coloured orange), the receptor forms a dimer (cyan and purple), thereby inducing interaction between the N-lobe of one kinase with the C-lobe of the other kinase and leading to allosteric kinase activation. Adapted from (Hubbard & Miller, 2007).


8

Serine/threonine kinases form the largest class (388) among the typical protein

kinases, which are related in their catalytic domain sequence. These kinases are

involved in a plethora of cellular processes ranging from cell cycle control and DNA

damage repair to the regulation of cellular metabolism and cytoskeletal

organisation (Cohen, 1978; Edwards et al., 1999; Ekim et al., 2011; Lee & Paull,

2007). Examples for serine/threonine kinases are cAMP-dependent protein kinase

(also referred to as protein kinase A or PKA), which is considered a prototypical

member of the protein kinase superfamily, and LIMK1, which represents the focus

of the current study.

For about one third of all human protein kinases, the structure of the catalytic

domain has been determined (SGC, 2011). All protein kinase domains share a

characteristic two-lobed fold (Knighton et al., 1991). It encompasses ~290

residues forming an N-terminal lobe composed of a twisted five-stranded anti-

parallel -sheet and a single -helix (also referred to as C helix), and a large C-

terminal lobe of predominantly α-helical secondary structure (Figure 1-3).


9

Figure 1-3: Crystal structure of the catalytic subunit of PKA in complex with

ATP and a peptide inhibitor.

The two lobed kinase fold is shown as a ribbon diagram based on PDB accession code 1ATP (Zheng et al., 1993). The N-lobe and C-lobe of the catalytic subunit are shown in blue and red, respectively, with -strands labelled 1-8 and -helices labelled A-J. The non-conserved αB helix is shown in turquoise, the protein kinase inhibitor peptide, PKI(5-24), is coloured green, and the structure of ATP is shown as ball-and-stick representation. Adapted from (Hodgson & Schroder, 2011).


10

In addition, the C-terminal lobe contains the activation segment, which is defined

as the region spanning the characteristic sequence motifs DFG and APE and forms

a crucial part of the substrate-binding site (Nolen et al., 2004; Skamnaki et al.,

1999).

A multitude of interactions contribute to the correct orientation of ATP in the

catalytically competent conformation of the kinase domain. These interactions are

outlined below using the example of protein kinase A. Nitrogen atoms N1 and N6

of the ATP adenine ring form hydrogen bonds with the peptide backbone of the

hinge region, while aspartate 184 of the conserved DFG motif in the activation

segment coordinates two Mg2+ ions, which in turn ensure that the triphosphate

points out of the adenosine pocket, facing the peptide substrate to enable transfer

of the -phosphate group (Figure 1-4) (Endicott et al., 2012). The orientation of

ATP in its binding pocket is supported by van der Waals contacts between the ATP

purine moiety and non-polar aliphatic groups lining the pocket. The ribose moiety

of ATP is held in place by hydrogen bonds between oxygen O2’ and glutamate 127,

and O3’ and the main chain carbonyl oxygen of glutamate 170. The correct

orientation of the α- and β-phosphate groups is assisted by glutamate 91 of the N-

terminal lobe and lysine 72 of β-sheet 3.


11

Figure 1-4: Schematic diagram of interactions between protein kinase

residues, ATP and peptide substrate prior to phosphoryl transfer.

The PKA residues involved in the binding and correct orientation of ATP and the peptide substrate are shown in black. ATP and peptide substrate are coloured blue and green, respectively. Red arrows indicate the mechanism of base-assisted nucleophilic attack. Adapted from (Hodgson & Schroder, 2011)


12

In the inactive conformation of the kinase domain, the activation segment is

partially disordered, which contributes to the structural diversity among inactive

kinases (Noble et al., 2004). In contrast, phosphorylation of a specific residue in the

activation loop (threonine 197 in PKA) induces complex structural changes that

promote closure of the two lobes to adopt the common catalytically competent

conformation of active kinases (Kornev et al., 2006). Most kinases recognise their

substrate by the unique sequence and structure surrounding the potential

phosphorylation site (Lowe et al., 1997). This region on the surface of the substrate

protein is usually less well-ordered, thereby allowing it to remodel for proper fit

into the catalytic site (Iakoucheva et al., 2004). The binding process is supported

by recognition pockets on the kinase domain to prevent promiscuous substrate

phosphorylation.

Phosphorylation can occur on the hydroxyl group of aliphatic (serine or threonine)

or aromatic (tyrosine) amino acid residues, depending on the kinase-specific

orientation of a sub-element of the activation segment that promotes preferential

binding of the bulkier aromatic or the smaller aliphatic residues (Nolen et al.,

2004). Aided by the activation segment and other conserved residues, the

substrate is oriented with the phosphoryl-accepting hydroxyl group facing the

catalytic aspartate (residue 166 in PKA). Following the binding and correct

orientation of ATP and the peptide substrate in the catalytic cleft, phosphoryl

transfer is achieved through base-assisted nucleophilic attack by the substrate’s

hydroxyl group on the γ-phosphoryl group of ATP (Valiev et al., 2007).

1.3 Protein kinase inhibitors

Mutations in protein kinases and defects in their regulation have been associated

with human disease, thus making this class of enzymes potential targets for

therapeutic intervention (Stenberg et al., 2000; Yang & Guan, 2007). The design of

selective small-molecule inhibitors binding to the conserved ATP-binding site and

less conserved surrounding pockets has been driven by differences in kinase

structure and pliability (Tibes et al., 2005). In recent years, numerous clinical trials


13

have been conducted to evaluate compounds that target kinases involved in

different stages of signal transduction (Noble et al., 2004).

Knowledge of relevant protein structures is the key to rational drug design and can

reduce the number of compound syntheses necessary for optimisation of a

potential drug (Breitenlechner et al., 2005a). A structure of the target protein

provides information on potential binding sites and substrate geometries and

enables a focused approach to screening for potential leads. In order to identify

alternate conformations that may be critical for target selectivity, multiple

structures of the target protein are needed. Solving the structure of the target

protein in a complex with inhibitor compounds can assist in increasing inhibitor

potency by optimising target-specific interactions and optimising pharmacokinetic

properties by targeted variation of moieties that are not essential for binding. In

addition, comparison of inhibitor binding modes in related proteins can enhance

target selectivity.

The microbial alkaloid staurosporine from Streptomyces staurosporeus is a potent

general inhibitor of protein kinases (Karaman et al., 2008). To establish a

structural model system for inhibitor binding to the kinase domain, the structure

of the bovine catalytic subunit of PKA has been solved in complex with

staurosporine (Prade et al., 1997). In the structure of the ternary complex with the

pseudo-substrate protein inhibitor peptide PKI(5-24) (PDB accession code 1STC),

staurosporine binds to the adenosine binding site in the cleft between the N-lobe

and C-lobe of the catalytic domain. However, the position of the triphosphate

group of ATP is left unoccupied (Figure 1-5).


14

Figure 1-5: Comparison of the structures of staurosporine and the non-

hydrolysable ATP analogue, AMP-PNP, when bound to the catalytic subunit of

PKA.

Orientation of bound staurosporine (thick lines) and AMP-PNP (5'-adenylyl- , -imidodiphosphate; thin lines) is shown in a stick representation derived from superimposing helices E and F of the staurosporine/PKA complex with the same helices of the AMP-PNP/PKA complex (PDB accession code 1CDK) (Bossemeyer et al., 1993). Adapted from (Prade et al., 1997).


15

Inhibitor binding is accompanied by extensive conformational changes as a result

of an induced fit mechanism, leading to a unique open conformation of the catalytic

domain (Figure 1-6). Tight binding of staurosporine is achieved by mimicking

aspects of the adenosine moiety of ATP and forming a larger total number of van

der Waals and polar interactions with residues lining the binding site than ATP.

This structure revealed a previously unknown degree of enzyme flexibility and

provided an explanation for the high inhibitory potential of staurosporine.


16

Figure 1-6: Rotation of the N-lobe of the PKA catalytic subunit in complex

with staurosporine.

Structures of the PKA catalytic subunit in complex with PKI (green Calpha atom trace; PDB accession code 2CPK (Knighton et al., 1991)), PKI in combination with AMP-PNP (blue C atom trace; PDB accession code 1CDK (Bossemeyer et al., 1993)), and PKI in combination with staurosporine (light purple C atom trace) were aligned using the central helices E and F from the C-lobe. Staurosporine is shown in ball-and-stick representation. Adapted from (Prade et al., 1997).


17

1.4 LIM kinases as regulators of actin dynamics

The serine/threonine kinase LIMK1 is a critical regulator of the actin cytoskeleton

and has been implicated in cancer cell invasion and metastasis. Despite the lack of

an atomic structural model for its kinase domain, several drug development

programs have been initiated with the aim of targeting the LIMK family.

1.4.1 Gene structure and expression pattern of LIMKs

The mouse Kiz-1 gene (now known as LIM kinase 1 or LIMK1), a homologue of the

human LIMK1 gene, was initially identified in a polymerase chain reaction (PCR)

screen for novel protein tyrosine kinases from immortalised olfactory epithelial

cell mRNA (Bernard et al., 1994). The gene is highly conserved between mouse and

human, and encodes a 647-amino acid protein containing two repeats of the

cysteine-rich LIM/double zinc finger motif at the N-terminus, one internal PDZ

domain and an unusual protein kinase domain at the C-terminus (Figure 1-7)

(Bernard et al., 1994; Okano et al., 1995; Yang et al., 1998). Being the first protein

kinase to contain the LIM motif, it was classified into a novel subfamily of

serine/threonine protein kinases, the LIM kinase family (Okano et al., 1995). The

LIM domain (named after the proteins LIN-11, Isl1 and MEC-3, which also contain

this functional domain) serves as modular protein-binding interface. It is found in

a large number of proteins with diverse cellular functions such as gene expression,

cell adhesion and signal transduction (Kadrmas & Beckerle, 2004). The PDZ

domain, whose name is derived from the proteins PSD-95, Discs-large and ZO-1, is

another protein-protein interaction module that is often found in multi-domain

scaffolding proteins (Tonikian et al., 2008).

In addition to the full-length LIMK1 transcript, there are two additional mRNA

species resulting from alternative splicing (Bernard et al., 1994). A 60-nucleotide

sequence located on a separate exon (exon 11) in the middle of the kinase domain

can be spliced out, resulting in the gene product LIMK1-short (LIMK1-s), which

lacks kinase activity due to modification of the ATP binding site (Bernard, 2007)


18

and acts as a dominant-negative for LIMK1 function (Arber et al., 1998). dLIMK1,

the second splice variant, is completely devoid of the kinase domain (Edwards &

Gill, 1999). LIMK1 protein is expressed in all embryonic and adult mouse tissues

with high levels found in brain, kidney, lung, stomach and testes.

Immunofluorescence analysis demonstrated that in cultured cells LIMK1 is

localised mainly in the cytoplasm, in particular at focal adhesions and in the

perinuclear region (Foletta et al., 2004).


19

Figure 1-7: Isoforms of LIMK1 resulting from alternative splicing.

The 647-amino acid protein, LIMK1, consists of two N-terminal LIM domains, a PDZ domain and a C-terminal kinase domain. The dominant-negative form, LIMK1-s (also referred to as LIMK1-short), lacks a short sequence of 20 amino acids in the ATP binding site of the kinase domain. The third isoform, dLIMK1, is devoid of the kinase domain.


20

LIMK1 has a paralog in the 638-amino acid protein LIMK2. The two LIMK family

members have an identical domain structure (Figure 1-8) and share 50% overall

amino acid identity (Okano et al., 1995). In the kinase domain, the sequence

identity is as high as 70%. Four mRNA species of LIMK2 have been reported. The

full-length transcript of the LIMK2 gene encoding the two LIM domains, one PDZ

domain and a kinase domain is called LIMK2a (Ikebe et al., 1997). A shorter

transcript, named LIMK2b, is generated from the same gene through usage of an

alternative promoter, whereby the first half of the 5’ LIM domain is replaced with a

LIMK2b-specific sequence. The LIMK2c isoform contains a frame shift-inducing

insertion in the kinase domain and gives rise to a protein lacking the entire kinase

domain (Ikebe et al., 1998), while LIMK2d contains only the LIM domain (Nunoue

et al., 1995). The fifth, testes-specific isoform, which is referred to as LIMK2t,

consists of half of the PDZ domain and the kinase domain.

In mice, LIMK2a mRNA is expressed in the brain, heart, lung, thymus, spleen,

kidney and stomach, while LIMK2b mRNA was detected only in the brain, heart,

lung and stomach (Koshimizu et al., 1997) (Table 1-1). Due to the lack of isoform-

specific antibodies, there is no direct evidence for the existence of LIMK2c, LIMK2d

and LIMK2t beyond transcript level.

The intracellular localisation of LIMK1 and LIMK2 differ significantly (Acevedo et

al., 2006; Foletta et al., 2004). While LIMK1 is localised mainly to focal adhesions,

LIMK2 appears to be localised to punctae resembling endosomes. It has been

suggested that LIMK2 may have cellular functions distinct from LIMK1 in addition

to their regulatory effect on actin polymerisation (Scott & Olson, 2007).


21

Figure 1-8: Isoforms of LIMK2.

The 638-amino acid protein, LIMK2a, has the same domain structure as LIMK1, consisting of two N-terminal LIM domains, a PDZ domain and a C-terminal kinase domain. As a result of alternative promoter usage, LIMK2b contains only one and a half LIM domains and a unique N-terminal sequence. The testes-specific isoform, LIMK2t, consists of a truncated PDZ domain and a kinase domain. Additional isoform of LIMK2, named LIMK2c and LIMK2d, have been identified, but are less well characterised.


22

Table 1-1: LIMK expression in mouse tissues.

LIMK1

- expressed in all mouse tissues

examined

- highest expression in brain, kidney,

lung, stomach and testes (Foletta et

al., 2004)

LIMK2

- widely expressed in all mouse

tissues

- high protein levels found in brain,

stomach, intestine, lung, heart, uterus

and skin (Acevedo et al., 2006)

- in testes exclusively expressed in

elongated spermatids

- LIMK2a mRNA expressed in the

mouse brain, heart, lung, thymus,

spleen, kidney and stomach

- LIMK2b mRNA expressed only in

the brain, heart, lung and stomach

(Koshimizu et al., 1997)

- LIMK2t mRNA exclusively expressed

in the testes (Takahashi et al., 1998)


23

1.4.2 Actin polymerisation and depolymerisation

Actin filaments (also referred to as F-actin) consist of globular monomeric actin

subunits (also called G-actin) that are joined together by non-covalent bonds

(Figure 1-9). Upon polymerisation, i.e. addition of actin subunits to the barbed end

of the filament, the ATP molecule bound to the subunit is hydrolysed, leading to a

conformational change that decreases the stability of the filament (Belmont et al.,

1999; Korn et al., 1987). As a result, the filament readily disassembles from the

pointed end to replenish the pool of monomeric actin. At steady state, filament

elongation at the barbed end balances filament depolymerisation at the pointed

end. This dynamic balance is referred to as treadmilling and results in propulsion

of the filament while maintaining its length (Bugyi & Carlier, 2010). Treadmilling

occurs in vitro at a relatively low speed and is accelerated and refined in vivo by a

multitude of actin-binding proteins (dos Remedios et al., 2003). Their functions

include monomer sequestration as well as filament end capping, severing and

cross-linking.


24

Figure 1-9: Polymerisation and depolymerisation of actin.

Actin filaments are assembled by addition of ATP-bound actin monomers (shown in red) to the barbed end. Hydrolysis of ATP into ADP and Pi leads to destabilisation of the filament, which depolymerises at the pointed end by releasing ADP-bound actin subunits (grey).


25

1.4.3 LIMK substrates

Both LIM kinases have been shown to regulate the organisation of the actin

network through phosphorylation of members of the actin-depolymerising factor

(ADF)/cofilin family of actin-binding proteins (Arber et al., 1998; Sumi et al., 1999;

Yang et al., 1998). Three forms of ADF/cofilin proteins are expressed in

mammalian cells: ADF (also called destrin), cofilin-1 and cofilin-2. Cofilin-2 is the

most abundant form in muscle, while expression of cofilin-1 prevails in non-muscle

tissues. These proteins consist of a single functional domain, which is termed ADF-

homology domain and mediates interactions with both monomeric and

filamentous actin. The different ADF/cofilin isoforms have qualitatively similar, yet

quantitatively different effects on actin dynamics (Van Troys et al., 2008). ADF

induces turnover of actin filaments most efficiently and has weaker nucleating

activity than cofilin. Cofilin-2 has the lowest actin depolymerising activity and

promotes filament assembly at steady state.

Importantly, knockout of cofilin-1 is embryonically lethal in mice, while ADF

knockout embryos survive and develop postnatal blindness (Gurniak et al., 2005;

Ikeda et al., 2003). Due to its essential role in embryonic development and cell

function, cofilin-1 has been more widely studied (Bernstein & Bamburg, 2010) and

will be herein referred to as cofilin.

The various effects of cofilin were found to be dependent on its concentration

(Andrianantoandro & Pollard, 2006). At low cofilin/actin ratios, binding of cofilin

to filamentous actin induces a conformational twist, which destabilises the

filament and causes persistent severing, as cofilin is recycled rapidly (Figure

1-10 A). Due to cofilin’s higher affinity for ADP-bound actin, severing occurs more

frequently at the pointed end of the filament. At high cofilin/actin ratios, several

cofilin molecules cooperatively bind to the same region of the filament, thereby

stabilising it in the twisted form (Figure 1-10 B). As cofilin is sequestered on this

region, filament severing is not persistent and actin nucleation prevails.

Spontaneous actin polymerisation is accelerated through binding of cofilin to actin

monomers. Accordingly, the presence of an intracellular cofilin gradient (referring


26

to both relative abundance and activity) may represent the “switch” in the various

activities of cofilin (Van Troys et al., 2008).

LIMK-mediated phosphorylation on serine-3 abolishes cofilin’s ability to bind to

actin, thereby inhibiting its depolymerising and severing effect on filamentous

actin (Agnew et al., 1995; Maciver et al., 1991; Moriyama et al., 1996). Cofilin can

be activated by dephosphorylation mediated by the chronophin and slingshot

phosphatases (Huang et al., 2006).


27

Figure 1-10: Concentration-dependent effect of cofilin on actin dynamics.

Cofilin (shown in purple) binds preferentially to ADP-actin in filaments, thereby inducing a twist. A: At low cofilin:actin ratios, cofilin causes persistent filament severing near the pointed end and cofilin is recycled rapidly. B: At high cofilin:actin ratios, cofilin binds cooperatively to the ADP-actin-containing region of the filament, leading to stabilisation of the saturated pieces upon severing. The stabilised filament fragments can serve as nucleus for actin polymerisation. Adapted from (Andrianantoandro & Pollard, 2006).


28

1.4.4 Regulators of LIMK activity

1.4.4.1 Rho GTPases

The family of Rho GTPases consisting of Rho, Rac and Cdc42 represents the major

regulators of cytoskeletal rearrangements (Hakoshima et al., 2003). Like other

GTPases, they serve as molecular switches in signal transduction pathways. In

their GTP-bound "on" state, they interact with their downstream effectors until

their intrinsic GTPase activity converts GTP to GDP and thus turns the GTPase

"off". This mechanism is fine-tuned through the action of GTPase-activating

proteins (GAPs), guanine nucleotide exchange factors (GEFs) and guanine

nucleotide dissociation inhibitors (GDIs). Post-translational modification of Rho

GTPases by addition of a lipid moiety mediates their localisation at the plasma

membrane. Upon interaction with GDIs, the GTPase is removed from the

membrane and translocates into the cytoplasm, where it remains inactive.

The individual members of the Rho GTPase family each have distinct functions. In

Swiss 3T3 cells, activated Rho, Rac and Cdc42 induce formation of stress fibres and

focal adhesions, lamellipodia, and filopodial protrusions, respectively (Nobes &

Hall, 1995). However, more recent studies suggest that the Rho GTPases are

involved in complex, inter-linked activation cascades resulting in coordinated

effects on actin organisation (Machacek et al., 2009).

Downstream of the Rho GTPases, the effector kinases ROCK (Rho-associated coiled

coil-containing protein kinase) and Pak (p21-activated kinases) 1 and 4 activate

LIMKs by phosphorylation of specific threonine residues located in the activation

loop of the kinase domain. LIMK1 is phosphorylated on threonine-508 by ROCK

and Pak 1 and 4 (Dan et al., 2001; Edwards et al., 1999; Ohashi et al., 2000),

whereas phosphorylation of LIMK2 on threonine-505 is mediated by ROCK (Sumi

et al., 2001a). It has also been suggested that both LIMKs are phosphorylated by

myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK ) downstream

of Cdc42 (Sumi et al., 2001b). Specifically, LIMK2 was phosphorylated by MRCK

on threonine-505 in vitro, supporting a role for LIMK2 in Cdc42-induced formation

of filopodia in addition to that in Rho-induced stress fibre formation (Amano et al.,

2001; Sumi et al., 1999).


29

1.4.4.2 Regulation of LIMK1 activity

Stimuli leading to the inactivation of cofilin through phosphorylation by LIMK1

include epidermal growth factor (EGF), transforming growth factor- (TGF ) and

stromal cell-derived factor 1 (SDF1), which have been implicated in the

stimulation of cell migration and correlate with tumour progression (Wang et al.,

2007). Upon ligand binding to the corresponding cell surface receptor, a signalling

cascade is initiated beginning at the level of the Rho family GTPases Rho, Rac and

Cdc42. LIMK1, which is activated by the Rho effector kinases ROCK and PAK 1 and

4, acts as a convergence point in this pathway to integrate a number of upstream

signals.

Phosphorylation of LIMK1 on threonine 508 promotes Hsp90-mediated

homodimer formation to enable further activation through trans-phosphorylation

of the kinase domain. Trans-phosphorylation greatly increases LIMK1 stability

(half-life of ~20 hours) compared to the non-phosphorylated monomer with a

half-life of ~4 hours (Li et al., 2006).

There is experimental evidence to suggest that the LIM domains are involved in the

self-association of LIMK1. It has been shown that the N-terminal LIM domain of

LIMK1 can bind to the protein kinase domain, providing a putative bimodal model

for LIMK1 self-association (Hiraoka et al., 1996). Intermolecular interactions

between these two domains may facilitate the formation of LIMK1 dimers. The

dimerisation or oligomerisation of protein kinases can lead to enhanced kinase

activity, as exemplified by the receptor tyrosine kinases (He & Hristova, 2008).

Intra-molecular association of the LIM domain and the kinase domain may occur in

the monomeric form, providing a potential mechanism for the regulation of the

catalytic function of LIMK1.

The phosphatase slingshot (SSH) serves a dual function in the regulation of actin

dynamics, as it is both a cofilin and LIMK phosphatase. Dephosphorylation of

cofilin results in re-activation of its actin-depolymerising and severing function,

whereas dephosphorylation of LIMK inhibits further cofilin phosphorylation

(Soosairajah et al., 2005). Furthermore, LIMK1 protein levels are regulated by

Rnf6, an E3 ubiquitin ligase that targets LIMK1 for proteasome-mediated

degradation in neurons (Tursun et al., 2005).


30

1.4.4.3 Regulation of LIMK2 activity

Activation of the GTPases Rho and Cdc42 induces LIMK2-mediated

phosphorylation of cofilin, whereby the individual Rho GTPases exert differential

effects on the organisation of the actin cytoskeleton (Sumi et al., 1999). Rho

activation induces the formation of stress fibres, whereas activation of Cdc42

induces the formation of filopodia. These cytoskeletal rearrangements are

dependent on LIMK2 activity as demonstrated by co-expression of kinase-dead

LIMK2, which acts as a dominant-negative form of the protein. Vardouli et al.

reported that transforming growth factor-beta 1 (TGF- 1) induces LIMK2-

mediated phosphorylation of cofilin through activation of RhoA and RhoB in

Swiss3T3 cells (Vardouli et al., 2005), thereby implicating the

Rho/ROCK/LIMK2/cofilin pathway in the co-ordination of cytoskeletal changes in

response to growth factor treatment.

It has been suggested that Hsp90 also promotes the formation of LIMK2 dimers, as

it contains the same amino acid sequence that is responsible for Hsp90 binding as

LIMK1 (Li et al., 2006). Furthermore, in vitro dephosphorylation studies suggest

that LIMK2 is dephosphorylated by SSH, albeit less efficiently than LIMK1

(Soosairajah et al., 2005). N-terminally truncated LIMK2 lacking both LIM domains

has ~3-fold higher catalytic activity toward cofilin than the full-length protein,

thereby providing further evidence for a negative regulatory effect of inter- or

intra-molecular interactions between the LIM domain and the protein kinase

domain on LIMK activity (Sumi et al., 1999).

1.4.5 LIMK knockout mice

LIMK1 knockout mice, generated by deletion of the exons containing the second

LIM domain and the PDZ domain, are largely normal and fertile (Meng et al., 2002).

They show a mild central nervous system phenotype, including abnormalities in

spine morphology and synaptic function as well as altered fear response and

spatial learning, thereby indicating a role for LIMK1 in dendritic spine

morphogenesis and brain function. However, these mice still express the kinase


31

domain and should therefore be considered hypomorphs. Similar to LIMK1

knockout mice, LIMK2 knockout mice are normal and fertile (Takahashi et al.,

2002). The only phenotypic abnormality was a reduction in testes size, associated

with partial degeneration of spermatogenic cells and increased apoptosis. The

LIMK1/LIMK2 double knockout mouse showed more severe defects in cofilin

phosphorylation and synaptic function in the brain compared to the single

knockout mice, suggesting a differential involvement of the two LIMK family

members in brain function (Meng et al., 2004). Recently, Rice et al. reported the

generation of a LIMK2 knockout mouse. In these mice the kinase domain was

deleted by homologous recombination. As a result, none of the LIMK2 isoforms are

expressed. These mice exhibited an “eyes open at birth” (EOB) phenotype due to

inhibited keratinocyte migration during eyelid development.

1.5 Regulation of actin dynamics in cell migration

1.5.1 Cofilin-mediated actin dynamics in cell motility

The ability to change cell shape is an important feature of migrating eukaryotic

cells (Pollard & Cooper, 2009). Polymerisation of monomeric actin into filaments

represents the driving force behind the formation of membrane protrusions,

entailing deformation of the plasma membrane and stabilisation through an array

of Y-shaped branches or linear bundles of actin filaments. Branched actin arrays

are commonly found in sheet-like structures such as lamellipodia (Campellone &

Welch, 2010), whereas linear bundles are involved in the formation of thinner

structures such as filopodia (Yang et al., 2007).

The dynamic architecture of the actin cytoskeleton is highly regulated both

temporally and spatially to allow quick adaptation in response to intracellular and

extracellular cues (Rafelski & Theriot, 2004). The formation of oligomeric actin

nuclei is the rate-limiting step in actin polymerisation in vitro (Cooper et al., 1983).

An efficient way of increasing the amount of filamentous actin is the severing of

existing filaments to create free high-affinity, or barbed ends, which can serve as

nuclei for actin polymerisation (Allen, 2003; Chen et al., 2001). These de novo actin


32

nucleation sites are created by non-phosphorylated cofilin upon binding to F-actin,

which changes the twist of the filament, leading to its destabilisation. Cofilin

binding to ADP-actin also increases the rate of depolymerisation at the pointed

end, thereby inducing the release of free monomers for barbed end growth. In

contrast, phosphorylation by LIMK inhibits cofilin’s actin severing and

depolymerising activity, leading to accumulation of filamentous actin (Yang et al.,

1998).

1.5.2 Regulation of cofilin activity in directed cell migration

Extracellular signals such as growth factors are involved in the regulation of the

growth and differentiation programmes of epithelial cells. Aberrant signalling has

been associated with malignant transformation (Zhang et al., 2010). Migration of

cells along a gradient of Epidermal Growth Factor is a well-established process

involved in the metastasis of mammary tumour cells and has been shown to

require cofilin activity (Mouneimne et al., 2004; Wyckoff et al., 2004). The activity

of cofilin is regulated by kinases and phosphatases that act in concert to influence

the architecture of the actin cytoskeleton in response to extracellular cues (Wang

et al., 2007). Three major processes are involved in the asymmetric regulation of

cofilin activity required for directional cell motility (Figure 1-11): First,

phospholipase C (PLC )-mediated hydrolysis of phosphatidylinositol-4,5-

bisphosphate (PIP2) (Mouneimne et al., 2006), leading to the release of active

cofilin at the site of stimulation; second, phosphorylation of cofilin on serine 3 by

LIMK1 and its related kinases (Zebda et al., 2000); and third, dephosphorylation of

serine 3 by slingshot and chronophin phosphatases (Gohla et al., 2005; Niwa et al.,

2002). Release of active cofilin adjacent to the plasma membrane facing the source

of EGF and global inhibition of cofilin by active LIMK1 produce an intracellular

gradient of active cofilin. Locally activated cofilin severs actin filaments, thereby

generating free actin filament barbed ends as sites for actin polymerisation. As a

result, directed protrusions are formed that determine the direction of cell

migration (Ghosh et al., 2004).


33

Figure 1-11: Localisation of cofilin activity in response to EGF

stimulation.

EGF-stimulated phospholipase C (PLC ) hydrolyses phosphatidylinositol-4,5-bisphosphate (PIP2), causing the release of active cofilin from its complex with PIP2 at the plasma membrane. Transient cofilin activity occurs as the result of the near simultaneous activation of cofilin-activating molecules (e.g. SSH: slingshot) and cofilin-inactivating molecules (e.g. LIMK) within the cofilin pathway. Activated cofilin severs filaments locally to start polymerisation and cell protrusion, while globally active LIMK inactivates cofilin diffusing from the initial site of activation. Adapted from (Wang et al., 2007).


34

Following the formation of a cell protrusion, cell-matrix adhesions are established

at the leading edge to connect the extracellular matrix to the actin network,

thereby forming an anchor that keeps the protrusion in place (Nobes & Hall, 1995).

In response to RhoA activation, myosin light chain (MLC) is phosphorylated by

ROCK, which induces assembly of myosin filaments (Amano et al., 1996). In

conjunction with anti-parallel F-actin, these filaments constitute contractile

bundles called stress fibres, which associate with focal adhesions and apply the

force that enables retraction of the rear of the cell (Chrzanowska-Wodnicka &

Burridge, 1996; Small & Resch, 2005).

1.5.3 The role of LIMK1 in cancer metastasis

Remodelling of actin filaments is essential for the formation and retraction of path-

finding structures used in the chemotaxis, cell migration and invasion of tumour

cells (Wang et al., 2007). There is strong evidence for a role of LIMK1 in cancer cell

invasion. The expression level and activity of LIMK1 is increased in highly invasive

breast and prostate cancer cell lines compared to non-invasive cells, and high

levels of LIMK1 expression have been observed in human prostate tumours (Davila

et al., 2003; Yoshioka et al., 2003). Further studies revealed that over-expression of

LIMK1 in non-invasive breast and prostate cell lines increases cell motility and

invasion, whereas over-expression of dominant-negative LIMK1 or siRNA-

mediated knock-down of LIMK1 results in decreased cell motility and impairs the

formation of osteolytic lesions in a mouse model of tumour invasion (Bagheri-

Yarmand et al., 2006; Davila et al., 2003; Horita et al., 2008; Yoshioka et al., 2003).

Pharmacologic inhibition of LIMK was also shown to antagonise TGF -induced

invasion of mouse mammary epithelial cells (Morin et al., 2011).

However, due to the requirement of a well-balanced cofilin pathway in order for

protrusion, cell migration and chemotaxis to occur optimally (Wang et al., 2007), it

is likely that the expression level of other regulatory or effector proteins within

this pathway is altered co-ordinately in vivo. Gene expression analysis of invasive

primary tumour cells demonstrated that both LIMK1 and cofilin mRNA are co-

ordinately up-regulated (Wang et al., 2004).


35

1.5.4 Inhibitors of the Rho GTPase/cofilin pathway

There is accumulating evidence that the overall activity of the cofilin pathway

determines the invasive and metastatic phenotype of tumour cells (Wang et al.,

2007). Therefore, it has been predicted that inhibitors directed at the output of this

pathway (i.e. the generation of actin filament barbed ends) may have therapeutic

benefit in combating the spread of cancer (Wang et al., 2004). Several different

approaches have been explored to control the regulation of the cofilin pathway at

the level of the Rho GTPases, their effector kinases and at the convergence point of

LIMK1 activity (Figure 1-12).

The Rac-specific inhibitor, EHT 1864 (developed by Exonhit Therapeutics, Paris,

France), binds to Rac1 and locks it in an inactive state (Shutes et al., 2007). The

inhibitor potently inhibits Rac1-mediated changes in cellular morphology and

cellular transformation. In addition to Rac1, EHT 1864 also binds to Rac1b and

Rac2 with similar affinity.

In contrast to most kinase inhibitors that bind to the conserved ATP-binding

pocket within the kinase domain, the inhibitor IPA-3 targets the auto-regulatory

domain of p21-activated kinase 1 (Pak1) (Deacon et al., 2008). Inhibitor binding

prevents the initiation of conformational changes and auto-phosphorylation events

that lead to kinase activation. The strategy of targeting auto-regulatory domains

within kinases may be advantageous in terms of selectivity towards a particular

kinase or class of kinases.

ROCK promotes actin-myosin-mediated contractile force generation and

morphological changes in cells through phosphorylation of LIMKs and other

downstream targets (Olson, 2008). Due to its role in processes like cell motility,

adhesion, smooth muscle contraction, neurite retraction and phagocytosis, ROCK

has attracted significant interest as a potential target for the treatment of a wide

range of pathological conditions including cancer, neuronal degeneration, kidney

failure, osteoporosis and cardiovascular diseases. In Japan, the small-molecule

ROCK inhibitor, fasudil (developed by Asahi Kasei Pharma, Tokyo, Japan), has been

in clinical use since 1995 to prevent cerebral vasospasms associated with

subarachnoid haemorrhage (Mueller et al., 2005). However, due to its broad

specificity it is still unclear whether its clinical effects result from the inhibition of


36

ROCK and/or other kinases. Even if inhibition of ROCK itself is responsible for

producing the clinical outcome, the truly important targets may be downstream of

ROCK, such as LIMK1 and LIMK2 (Olson, 2008). This example emphasizes the

potential scope of clinical application of specific LIMK inhibitors (i.e. treatment of

pathological conditions other than cancer) and highlights the importance of such

inhibitors as research tools to elucidate the role of other kinases in signal

transduction and evaluate their potential as molecular targets for the treatment of

human diseases. Although not approved for clinical use, the ROCK inhibitor, Y-

27632 (developed by Mitsubishi Pharma, Tokyo, Japan), has found wide

application in cell culture and animal studies addressing the role of ROCK in insulin

resistance, cancer cell invasion and cardiovascular disease (Jeong et al., 2012;

Nakamura et al., 2006; Wang et al., 2012).


37

Figure 1-12: Inhibitors of the Rho GTPase/cofilin pathway.

Dysregulation of actin dynamics has been implicated in various diseases and inhibitors have been developed against several targets upstream of LIMK. Important molecules in the Rho GTPase/cofilin pathway are abbreviated as follows: Rho, Rac (Ras-related C3 botulinum toxin substrate), Cdc42 (cell division cycle 42), ROCK (Rho-associated coiled coil-containing protein kinase), Pak (p21-activated kinase), SSH (Slingshot). Actin filaments are shown in red as polymers made up of actin monomers (red rectangles). Cofilin molecules are displayed as yellow circles. Phosphorylation (P) is indicated as a blue circle.


38

Early compounds developed by Bristol-Myers Squibb to target LIMKs inhibited

LIMK kinase activity in vitro and exhibited anti-proliferative and cytotoxic effects

in vivo (Ross-Macdonald et al., 2008). However, as subsequent compounds were

evaluated, it became apparent that the cytotoxic mechanism of one group of

compounds was unrelated to the inhibition of LIMK. This finding suggested the

existence of a second molecular target, which was later identified as tubulin. The

cytotoxic mechanism was shown to be microtubule depolymerisation, which is

also used by known microtubule-targeting agents such as nocodazole (Mollinedo &

Gajate, 2003).

Triggered by the finding that LIMK2 knockout mice had lower intra-ocular

pressure than their wild-type littermates, Lexicon Pharmaceuticals developed the

pyrrolopyrimidine class of LIMK2 inhibitors (Harrison et al., 2009). These

compounds had similar activity against LIMK1 and LIMK2 in vitro (IC50 in the low

nanomolar range), but had a ~100 and 900 times higher selectivity for LIMK2

compared to ROCK1 and ROCK2, respectively. In vivo testing in a steroid-induced

mouse model of ocular hypertension demonstrated that topical application

reduced intra-ocular pressure to the baseline level measured in normotensive

mice. Therefore, LIMK2 is considered a promising target for the treatment of

glaucoma and disorders associated with ocular hypertension. Using the BMS

pyrazolo compound, inhibition of LIMK was shown to impair matrix-remodelling

activities that are required for path generation by leading cells in collective

invasion (Scott et al., 2010). However, cell motility was not affected by this

inhibitor.

An additional class of LIMK inhibitors was developed by the Cancer Therapeutics

CRC in a campaign focussing specifically on potential anti-metastatic compounds

(Sleebs et al., 2011). The resulting lead series had low micromolar inhibitory

activity and showed potential for optimisation of drug-like properties.

A recent publication on the development of a high-throughput assay for the

identification of LIMK inhibitors further emphasises the current interest in the

therapeutic targeting of the LIMK family (Mezna et al., 2011).

The most recently published LIMK inhibitor, Pyr1, showed a microtubule-

stabilising effect in addition to inhibition of actin dynamics and cell motility in vitro


39

(Prudent et al., 2012). Interestingly, it retained its activity in multidrug-resistant

cancer cells.

1.6 Protein structure determination

Given the usefulness of structural models for the development of kinase inhibitors,

an ever-increasing number of protein kinases is subjected to structure

determination, either in individual efforts or as part of the high-throughput

pipeline of structural genomics consortia. Several techniques are available to solve

protein structures at high resolution, including electron microscopy and nuclear

magnetic resonance (NMR) spectroscopy. However, protein X-ray crystallography

has been the most successful method, resulting in ~78.8% of protein kinase

structures deposited in the protein data bank (PDB).

1.6.1 Protein crystallography

Protein crystallisation is a trial-and-error procedure in which the purified protein

of interest is slowly precipitated from its solution (Chayen, 2004). Precipitation of

the protein can be achieved in many ways, for example by increasing the effective

concentration of the protein or by diminishing the repulsive forces between the

protein molecules (Durbin & Feher, 1996). The purity requirements for protein

crystallographic studies are different and more stringent than those for

biochemical studies. As a general rule, the higher the purity of the protein, the

better the chances to grow crystals (Drenth, 1999).

The most popular type of crystallisation assay is the vapour diffusion experiment

(Wiencek, 1999). The commonly used hanging drop method involves preparing a

mixture of equal volumes of protein and precipitant solution on a microscope

coverslip, which is subsequently placed upside-down over a reservoir of higher

concentrated precipitant solution. Equilibration of the water in the hanging drop

with the water in the reservoir solution leads to the simultaneous increase in

protein concentration and precipitant concentration in the drop.


40

In order for crystals to grow, an aqueous protein solution must be brought into

supersaturation, which can be achieved by variation of parameters such as the

concentration of protein, precipitant and additives as well as temperature and pH

(Chayen, 2004). The phase diagram of protein crystallisation consists of four

zones: 1) the zone of undersaturation, which is represented by the area under the

solubility curve; 2) the zone of low supersaturation between the solubility curve

and the supersolubility curve; 3) the zone of moderate supersaturation, where

crystal nucleation occurs spontaneously; and 4) the zone of high supersaturation,

in which the protein precipitates (Figure 1-13). The solubility curve is defined by

the conditions under which protein in the aqueous phase (fully dissolved) is in

equilibrium with the crystallised protein. The supersolubility curve, on the other

hand, separates conditions that give rise to crystal nuclei or protein precipitates

from those under which a protein solution (despite supersaturation) remains clear

if left undisturbed. Ideal conditions for the growth of well-ordered protein crystals

are represented by the metastable zone 2, as it provides a growth-promoting and

stabilising environment without allowing further nucleation.

The molecules in a protein crystal are arranged in a periodic fashion and diffract X-

ray radiation that passes through the crystal. The crystal-specific diffraction

pattern (characterised by the direction and intensities of the diffracted X-ray

beams), is determined by the molecules’ specific structure and arrangement in the

crystal. X-rays are scattered almost exclusively by the electrons in the atoms and

not by the nuclei. Thus, the relationship between the diffraction data and electron

density provides some of the information that is needed to solve the protein

structure. Each diffracted beam (also called reflection) is characterised by its

amplitude and phase. However, only the reflection amplitudes can be obtained

from the measured intensities, while the phases are lost in the experiment (Taylor,

2003). The latter fact is also referred to as the phase problem. Several methods can

be used to recover the phases and all of them are based on some prior knowledge

of the electron density or structure. Molecular replacement is commonly used

when the structure of a very similar molecule is available, from which approximate

phases for the new target can be calculated (Evans & McCoy, 2008). Another

approach involves the generation of isomorphous heavy-atom derivatives of the


41

target protein, which produces changes in the intensity of some reflections and

thereby allows deduction of the position of the heavy atoms (Terwilliger &

Berendzen, 1996). Combining the calculated phases with the measured amplitudes

leads to an initial approximate electron density distribution, which can be

improved in an iterative fashion, eventually converging at a faithful electron

density map (Wlodawer et al., 2008). The electron distribution is then interpreted

in terms of individual atoms and molecules, and the atomic model is refined to

achieve the best agreement between the observed reflection amplitudes and those

calculated from the model.


42

Figure 1-13: Crystallisation phase diagram.

The transition of a protein from an aqueous solution (black dot) to supersaturation (dotted line) forms the rational basis of crystal nucleation and growth in a vapour diffusion experiment. Following nucleation, ideal growth conditions for well-ordered crystals are represented by the metastable zone located between the solubility and the supersolubility curve. Adapted from (Chayen, 2004).


43

1.6.2 High-resolution structures of LIM kinases

Despite the large number of protein kinases that have been identified through

sequencing of the genome, only a relatively small number of high-resolution kinase

structures are available (Marsden & Knapp, 2008). Several structures of individual

domains of LIMK2 have been deposited in the Research Collaboratory for

Structural Bioinformatics (RCSB) Protein Data Bank (PDB), including the solution

structures of the second LIM domain of human LIMK2 (PDB accession code 1x6A)

and of the PDZ domain of mouse LIMK2 (PDB accession code 2YUB). However,

until recently the structure of the LIMK kinase domain has remained elusive. As

the kinase domain represents the most relevant domain as far as drug discovery is

concerned, a homology model of the LIMK1 kinase domain has been generated by

Li et al. based on the crystal structure of the kinase domain of the human tyrosine

kinase c-Src in complex with the inhibitor compound AMP-PNP (PDB accession

code 2SRC) (Li et al., 2006). As protein structure shows a higher degree of

evolutionary conservation than sequence, an amino acid identity of 35% between

both kinase domains was sufficient to enable an alignment of their sequences and

the generation of a structural model of the target protein based on the structure of

the template kinase (Marti-Renom et al., 2000; Scheeff & Bourne, 2005). However,

due to the relatively low sequence identity between the kinase domains of LIMK1

and c-Src, and the presence of unique features in the LIMK1 kinase domain, further

studies were required to generate a more reliable model of the LIMK1 kinase

domain.

1.7 Hypothesis and aims

LIMK1 is considered a promising therapeutic target and recent drug discovery

efforts have yielded several inhibitor compounds with potential for optimisation in

terms of potency and selectivity. This study aims to determine the crystal structure

of the LIMK1 kinase domain in the presence and absence of available inhibitor

compounds to provide a structural basis for the design of the next generation of


44

LIMK inhibitors with increased potency and selectivity. Figure 1-14 outlines the

series of experimental steps leading to detailed models of the protein structure

from which important clues can be derived relating to the geometry of the

compound-binding site and to LIMK residues participating in interactions with the

compound.


45

Figure 1-14: Protein crystallography pipeline from gene to structure and

beyond.

The flow chart shows the tasks involved in the determination and analysis of a protein’s atomic structure. The major bottlenecks in the process are shaded in grey. Adapted from (Chayen & Saridakis, 2008).

47

CHAPTER 2 − MATERIALS &

METHODS

Chapter 2 − Materials & Methods

48


49

2.1 Generation of expression constructs

2.1.1 Bioinformatic analysis

Multiple sequence alignment and sequence comparison of protein kinase domains

were performed using Clustal Omega (version 1.1.0) (Sievers et al., 2011) and

FASTA (Pearson & Lipman, 1988), respectively. Intrinsic protein disorder was

predicted using DisEMBL (version 1.5), applying the “hot loops” definition to

identify highly dynamic coils (Linding et al., 2003). Secondary structure prediction

was performed using PSIPRED (version 3.0) (Jones, 1999).

2.1.2 DNA constructs

Human LIMK1 kinase domain cDNA encoding residues 309-647 was obtained from

Life Technologies (Grand Island, NY, USA) as chemically synthesised sequence that

was codon-optimised for expression in insect cells, from which a truncated form of

the human LIMK1 kinase domain sequence (encoding residues 332-607) was

derived. The cDNA sequences for mouse LIMK1 kinase domain (residue 309-647)

and rat LIMK2 kinase domain (residues 333-638) were subcloned from previously

generated expression constructs. Truncated rat LIMK2 kinase domain (residues

324-562) and mouse LIMK1 kinase domain (residues 332-607 and 332-647) were

derived from the cDNA sequence for full-length LIMK2 and LIMK1, respectively. An

additional construct encoding His6-tagged mouse LIMK1 kinase domain (residue

309-647) was kindly provided by Dr. Rong Li. Analysis of codon usage in the cDNA

sequences was performed using the Graphical Codon Usage Analyser software

version 2.0. The fraction of usage of each codon in the query sequence was

computed and plotted against the fraction of usage of the codon in Spodoptera

frugiperda. Mean differences in relative adaptiveness were used to compare

sequences from different species.


50

2.1.3 Polymerase chain reaction

PCR was used to amplify cDNA sequences for the LIMK kinase domains and to

incorporate suitable endonuclease restriction sites flanking the fragment of

interest. The oligonucleotides were purchased from Sigma-Aldrich (St. Louis, MO,

USA). The lyophilised powder was resuspended in 1X TE buffer (10 mM Tris-HCl, 1

mM EDTA, pH 8.0) at 100 µM final concentration and was diluted to a working

concentration of 50 µM. The following combinations of primers were used to

amplify LIMK kinase domain cDNA flanked by endonuclease restriction sites for

the enzymes shown in parentheses:


51

Table 2-1: PCR primers for subcloning of LIMK kinase domain constructs.

The cDNA sequences for human and mouse LIMK1 kinase domain and rat LIMK2 kinase domain were amplified by PCR using the following sense and antisense primers. Restriction sites for the endonucleases specified in parentheses are shown in red.

Human LIMK1 kinase domain (residues 332-607)

Sense: 5’-TTATTAGGATCCCGCATCTTCCGCCCCAG-3’ (BamHI)

Antisense: 5’-ATGTCGAAGCTTATTACAGGGTCTCCAGCCAGTGCTC-3’ (HindIII)

Mouse LIMK1 kinase domain (residue 309-647)

Sense: 5’-GACCAGAGCTAGCAGGATCTACAATGGCCCCTATAC-3’ (NheI)

Antisense: 5’-CTGGTCTGCTAGCTCAGTCAGGACCTCGGGGTG-3’ (NheI)

Mouse LIMK1 kinase domain (residues 332-607)

Sense: 5’-GACTAGTCGAATCTTCCGGCCATCTG-3’ (SpeI)

Antisense: 5’-GAATGCGGCCGCTTAAAGTGTTTCTAGCCATTGTTCCAG-3’ (NotI)

Mouse LIMK1 kinase domain (residues 332-647)

Sense: 5’-GACTAGTCGAATCTTCCGGCCATCTG-3’ (SpeI)

Antisense: 5’-CTGGTCTGCTAGCTCAGTCAGGACCTCGGGGTG-3’ (NheI)

Rat LIMK2 kinase domain (residues 333-638)

Sense: 5’-GACCAGAGCTAGCAGGATCTACAATGGCCCCTATAC-3’ (NheI)

Antisense: 5’-CTGGTCTGCTAGCTCAGGGTGGCGAGTCCCGGGT-3’ (NheI)

Rat LIMK2 kinase domain (residues 324-562)

Sense: 5’-TTATATGGATCCCAGATCTTCCGGCCCTGTGAC-3’ (BamHI)

Antisense: 5’-AACTTCAAGCTTATTAGAGCGCCTCGAATGAGTCCTCCAG-3’ (HindIII)


52

The 50-µl PCR reactions included 8 ng DNA template and 300 nM of sense and

antisense oligonucleotide primer in 1X Pwo reaction buffer containing

2 mM MgSO4 and 200 µM of each dNTP (Invitrogen, Carlsbad, CA, USA). Upon

addition of 2.5 units of Pwo DNA polymerase (Roche, Basel, Switzerland),

enzymatic DNA synthesis was performed using an Eppendorf Mastercycler thermal

cycler (Eppendorf, Germany) with the following program parameters:

Denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for

30 s, annealing at 55°C for 30 s and extension at 72°C for 2 min prior to final

extension at 72°C for 7 min.

2.1.4 Polyadenylation and cloning of His6-GST-tagged constructs

A poly(A) tail was attached to the PCR product by incubation of a 10-µl reaction in

1X Taq buffer containing 2.5 mM MgCl2, 200 µM dATP and 5 units Taq polymerase

(Invitrogen) at 72°C for 10 min. The resulting DNA was inserted into the cloning

vector pGEM-T (Promega, Madison, WI, USA).

2.1.5 Agarose gel electrophoresis

Agarose gel electrophoresis was used to separate DNA molecules according to

their size. An agarose gel solution of 0.5% to 3% w/v agarose in 0.5X TBE (45 mM

Tris-borate, 1 mM EDTA) was prepared and the nucleic acid stain GelRed (Biotium,

Hayward, CA, USA) was added at a 1:10,000 dilution before the gel was cast and

allowed to solidify. 6X DNA loading buffer (containing 0.15% w/v Orange G, 0.03%

w/v xylene cyanol and 60% v/v glycerol) was added to the DNA samples to yield

1X final concentration. 4 µl of 1 kb DNA ladder (Invitrogen) were loaded and

separated alongside the DNA samples to allow estimation of molecular weight and

DNA concentration. Following gel electrophoresis, DNA bands stained with GelRed

dye were visualised using a UV transilluminator and photographed.


53

2.1.6 Purification and preparative digestion of DNA

Following gel electrophoresis, the PCR products were excised and extracted from

the gel using the QIAquick gel extraction kit (Qiagen, Hilden, Germany) according

to the manufacturer’s instructions. Subsequently, 15 µl of the purified PCR product

and 2 µg of vector DNA were incubated with 20 units of restriction enzyme

(Promega) in the appropriate 1X restriction buffer as specified by the

manufacturer, and the 50-µl sample was purified by addition of 5 volumes of

Buffer PB (from the QIAquick gel extraction kit) prior to binding to a QIAquick

column. After washing with 750 µl Buffer PE, the purified product was eluted in

40 µl Milli-Q water.

For generation of N-terminally His6-GST-tagged constructs, the LIMK kinase

domain sequence was cloned into the pGAT2 vector using the BamHI and HindIII

restriction sites, followed by subcloning of the tagged sequence into the pFastBac

Dual vector (into multiple cloning site 1 downstream of the AcMNPV polyhedrin

promoter) using the restriction enzymes XbaI and HindIII. N-terminally GST-

tagged constructs were generated by cloning of the LIMK kinase domain into the

pEBG vector using the restriction sites for BamHI and NotI prior to subcloning of

the tagged sequence into the pFastBac Dual vector via EcoRI and NotI.

Alternatively, the GST-LIMK kinase domain sequence was non-directionally cloned

into the pFastBac Dual vector (into multiple cloning site 2 downstream of the

AcMNPV p10 promoter) using the restriction enzyme NheI.

2.1.7 DNA ligation

To insert the purified DNA fragment into the target vector, ligation with the

endonuclease-treated vector was performed at a molar ratio of 4:1 (insert:vector)

using the T4 DNA ligase kit (New England Biolabs, Ipswich, MA, USA) according to

the manufacturer’s instructions. As a vector re-ligation control, an additional

ligation reaction was prepared containing only the vector DNA.


54

2.1.8 Heat-shock transformation

50-µl aliquots of chemically competent cells (E. coli strain DH5a) were thawed on

ice for 10 min, followed by addition of the DNA ligation reaction and incubation on

ice for 30 min. Subsequently, the cells were incubated in a 42 C water bath for

exactly 45 s and then put on ice for 2 min. The cells were allowed to recover in 1 ml

SOC medium (0.5% w/v yeast extract, 2% w/v tryptone, 10 mM NaCl, 2.5 mM KCl,

10 mM MgCl2, 10 mM MgSO4 and 20 mM glucose) at 37 C for 1 h before plating

them on LG agar plates (0.5% w/v yeast extract, 1% w/v tryptone, 200 mM NaCl,

1.5% w/v agar) containing 100 µg/ml ampicillin or 50 µg/ml kanamycin (Sigma-

Aldrich). The agar plates were incubated overnight at 37 C to select for bacterial

clones that successfully acquired the antibiotic resistance gene present on the

transfected plasmid.

2.1.9 Amplification and purification of plasmid DNA

For a small-scale purification of plasmid DNA, 4-15 colonies were picked up from

the agar plate using a fine pipette tip and transferred into 2 - 3 ml of LB medium.

After overnight incubation in a horizontal shaker at 37 C, the cells were pelleted

by centrifugation at 16,100 x g for 1 min and plasmid DNA was purified using a

Plasmid Mini kit (Qiagen) according to the manufacturer’s instructions. Large-scale

purification of plasmid DNA was performed using a Plasmid Maxi kit (Qiagen).

2.1.10 Analytic digestion of plasmid DNA

After non-directional insertion of the GST-LIMK kinase domain sequences into the

pFastBac Dual vector, the plasmid was digested with BamHI, which, depending on

the orientation of the insert, released either a ~1700 bp or a ~100 bp fragment

between the recognition site within the kinase domain and that located in the

multiple cloning site region of the vector. Plasmids with the correct insert

orientation were further analysed by DNA sequencing.


55

2.1.11 DNA sequencing

The DNA fragment of interest was amplified and fluorescently labelled by PCR

using a suitable 3’ primer and the BigDye Terminator mix (Applied Biosystems,

Carlsbad, CA, USA). One reaction contained 1-2 µl mini DNA, 4 µl ¼ BDT mix, 1 µl

5 µM primer and 6 µl dH2O, and the PCR program parameters were as follows:

Denaturation at 96°C for 1 min prior to 25 cycles of denaturation at 96°C for 10 s,

annealing at 50°C for 5 s and extension at 60°C for 4 min.

DNA fragments cloned into the pFastBac Dual vector downstream of the

polyhedrin promoter were amplified for DNA sequencing using the following

antisense primer complementary to parts of the SV40 polyadenylation signal: 5’-

ACATTGATGAGTTTGGACAAACC-3’. Alternatively, the same antisense primers as

used for the subcloning of the LIMK kinase domain were employed for sequencing

(see Chapter 2.1.3).

The amplified DNA was precipitated by addition of 3 µl of 3 M sodium acetate (pH

5.2), 62.5 µl 96% v/v ethanol and 14.5 µl dH2O, followed by thorough mixing and

incubation at room temperature for 15 min. After centrifugation at 16,100 x g for

30 min, the supernatant was carefully removed and the pellet was washed with

200 µl 70% v/v ethanol. The sample was centrifuged at 16,100 x g for 5 min and

the supernatant was carefully removed and discarded. The DNA pellet was dried at

90 C for 1 min and was subsequently allowed to cool down to room temperature.

Sequencing by capillary electrophoresis was performed at the Micromon DNA

sequencing facility, Department of Microbiology, Monash University. Alternatively,

plasmid DNA and primers were submitted to the sequencing facility at the

Department of Pathology, University of Melbourne, where DNA amplification by

PCR and capillary electrophoresis were carried out. The resulting nucleotide

sequences were entered into the BLAST (Basic Local Alignment Search Tool)

database to confirm the identity and integrity of the sequence.


56

2.1.12 Generation of recombinant bacmid DNA

To allow site-specific transposition of the GST-tagged or His6-GST-tagged LIMK

kinase domain sequences from the pFastBac Dual vector into the baculovirus

shuttle vector bMON14272, transformation of DH10Bac competent cells was

performed as described above for DH5 competent cells (see Chapter 2.1.8). The

cells were allowed to recover in 900 µl SOC medium at 37 C for 4 - 6 h before

plating them as serial dilutions (1, 1:10, 1:100) on LG agar plates containing 50

µg/ml kanamycin, 7 µg/ml gentamycin and 10 µg/ml tetracyclin in addition to 100

µg/ml X-Gal and 35 µM IPTG. The plates were incubated at 37 C for 2 - 3 days until

clear colour differences were observed between blue (negative) and white

(positive) colonies. 10 white colonies as well as one blue clone were re-streaked on

fresh selection plates and were further incubated at 37 C to confirm the

preliminary blue/white selection results and exclude cross-contamination while

picking up individual colonies.

2.1.13 Isolation of recombinant bacmid DNA

Recombinant bacmid DNA was purified from DH10Bac transformants according to

the protocol provided in the Invitrogen Bac-to-Bac Baculovirus Expression System

manual (Version D, 6 April 2004, pages 51-52). Individual bacterial colonies were

inoculated into 2-3 ml of LB medium containing 50 µg/ml kanamycin, 7 µg/ml

gentamicin, and 10 µg/ml tetracycline. The cultures were incubated overnight at

37°C prior to centrifugation of 1.5 ml of culture at 14,000 x g for 1 min. The

supernatant was discarded and the cell pellet was resuspended in 300 µl of Buffer

P1 (from the Qiagen Plasmid Maxi kit), followed by addition of 300 µl of Buffer P2,

gently mixing and incubation at room temperature for 5 min. Immediately after

addition of 300 µl of Buffer P3, the sample was mixed gently and placed on ice for

10 min. Following centrifugation at 14,000 x g for 10 min, the supernatant was

transferred into an Eppendorf tube containing 800 µl isopropanol and the tube

was inverted several times and placed in ice for 10 min. After a 15-min

centrifugation at 14,000 x g, the supernatant was discarded and the DNA pellet


57

was washed with 500 µl 70% v/v ethanol, followed by another 5-min

centrifugation at 14.000 x g. The supernatant was discarded and the pellet was air-

dried at room temperature for around 10 min. The dried pellet was dissolved in 40

µl 1X TE Buffer (pH 8.0) aided by occasional gentle tapping of the tube. The

purified bacmid DNA was stored at 4°C and was used for analytical purposes and

transfection of insect cells within one week.

2.1.14 Analysis of recombinant bacmid DNA

Due to its large size of >135 kb, recombinant bacmid DNA was analysed by PCR

rather than enzymatic digestion to verify the presence of the gene of interest. The

combination of M13 Forward (5’-GTTTTCCCAGTCACGAC-3’) and M13 Reverse

primers (5’-CAGGAAACAGCTATGAC-3’) was used to amplify the sequence that was

inserted into the mini-attTn7 attachment site of the bacmid. Another combination

of either M13 Forward or M13 Reverse primer and a primer specific for the

inserted sequence (see Chapter 2.1.3) was employed to confirm the correct

orientation of the insert.

20-µl PCR reactions containing ~20 ng of recombinant bacmid DNA, 1.5 mM MgCl2,

250 nM forward and reverse primer, 200 µM dNTPs and 1 unit Taq polymerase

(Invitrogen) in 1X Taq polymerase buffer were prepared and PCR was performed

using the following parameters: Denaturation at 94°C for 3 min, followed by 30

cycles of denaturation at 94°C for 45 s, annealing at 55°C for 45 s and extension at

72°C for 2.5 min prior to final extension at 72°C for 7 min.

2.2 Insect cell culture and baculovirus production

2.2.1 Cell culture materials

Sf-900 II Serum-free Medium, 1X phosphate-buffered saline (PBS), 1.3x Sf-900 II

Serum-free Medium, 4% w/v agarose and Cellfectin II transfection reagent were

purchased from Invitrogen. All disposable plastic cell culture flasks and dishes


58

were purchased from Corning Life Sciences (Lowell, CA, USA). Glass Erlenmeyer

flasks were obtained from Schott (Mitterteich, Germany).

2.2.2 Cell lines & culture maintenance

Spodoptera frugiperda Sf9 and Sf21 and Trichoplusia ni High-Five (BTI-Tn-5B1-4)

insect cells were cultured in Sf-900 II Serum-free Medium (SFM) without added

antibiotics as suspension or adherent cultures at temperatures between 22 C and

27 C. Suspension cultures were incubated on a horizontal rotary shaker and were

split every 3-4 days to maintain cell densities between 0.5 x 106 and 6 x 106 cells

per ml.

2.2.3 Transfection

0.8-1.2 x 106 Sf9 or Sf21 cells in 2 ml Sf-900 II SFM were plated in each well of a 6-

well tissue culture dish and the cells were allowed to adhere for at least one hour.

In two Eppendorf tubes, 8 µl Cellfectin II transfection reagent and ~1 µg bacmid

DNA were diluted separately in 100 µl Sf-900 II SFM each and the two dilutions

were combined, followed by gentle mixing and incubation at room temperature for

30 minutes. The transfection mixture was slowly added onto the cells. After four to

five hours of incubation at 27 C the transfection medium was replaced with 2 ml

Sf-900 II SFM and the cells were incubated at 27 C for three to four days until signs

of viral infection become visible. Subsequently, the supernatant (also referred to as

first-generation or P1 baculovirus) was harvested and stored at 4 C.

2.2.4 Analysis of recombinant protein expression

Sf9 cells were seeded in a 96-well plate at a density of 3-5 x 104 cells per well in

100 µl of Sf-900 II SFM and were allowed to adhere for at least one hour.

Subsequently, 2-100 µl of recombinant baculovirus was added and the plate was


59

sealed with Parafilm (Pechiney, Chicago, IL, USA) to prevent evaporation of the

medium. Following incubation at 27 C for 2-3 days, the medium was discarded and

the cells were lysed in 100 µl 1X Laemmli buffer. The samples were analysed by

SDS polyacrylamide gel electrophoresis and Western blot using a mouse

monoclonal anti-GST antibody.

2.2.5 Baculovirus plaque assay

Sf9 cells were plated in a 6-well tissue culture dish at a density of 0.25 x 106 cells

per well and were allowed to adhere for at least one hour. The cells were infected

with 100 µl of a serial dilution of P1 baculovirus ranging from 10-3 to 10-8.

Following incubation at 27 C for two hours, the medium was removed and 2 ml of

a 1% w/v agarose in Sf-900 II SFM suspension were added on top of the cell layer

and allowed to set at room temperature for 10 min. To prevent the agarose from

drying out, 1 ml Sf-900 II SFM was put on top. The plate was sealed with parafilm

and incubated at 27 C for 6-10 days. The majority of spatially isolated plaques

appeared in the wells containing cells infected with the 10-5 and 10-6 baculovirus

dilutions.

2.2.6 Baculovirus clone amplification

Sf9 cells were plated in a 6-well tissue culture dish at a density of 0.5 x 106 cells per

well and were allowed to adhere for at least one hour. The agarose layer covering

the area of a plaque was mashed up with a 1-ml pipette and the liquefied agarose

was transferred into 250 µl Sf-900 II SFM. 50-100 µl of the resulting virus

suspension were used to infect one well of Sf9 cells. After incubation for three to

four days at 27 C, the supernatant (also referred to as plaque-purified P1 virus or

ppP1 virus) was harvested and stored at 4 C. The second-generation (P2)

baculovirus was generated from either the P1 baculovirus or the plaque-purified

P1 virus. 3-12 x 106 Sf9 or Sf21 cells were plated in a 75-cm2 tissue culture flask

and were allowed to adhere for at least one hour. The cells were infected with 250-


60

300 µl of P1 or ppP1 virus and incubated at 27 C for 5-8 days. The medium

containing the P2 baculovirus was centrifuged at 1,000 x g for 5 min and the

supernatant was stored at 4 C. The third-generation (P3) virus was generated

from P2 baculovirus in a suspension culture. Sf9 or Sf21 cell suspension was

transferred into 250 or 500 ml shaker flask (glass or disposable plastic) and

diluted with Sf-900 II SFM to yield a total volume of 50-100 ml or 100-200 ml,

respectively, at a cell density of 1 x 106 cells per ml. The culture was infected with

500 µl P2 baculovirus per 100 ml culture volume and incubated at 27 C for 7-10

days. The medium containing the P3 baculovirus was centrifuged at 1,000 x g for 5

min and the supernatant was stored at 4 C.

2.3 Protein expression in insect cells

2.3.1 Micro-scale expression

Sf9 cells plated in a 96-well tissue culture dish at a density of 5 x 104 cells per well

were allowed to adhere for at least one hour. The cells were infected with 50 µl of

baculovirus and incubated at 27 C for three days. For Western blot analysis, cell

pellets were incubated with 100 µl of 1X Laemmli buffer at 95 C for 5 min.

2.3.2 Small/medium-scale protein expression

Sf9, Sf21 or High-Five cell suspension was transferred into 500 ml or 1800 ml

culture flasks and diluted with Sf-900 II SFM to yield a total volume of 200 ml or

800-1000 ml, respectively, at a cell density of 2 x 106 cells per ml. The culture was

infected with P2 or P3 baculovirus at a 1:40-1:100 dilution. After incubation at

27 C for three days the culture was transferred into centrifuge bottles and

centrifuged at 1,000 x g at 4 C for 5-7 min. The pellet was washed with cold (4 C)

PBS and centrifuged again as described above. The washed cell pellets were stored

in aliquots at -80 C.


61

2.4 Protein purification

2.4.1 Ammonium sulphate precipitation, glutathione-affinity

chromatography and GST tag removal

Pellets of insect cells infected with recombinant baculoviruses were thawed and

resuspended in ice-cold homogenisation buffer (50 mM Tris-HCl [pH 8.0], 150 mM

NaCl, 10 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 0.1% v/v 2-mercaptoethanol, 1X

protease inhibitors), followed by lysis using a chilled EmulsiFlex high-pressure

homogeniser (Avestin, Ottawa, ON, Canada). DNase I was added to the cell lysate to

a final concentration of 25 µg/ml to lower its high viscosity. After centrifugation at

25,000 x g at 4 C for 30 min, the cleared lysate was passed through an empty

chromatography column to remove further particulates and ensure proper flow

during the chromatography step. Cold saturated ammonium sulphate solution was

slowly added to the filtered lysate to a final concentration of 40% v/v with gentle

stirring on ice, and precipitation of protein was allowed to occur at 4°C for 30 min.

The precipitated protein fraction was pelleted by centrifugation at 12,000 x g at

4 C for 15 min and was resuspended in cold homogenisation buffer, followed by

filtering through an empty chromatography column. Glutathione Sepharose beads

were washed several times with PBS and equilibrated in homogenisation buffer

prior to their addition to the resuspended high-molecular weight protein fraction

(at a beads:protein volume ratio of 1:25) and incubation at 4°C for 1 hour. The

mixture of beads and cell lysate was transferred into a chromatography column.

The flow-through was put aside (eventually discarded) and the beads were

washed with 50 volumes of homogenisation buffer, followed by washing with 30

volumes of low-salt wash buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% v/v

2-mercaptoethanol), 50 volumes of high-salt wash buffer (50 mM Tris-HCl [pH

8.0], 500 mM NaCl, 0.1% v/v 2-mercaptoethanol) and an additional 20 volumes of

low-salt buffer. The washed beads were incubated overnight at 4°C with elution

buffer containing 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 10 mM reduced

glutathione, 3 mM TCEP (Pierce, Rockford, IL, USA) and 1X Complete protease

inhibitor cocktail (Roche). The eluate was collected and another two elutions were

performed. The remaining protein that was bound to the beads was treated with


62

thrombin cleavage buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 2.5 mM CaCl2,

0.1% v/v 2-mercaptoethanol) containing 1 mg/ml of a carbohydrate-based

polymer additive (NV10; Expedeon Protein Solutions, Cambridge, UK) and 10

units/ml thrombin (Sigma-Aldrich) at room temperature. The eluate was passed

through a thin layer of Benzamidine Sepharose matrix (GE Healthcare, Piscataway,

NJ, USA) to remove thrombin from the solution and protease inhibitors were

added to 1X final concentration.

2.4.2 Ion exchange chromatography

GST-tagged LIMK1 kinase domain was further purified by ion exchange

chromatography on a Mono Q 5/50 GL anion exchanger column (GE Healthcare).

The sample was filtered using an Ultrafree-MC centrifugal filter unit (Millipore,

Billerica, MA, USA) to remove protein aggregates and was then applied to the

column at a flow rate of 1 ml/min. Proteins were eluted with a linear salt gradient

ranging from 25 mM to 600 mM NaCl in 8 ml 20 mM Bistris propane buffer at pH

10.0. The eluate was collected in fractions of 0.2 ml volume.

2.4.3 Size exclusion chromatography

Prior to separation of proteins according to size, the sample was filtered as

described above. The sample containing 0.3 mg/ml NV10 was applied to a

Superdex 200 10/300 GL column (GE Healthcare) that was pre-equilibrated with

gel filtration buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% v/v 2-mercaptoethanol, 5

mM TCEP, pH 8.0). 250-µl fractions were collected at a flow rate of 0.5 ml/min.

2.4.4 Facilitation of protein solubility

To remove the GST tag, the GST fusion protein bound to the glutathione Sepharose

beads was incubated with thrombin, which recognises a specific cleavage site


63

located in the linker region between the N-terminal tag and LIMK. Thus, a varying

percentage of total LIMK (approximately 20-60%) can be converted into the tag-

free form and collected in the eluate. Incubation with thrombin at room

temperature for one hour resulted in less LIMK degradation due to non-specific

thrombin activity than incubation at 4°C for 16 hours and was therefore used for

tag removal.

To prevent aggregation and precipitation after removal of the solubility-enhancing

GST-tag, a carbohydrate-based polymer additive (NV10; Expedeon) was added to

the thrombin cleavage buffer (50 mM Tris-HCl, 150 mM NaCl, 2.5 mM CaCl2, 0.1%

v/v 2-mercaptoethanol, pH 8.0).

2.5 Protein characterisation

2.5.1 Determination of protein concentration

Protein concentration was determined according to the method of Bradford

(Bradford, 1976) using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA) on a

96-well microplate scale. Absorbance was measured at 620 nm using a Polarstar

Optima microplate reader (BMG Labtech, Ortenberg, Germany) and was

standardised to known amounts (1 to 8 µg) of BSA (Promega). All samples were

measured in duplicates or triplicates.

2.5.2 SDS-polyacrylamide gel electrophoresis

Samples of soluble protein were combined with 5X Laemmli buffer (250 mM Tris-

HCl [pH 6.8], 4% v/v 2-mercaptoethanol, 2% w/v SDS, 0.05% w/v bromophenol

blue, 20% v/v glycerol) at a ratio of 4:1, whereas samples of protein precipitate or

protein bound to glutathione Sepharose beads were mixed with an appropriate

volume of 1X Laemmli buffer prior to incubation at 95 C for 5 min. Denatured low-

molecular weight proteins were resolved on Novex 4-20% Tris-Glycine SDS

polyacrylamide gels (Invitrogen), while all other protein samples were separated


64

on 10 to 12% polyacrylamide gels. As a molecular weight marker, 10 to 12 µl of

Precision Plus All Blue protein standard or Kaleidoscope pre-stained protein

standard (Bio-Rad) were loaded on the gel. The gel running buffer was composed

of 25 mM Tris, 192 mM glycine and 0.1% w/v SDS (pH 8.3). Preparative gels were

incubated with Coomassie Blue staining buffer (0.05% w/v Coomassie Blue G-250,

40% v/v methanol, 10% v/v acetic acid) for approximately 20 min, followed by

destaining overnight in water.

2.5.3 Western blot

The separated proteins were transferred from the gel to an 8 cm x 6 cm Hybond-C

Extra nitrocellulose membrane (GE Healthcare) using the wet transfer method at

90 V for 1.5 h to 2 h or overnight at 30 V. The transfer buffer was composed of 25

mM Tris, 192 mM glycine and 10% v/v methanol. Following transfer, the non-

specific binding sites on the membrane were blocked by incubation with 5 % w/v

skim milk powder in PBS-T (PBS containing 0.1% v/v Tween-20) for at least 30

min. The membrane was then incubated with polyclonal rabbit anti-LIMK1

antibody (Cell Signaling, Beverly, MA, USA) at a dilution of 1:1000 or monoclonal

rat anti-GST antibody (WEHI Monoclonal Antibody Facility, Bundoora, Australia) at

a dilution of 1:3000 in blocking buffer at room temperature for 1 h or overnight at

4 C. After three washes with PBS-T for at least 5 min, the membrane was incubated

with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-rat

antibody (Invitrogen) at a dilution of 1:5000 in blocking buffer at room

temperature for 1 h, followed by washing with PBS-T at least three times for 5 min.

Enhanced chemiluminescence reaction (Thermo Fisher Scientific, Boston, MA,

USA) was performed to detect immunoreactive protein bands. These signals were

recorded on Hyperfilm ECL X-ray films (GE Healthcare) after exposure times

ranging from 1 s to 5 min. The exposed film was developed using a Fuji Medical X-

ray film processor (Fujifilm, Tokyo, Japan). The position of the protein maker

bands as seen on the membrane was marked on the X-ray film for molecular

weight determination. Quantification and normalisation of protein levels was


65

performed using the ImageQuant 7 software (Molecular Dynamics, Sunnyvale, CA,

USA).

2.5.4 In vitro kinase assay

The catalytic activity of the purified LIMK1 kinase domain toward its substrate

cofilin was determined in an in vitro kinase assay. 20 µl of either purified LIMK1

kinase domain or GST-LIMK1 (equivalent to approximately 1-2 µg of protein) were

incubated in the presence or absence of 4 µg GST-cofilin with 8.9 µl kinase assay

buffer (20 mM HEPES [pH 7.4], 150 mM KCl, 10 mM MgCl2, 10 mM MnCl2, 50 mM

NaF, 1 mM Na3VO4, 10 mM NaP2O7) supplemented with 0.6 µl 1 mM ATP and 0.5 µl

10 mCi/ml [ -32P]ATP (Perkin Elmer, Waltham, MA, USA) in a total volume of 32 µl

at 30 C for 30 min. The kinase reaction was stopped by addition of 8 µl 5X Laemmli

buffer and boiling for 5 min. Subsequently, the samples were separated on a 12%

SDS polyacrylamide at 90-150 V. The gel was stained with Coomassie and the

radioactive signal detected after 3-day exposure of a phosphoimaging screen (GE

Healthcare) and scanning on a Storm 820 Phosphorimager (GE Healthcare).

2.5.5 Limited proteolysis

Purified GST-LIMK1 kinase domain was incubated with Proteinase K at a weight

ratio of 1000:1 in the presence of 1 mM ATP at 25 C and after 0, 0.5, 1, 2, 5, 10, 30

and 60 min, aliquots were collected and 5X Laemmli buffer was added to stop the

proteolysis reaction. Proteins were resolved on 4-20% polyacrylamide gels and

analysed by staining with Coomassie Blue or Western blot using an anti-LIMK1

antibody. A protease-resistant fragment of approximately 37 kDa was excised from

the Coomassie-stained gel for analysis by mass spectrometry.


66

2.5.6 In-gel digestion and mass spectrometry

In-gel digestion with trypsin was performed according to the method of

Shevchenko (Shevchenko et al., 2006) and peptides were analysed by liquid

chromatography using a Dionex UltiMate 3000 nano LC system (Thermo Fisher

Scientific) coupled to a QSTAR Pulsar i mass spectrometer with micro-ion spray

source (Applied Biosystems). Peptide mass fingerprint data were analysed using

the MASCOT software (Matrix Science, Boston, MA, USA).

2.5.7 Concentrating proteins

Purified proteins were concentrated using an Amicon Ultra-4 centrifugal filter unit

with a 10-kDa molecular weight cut-off (Millipore). Up to 4 ml of sample were

centrifuged at 4,000 x g at 4 C for up to 12 minutes in 2 to 4-minute intervals, after

each of which the sample was assessed for macroscopic precipitation. In addition,

the sample was mixed gently after each interval by use of a pipette to prevent the

formation of a steep concentration gradient within the sample.

2.6 Screening for crystallisation conditions

Purified LIMK1 kinase domain at a concentration of approximately 3 mg/ml in 50

mM Tris (pH 8.0), 150 mM NaCl, 2.5 mM CaCl2, 4 mM TCEP was subjected to

crystallisation trials in the presence and absence of 1 mM ATP. Robotic

crystallisation screens were performed at the Collaborative Crystallisation Centre

(Melbourne, Australia) using the following sparse matrix and grid screens: Crystal

Screen I and II, PEG/Ion Grid Screen, MPD Grid Screen, Index Screen (Hampton

Research, Aliso Viejo, CA, USA), Wizard I and II (Emerald BioStructures, Bainbridge

Island, WA, USA), Nextal PACT and Anions screens (Qiagen).


67

2.7 Protein structure analysis

Atomic structures from the protein databank were visualised and analysed using

Jmol (version 12.2.15) or PyMOL (version 1.2).

69

CHAPTER 3 − EXPRESSION,

PURIFICATION AND

CRYSTALLISATION OF THE LIMK1

KINASE DOMAIN

Chapter 3 − Expression, purification and crystallisation of the LIMK1 kinase domain

70


71

3.1 Protein expression for structural studies

X-ray crystallography refers to a high-resolution structural technique employing X-

ray radiation to determine the atomic coordinates of a crystallised protein in

three-dimensional space. As a requirement for such studies, milligram amounts of

purified protein are required, most of which are used during the optimisation of

crystallisation conditions.

Large-scale expression of LIMK proteins in both mammalian and bacterial cells has

been attempted previously in our laboratory. While LIMK has been expressed in

and purified from transiently transfected HEK293 cells, the protein yield was not

sufficient for crystallisation. Expression in Escherichia coli has yielded only small

amounts of soluble protein, while the majority of the expressed protein was

insoluble in inclusion bodies. Furthermore, an in vitro translation method was

explored, however, with poor yields.

The baculovirus/insect cell system has become one of the most widely used

systems for expression of recombinant proteins (Kost et al., 2005). Its advantage

over bacterial expression systems lies in the ability to incorporate post-

translational modifications and correctly formed disulphide bonds (Unger & Peleg,

2012; Vrljic et al., 2011). The virus-mediated mode of DNA introduction into insect

cells is more efficient and more economical than transfection of mammalian cells

(Maruniak, 1996). Furthermore, culturing insect cells in suspension cultures offers

the advantage of easy scale-up to increase protein yields. For these reasons, the

baculovirus/insect cell system was chosen for the production of milligram

amounts of purified LIMK kinase domain for structural studies.

3.2 Construct design

A large number of kinase domain structures have been solved by X-ray

crystallography and have been deposited in the Protein Data Bank (PDB) together

with the amino acid sequence of the crystallised proteins. Alignment of the human

LIMK1 kinase domain sequence (residues 309-647) with other protein kinase


72

constructs of similar length (encoding the catalytic domain of casein kinase 2,

hepatocyte growth factor receptor [c-Met], p21-activated kinase 6,

phosphoinositide-dependent protein kinase 1 and Polo-like kinase 1) revealed a

wide variety of N-terminal sequences with diverse secondary structure (Eathiraj et

al., 2011; Eswaran et al., 2007; Ferguson et al., 2011; Kothe et al., 2007; Lopez-

Garcia et al., 2011) (Figure 3-1).


73


74

Figure 3-1: Sequence alignment of the catalytic domain of LIMK1 and other

crystallised protein kinases.

The sequence of the human LIMK1 kinase domain construct was aligned with the recently crystallised constructs encoding casein kinase II subunit (CK2; PDB accession code 3NGA), hepatocyte growth factor receptor (also known as c-Met; 3RHK), p21-activated kinase 6 (PAK6; 2C30), phosphoinositide-dependent protein kinase 1 (PDK1; 4A06) and Polo-like kinase 1 (PLK1; 2OU7). Asterisk (*) indicates fully conserved residues; colon (:) indicates strong similarity; period (.) indicates weak similarity.


75

Prediction of disorder in the secondary structure of the human LIMK1 kinase

domain suggested the presence of flexible regions at the N-terminus and C-

terminus of the kinase domain construct as well as within the catalytic domain

(Figure 3-2). These regions were comprised of residues 309-344, 400-408, 482-

518 and 635-647. The internal flexible region 482-518 represents the activation

segment of the LIMK1 kinase domain flanked by the characteristic DFG and APE

motifs. Despite its intrinsic flexibility, the activation loop may be stabilised by

interactions with other kinase domain residues or bound inhibitor compounds, as

demonstrated in the crystal structure of p21-activated kinase 4, 5 and 6 (Pak4, 5

and 6) and Janus kinase 3 (Jak3) (Boggon et al., 2005; Eswaran et al., 2007). The

native C-terminus of LIMK1 (residues 635-647) was intentionally not truncated in

the human and mouse kinase domain construct as it may serve a regulatory

function similar to the C-terminal regulatory region found in other protein kinases

such as calcium/calmodulin-dependent kinase 1 (Goldberg et al., 1996). In

addition to the human and mouse LIMK1 kinase domain, an N-terminally and C-

terminally truncated rat LIMK2 kinase domain construct was cloned, thereby

producing a potentially less flexible kinase domain.

The sequence identity between the human and mouse LIMK1 kinase domain was

96.8% within a 309-amino acid overlap. Taking into account the chemical

similarity between non-identical residues, the two sequences showed 99.4%

similarity. In contrast, sequence identity and similarity were considerably lower

between the human LIMK1 and rat LIMK2 kinase domain constructs (68.6% and

85.7%, respectively, within a 306-amino acid overlap). Interestingly, the rat LIMK2

kinase domain contains a stretch of 6 additional residues in the activation loop

when compared to the LIMK1 kinase domain.


76

Figure 3-2: Multiple sequence alignment of LIMK kinase domain constructs.

The amino acid sequences of the kinase domain constructs for human LIMK1, mouse LIMK1 and rat LIMK2 were aligned using the FASTA algorithm (Pearson & Lipman, 1988). Asterisk (*) indicates fully conserved residues; colon (:) indicates strong similarity; period (.) indicates weak similarity. Disordered regions in the sequence for human LIMK1 kinase domain were predicted using the software DisEMBL and are underlined in red.


77

To assess the structural similarity between the LIMK paralogues, the human

LIMK1 kinase domain and rat LIMK2 kinase domain sequences were subjected to

secondary structure prediction (Figure 3-3). Despite only moderate sequence

identity, the two paralogues were predicted to have high similarity in regard to

their secondary structure elements (10 -helices and 8 -strands in human LIMK1

kinase domain versus 9 -helices and 7 -strands in rat LIMK2 kinase domain).


78

Figure 3-3: Secondary structure prediction for the kinase domains of human

LIMK1 and rat LIMK2.

The 306-amino acid sequence overlap between the human LIMK1 and rat LIMK2 kinase domain constructs was subjected to secondary structure prediction by the Protein Structure Prediction Server (PSIPRED). Confidence of prediction is shown as blue bars above the predicted secondary structure elements. indicated by purple barrels, strands (E) by yellow arrows and unstructured coils (C) by black lines. Amino acid sequence (AA) is shown at the bottom.


79

3.3 Generation of expression constructs and baculoviruses

Protein expression systems vary in the preference of codon usage during

translation of the mRNA sequence into amino acids that form the growing

polypeptide chain. This is due to differences in the relative quantities of tRNAs

employed by different species. To maximise the potential protein yield of the

human LIMK1 kinase domain expressed in insect cells, a synthetic, codon-

optimised form of the nucleotide sequence was obtained from a commercial source

and was fused with the sequence encoding an N-terminal GST tag. In addition, GST

fusion constructs containing the non-optimised sequences for mouse LIMK1 kinase

domain and rat LIMK2 kinase domain were generated as well as a His6-tagged

mouse LIMK1 kinase domain construct (Table 3-1).


80

Table 3-1: List of affinity-tagged kinase domain constructs for LIMK1 and

LIMK2.

Species Tag Kinase domain Residues

Human GST LIMK1 309-647

Mouse GST LIMK1 309-647

Mouse His6 LIMK1 309-647

Rat GST LIMK2 333-638


81

All sequences were inserted into the pFastBac Dual vector for the generation of

bacmid DNA through site-specific transposition using DH10Bac competent cells.

The isolated, recombinant bacmid DNA was transfected into Sf9 insect cells to

generate infectious recombinant baculovirus particles. Initially, the baculoviruses

were amplified to third-generation (P3) viruses in Sf9 cells without plaque

purification. However, there was a noticeable drop in the expression level from the

second-generation (P2) to P3 viruses. Purification of recombinant baculovirus

clones by plaque assays and amplification of virus clones (ppP1) to P3 viruses

resulted in increased expression, facilitating the production of large volumes of

high-titre baculoviruses. The expression level of His6-tagged mouse LIMK1 kinase

domain, however, was insufficient for large-scale protein production. Figure 3-4

shows the expression levels of the GST-tagged LIMK kinase domain proteins in Sf9

cells as determined by Western blotting using an anti-GST antibody.


82

Figure 3-4: Expression of GST-LIMK kinase domain proteins.

Sf9 insect cells were infected with baculovirus expressing GST-tagged mouse LIMK1 kinase domain, rat LIMK2 kinase domain and human LIMK1 kinase domain. After three days of incubation at 27°C, cells were lysed in 100 µl of 1X Laemmli buffer and 45 µl of each lysate was analysed by Western blotting with an anti-GST antibody.


83

GST-tagged rat LIMK2 kinase domain was expressed at lower levels than its mouse

and human LIMK1 counterparts, which were expressed at similar levels. The codon

usage bias in the rat LIMK2 kinase domain and mouse LIMK1 kinase domain

sequence compared to the codon usage table specific for Spodoptera frugiperda

shows a mean difference in relative adaptiveness of 24.55% and 28.14%,

respectively, suggesting that the difference in expression levels between the

sequences from rat and mouse was not driven by codon usage bias. The vast

majority of the immuno-reactive protein in the GST-kinase domain samples

appeared to be undegraded prior to purification.

3.4 Optimisation of baculovirus amplification and protein expression

The three insect cell lines Sf9, Sf21 and High-Five are commonly used for

expression of intracellular, membrane-bound or secreted recombinant proteins.

Sf9 and Sf21 cells are the preferred cell lines for transfection of bacmid DNA and

production of baculoviruses, while Sf9, Sf21 or High-Five cells are used for protein

production. The protein yield, however, may differ between cell lines depending on

the amino acid sequence and other factors such as the cellular localisation of the

protein (cytosolic, membrane or secreted).

In collaboration with the Fermentation group at CSIRO Molecular and Health

Technologies (Parkville, Victoria), baculovirus encoding GST-tagged human LIMK1

kinase domain was amplified in Sf9 cells from a plaque-purified clone to the fourth

virus generation. Sf21 and High-Five cells were infected with these viruses and the

amounts of the expressed protein were compared by Western blot using an anti-

GST antibody. As shown in Figure 3-5, LIMK1 kinase domain expression was

generally higher in High-Five cells (lanes 1-4) than in Sf21 cells (lanes 5-8), and

more degradation products were evident in High-Five cells after 4-day incubation

than after 3 days. Doubling the amount of baculovirus used for infection did not

result in increased LIMK1 kinase domain expression levels.


84

Figure 3-5: Optimisation of protein expression.

Baculovirus clones expressing GST-tagged human LIMK1 kinase domain were amplified to high-titre viruses in Sf9 cells, and were used to infect High-Five and Sf21 cells at a multiplicity of infection (MOI) of 1 and 2. Aliquots of the infected cultures were analysed after 72-h and 96-h incubation by SDS-PAGE and Western blotting using an anti-GST antibody.


85

These results indicate that the expression level of GST-tagged human LIMK1 kinase

domain is highest in High-Five cells when infected with baculovirus that was

amplified in Sf9 cells. A 3-day incubation period appeared to be more favourable

for less protein degradation.

For large-scale protein production, High-Five insect cells were grown in

suspension to a density of 2 x 106 cells per ml (logarithmic growth phase and

maximum viability) and were subsequently infected with P3 or P4 baculovirus at a

40- to 100-fold dilution of the baculovirus stock. After incubation at 27°C for three

days, insect cell pellets were harvested and stored at -80°C until use. Prior to

freezing, 50 µl aliquots of each culture were lysed in Laemmli buffer and analysed

by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting to

estimate the expression level and to determine protein integrity. Estimated

amounts of 2-10 mg of GST-LIMK kinase domain per litre of culture were

considered sufficient for purification.

3.5 Cell lysis and affinity chromatography

Cells were lysed using a cooled high-pressure homogeniser in preference to lysis

by sonication, which heated up the sample excessively despite constant cooling

and short burst periods. The lysate was cleared from large particulates by

centrifugation and filtration prior to incubation with glutathione Sepharose beads

and thorough washing. Subsequently, small aliquots of beads were boiled in 1X

Laemmli buffer and the purified proteins were separated on a 4-20%

polyacrylamide gel. Surprisingly, the majority of protein purified by glutathione-

affinity chromatography was contained in a small-molecular weight band around

25 kDa that was stained strongly by Coomassie Blue. To determine the expression

level of this contaminant that seemed to compete with the GST-tagged

recombinant protein for binding to the glutathione Sepharose beads, lysates of

uninfected Sf9, Sf21 and High-Five cells were prepared and subjected to the same

protocol. SDS-PAGE and Coomassie staining of the proteins bound to the

glutathione Sepharose beads revealed large amounts of a 25-kDa endogenous

protein in all three insect cell lines (Figure 3-6). This protein was most likely


86

endogenous GST-like protein, which is expressed in insect cells in large amounts,

as previously suggested (Bichet et al., 2000). Surprisingly, this protein was not

detected by Western blotting with our anti-GST antibody.


87

Figure 3-6: Expression of endogenous GST-like proteins in insect cells.

Lysates of Sf9, High-Five (Hi5) and Sf21 insect cells were incubated with glutathione Sepharose beads and aliquots of the beads were analysed by SDS-PAGE and Coomassie staining. Large amounts of endogenous GST-like proteins that bind to glutathione Sepharose beads are present in the insect cell lysates.


88

To separate the high-molecular weight protein fraction of the cell lysate (including

GST-LIMK kinase domain) from the low-molecular weight components including

the endogenous GST-like protein, ammonium sulphate precipitation was

performed. Ammonium sulphate competes with proteins for binding to water

molecules in the solution and increasing dehydration of proteins leads to their

aggregation and precipitation depending on their molecular weight. Increasing

amounts of ammonium sulphate were added to insect cell lysate containing GST-

tagged mouse LIMK1 kinase domain and aliquots were analysed by Western

blotting using an anti-GST antibody. Efficient precipitation of the kinase domain

was observed at a final ammonium sulphate concentration of at least 40% v/v

(Figure 3-7). The second immuno-reactive band at an apparent molecular weight

of ~31 kDa most like represents a small degradation product containing the GST

tag. Other proteins of lower molecular weight including insect cell GST remained

soluble in the presence of 40% v/v ammonium sulphate and were discarded, while

the precipitated protein was resuspended in homogenisation buffer and was

further subjected to glutathione-affinity chromatography.


89

Figure 3-7: Ammonium sulphate precipitation of LIMK1 kinase domain.

Lysates of insect cell cultures expressing GST-tagged mouse LIMK1 kinase domain were incubated with 0, 30, 40 and 50% v/v ammonium sulphate (AS). The precipitates were analysed by SDS-PAGE and Western blotting using an anti-GST antibody. The diffuse migration of the protein through the gel is most likely caused by the elevated salt content of the samples.


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3.6 Proteolytic tag removal

Affinity tags such as His6, Flag, MBP (maltose binding protein) and GST are

commonly fused to the protein of interest to enable single-step enrichment of the

target protein using tag-specific affinity chromatography columns. While the size

of the His6 and Flag tag is very small (6 and 8 amino acids, respectively), MBP and

GST are relatively large proteins (42 kDa and 26 kDa, respectively). Both MBP and

GST promote the solubility of their fusion partner (Kapust & Waugh, 1999; Luo et

al., 2007), which can result in higher recovery yields. Fusion of the GST tag to the

LIMK kinase domain may be beneficial for solubility, yield and ease of purification.

However, for structural studies, the presence of a large tag is unfavourable because

of its own spatial requirements and potential structural flexibility of the linker

region connecting the tag and the fusion partner. Furthermore, there is a potential

for tag-mediated oligomerisation, which can be beneficial or detrimental to the

formation of highly structured protein aggregates.

The linker region between the amino-terminal GST tag and the LIMK fusion

partner was designed to contain a cleavage site that is specific for the protease

thrombin. The consensus sequence P2-Pro, P1-Arg, P1′ -Ser/Ala/Gly/Thr, P2′ -not

acidic and P3′ -Arg (Gallwitz et al., 2012) is absent from the LIMK kinase domain

sequence. Therefore, it was concluded that the likelihood of proteolytic LIMK

kinase domain degradation by thrombin is relatively low.

GST-tagged mouse LIMK1 kinase domain was purified by glutathione-affinity

chromatography (Figure 3-8, lane 1) and the glutathione Sepharose beads were

incubated overnight with thrombin at 4°C. Analysis of the eluate (lanes 2 and 3) by

SDS-PAGE and staining with Coomassie Blue demonstrated the presence of a

cleaved product of about 39 kDa, which corresponds to the predicted molecular

weight of the mouse LIMK1 kinase domain. Considerable amounts of GST-tagged

LIMK1 kinase domain remained bound to the beads, as indicated by the dominant

high-molecular weight band in lane 4. Successful cleavage of a smaller amount of

fusion protein was confirmed by the presence of a band at around 25 kDa

representing the cleaved GST tag. Western blotting with an anti-LIMK1 antibody


91

that recognises the protein’s carboxyl-terminus confirmed the identity of the

eluted 39-kDa fragment.


92

Figure 3-8: Thrombin-mediated GST tag removal.

Affinity-purified GST-LIMK1 kinase domain was incubated with thrombin for on-column tag removal. Aliquots of immobilised protein before and after thrombin treatment as well as eluted soluble fractions were analysed by SDS-PAGE and Coomassie staining (A) or by Western blotting using an anti-LIMK1 antibody (B).


93

To assess the effect of prolonged exposure of the LIMK kinase domain to thrombin,

aliquots of purified LIMK1 kinase domain were collected before incubation with

the protease (GST-tagged protein immobilised on beads; Figure 3-9, lane 1) and

after 1- or 3-day incubation at 4°C (lanes 2 and 3) as well as after 3-day incubation

at room temperature (lane 4). Increasing amounts of protein fragments smaller

than 39 kDa were evident after 1 and 3 days incubation, suggesting that prolonged

incubation with thrombin leads to progressive degradation of the mouse LIMK1

kinase domain. The extent of protein degradation after overnight incubation with

the protease at 4°C was already considerable, as evidenced by the presence of two

degradation products representing two LIMK1-specific fragments that were

smaller than the kinase domain (approximately 35 kDa and 20 kDa, respectively).

This indicates the need for a shorter incubation time with thrombin as well as for

protection from other proteases that may have been co-purified. Incubation with

thrombin at 25 C for one hour cleaved the fusion protein with similar efficiency to

overnight incubation at 4°C. Following tag removal, the eluate was incubated with

Benzamidine Sepharose to remove serine proteases such as thrombin from the

protein preparation. In addition, a mixture of protease inhibitors was added to the

purified protein sample to inhibit residual protease activity.


94

Figure 3-9: Degradation of LIMK1 kinase domain.

Following on-column tag removal, aliquots of the eluate were stored for 1 or 3 days at 4°C and for 3 days at room temperature to assess protein stability. Samples were analysed by SDS-PAGE and Western blotting using an anti-LIMK1 antibody.


95

To determine the minimum concentration of thrombin required for efficient

cleavage of GST-LIMK1 kinase domain without causing excessive degradation of

the protein, glutathione Sepharose beads were saturated with GST-tagged human

LIMK1 kinase domain and were incubated with increasing amounts of thrombin

for one hour. The proteins in the eluate and in the immobilised fraction (beads)

were separated by SDS-PAGE and stained with Coomassie Blue. In the absence of

thrombin, no protein was detected in the soluble fraction, while large amounts of

GST-LIMK1 kinase were immobilised on the beads (Figure 3-10). With increasing

amounts of thrombin, a gradual increase in kinase domain protein was present in

the eluate. Similarly, increasing amounts of the second cleavage product, GST, were

found in the beads fraction. After incubation with 0.1 and 0.2 units of thrombin per

ml of beads, two major degradation products appeared in the soluble fraction (see

small arrows in Figure 3-10 at approximately 19 kDa and 24 kDa), indicating that

incubation with large amounts of thrombin can cause degradation of the LIMK1

kinase domain protein.


96

Figure 3-10: Titration of thrombin used for GST tag removal.

GST-tagged LIMK1 kinase domain was immobilised on glutathione Sepharose beads, followed by on-column tag removal using increasing amounts of thrombin (expressed as units per µl of beads). Aliquots of soluble and immobilised proteins were separated by SDS-PAGE and stained with Coomassie Blue. Small arrows indicate degradation products of approximately 19 kDa and 24 kDa, respectively.


97

3.7 Limited proteolysis

The size of the LIMK1 kinase domain used in this study (amino acids 309-647) was

considerably larger than the kinase domain fragments of other proteins that have

been successfully crystallised. For example, the kinase domains of the crystallised

human Src (PDB accession code 1YOJ; 283 residues; 34% sequence similarity)

(Breitenlechner et al., 2005b), Abl (3CS9; 277 residues; 30% sequence identity)

(Weisberg et al., 2005), Hck (2HK5; 270 residues; 33% sequence identity) (Sabat et

al., 2006) and Lck (3BYM; 272 residues; 31% sequence identity) (Martin et al.,

2008) are considerably shorter than the LIMK1 kinase domain of 339 residues.

The multiple sequence alignment of the above-mentioned kinase domains as

generated by the software Clustal Omega is shown in Figure 3-11.


98

Figure 3-11: Multiple sequence alignment of LIMK1 kinase domain and previously crystallised kinase domains. The amino acid sequences of the kinase domains of human Src, Hck, Lck, Abl and LIMK1 were aligned using the software Clustal Omega. Asterisk (*) indicates fully conserved residues; colon (:) indicates strong similarity; period (.) indicates weak similarity. Secondary structure prediction of LIMK1 kinase domain (residues 309-647) was performed using PSIPRED. Purple barrels and yellow arrows represent

-helices and -strands, respectively. Black lines represent non-structured coil regions.


99

The secondary structure prediction indicates that the LIMK1 kinase domain

construct is likely to contain extensive non-structured regions flanking the

catalytic domain at both the N-terminus and the C-terminus. Due to their high

degree of flexibility, these regions have a potential to interfere with the growth of

highly ordered protein crystals. Limited proteolysis and subsequent mass

spectrometry analysis offer an experimental approach to narrow down the

boundaries of a protein’s structured folding domains and exclude the surrounding

flexible regions, which due to their lack of structure are more susceptible to the

activity of proteases.

Recombinant GST-tagged human LIMK1 kinase domain was purified and incubated

with Proteinase K at 25 C, and aliquots were collected after incubation for 0, 0.5, 1,

2, 5, 10, 30 and 60 min.

Figure 3-12 shows the limited proteolysis samples separated by SDS-PAGE and

stained with Coomassie Blue. Despite the high extent of degradation of the protein

sample prior to addition of Proteinase K, efficient degradation of the ~70-kDa GST-

LIMK1 kinase domain was noticeable. During the first five minutes of incubation,

the amounts of high-molecular weight fragments decreased rapidly and an

increase in the intensity and number of bands representing lower-molecular

weight degradation products became apparent (Figure 3-12 A). Interestingly, a

protein band of approximately 37 kDa (see arrow) was found in all samples

collected between 0 and 10 min after addition of protease, indicating the presence

of a protease-resistant fragment with a molecular weight similar to that expected

for the LIMK1 kinase domain. Analysis of the same set of samples by Western

blotting using an anti-LIMK1 antibody confirmed the identity of that fragment. The

protein band was excised from the polyacrylamide gel and subjected to tryptic

digest and mass spectrometric analysis (peptide mass fingerprinting).


100

Figure 3-12: Limited proteolysis of GST-tagged LIMK1 kinase domain using Proteinase K. Recombinant GST-LIMK1 kinase domain was incubated with Proteinase K and aliquots of the reaction mixture were collected after 0, 0.5, 1, 2, 5, 10, 30 and 60 min. A protease-resistant fragment (arrow) was present until 30 min after addition of protease. A: Coomassie staining; B: anti-LIMK1 (C-terminus) Western blot.


101

The identified peptides (Figure 3-13; shown in bold red) matched 41% of the

amino acid sequence of human LIMK1 and overlapped with the LIMK1 kinase

domain sequence as listed in the Universal Protein Resource database

UniProtKB/Swiss-Prot (query: LIMK1_HUMAN; accession code P53667; amino

acids 339-604). The most N-terminal amino acid of the LIMK1 kinase domain

fragment as detected by peptide mass fingerprinting was isoleucine-333.

Interestingly, the most C-terminal amino acid identified in the sequence of the

LIMK1 kinase domain fragment was aspartate-647, which also represents the most

C-terminal residue in the full-length LIMK1 protein. This observation suggests that

the C-terminus of the protein was not exposed to the activity of Proteinase K and

may therefore form an integral part of the compact folding domain.

Integration of the experimental results and the theoretical considerations

concerning the boundaries of the LIMK1 kinase domain led to the design of a new

candidate fragment comprising residues 332-607 (Figure 3-14; sequence shown in

red). The truncated kinase domain sequence was fused to a cleavable N-terminal

His6-GST tag to increase the number of options for protein purification.


102

Figure 3-13: Peptides resulting from the tryptic digestion of a LIMK1 kinase domain fragment resistant to Proteinase K. Oligopeptide sequences coloured in red represent the LIMK1 peptides identified by the peptide mass fingerprinting. The remaining sequence of full-length LIMK1 is shown in black.


103

Figure 3-14: Amino acid sequence of the truncated LIMK1 kinase domain

designed for structure determination by x-ray crystallography.

The sequence in black represents the His6-GST tag and the LIMK1 kinase domain sequence (amino acids 332-607) is shown in red.


104

Similarly, expression constructs were generated for GST-tagged mouse LIMK1

kinase domain (residues 332-607 and 332-647). Based on the high similarity in the

predicted secondary structure between the LIMK1 and LIMK2 kinase domain (as

shown for the sequences for human LIMK1 kinase domain and rat LIMK2 kinase

domain in Figure 3-3), a His6-GST-tagged rat LIMK2 kinase domain construct

encoding residues 324-562 was cloned to further increase the number and

diversity of the related LIMK constructs available for structure determination

(Table 3-2).

The kinase domain fragments were expressed using the baculovirus/insect cell

system as described above. The expression levels of the His6-GST-tagged human

LIMK1 kinase domain and rat LIMK2 kinase domain as well as those of the GST-

tagged mouse LIMK1 kinase domain (residues 332-607) were very low compared

to mouse LIMK1 kinase domain without C-terminal truncation. Because of the

higher expression level, the longer construct encoding amino acids 332-647 was

used for the second round of protein expression and purification.


105

Table 3-2: Extended list of affinity-tagged kinase domain constructs for

LIMK1 and LIMK2.

Species Tag Kinase domain Residues

Human GST LIMK1 309-647


Mouse His6 LIMK1 309-647

Rat GST LIMK2 333-638

Human His6-GST LIMK1 332-607



Rat His6-GST LIMK2 324-562


106

3.8 Purification of the N-terminally truncated LIMK1 kinase domain

Recombinant bacmid DNA encoding the protease-resistant core of the LIMK1

kinase domain was generated and recombinant protein was expressed in High-

Five insect cells infected with P3 baculovirus. The protein was purified employing

ammonium sulphate precipitation and glutathione-affinity chromatography,

followed by thrombin-mediated removal of the GST tag. Samples were collected

throughout the purification process and were analysed by SDS-PAGE and

Coomassie staining (Figure 3-15). GST-tagged LIMK1 kinase domain is represented

by the ~65 kDa band that was present in the whole cell lysate (lane 1), cleared

lysate (lane 2) and in the protein pellet after incubation with ammonium sulphate

(lane 3), but not in the corresponding supernatant (lane 4). After glutathione-

affinity chromatography, unbound protein of the size of GST-LIMK1 kinase domain

was present in the flow-through (lane 5), yet large amounts of protein were

retained on the beads and were subsequently eluted with 10 mM reduced

glutathione (lanes 6 and 7). More purified protein was obtained by washing the

beads and collecting the wash fractions (lanes 8 and 9). After elution of the protein,

there was still a considerable amount of GST-tagged LIMK1 kinase domain bound

to the beads (lane 10), which was incubated with thrombin to remove the kinase

domain from the immobilised GST tag. The kinase domain fraction was collected

(lane 11) and further soluble protein was washed off the beads (lane 12). The

glutathione Sepharose beads retained only a small amount of tagged and untagged

LIMK1 kinase domain as well as the GST tag (lane 13).


107

Figure 3-15: Purification of truncated GST-tagged and tag-free LIMK1 kinase

domain.

The LIMK1 kinase domain was purified by glutathione-affinity chromatography followed by thrombin-mediated removal of the GST tag. Samples collected throughout the purification procedure were separated by SDS-PAGE and stained with Coomassie Blue. (NH4)2SO4: ammonium sulphate; GSH: reduced glutathione.


108

To test the stability of the tagged and tag-free forms of the LIMK1 kinase domain,

aliquots of the purified proteins were stored at room temperature, 4°C or -20°C for

a 3-day period and were subsequently analysed by SDS-PAGE and Coomassie

staining. Small-molecular weight degradation products were found in the tag-free

sample when stored at room temperature (Figure 3-16), while less degradation

was observed after storage at 4°C or -20°C. Surprisingly, the tag-free kinase

domain sample contained a considerable amount of white precipitate. Separation

of the soluble and insoluble fractions by centrifugation demonstrated that the

amount of soluble kinase domain protein was greatly reduced after removal of the

GST tag, while most of the protein appeared in the precipitated fraction.


109

Figure 3-16: Stability and solubility of truncated LIMK1 kinase domain.

Aliquots of purified GST-tagged LIMK1 kinase domain (GST-Kinase) and tag-free LIMK1 kinase domain (Kinase) were stored at room temperature, 4°C and -20°C for 3 days. Tag-free LIMK1 kinase domain was further separated into soluble and insoluble (precipitate) fractions. Samples were analysed by SDS-PAGE and Coomassie staining.


110

3.9 Solubility of LIMK1 kinase domain

The removal of the GST tag from a fusion protein is a critical step in the

purification procedure, as this may change the solubility characteristics of the

fusion partner. To try to increase the solubility of the tag-free LIMK1 kinase

domain, further GST-LIMK1 kinase domain was purified by glutathione-affinity

chromatography. The glutathione Sepharose beads were incubated with thrombin

and the eluate as well as the immobilised protein was separated by SDS-PAGE

prior to staining with Coomassie Blue. As shown in Figure 3-17, most of the LIMK1

kinase domain protein that was released from the GST tag and eluted from the

column appeared in the insoluble precipitated form (Ins). Only a small proportion

of the LIMK1 kinase domain was found in the soluble fraction (Sol). This indicates

a low solubility of the tag-free kinase domain under the tested buffer conditions

(pH 7.6-8.0, 100-150 mM NaCl, 1 mM DTT or 0.1% v/v 2-mercaptoethanol).

Addition of the carbohydrate-based polymer additive, NV10, to the cleavage buffer

efficiently prevented the precipitation of LIMK1 kinase domain after tag removal

and greatly increased the amount of kinase domain found in the soluble form. This

is most likely due the additive’s potential to mask hydrophobic patches on the

protein surface, thereby increasing its solubility and decreasing its tendency to

aggregate. In addition to the LIMK1 kinase domain protein, there were small

amounts of several other fragments present in the soluble fraction, indicating a

need for further purification of the protein.


111

Figure 3-17: On-resin cleavage of GST-hLIMK1 kinase domain in the presence

and absence of NV10.

The following samples were analysed by SDS-PAGE and Coomassie staining: protein bound to glutathione Sepharose beads before (B0) and after (B) incubation with thrombin; soluble protein (Sol) after incubation with thrombin; insoluble precipitate (Ins) formed during incubation with thrombin. NV10: solubilising polymer additive.


112

3.10 Purification of the LIMK1 kinase domain by ion-exchange and size-

exclusion chromatography

Ion-exchange chromatography and size-exclusion chromatography are commonly

used to improve the purity of protein preparations pre-purified by affinity

chromatography. Following removal of the GST tag (Figure 3-18 B, lane 1) in the

presence of the solubilising agent, NV10, the LIMK1 kinase domain sample was

dialysed against the gel filtration buffer (lane 2), filtered to remove particles and

precipitates (lane 3) and subjected to ion-exchange chromatography using a

MonoQ anion exchange column. However, the additive that afforded the protein

enhanced solubility seemed to interfere with the binding of the kinase domain to

the chromatography column, leading to its exclusion in the void volume (lane 4).


113

Figure 3-18: Purification of LIMK1 kinase domain by ion-exchange

chromatography.

A, Elution profile of the LIMK1 kinase domain sample separated using a MonoQ 5/50 GL ion-exchange chromatography column. Arrows indicate peaks of unbound protein as detected by absorbance measurement at 280 nm (blue line). Brown and yellow lines show conductivity and approximate eluent concentration, respectively. B, Western blot analysis of LIMK1 kinase domain sample after glutathione-affinity chromatography (1), after dialysis (2) and after filtration (3), as well as of the unbound fraction (4).


114

As separation according to protein size does not require binding to the column

matrix, size-exclusion chromatography was used to separate the soluble LIMK1

kinase domain from contaminants of higher and lower molecular weight. Aliquots

of LIMK1 kinase domain were collected after affinity chromatography and tag

removal (Figure 3-19 A, lane 1), after concentrating the sample with a 10-kDa cut-

off filtration column (lane 2) and after separation by size-exclusion

chromatography (lane 3). The aliquots were analysed by SDS-PAGE and staining

with Coomassie Blue. As displayed in Figure 3-19 A, lanes 1 and 2, the LIMK1

kinase domain sample included four major contaminant proteins, two at lower

molecular weight and two at higher molecular weight than the kinase domain. The

purity of the sample increased through size-exclusion chromatography as it

efficiently removed the high-molecular weight contaminants (lane 3). However,

the low-molecular weight contaminants still remained in the sample. Figure 3-19 B

shows the elution profile of the LIMK1 kinase domain sample when separated

according to protein size. As the buffer conditions were selected to separate

proteins in their native state, the major peak in the elution profile (marked by the

arrow) indicates that LIMK1 kinase domain protein was present predominantly in

one oligomeric form eluted at a buffer volume of approximately 16.5 ml.

Comparison with the elution profile of a protein size standard (separated on the

same model of column; Figure 3-19 C) indicates that this dominant form was

similar in size to the protein -lactoglobulin (molecular weight of 35 kDa) and

therefore was the LIMK1 kinase domain monomer.


115

Figure 3-19: Purification of the LIMK1 kinase domain by size-exclusion

chromatography.

A, Elution profile of a protein preparation containing LIMK1 kinase domain (see arrow). Molecular weight of a protein standard is shown below (adapted from GE Healthcare instructions booklet 71-5017-96 AF). B, 1: Sample after glutathione-affinity chromatography and removal of GST tag. 2: Sample after concentration using a filtration column with 10-kDa cut-off. 3: Sample after size-exclusion chromatography. Note that high-molecular weight contaminants were efficiently removed. Arrow indicates LIMK1 kinase domain.


116

3.11 Catalytic activity of the purified LIMK1 kinase domain fragment

A protein’s ability to perform a catalytic function is dependent on its proper

folding. In the context of protein kinases, a catalytically active tertiary structure

must be able to accommodate a phosphoryl group donor (e.g. ATP) as well as a

phosphoryl group acceptor, and to facilitate the transfer of the -phosphoryl group.

To test the catalytic activity of the purified LIMK1 kinase domain, an in vitro [ -

32P]ATP kinase assay was performed using GST-tagged cofilin as a LIMK1

substrate.

When LIMK1 was omitted from the reaction mixture, GST-cofilin was not

phosphorylated (Figure 3-20). In the absence of cofilin, only a faint band of auto-

phosphorylated LIMK1 kinase domain was observed. The LIMK1 kinase domain

efficiently phosphorylated its substrate, GST-cofilin, to a similar level as the full-

length LIMK1 protein. This result shows that the purified LIMK1 kinase domain

retained its catalytic activity and suggests that the tertiary structure of the catalytic

domain was not compromised by the purification procedure.


117

Figure 3-20: In vitro phosphorylation of cofilin by purified LIMK1.

[ -32P]ATP kinase assay was performed using either LIMK1 kinase domain or GST-tagged full-length LIMK1 protein, and GST-cofilin as a substrate. Samples were analysed by SDS-PAGE followed by autoradiography.


118

3.12 Crystallisation trials

At several stages in the course of this study, the LIMK kinase domain samples were

subjected to crystallisation screening at the Bio21 Collaborative Crystallisation

Centre, Melbourne. Despite suboptimal sample purity, these trials were conducted

knowing that the process of crystallisation may be considered a purification step

itself (Benvenuti & Mangani, 2007; Judge et al., 1998).

The LIMK1 kinase domain was purified in either a two-step procedure involving

ammonium sulphate precipitation and glutathione-affinity chromatography or in

three steps with the addition of a size-exclusion chromatography step. The sample

was concentrated in the presence or absence of the solubilising agent, NV10, and

was supplemented or not with 1 mM ATP prior to performing the automated 96-

well based crystallisation assays. Nucleotide binding to the kinase domain was

hypothesised to stabilise a specific conformation, thereby increasing the degree of

order in the protein sample. However, none of the conditions tested were suitable

for the growth of LIMK1 kinase domain protein crystals or the formation of

microcrystalline precipitation. This may be due to persistent impurities in the

sample caused by continuous protein degradation or to insufficiently high protein

concentration.

These issues were overcome by Beltrami et al. from the Structural Genomics

Consortium, who deposited the structure of the human LIMK1 kinase domain in

complex with the non-selective protein kinase inhibitor staurosporine in the

Protein Data Bank (PDB accession code 3S95).

119

CHAPTER 4 − DISCUSSION

Chapter 4 − Discussion

120


121

4.1 Optimisation of baculovirus production

Previous work performed in our laboratory indicated that large-scale expression

of soluble LIMK protein in bacteria and mammalian cells was rather inefficient and

uneconomical. In contrast, using the baculovirus/insect cell system appeared to be

a viable approach for the expression of LIMK protein as it is used successfully to

produce commercial recombinant LIMK proteins (e.g. Millipore).

Despite the theoretical advantage of the Bac-to-Bac Baculovirus Expression System

in avoiding contamination with non-recombinant parental virus (Luckow et al.,

1993), baculovirus plaque assays had to be performed to prevent a decrease in

titre during virus amplification. The resulting high-titre viruses were used at a

virus:culture ratio between 1:40 and 1:100 to yield varying levels of protein

expression depending on the specific expression construct. The expression level of

recombinant mouse and human LIMK1 kinase domain proteins was highest in the

Trichoplusia ni cell line, High Five (see Figure 3-5). In favour of protein integrity

over maximum expression level, an incubation period of 72 hours was chosen.

In the literature, there is no consensus on which insect cell lines produce the

largest quantity of recombinant proteins, as protein expression depends on a

variety of factors including the size and sequence of the specific gene product, the

expression vector and other parameters influencing the rate of cell growth such as

pH, oxygen level, medium supplements and cell density (Maruniak, 1996).

Therefore, careful optimisation of expression conditions has a potential for

considerable gains in recombinant protein yield.

4.2 Construct design

The initial set of constructs used to establish suitable conditions for the expression

of large quantities of recombinant LIMK kinase domain included a variety of

sequences from different species in combination with different affinity tags.

Despite a sequence similarity of ~70% in the kinase domain of LIMK1 and LIMK2,

there was a large difference in the expression levels of the recombinant LIMK1 and


122

LIMK2 kinase domain proteins (see Figure 3-4). This may be attributed to the

difference in amino acid sequence or to the fact that the cDNA encoding the human

LIMK1 kinase domain was codon-optimised for expression in insect cells.

However, the effect of codon optimisation on the expression level may be only

minor as the non-optimised mouse LIMK1 kinase domain sequence was expressed

at equally high levels as the human LIMK1 kinase domain.

As far as the choice of affinity tag is concerned, there is a multitude of options

available for facilitating the purification and detection of the protein of interest.

The most widely used tags include the His6 and GST tag, both of which can be

added to either the N- or C-terminus of the recombinant protein and have been

used successfully in baculovirus-infected insect cells (Beekman et al., 1994;

Schmidt et al., 1998). The 26-kDa GST tag derived from Schistosoma japonicum is

known for its solubility-enhancing effect on its fusion partner. In addition, it can

also help to protect the recombinant protein against cleavage by intracellular

proteases (Terpe, 2003). In contrast, the small His6 tag is less likely to affect any

critical characteristics of the fusion partner such as solubility, protein folding and

function. This provides a possible explanation for the low yield of His6-tagged

mouse LIMK kinase domain compared to the GST-tagged proteins, which most

likely derived a benefit from the protective and solubilising effects of the larger

affinity tag.

Following affinity purification, the GST tag was removed by proteolytic cleavage as

it may induce dimerisation and interfere with the native conformation of the

LIMK1 kinase domain. It also represents a potential source of undesirable

structural flexibility due to the presence of a flexible linker region between the GST

tag and the kinase domain, in which a recognition sequence for proteolytic

cleavage is located. Despite its lower sequence specificity compared to other

commonly used proteases such as the TEV protease (Phan et al., 2002), thrombin

has a lower tendency to bind non-specifically to column materials, which makes it

more suitable for on-column tag removal.


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4.3 Ammonium sulphate precipitation and catalytic activity

The first step in the purification of the LIMK1 kinase domain, namely its

precipitation induced by addition of ammonium sulphate (see Figure 3-7),

successfully removed the high levels of endogenous glutathione-binding proteins

that are expressed in insect cells and interfered with the affinity purification on

immobilised glutathione (see Figure 3-6). Bichet et al. estimated that these

endogenous proteins are expressed at levels as high as 5-10 mg per litre of culture

(Bichet et al., 2000). They also showed that their amino acid sequences were

different from that of the GST tag used in fusion proteins, which provides an

explanation for why they were not detectable by immunoblotting for GST.

However, due to their similarity to glutathione S-transferase, these previously

uncharacterised, endogenous proteins have been named GST-like proteins.

The two ions, ammonium and sulphate, that form the precipitating agent,

ammonium sulphate, both rank high in the Hofmeister series, a classification of

ions in order of their influence on the physical behaviour (e.g. solubility) of

macromolecules (Zhang & Cremer, 2006). The salting-out process is based on the

recruitment of solvent molecules to form solvent shells around salt ions. This

leaves less solvent molecules available for the hydration of proteins. The increased

surface tension of the solution stabilises the protein structure, while hydrophobic

regions on the protein surface are encouraged to interact, resulting in protein

aggregation. At a given ammonium sulphate concentration, proteins with a larger

hydrophobic surface area precipitate, whereas those with a less hydrophobic

surface character remain in solution.

The pellet of the ammonium sulphate-precipitated protein (containing the protein

of interest) was readily re-dissolved prior to purifying the LIMK1 kinase domain.

The final product retained its catalytic activity toward its substrate cofilin (see

Figure 3-20). It is well established that protein function, such as phosphoryl-group

transfer in the case of kinases, is dependent on the correct folding of the

polypeptide chain (Berg et al., 2002a). Consistent with the structure-stabilising

effect of “salting-out” (Timasheff & Arakawa, 1997), the LIMK1 kinase domain

maintained its catalytic activity toward cofilin, indicating that its three-


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dimensional structure was preserved and unaffected by the multi-step purification

procedure.

4.4 Protein stability

Following on-column tag removal by thrombin, the LIMK1 kinase domain protein

containing residues 309-647 appeared to be prone to degradation (see Figure 3-9).

The observed structural instability did not improve by handling and storing the

sample on ice or at 4°C, or by addition of a cocktail of protease inhibitors. Removal

of thrombin by incubation with immobilised Benzamidine did not inhibit protein

degradation. One potential explanation may be bacterial contamination of the

sample associated with protein degradation by bacterially produced proteases.

Nonetheless, low protein stability can be considered a valuable indicator for an

unfavourable inherent characteristic of the construct that requires optimisation.

Regions of high flexibility in the polypeptide chain were eliminated by limited

proteolysis (see Figure 3-11). The broad specificity of the protease, Proteinase K,

was utilised to induce proteolytic events that are determined by the

stereochemistry and flexibility of the substrate, and are independent of the

specificity of the attacking protease (Fontana et al., 2004; Sweeney & Walker,

1993). Significant conformational changes in the polypeptide backbone are

necessary for proteolytic cleavage, involving local unfolding of a stretch of residues

according to the requirements of an induced-fit mechanism (Hubbard et al., 1994).

Therefore, only sites demonstrating inherent chain flexibility, such as the

connecting segments between protein domains, are cleaved, while the rigid protein

core is resistant to proteolytic activity.

Despite a relatively low enzyme:substrate ratio (Proteinase K:GST-LIMK1 kinase

domain) of 1:1000 (w/w), the parental GST-LIMK1 kinase domain protein was

undetectable by Western blot only 5 min after addition of the protease.

Considering that commonly used enzyme:substrate ratios are in the range of 1:50

to 1:100 (Fontana et al., 2004), the rapid degradation of GST-LIMK1 kinase domain

is an indicator for the low stability of this protein. This is also supported by the

extent of degradation of the sample before addition of the protease despite careful


125

preparation under temperature-controlled and protease-inhibiting conditions. It

should be noted that the only antibody available for detecting the kinase domain

by Western blot was an anti-LIMK1 antibody raised against the protein’s C-

terminus. Consequently, only fragments containing this epitope could be detected

by Western blot.

The sequences of all peptides that were identified by mass spectrometry (see

Figure 3-13) were then aligned with a secondary structure prediction for the

LIMK1 kinase domain. (see Figure 3-11) Both the limited proteolysis results and

the secondary structure prediction suggested the absence of the N-terminal region

of the LIMK1 kinase domain from the compactly folded core. However, while the in

silico prediction indicates a large unstructured region at the protein’s C-terminus,

the results of the limited proteolysis experiment suggest that the LIMK1 C-

terminus is protected from proteolytic degradation. This raises the possibility that

the C-terminal tail of LIMK1 may fold back into the kinase domain, thereby making

it inaccessible to proteolytic cleavage. A similar mechanism has been reported for

the C-terminal tail of the tyrosine kinase c-Src, and has been implicated in the

regulation of its kinase activity (Cowan-Jacob et al., 2005).

From these results, two truncated constructs containing residues 332-647 and

residues 332-607 were derived as promising candidates for crystallisation trials.

Due to the low expression level of the C- and N-terminally truncated construct

(residues 332-607), only the N-terminally truncated construct was expressed at

large enough quantities for structural studies.

4.5 Protein solubility

The “sticky” nature of the LIMK1 kinase domain represented a major challenge

during protein purification, as it impedes the use of chromatographic methods in

the absence of an efficient strategy to neutralise hydrophobic patches on the

protein surface and prevent non-specific interactions with column materials and

other kinase domain molecules. Addition of the NVoy polymer NV10 to the tag-free

LIMK1 kinase domain sample increased protein solubility and prevented protein

precipitation (Figure 3-17). This neutral, linear polymer with a molecular weight of


126

5 kDa binds to hydrophobic patches on the surface of proteins through its

hydrophobic side chains, which are attached to a hydrophilic carbohydrate

backbone. Thereby, it reduces protein aggregation induced by hydrophobic

interactions. Similar beneficial effects have been reported for the use of NVoy

NV10 as an additive in a cell-free protein expression system, where it increased the

quantities of soluble protein yield without substantially reducing the total protein

yield of the in vitro translation reaction (Guild et al., 2011). It has also been used

for the cell-free expression and solubilisation of integral membrane proteins

(Klammt et al., 2011). This strategy for enhancing protein solubility was chosen in

favour of the use of detergents, as these agents at too high concentrations can

inhibit crystal growth.

In contrast, Beltrami and co-workers, who solved the atomic structure of the

human LIMK1 kinase domain in complex with the inhibitor staurosporine, used L-

arginine and L-glutamate at a concentration of 5 mM each as well as 5% v/v

glycerol as solubility-enhancing additives, allowing them to efficiently purify and

concentrate the protein to 8 mg/ml (Beltrami et al., 2011). Simultaneous addition

of the charged L-amino acids, arginine and glutamate, has been shown to increase

solubility and long-term stability of purified proteins (Golovanov et al., 2004). The

mechanism has been speculated to involve both electrostatic and hydrophobic

components, leading to a reduced “stickiness” of the protein and therefore less

protein aggregation. In addition, the presence of L-glutamate and L-arginine

provide protection from proteolytic degradation, presumably due to competitive

inhibition or masking of recognition sites for proteases. Similarly, proteins are

preferentially hydrated in the presence of glycerol, which stabilises the native

structure of globular proteins (Gekko & Timasheff, 1981). At high concentrations

(>>0.1 M), L-glutamate and L-arginine may have an increased osmolytic effect on

proteins, thereby weakening protein-ligand interactions. This is consistent with

our observation that concentrations of 0.5 M cause elution of GST-tagged LIMK

protein from glutathione Sepharose beads in the absence of reduced glutathione.

It has been estimated that about 33-50% of all expressed proteins have

unfavourable solubility characteristics and that 25-57% of the remaining soluble

proteins cannot be concentrated, as they tend to aggregate or precipitate


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(Golovanov et al., 2004). Based on the higher protein concentration that was

achieved by the Beltrami and co-workers, their solubilisation strategy was more

effective, thereby increasing the likelihood of obtaining protein crystals in

crystallisation trials (Schmit & Dill, 2012).

4.6 Crystallisation propensity

Crystallisation trials in the presence and absence of ATP were performed using

sparse matrix and grid screens to test a wide range of crystallisation conditions

with varying pH, concentrations and combinations of ions, polymers and organic

additives. While most drops remained clear, amorphous precipitates were

observed only in a small number of wells, indicating that conditions were

unfavourable for protein nucleation and crystal growth.

The lack of hits in the performed crystallisation screens was most likely due to

high sample heterogeneity caused by protein degradation, and lack of

monodispersity due to low solubility of the LIMK1 kinase domain. Sample

homogeneity, stability and solubility strongly correlate with the probability of

yielding protein crystals (Ericsson et al., 2006). Addition of the solubilising agent,

NV10, enhanced protein solubility, but was not sufficient to prevent precipitation

of the concentrated protein sample.

Addition of ATP, the natural nucleotide substrate that is mimicked by most

synthetic kinase inhibitor compounds, was predicted to potentially have a

stabilising effect on the structure of the LIMK1 kinase domain (Niesen et al., 2007).

In a typical kinase domain structure, ATP binds in a deep cleft that is located at the

interface of the two lobes of the catalytic core (Johnson et al., 1996). Due to the

central location of the nucleotide docking site, it seems plausible that ATP binding

may have a stabilising effect on the relative orientation of the two lobes or on other

elements that affect protein conformation. For example, it has been shown that

occupancy of the nucleotide-binding pocket of Akt enables intra-molecular

interactions that restrict access of phosphatases to threonine-308 in the activation

loop (Chan et al., 2011). This loop, which is flanked by the characteristic DFG and

APE motifs, can block the substrate-binding site or allow access depending on its


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phosphorylation state (Frankel et al., 1999). For the pseudo-kinase STRAD , it has

been demonstrated that binding of ATP and the scaffolding protein MO25

stabilises the closed conformation that is essential for STRAD -mediated

activation of the LKB1 tumour suppressor (Zeqiraj et al., 2009).

Similar to ATP, addition of the non-specific kinase inhibitor, staurosporine, to the

LIMK1 kinase domain, as performed by Beltrami et al., may have had a major

impact on the protein’s crystallisability as the inhibitor is likely to stabilise the

kinase domain in a certain conformation (Vedadi et al., 2006). When bound to their

molecular target, kinase inhibitors can stabilise the active or inactive form of the

protein (Jura et al., 2011; Xu et al., 2011). Trapping the enzyme in one out of

multiple possible conformations may facilitate protein cystallisation by enhancing

protein rigidity and inducing a higher degree of homogeneity among the molecules

in the sample.

In contrast, other studies suggest that crystallisation propensity is not strongly

influenced by overall thermodynamic stability (Price et al., 2009). Instead, the

prevalence of well-ordered surface epitopes capable of mediating protein-protein

interactions has been proposed to be the major determinant for crystallisability. As

demonstrated by Beltrami and co-workers, crystallisation of the human LIMK1

kinase domain can be achieved without prior mutation of surface residues.

Nonetheless, reduction of the conformational entropy of surface residues may be

necessary for proteins that are notoriously difficult to crystallise (Cooper et al.,

2007; Longenecker et al., 2001).

The optimised LIMK1 kinase domain construct (residues 332-647) had a

promising 97.5% overlap with the slightly shorter construct (residues 330-637)

used by Beltrami and co-workers. However, even small differences in sequence can

potentially have a large impact on a protein’s propensity to form crystals.

4.7 Structure of the LIMK1 kinase domain/staurosporine complex

The structure of the human LIMK1 kinase domain in complex with staurosporine

as determined by Beltrami et al. to 1.65-Å resolution shows the typical bi-lobal


129

kinase fold with the inhibitor occupying the ATP binding pocket at the interface

between the two lobes (Beltrami et al., 2011) (Figure 4-1).

The inhibitor interacts with the catalytic cleft through hydrogen bonds between

the backbone carbonyl groups of E414 and H464, and nitrogen atoms N1 and N4 of

staurosporine, respectively (Figure 4-2). An additional hydrogen bond is formed

between the oxygen atom O5 of staurosporine and the amide nitrogen of I416, in

combination with a dipolar interaction between the amide carbonyl group of I416

and the amide carbon of staurosporine.


130

Figure 4-1: Structure of the LIMK1 kinase domain/staurosporine complex.

The two-lobed structure of the LIMK1 catalytic domain is shown as cartoon representation with staurosporine (STU) bound in the catalytic cleft (PDB accession code 3S95). Yellow arrows indicate -strands and -helices are coloured pink. Critical loops in the vicinity of the substrate-binding site as well as the loop insertion replacing the G helix are indicated.


131

Figure 4-2: Polar interactions between staurosporine and residues in the

catalytic cleft of LIMK1 kinase domain.

The catalytic cleft of LIMK1 kinase domain is shown as cartoon representation (strands coloured purple; helices in green; coils in brown) and staurosporine is depicted as a stick model. Hydrogen bonds and dipolar interactions are represented by black dotted lines. The catalytic aspartate (D460) and the non-phosphorylated threonine residue 508 (T508) are shown as reference points.


132

The αC helix represents an important structural element in the regulation of kinase

activity (Taylor & Kornev, 2011). In the active conformation of the kinase domain,

a characteristic salt bridge is formed between a lysine residue in the 3 strand and

a glutamate residue in the C helix. This enables the conserved lysine residue to

coordinate the - and -phosphate groups of ATP. In the LIMK1/staurosporine

complex, a 2.8-Å salt bridge is formed between K368 and E384, indicating that

compound binding stabilises the active conformation of the kinase domain (Figure

4-3) (Beltrami et al., 2011).

Correct positioning of the -phosphate of ATP is achieved in serine/threonine

kinases through contact with a lysine residue located two residues C-terminal to

the catalytic aspartate (D460 in LIMK1) (Endicott et al., 2012). In tyrosine kinases,

the stabilising residue is an arginine located four residues C-terminal to the

catalytic aspartate. Despite being a serine/threonine kinase, LIMK1 does not fit

either category, with residues 462 and 464 being an asparagine and a histidine,

respectively (Okano et al., 1995).

The unusual additional loop replacing the G helix is a common feature of the LIM

and TES (testis-specific) kinase families (Figure 4-4), both of which are tyrosine

kinase-like serine/threonine kinases and have cofilin as a substrate (Toshima et

al., 1995; Toshima et al., 2001a; Toshima et al., 2001b). The loop insertion has been

suggested to contribute to substrate specificity through recognition of cofilin-

specific surface features (Beltrami et al., 2011). The sequence forming the

additional helix J at the C-terminus of the LIMK1 kinase domain has no conserved

equivalent in the TES kinases and appears to be LIMK-specific (Figure 4-4).


133

Figure 4-3: Active conformation of LIMK1 kinase domain in complex with

staurosporine.

The -sheet of the N-lobe and the catalytic cleft of the LIMK1 kinase domain are shown as cartoon representation (strands in purple; helices in green; coils in brown). Staurosporine is shown as a stick model. A salt bridge (black dotted lines) formed between two residues (K368 and E384) located in -strand 3 and the C helix stabilises the catalytic domain in an active conformation. The catalytic aspartate (D460) and the non-phosphorylated threonine residue 508 (T508) are shown as reference points.


134

A large part of the activation loop (residues 487-506) was not defined in the

electron density and is therefore not shown in the structure (Figure 4-1, 4-3 and 4-

4). This suggests high flexibility in this region, which may be associated with the

absence of phosphorylation on threonine residue 508 in the activation loop (Figure

4-3).

Further studies aimed at determining the structure of the LIMK1 kinase domain by

itself or in complex with more specific inhibitors are required to gain further

insight into the function of LIMK-specific features. Crystallisation of the

phosphomimetic mutant, T508EE, will be of particular interest with regard to

stabilisation of the activation loop. These studies also promise new avenues to

optimising the potency and specificity of small-molecule LIMK inhibitors.


135

Figure 4-4: Sequence alignment of LIM and TES kinases.

Multiple sequence alignment of human LIMK1, LIMK2, TESK1 and TESK2 was generated based on the sequence of the LIMK1 kinase domain construct used by Beltrami et al. (Beltrami et al., 2011). Asterisk (*) indicates fully conserved residues; colon (:) indicates strong similarity; period (.) indicates weak similarity. The part of the activation segment missing in the LIMK1/staurosporine structure (PBS accession code 3S95) is underlined in green. Red box indicates the loop insertion replacing the G helix. The additional helix J in LIMK1 is underlined in purple.

137

CHAPTER 5 − CONCLUSION

Chapter 5 − Conclusion

138


139

The LIMK family of proteins plays a critical role in the regulation of actin re-

organisation, which has sparked significant interest in its suitability as a drug

target. Several commercially and publicly funded drug development campaigns

have since been initiated, yielding promising lead compounds and more refined

inhibitor compounds. However, the absence of a three-dimensional structural

model for the kinase domain has hindered the rapid progress that can be enabled

by structure-based optimisation of LIMK inhibitors.

In this thesis, the benefits of employing the baculovirus/insect cell system are

demonstrated for large-scale expression of LIMK kinase domain proteins, which

have previously been expressed using other systems at levels that were

insufficient for structural studies. Associated with the choice of expression system

are specific technical challenges such as expression of endogenous proteins that

interfere with the purification of the protein of interest. In particular, endogenous

GST-like proteins had to be removed from the cell lysate by ammonium sulphate

precipitation prior to glutathione affinity purification. The relatively large, N-

terminally fused GST moiety had a significant solubility-enhancing effect on the

LIMK kinase domain. Accordingly, its removal resulted in protein precipitation,

which was alleviated by addition of the solubilising agent, NV10. Identification of a

protease-resistant fragment of the human LIMK1 kinase domain led to an

improved definition of its domain boundaries. This fragment retained its catalytic

activity toward its in vivo substrate, cofilin. In addition to the protein’s propensity

to precipitate, it also demonstrated limited amenability to common

chromatographic purification methods due to non-specific hydrophobic

interactions with column matrices.

Unfavourable solubility characteristics are well known to be a major hurdle in the

process of protein structure determination and require effective and case-specific

solutions. Addition of the solubilising polymer, NV10, proved to be inferior to the

solubilisation strategy developed by Beltrami and co-workers, allowing them to

achieve a much higher final concentration of purified protein (8 mg/ml, as

compared to approximately 3 mg/ml in the current study) and to crystallise the

human LIMK1 kinase domain in the presence of 24% v/v 2-methyl-2,4-

pentanediol, 100 mM Tris (pH 7.2) and 10 mM phenol.


140

Protein crystallisation still remains the bottleneck in the process of structure

determination. However, regardless of the outcome of crystallisation trials,

systematic characterisation of the protein of interest (prior to crystallisation)

provides vital information for future attempts to solve the structure of related

molecules. The importance of detailed empirical data on proteins such as LIMK

becomes even clearer when it is placed in the context of the development of

algorithms used to predict a protein’s experimental behaviour. This development

is driven by structural genomics consortia, which, in a coordinated international

effort, produce large numbers of atomic protein structures through a pipeline of

successive experimental stages ranging from cloning to structure determination

and deposition (Smialowski & Frishman, 2010). As a result of this high-throughput

approach, an abundance of positive and negative experimental data has become

available. One of the most striking observations was that only about three percent

of the initially selected targets reach the end of the pipeline and are deposited as

structures in the protein data bank. Despite the relatively low success rate,

detailed analysis of the success and failure data has extended our limited

understanding of how specific sequence features impact on a protein’s

experimental behaviour, thereby linking physical and chemical properties to the

amenability to structure determination (Babnigg & Joachimiak, 2010; Mizianty &

Kurgan, 2011). This preliminary knowledge has been incorporated into improved

algorithms to more reliably predict important protein features such as domain

boundaries, secondary structure, regions of high entropy and hydrophobic

patches, and to guide the design of crystallisable protein constructs.

The determination of the first atomic structure of the LIMK1 kinase domain by

Beltrami et al. represents a milestone in the development of LIMK-specific

inhibitors and warrants further structural studies that will lead to a more accurate

and representative molecular model. Such a model, in turn, will enable in silico

docking studies using large compound libraries to identify new lead structures for

competitive inhibitors. Structures of the LIMK kinase domain in complex with

currently available LIMK inhibitors are likely to emerge in the near future,

allowing the identification of possible candidates for residues contributing to

interactions with these compounds. Site-directed mutagenesis targeting relevant


141

residues and subsequent functional studies will provide data that will form the

rational basis of how the potency of LIMK inhibitors can be improved. Ultimately,

identification of potential sequence differences in the less conserved loop regions

around the compound binding sites in LIMK1 and LIMK2 may even provide clues

for the development of more selective inhibitors.

Highly potent and selective LIMK inhibitors do not only represent promising

research tools that can be used to delineate the complex signalling cascades

leading to actin re-organisation, but may also be developed into therapeutic drugs

targeting actin dysregulation as the underlying cause of human diseases. First and

foremost, LIMK inhibitors have a potential to satisfy the currently unmet clinical

need for anti-metastatic drugs. Interestingly, pharmacological targeting of a

different regulator of the actin cytoskeleton, fascin, has recently been

demonstrated to inhibit tumour cell migration, invasion and metastasis (Chen et

al., 2010). The underlying mechanism involves inhibition of the actin-bundling

activity of fascin, which is required for the cross-linking of actin filaments in

filopodia (Vignjevic et al., 2006). In addition to its potential application in cancer

therapy, LIMK inhibitors may also prove useful for the treatment of ocular

hypertension in patients who are at risk of developing glaucoma (Harrison et al.,

2009).


142

143

CHAPTER 6 − LITERATURE REVIEW


144


145

6.1 Introduction

The LIMK family of protein kinases plays an important role in the regulation of

actin dynamics (Bernard, 2007) and has been implicated in pathological conditions

such as cancer cell invasion and increased intraocular pressure (Harrison et al.,

2009; Morin et al., 2011; Scott et al., 2010; Yoshioka et al., 2003). No phenotypic

abnormalities in postnatal growth and development were found in the LIMK2

knockout mice generated by Takahashi and co-workers, except for degeneration of

spermatogenic cells in the testes, which can be attributed to the lack of the testis-

specific isoform, LIMK2t (Takahashi et al., 2002). To investigate the individual

contributions of the other LIMK2 isoforms, in particular LIMK2a and LIMK2b, to

the regulation of actin dynamics, a LIMK2a-deficient mouse was generated.

Disruption of only the longest transcript of the LIMK2 gene gave rise to an

unexpected obesity phenotype in adult male mice. This finding suggested a

previously uncharacterised role for LIMK2a in the development or function of

adipose tissue. Based on its regulatory function in cofilin-mediated actin re-

organisation, we hypothesised that LIMK2a may be involved in the process of

adipogenic differentiation, which is known to entail extensive restructuring of the

actin network (Kawaguchi et al., 2003; Spiegelman & Farmer, 1982).

Obesity as a health condition is not restricted to adipose tissue, but has been

recognised to affect a variety of tissues, some of which are of great significance for

systemic energy homeostasis. Consequently, this complex disease has been

associated with a host of co-morbidities including cardiovascular disease (Klein et

al., 2004), type 2 diabetes (Arai et al., 2010), hypertension (Dorresteijn et al.,

2011), sleep apnoea (Foster et al., 2009) and cancer (Calle & Kaaks, 2004).

Unravelling the mechanism that links LIMK2a to the pathogenesis of obesity will

further our understanding as to how genetic factors predispose individuals to

developing the disease and may help to discover novel treatment strategies for

patients suffering from obesity-related morbidities.


146

6.2 Overweight and obesity

The rising prevalence of overweight and obesity in the population of both

developed and developing countries is recognised as a health concern of global

magnitude (Prentice, 2006). Data collected in the Australian National Health

Survey in 2007 and 2008 (ABS, 2009) suggests that 37% and 25% of the

population were overweight (BMI1 ≥ 25 kg/m2 < 30 kg/m2) and obese (BMI ≥ 30

kg/m2), respectively, with numbers predicted to rise steadily. The so-called obesity

epidemic places a considerable burden on the public health system as well as on

the economy in general through decreased work productivity (Wang et al., 2011;

Withrow & Alter, 2010). Critical determinants of the epidemic are behavioural and

environmental changes such as increased calorie intake, larger portion size and

unfavourable nutritional composition of meals as well as lack of physical activity as

a result of an increasingly sedentary lifestyle (Hernandez et al., 2006). Genetic

factors, on the other hand, appear to determine an individual’s susceptibility to the

effects of environmental factors (O'Rahilly & Farooqi, 2006). In most cases, obesity

is caused by several genetic factors that each contribute a small degree to the

cumulative effect (i.e. weight gain) (Clement, 2005; Li et al., 2010).

6.3 Obesity-induced adipocyte dysfunction

White adipose tissue is specialised in the storage and release of chemical energy

(Berg et al., 2002b). During times of high energy intake, glucose and free fatty acids

are converted to glycerol-3-phosphate and fatty acyl-coA, which are esterified to

yield triacylglycerol in a process called lipogenesis (Figure 6-1). The energy

content per gram of stored lipid is considerably higher compared to other sources

of chemical energy, such as carbohydrates and amino acids. Moreover, due to their

hydrophobic character, lipids can be stored in the absence of water molecules.

When the supply of glucose is insufficient, fatty acids and glycerol are released

1 Body mass index (BMI) is defined as the ratio of body weight in kilograms to the square of height in

metres, and is commonly used as an approximation for body fat content.


147

from adipocytes to provide other tissues with chemical energy (Berg et al., 2002b;

Ducharme & Bickel, 2008). The hydrolytic mobilisation of fatty acids from

triglycerides is called lipolysis and involves a series of reactions regulated by

lipases, co-lipases and lipid droplet coat proteins. The sequential hydrolysis of

triglycerides, diglycerides and monoglycerides is catalysed by adipose triglyceride

lipase (ATGL), hormone-sensitive lipase (HSL) and monoglyceride lipase,

respectively. The lipid droplet surface protein, perilipin 1, plays an essential role in

mediating the response of ATGL action to -adrenergic signals (e.g. isoproterenol)

(Lass et al., 2011; Miyoshi et al., 2007). Upon phosphorylation by PKA, perilipin

releases CGI-58, a co-activator of ATGL, leading to enhanced ATGL-mediated

lipolysis (Granneman et al., 2009).

A prolonged positive energy balance caused by high calorie intake that exceeds

energy expenditure leads to adipose tissue expansion through increase in cell size

(adipocyte hypertrophy) and/or cell number (adipose tissue hyperplasia). It has

been proposed that the maximum fat storage capacity of an individual is limited by

environmental and genetic factors (Slawik & Vidal-Puig, 2007). Once the lipid

storage capacity of the adipose tissue is exhausted because of an inability to

further expand, ectopic lipid deposition occurs in organs such as liver and muscle,

where it causes lipid-induced toxicity through oxidative and non-oxidative

metabolic pathways (Unger & Zhou, 2001).

In addition to its lipid storage function, adipose tissue has been recognised for its

contribution to the regulation of energy homeostasis through production and

secretion of a large number of adipose tissue-specific hormones, which are

collectively termed adipokines (Fruhbeck et al., 2001; Kershaw & Flier, 2004).

These adipocyte-derived secretory factors can act in an autocrine, paracrine or

endocrine fashion on a host of target tissues involved in whole body energy

homeostasis (Hauner, 2005). Imbalance in the adipokine secretion profile, as

present in many obese individuals, affects insulin sensitivity and other metabolic

parameters (Ouchi et al., 2011). For example, the adipokine adiponectin is

recognised for its potency as insulin-sensitising hormone in addition to its anti-

inflammatory, anti-apoptotic and pro-angiogenic activities (Landskroner-Eiger et

al., 2009; Rajala & Scherer, 2003; Yamauchi et al., 2001). In adipose tissue from


148

obese mice and humans, adiponectin levels are significantly lower than in lean

animals or humans (Hu et al., 1996).

As another consequence of obesity, adipose tissue is in a low-grade inflammatory

state triggered by hypertrophic adipocytes secreting proportionally more pro-

inflammatory cytokines than normal adipocytes (Skurk et al., 2007) as well as

chemoattractants responsible for macrophage infiltration (Jiao et al., 2009).

Infiltrating macrophages are activated by elevated levels of free fatty acids present

in the serum of obese individuals with exhausted lipid storage capacity (Nguyen et

al., 2007).


149

Figure 6-1: Lipid metabolism.

Glucose is converted to triacylglycerides in a process called lipogenesis. Triacylglycerides are stored in adipose tissue in an anhydrous form until they are broken down into fatty acids and glycerol (lipolysis) for utilisation by other tissues.


150

6.4 Obesity-induced insulin resistance

The concentration of circulating substrates (e.g. glucose, amino acids and fatty

acids) that serve as energy sources for various tissues is tightly regulated. This

requires a high degree of coordination between the organs involved in energy

sensing, storage and mobilisation including brain, liver, muscle, pancreas and

adipose tissue (Thorens, 2011). Upon energy intake, energy homeostasis is

achieved through sensing elevated blood fuel concentrations by and triggering the

secretion of insulin from pancreatic -cells. Insulin-sensitive tissues such as muscle

and adipose tissue respond to this hormone-mediated signal by taking up glucose

from the blood to maintain a plasma glucose concentration of ~5 mM. Under

insulin-stimulated conditions, glucose disposal in muscle and adipose tissue

account for ~80-85% and ~4-5% of the glucose clearance from the blood,

respectively (DeFronzo, 2004). This is mediated to a large extent by the glucose

transporter, GLUT4, which translocates to the plasma membrane in response to

insulin (Czech & Corvera, 1999) (Figure 6-2).

Binding of insulin or insulin-like growth factor (IGF) to the extracellular -

subunits of the insulin receptor (IR) triggers the tyrosine kinase activity of the

receptor’s intracellular -subunits (Ullrich & Schlessinger, 1990). Following trans-

phosphorylation, tyrosine phosphorylation of insulin receptor substrate (IRS)

occurs. This induces binding and activation of phosphatidylinositol 3-kinase (PI3K)

and subsequent activation of Akt, atypical protein kinase C (aPKC) and mammalian

target of rapamycin (mTOR) pathways (Tanti & Jager, 2009). Together, these

signalling pathways coordinate the anabolic actions of insulin. Further fine-tuning

of insulin receptor signalling is enabled by phosphorylation of serine residues in

IRS.


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Figure 6-2: Insulin-induced glucose uptake in adipocytes.

Glucose uptake into adipocytes is facilitated by the hexose transporter, GLUT4, which translocates to the plasma membrane in response to insulin signalling through the IR/IRS/PI3K/Akt pathway. Mediators of adipose tissue inflammation such as tumour necrosis factor (TNF) inhibit insulin signalling in hypertrophic adipocytes by inducing serine phosphorylation of insulin receptor substrate, IRS.


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Insulin resistance is a central theme in the context of obesity-related diseases as it

is believed to be the mechanism that cripples the physiological functions of both

adipose tissue and other tissues of metabolic significance. Disruption of the insulin

signalling pathway in adipose tissues is triggered by obesity-induced inflammatory

and metabolic stresses. First evidence for obesity-induced inflammation was

obtained from experiments demonstrating increased levels of TNF- in adipose

tissue of obese mice when compared to lean controls (Hotamisligil et al., 1995).

This was followed by the identification of c-jun N-terminal kinase (JNK), inhibitor

of kappa kinase (IKK) and protein kinase R (PKR) as intracellular mediators of the

inflammatory response (Nakamura et al., 2010; Solinas & Karin, 2010).

Furthermore, adipose tissue of obese animals showed a higher degree of

macrophage infiltration compared to lean controls, indicating the presence of a

second source of cytokine expression in adipose tissue (Weisberg et al., 2003).

Similar observations relating to obesity-induced adipose tissue inflammation have

been made in humans (Bashan et al., 2007; Harman-Boehm et al., 2007;

Hotamisligil et al., 1995). The stress kinases JNK, IKK and PKR are activated in

response to excess nutrients (e.g. by the lipid metabolites ceramide and

diacylglycerol) and reactive oxygen species, resulting in inhibitory serine

phosphorylation of insulin receptor substrate 1 (IRS-1) (Boura-Halfon & Zick,

2009). In addition to inhibition of insulin signalling, stress kinase activation also

initiates transcriptional programmes controlled by the transcription factors

activator protein-1 (AP-1), nuclear factor -

factor (IRF), leading to increased expression of pro-inflammatory cytokines, thus

creating a positive feedback loop to further disrupt insulin signalling (Tilg &

Moschen, 2008). Inflammatory signalling can down-regulate the activity of the

master transcriptional regulator, PPAR , thereby affecting adipogenesis as well as

the maintenance of adipocyte-specific gene expression and function (Guilherme et

al., 2008). Associated with decreased PPAR activity is a reduction in adiponectin

expression, resulting in impaired systemic insulin sensitivity (Trujillo & Scherer,

2005).

Another physiological effect of insulin on adipose tissue is the suppression of

lipolysis, which has been proposed to involve the reduction of cAMP levels and


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thus PKA activity (Choi et al., 2010). In contrast, obesity-induced insulin resistance

leads to increased release of fatty acids into the circulation. Over-supply of free

fatty acids causes increased uptake into muscle and liver, where they are stored as

triacylglycerol and compete with the main physiological substrate, glucose (Bays et

al., 2004). Increased levels of fatty acid metabolites generated by fatty acid

oxidation impair intracellular insulin signalling, thereby decreasing glucose uptake

into muscle in response to insulin (Summers, 2006). In the liver, impaired insulin

signalling results in increased production of glucose by gluconeogenesis, which is

fueled by the surplus of energy generated by the oxidation of fatty acids (DeFronzo

et al., 1989; Magnusson et al., 1992). Normoglycaemia can only be maintained as

long as sufficient amounts of insulin are produced and secreted to compensate for

the reduced insulin sensitivity in insulin-responsive tissues. However, fatty acid

overload can also have detrimental effects on the insulin-secreting -cells in the

pancreas, causing -cell dysfunction, apoptosis and ultimately type 2 diabetes

(Unger & Zhou, 2001). The above-mentioned mechanisms plausibly explain why

chronic excess adiposity is the strongest risk factor for type 2 diabetes (Day &

Bailey, 2011).

6.5 Regulation of adipose tissue development and function

White adipose tissue is a complex and dynamic organ that serves not only lipid

storage and endocrine functions, but also provides structural support and

cushioning as well as insulation from heat and cold (Wronska & Kmiec, 2012).

Adipose tissue is found in different parts of the body and there are considerable

differences in the gene expression profiles between individual depots (Laplante et

al., 2003). Understanding the regulatory mechanism underlying this functional

plasticity is one of the key challenges in defining the patho-physiological processes

that are responsible for the devastating metabolic consequences of obesity.


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6.5.1 Adipose tissue development and composition

Although the majority of the adipose tissue volume is occupied by mature, lipid-

laden adipocytes, a multitude of other cell types contributes to the structure and

function of this tissue (Figure 6-3) (Zuk et al., 2001). It also contains pre-

adipocytes, which are committed to the adipogenic fate, as well as other adipocyte

progenitors, such as the morphologically indistinguishable adipoblasts

(Tchoukalova et al., 2004). The adipose lineage arises from multi-potent stem cells

of mesodermal origin (Gesta et al., 2007; Hwang et al., 1997), which also have the

potential to differentiate into osteoblasts, chondrocytes and myoblasts.

Mesenchymal stem cells (MSCs) residing in the stroma of adipose tissue have an

inherent ability for self-renewal, proliferation and differentiation toward mature

tissues depending on the microenvironment (Sarugaser et al., 2009). In addition to

cell types of the adipogenic lineage, the stromovascular fraction of adipose tissue

consists of fibroblasts, endothelial and smooth muscle cells, leukocytes and

macrophages as well as sympathetic neuronal projections that innervate the tissue

(Bartness & Bamshad, 1998; Riordan et al., 2009; Schaffler et al., 2005).


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Figure 6-3: Adipose tissue composition.

Adipose tissue is composed of a multitude of different cell types including mature adipocytes, pre-adipocytes, fibroblasts, T lymphocytes, macrophages and endothelial cells. Adapted from (Ouchi et al., 2011).


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6.5.2 Adipogenic differentiation in vitro

One cell type characteristic that obstructs the application of traditional cell culture

methods on mature adipocytes is their increased buoyancy due to the presence of

intracellular lipid droplets (Poulos et al., 2010). As a substitute for terminally

differentiated adipocytes, fibroblast-like committed pre-adipocytes are commonly

used as a cell culture model for studying adipogenic differentiation and adipocyte

function. For example, 3T3-L1 cells, a well-established and widely used unipotent,

pre-adipocyte cell line derived from embryonic mouse tissue can be stimulated to

efficiently differentiate into adherent, lipid-laden adipocytes (Green & Kehinde,

1975).

More similar to the conditions found in vivo is the differentiation of mouse

embryonic fibroblasts (MEFs) prepared from 13-15 days old embryos (E13-15).

These cells have the potential to undergo adipogenic, osteogenic and chondrogenic

differentiation (Garreta et al., 2006; Lengner et al., 2004; Noguchi et al., 2007).

Heterogeneous cultures such as the stromovascular cell fraction of adipose tissue

are considered to be even more predictive of in vivo conditions as they allow

potential interactions between different cell types that may be involved in

regulating differentiation (Poulos et al., 2010).

The differentiation of adipocyte progenitors into mature, lipid-laden adipocytes

has been studied extensively in vitro. It is regulated through a transcriptional

cascade that governs the characteristic cell morphological changes and

accumulation of lipids, and coordinates the expression of genes responsible for

adipocyte function (Farmer, 2006). Induction of growth-arrested cells using an

adipogenic differentiation cocktail consisting of insulin, serum mitogens, isobutyl

methylxanthine (an inducer of cAMP signalling) and dexamethasone (a

glucocorticoid) stimulates the expression of CCAAT/enhancer-binding protein

(C/EBP ). Its DNA binding activity, however, is repressed until the cells

synchronously re-enter the cell cycle and begin to undergo mitotic clonal

expansion (Tang et al., 2004). Subsequently, C/EBP induces the expression of the

master transcriptional regulators of adipogenesis, C/EBP and peroxisome

proliferator-activated receptor (PPAR ), which coincides with terminal growth


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arrest. Terminal differentiation of adipocytes involves formation of lipid droplets

and transcription of genes responsible for expression of the adipocyte phenotype

(e.g. lipid transport and metabolism) (Gesta et al., 2007; Hausman et al., 2001;

Rosen & MacDougald, 2006).

Morphologically, adipogenic differentiation is associated with marked changes in

cell shape. The transition from a flattened, fibroblastic morphology to a more

rounded appearance is facilitated by reorganisation of actin filaments into a

cortical network that is packed densely beneath the plasma membrane (Kanzaki &

Pessin, 2001). The levels of fibronectin, integrins, actin and other cytoskeletal

proteins have been reported to be down-regulated during adipogenesis (Rodriguez

Fernandez & Ben-Ze'ev, 1989; Spiegelman & Farmer, 1982), and disassembly of

contacts with the extracellular matrix was shown to be essential for efficient

differentiation (Spiegelman & Ginty, 1983).

6.5.3 Lineage commitment of adipocyte progenitor cells

At stem cell level, both cytoskeleton and cell-matrix adhesions have been involved

in the regulation of adipogenesis through their critical role in defining cell shape.

Treatment of bone marrow-derived mesenchymal progenitor cells with heparin

disrupts cell-matrix adhesion, thereby leading to cell rounding and enhanced

adipogenic potential (Luo et al., 2008). In mouse embryonic stem cells, disruption

of stress fibres and inhibition of focal adhesion formation by cytochalasin D also

caused cell rounding and increased adipogenic potential (Feng et al., 2010).

In addition to structural disruption, activation of RhoA has been implicated in the

cell lineage commitment of human mesenchymal stem cells. Expression of

constitutively active RhoA leads to osteoblastic differentiation, whereas expression

of dominant-negative RhoA leads to adipogenic differentiation (McBeath et al.,

2004). In contrast, treatment of human mesenchymal stem cells with the specific

ROCK inhibitor, Y-27632, increases adipogenic differentiation (Kunisada et al.,

2012). The role of Rho in adipogenesis, which has been attributed to ROCK

activation and subsequent phosphorylation of insulin receptor substrate (IRS),

appears to be independent of its ability to affect cytoskeletal organisation (Sordella


158

et al., 2003). Instead, it is believed to act through regulation of PPAR expression

(Li et al., 2011). Recent data suggest that adipogenesis can be induced through

PPAR -dependent and -independent pathways (Hung et al., 2008; Peng & Liou,

2012).

6.5.4 Actin cytoskeletal regulators in adipocytes

Cell rounding driven by actin reorganisation is essential for efficient adipogenesis.

However, only few studies have explored the detailed mechanism underlying these

morphological changes in adipocytes. The actin-depolymerising protein, cofilin, is

an important regulator of actin organisation, and is differentially expressed in

different fat depots (Choi et al., 2003). Cofilin mRNA is more abundant in adipose

tissue located inside the abdominal cavity (also referred to as visceral adipose

tissue) than in subcutaneous adipose tissue. This difference in cofilin levels is even

more pronounced in mice fed a high-fat diet. As excessive lipid deposition in

visceral adipose tissue, i.e. visceral obesity, has been associated with more severe

metabolic consequences (Fox et al., 2007; Jensen, 2008), it can be speculated that

cofilin may be functionally involved in the pathogenesis of obesity, acting most

likely through its destabilising effect on filamentous actin.

In addition to its importance during adipogenic differentiation, actin remodelling is

also critically involved in the regulation of insulin-induced glucose uptake, which is

mediated by the glucose transporter, GLUT4 (Kanzaki & Pessin, 2001; Khan &

Pessin, 2002). Recently, it was demonstrated that cofilin phosphorylation in

adipocytes occurs in a ROCK1-dependent manner and facilitates GLUT4

translocation to the plasma membrane through actin cytoskeleton remodelling,

which is likely to be mediated by LIMK (Chun et al., 2012). Glucose uptake by

adipocytes represents a critical event in energy homeostasis as it removes glucose

from the blood and leads to its storage in the form of intracellular lipids.

Monomeric -actin was shown to be associated with lipid droplets in adipocytes

(Fong et al., 2001). It has been speculated that this pool of -actin may be involved

in directing or accelerating intracellular lipid transport along actin filaments in

response to hormone stimulation.


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Despite the lack of reports directly implicating LIMKs in the regulation of actin-

dependent processes in adipocytes, there is an abundance of potential roles for

LIMK2 in adipose tissue development and function, including cell shape-induced

adipogenesis, insulin-stimulated glucose uptake and lipid metabolism.

6.6 Hypothesis and aims

This thesis reports for the first time an obesity-related phenotype as a

consequence of disruption of the LIMK2 gene in mice. Based on the critical role of

LIMKs in regulating cofilin-mediated actin reorganisation, we hypothesised that

LIMK2a may play a role in adipogenic differentiation, a process known to involve

extensive restructuring of the F-actin network (Figure 6-4).

The present study aims to characterise the phenotype of the LIMK2a knockout

mouse with focus on systemic and local (adipose tissue, muscle, liver) energy

homeostasis. Furthermore, it investigates the regulation and function of LIMK2 in

adipose tissue and cultured adipocytes, in particular in the context of adipogenesis.

The third aim of this study is to identify novel substrates of LIMK2, which may be

involved in the mechanism underlying the development of obesity in the LIMK2a

knockout mouse.


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Figure 6-4: Potential role of LIMK2a in the pathogenesis of obesity and

insulin resistance.

LIMK2a deficiency in mice results in adipocyte hypertrophy and obesity. Potential mechanisms include dysregulation of cofilin-mediated actin reorganisation and altered efficiency of adipogenic differentiation. Enlargement of adipocytes leads to functional impairment of adipose and non-adipose tissues due to inhibition of insulin signalling.

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CHAPTER 7 − MATERIALS &

METHODS

Chapter 7 − Materials & methods

162


163

7.1 Animals and animal care

LIMK2a knockout mice were generated by injecting E14Tg2a.4 embryonic stem

cells with one disrupted allele of the LIMK2 locus (BayGenomics Consortium, CA,

USA) into blastocysts of C57BL/6 mice (Figure 7-1). Knockout and wildtype mice

were obtained by breeding heterozygous mice for the deleted allele. Male LIMK2a

knockout and wildtype littermates were used for all experiments unless indicated

otherwise. Leptin-deficient ob/ob mice and wildtype littermate controls (both of

the C57BL/6 genetic background) were obtained from Monash Animal Services

(Clayton, Australia) at 10 weeks of age. Mice were fed a standard rodent chow diet

consisting of 5% of calories from fat (composed of 15.6% saturated, 45.2%

monounsaturated and 39.2% polyunsaturated fatty acids; Specialty Feeds, Glen

Forrest, WA, Australia) or a high-fat diet consisting of 59% of calories from fat

(composed of 60.3% saturated, 32.9% monounsaturated and 6.8%

polyunsaturated fatty acids) for 12 weeks. Animals were housed in a pathogen-free

facility with a 12-h light, 12-h dark cycle and were given free access to food and

water. Mice were anaesthetised with an intraperitoneal injection of pentobarbital

sodium (60 mg/kg Nembutal; Boehringer Ingelheim, Sydney, Australia) at 11:00

AM after fasting for 1-3 hours. All procedures were approved by the St. Vincent’s

Hospital Animal Ethics Committee and the Monash University School of Biomedical

Sciences Animal Ethics Committee and conformed to the National Health and

Medical Research Council of Australia (NHMRC) Code of Practice for the Use and

Care of Animals for Scientific Purposes.


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Figure 7-1: Genomic organisation and isoforms of LIMK2.

LIMK2a knockout mice were generated by insertion of a plasmid (red arrow) between exon 2 and 2b of the LIMK2 gene. The initiation codons for LIMK2a and LIMK2b are labeled “ATG” and differential splicing of the two transcripts is indicated by thin black lines above (LIMK2a) and below (LIMK2b) the schematic representation of the LIMK2 gene. The 5’-terminal region of the LIMK2a and LIMK2b transcripts is shown in the lower panel. Adapted from (Ikebe et al., 1997).


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7.2 Genotyping of mouse tails

Genomic DNA was extracted from mouse tail tips using the REDExtract-N-Amp

Tissue PCR kit (Sigma). The tips were incubated in a mixture of 100 µl Extraction

Solution and 25 µl Tissue Preparation Solution at 50 C for 30 min. Subsequently,

the samples were incubated at 95 C for 4 min, followed by addition of 100 µl

Neutralisation Solution. PCR reactions were set up by adding 4 µl of extracted DNA

to a mixture of 4.5 µl H2O, 10 µl 2X PCR Reaction Mix and 0.5 µl of a 50 µM dilution

of each of the following primers:

Table 7-1: Nucleotide sequences of PCR primers used for genotyping.

Primer name Nucleotide sequence

LIMK2a Forward 5’-ATTGGCCAGAGAGAGCAGAA-3’

LIMK2a Reverse 5’-CGCTCACAGGCACATACCTA-3’

pGT21xfGeneTrapVector 5’-CAACCTCCGCAAACTCCTAT-3’

LIMK2 Forward 5’-AAATGGCGTTACTTAAGCTAGCTTGC-3’

LIMK2 Reverse 5’-GTTTGCTGATCCCACTTTACAACG-3’

LTR 5’-TGGCAGGAGACACAATGCTGGAAC-3’


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PCR was performed using the following program parameters: Denaturation at 94°C

for 2 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C

for 30 s and extension at 72°C for 30 s prior to final extension at 72°C for 10 min.

The PCR products were separated by gel electrophoresis on a 3% w/v agarose gel

containing GelRed, followed by visualisation using a UV transilluminator.

7.3 Indirect calorimetry and feeding

Animals were acclimated to the Oxymax open-circuit calorimeter (Columbus

o2

co2

co2 o2), activity and feeding were recorded over the subsequent 48 h.

7.4 Plasma metabolite analysis

Venous blood samples were obtained from the pleural cavity of anaesthetised mice

and whole blood was centrifuged at 8,000 x g for 5 min. Plasma glucose was

measured using a glucose oxidase assay (Sigma) and free fatty acid concentration

was determined by NEFA-C enzymatic colorimetric assay (Wako Chemicals,

Richmond, VA, USA). Plasma triacylglycerol was assessed by an enzymatic

colorimetric method using the GPO-PAP kit (Roche Diagnostics, Mannheim,

Germany).

7.5 Tissue metabolite analysis

Lipids were extracted from epididymal adipose tissue, mixed quadriceps and liver

using chloroform:methanol (2:1) and the organic phase obtained and dried under

N2. Triacylglycerol content was assessed using the GPO-PAP assay. Diacylglycerols

and ceramides were extracted with chloroform:methanol:PBS (1:2:0.8) and 0.2%

w/v SDS and assayed according to the methods of Preiss et al. (Preiss et al., 1986).


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7.6 Lipogenesis and lipolysis

Epididymal adipose tissue was extracted from mice subjected to a fasting period of

4 h, and rinsed in PBS containing 0.1% w/v BSA. Modified Krebs-Henseleit buffer

(containing 4.5% w/v NaCl, 5.75% w/v KCl, 6.1% w/v CaCl2, 10.55% w/v KH2PO4,

19.1% w/v MgSO4 7H2O, 1.3% w/v NaHCO3, pH 7.4) was gassed for 30 min with

95% O2/5% CO2, followed by addition of glucose (8 mM) and fatty acid-free BSA

(4% w/v) immediately before experiments were performed in a shaking water

bath at 37°C.

For measurement of lipogenesis, D-[3-3H]glucose (GE Healthcare) was added to

the buffer to give a final concentration of 0.5 μCi/ml. Adipose tissue explants (50-

70 mg) were incubated for 2 h, and the medium was discarded. The tissue was

washed in PBS and then homogenized in 1 ml PBS. Lipids were extracted in 2:1

chloroform-methanol. A 1-ml aliquot of the organic phase was added to 20 ml

scintillation fluid and radioactivity was counted in a Tri-Carb 2000 liquid

scintillation counter (Perkin Elmer).

For lipolysis experiments, adipose tissue explants were placed in 2 ml gassed

modified Krebs-Henseleit buffer containing 5 mM glucose and 4% w/v fatty acid-

free BSA. The medium was collected after 2 h of incubation in the presence or

absence of 1 µM isoproterenol hydrochloride (Sigma) for determination of glycerol

by an enzymatic colorimetric method (Sigma).

7.7 Insulin tolerance test

Mice were fasted for 4 h, followed by intraperitoneal injection of 0.5 U insulin

(Actrapid; Novo Nordisk, Bagsværd, Denmark) per kg body mass. Blood samples

were obtained from the tail at 0, 15, 30, 45, 60 and 90 min, and blood glucose was

measured using a glucometer (Accu-Chek; Roche Diagnostics).


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7.8 Haematoxylin and eosin staining

Epididymal adipose tissue was harvested and washed with PBS prior to fixation

with 4% w/v paraformaldehyde, processing and embedding in paraffin. 10-μm

sections were stained with hematoxylin and counterstained with eosin. Analysis

was performed at 400X magnification using an Olympus BX50 microscope

(Olympus, Tokyo, Japan).

7.9 Glucose uptake

2-deoxy-D-glucose uptake in adipose tissue and muscle was measured after 15 min

of pre-incubation with 10 nM insulin over 10 min in modified Krebs-Henseleit

buffer containing 2 mM pyruvate, 8 mM mannitol and 0.1% w/v BSA as well as 0.5

μCi/ml 2-[2,6-3H]-deoxy-D-glucose, 10 µM 2-deoxy-D-glucose and 0.2 μCi [1-14C]-

mannitol/ml. Radioactivity in tissue lysates was measured using a Tri-Carb 2000

liquid scintillation counter (Perkin Elmer).

7.10 RNA extraction, reverse transcription and quantitative real-time PCR

Total RNA was extracted from 20-30 mg tissue or 0.5 x 106 cultured cells using the

RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions.

Homogenisation of the sample was performed using a 1-ml syringe and a 20-G

needle. Following binding of the RNA to the column and washing, the RNA was

eluted in 30 µl DEPC-treated dH2O. The RNA concentration was determined by

measuring a 1:10 dilution in DEPC-treated dH2O using a NanoDrop ND-1000

spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). To remove

any contaminating genomic DNA, 1.5 µg of RNA were incubated with 3 units

RNase-free DNaseI (Roche) in DNaseI buffer diluted with DEPC-treated dH2O to 1X

final concentration in a total volume of 30 µl. The reaction mix was incubated at

37 C for 10 min, followed by addition of 1.5 µl 0.1 M EDTA and inactivation of the

enzyme at 75 C for 10 min. cDNA synthesis was performed using the SuperScript II


169

First-Strand Synthesis Kit (Invitrogen). For reverse transcription of one sample,

0.5 µl of 20 ng/µl random hexamers was diluted with 0.5 µl DEPC-treated dH2O

and added to 5 µl of diluted RNA containing 200 ng RNA. The RNA/random

hexamer mixture was incubated at 65 C for 5 min and put on ice for another 5 min.

In a separate tube, 2 µl 5X First Strand Synthesis buffer, 1 µl 100 mM DTT, 0.5 µl 10

mM dNTPs and 0.5 µl (100 units) SuperScript II Reverse Transcriptase were

combined and subsequently added to the RNA/random hexamer mixture. No-

reverse transcriptase control was prepared as described above for the other

samples except that the SuperScript II enzyme was omitted. Similarly, a no-

template control was set up without addition of template RNA. Reverse

transcription was performed at 42 C for 50 min, followed by incubation at 70 C for

15 min. Quantitative real-time PCR was performed using the SYBR Green method

(Ponchel et al., 2003). Each sample containing 12.1 µl 2X premix (50 U/ml

AmpliTaq Gold [Invitrogen], 2X SYBR Green/DMSO [Sigma], 400 nM of each dNTP

and 16% v/v DMSO) was diluted with 10.9 µl DEPC-treated dH2O prior to addition

of 1 µl (20 ng) cDNA template and 1 µl oligonucleotides primer pool (5 µM each,

see Table 7-2). Each reaction was performed in triplicate or quadruplicate in

individual wells of a clear 96-well microplate (Axygen, Union City, CA, USA) using a

Stratagene Mx3000P qPCR System (Stratagene, La Jolla, CA, USA). The real-time

PCR program included a 10-minute denaturation step at 95 C, followed by 40

cycles of denaturation at 95 C for 15 s and amplification at 60 C for 1 min.

Subsequently, a dissociation curve was generated using the following program:

95°C for 1 min and 60°C for 30 s prior to a temperature ramp from 60°C to 95°C at

2°C/s. Only PCR reactions yielding one gene-specific amplicon were used for

analysis. Measured transcript abundance was normalised to the expression of the

housekeeping genes, hypoxanthine-guanine phosphoribosyl transferase-1

(HPRT1) or ribosomal protein L32, and the relative quantities of each transcript

were calculated using the comparative critical threshold (Ct) method. Gene

expression of C/EBP , PPAR and aP2 was measured by quantitative real-time PCR

as described in (Ribas et al., 2010).


170

Table 7-2: Sequences of oligonucleotides used for real-time PCR.

Primer name Nucleotide sequence

LIMK2a Forward QuantiTect Primer Assay *

LIMK2a Reverse QuantiTect Primer Assay *

LIMK2b Forward QuantiTect Primer Assay *

LIMK2b Reverse QuantiTect Primer Assay *

HPRT1 Forward 5’-TGATTAGCGATGATGAACCAG-3’

HPRT1 Reverse 5’-AGAGGGCCACAATGTGATG-3’

L32 Forward 5’-CAGGGTGCGGAGAAGGTTCAAGGG-3′

L32 Reverse 5’-CTTAGAGGACACATTGTGAGCAATC-3′

* Validated oligonucleotide primers specific for LIMK2a and LIMK2b were purchased from Qiagen.


171

7.11 Adipogenic differentiation of 3T3-L1 pre-adipocytes

3T3-L1 cells were grown as adherent cultures in Dulbecco’s Modified Eagle

Medium (DMEM) supplemented with 10% v/v FBS and 1X penicillin/streptomycin

until 100% confluent. The cultures were kept at 100% confluence for another 2-3

days prior to initiating adipogenic differentiation with DMEM/10%

FBS/penicillin/streptomycin containing 5.7 µg/ml insulin, 0.5 mM IBMX and 1 µM

dexamethasone. Three days later, the medium was changed to DMEM/10%

FBS/penicillin/streptomycin supplemented with 5.7 µg/ml insulin. After another

three days, the medium was replaced with DMEM/10%FBS/penicillin/strepto-

mycin and the cells were allowed to differentiate until 8 days post-induction.

7.12 Preparation of mouse embryonic fibroblasts

Pregnant female mice were culled by cervical dislocation between day 13.5 and 16

of gestation (E13.5-16) and the uterine horns were harvested and washed twice

with 2 ml PBS. Embryos were separated from their placenta, followed by

decapitation and removal of limbs, tail and visible red organs. After washing with

PBS, the individual carcasses were finely minced using fine scissors, and were

subsequently incubated with 1 ml trypsin-EDTA at 37°C for 30-40 min with

occasional gentle shaking. The resulting cell suspension was diluted with 7 ml of

growth medium and mixed gently by pipetting up and down. Remaining pieces of

tissue were allowed to settle down to the bottom of the tube and the supernatant

was transferred into a T25 tissue culture flask. Genotyping of embryos was

performed as described for mouse tails in Chapter 7.2 with the exception of using

small aliquots of pelleted MEFs.


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7.13 Western blot

Protein separation by gel electrophoresis and Western blotting were performed as

outlined in Chapter 2.5.3. The following primary and secondary antibodies were

used to probe for LIMK1, LIMK2, phosphorylated and total cofilin, and GAPDH (as a

loading control):

Table 7-3: Primary and secondary antibodies used for Western blotting.

Primary antibody Dilution Source

Rat anti-LIMK1 1:1,000 Generated as described in

(Foletta et al., 2004)

Rabbit anti-LIMK2 1:1,000 Cell Signaling

Rabbit anti-cofilin 1:20,000 Cytoskeleton Inc., Denver,

CO, USA

Rabbit anti-phospho-cofilin 1:5,000 Gift from James R. Bamburg

(Colorado State University)

Rabbit anti-GAPDH (HRP-conjugated) 1:5,000 Cell Signaling

Secondary antibody Dilution Source

Rabbit anti-rat (HRP-conjugated) 1:3,000 Invitrogen

Goat anti-rabbit (HRP-conjugated) 1:5,000 Invitrogen


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Determination of protein levels for Perilipin A, ATGL and CGI-58 was performed

using Western blot analysis as described in (Huijsman et al., 2009).

7.14 Oil Red O staining

Staining of intracellular neutral lipids was performed in a 6-well plate format using

the lysochrome Oil Red O (Sigma). Cells were washed with 2 ml cold PBS, followed

by fixing with 800 µl 10% v/v NBF at room temperature for 10 min. Subsequently,

the fixative was removed and cells were stored in 1 ml 10% v/v NBF at 4 C until

staining. Fixed cells were washed twice with 2 ml dH2O prior to incubation with 1

ml 60% v/v isopropanol for 5 min. The isopropanol was discarded and cells were

dried completely by use of a hairdryer. Cells were incubated with 1 ml 0.21% m/v

Oil Red O in 60% v/v isopropanol for 15-20 min, followed by at least 5 washes

with dH2O to remove unbound dye. Stained cells were visualised using a Zeiss

Axiovert 25 microscope (Carl Zeiss, Jena, Germany) with a 10X and 40X objective

lens. Representative images were acquired using an AxioCam MRc camera in

combination with the AxioVision Rel. 4.6 software (Zeiss). After microscopic

analysis, the cells were dried completely prior to incubation with 800 µl

isopropanol for 10 min. Absorbance of the eluted dye was measured at 490 nm or

530 nm using a Polarstar Optima microplate reader (BMG Labtech, Ortenberg,

Germany). The measured absorbance was normalised to the blank value for

isopropanol. For staining of cells in a 12-well format, the reagent volumes shown

above were downscaled at a 1:2.5 ratio.

7.15 siRNA-mediated knockdown of LIMK2

3T3-L1 cells were seeded in 10-cm tissue culture dishes at a density of 0.4 x 106

cells per dish one day before transfection. One hour before transfection, the

medium was discarded and replaced with 6 ml DMEM + 10% v/v FBS without

antibiotics. Transfection of 3T3-L1 cells was performed using Lipofectamine 2000

(Invitrogen) according to the manufacturer’s recommendations. In brief, 30 µl of


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Lipofectamine 2000 diluted in 1.5 ml Opti-MEM (Invitrogen) was added to 1.5 ml

Opti-MEM containing either 0.7 µl 20 µM Block-iT fluorescently labelled siRNA

(Invitrogen) or 5.9 µl mouse LIMK2 siRNA (250 µM final concentration; Thermo

Scientific). After 20 min of incubation, the growth medium was replaced again with

3 ml DMEM + 10% v/v FBS without antibiotics and the total volume of the

transfection mixture was added to the cells, followed by gently shaking of the dish.

Cells transfected with fluorescently labelled siRNA were visualised one day post-

transfection by fluorescence microscopy. The estimated transfection efficiency was

~90%. Prior to induction of adipogenic differentiation, transfected cells were

trypsinised, plated in 24-well plates at 9 x 104 cells per well and maintained at

100% confluence for 2 days.

7.16 In vitro phosphorylation of LIMK2 using ROCK1

Recombinant GST-tagged rat LIMK2 was expressed in High-Five cells using the

baculovirus/insect cell system as described in Chapter 2.3. Protein purification

was performed as outlined for LIMK kinase domain protein in Chapter 2.4.1. The

only procedural modification concerned the ammonium sulphate precipitation

step, which was adjusted to a final ammonium sulphate concentration of 30% v/v

to allow for the higher molecular weight of the GST-tagged full-length protein. 400

µl of purified GST-LIMK2 immobilised on glutathione Sepharose beads were

incubated with 120 µl of 50 µg/ml recombinant Flag-ROCK, 120 µl 10X activation

buffer (500 mM Tris-HCl [pH 7.5], 1 mM EGTA, 1% v/v 2-mercaptoethanol), 160 µl

magnesium acetate/ATP buffer (2.5 mM HEPES [pH 7.4], 50 mM Mg(CH3CO2)2 and

0.5 mM ATP) and 600 µl dH2O at 30 C for 40 min. The beads were washed with

lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 100 mM NaF, 10 mM Na4P2O7,

1 mM Na3VO4, 0.1% v/v Triton X-100, 1X COMPLETE protease inhibitors) and

high-salt wash buffer (50 mM Tris [pH 8.0], 1 M NaCl, 1 mM DTT), and were

subsequently resuspended in storage buffer (20 mM Tris [pH 7.6], 150 mM NaCl, 1

mM DTT, 20% v/v glycerol) at a volume ratio of 1:1. The activity of activated and

non-activated GST-LIMK2 was compared by in vitro kinase assay using cofilin as a

substrate. 5 µl of immobilised GST-LIMK2 was incubated with 0.6 µl 1 mM ATP,


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18.4 µl kinase assay buffer (20 mM HEPES [pH 7.4], 10 mM MgCl2, 10 mM NaF, 1

mM Na3VO4, 1X COMPLETE protease inhibitors) and 1 µl 10 mCi/ml [ -32P]ATP in

the presence and absence of 5 µl 2 mg/ml GST-cofilin in a total reaction volume of

30 µl at 37 C for 15 min. The kinase reaction was stopped by addition of 8 µl 5X

Laemmli buffer and incubation at 95 C for 5 min. Samples were separated by SDS-

PAGE using a 12% polyacrylamide gel prior to analysis by autoradiography.

7.17 Fractionation of adipose tissue extract

Adipose tissue extracts were prepared from epididymal adipose tissue of wildtype

mice. Tissues were homogenised on ice in 400 µl lysis buffer (50 mM HEPES [pH

7.4], 150 mM NaCl, 100 mM NaF, 10 mM Na4P2O7, 5 mM EDTA, 250 mM sucrose, 1

mM DTT, 1% v/v Triton X-100, 1 mM Na3VO4, 1X COMPLETE protease inhibitors)

using a Tissue-Tearor homogeniser (BioSpec, Bartlesville, OK, USA). The

homogenates were centrifuged at 16,100 x g at 4 C for 10 min to separate the

soluble fraction from cell debris and the floating lipid layer. The soluble fraction

was carefully transferred into a fresh tube using a syringe and 23-G needle. Lipid-

depleted adipose tissue extract was subjected to buffer exchange (50 mM Tris-HCl

[pH 7.4]) using a PD-10 desalting column (GE Healthcare) according to the

manufacturer’s instructions. The desalted sample was loaded onto a 7-ml Q

Sepharose FastFlow column (GE Healthcare) at a flow rate of 1 ml/min. Unbound

proteins were collected in the flow-through and bound proteins were eluted in 1-

ml fractions by applying a 0-100% gradient of 50 mM Tris-HCl (pH 7.4) containing

1 M NaCl over a 20-min period.

7.18 Fractionation of NIH3T3 cell extract

Confluent NIH3T3 cells grown in 15-cm dishes were washed with PBS and

collected in lysis buffer (50 mM Tris-HCl [pH 7.3], 20 mM NaCl, 1 mM DTT, 0.1%

v/v Triton X-100, 1X COMPLETE protease inhibitors) using a cell scraper. Cells

were lysed by sonication and cell debris were removed by centrifugation at 16,100


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x g at 4 C for 15 min. The lysate was loaded onto a pre-packed 1-ml SP Sepharose

FastFlow column (GE Healthcare) equilibrated with 50 mM Tris-HCl (pH 7.4) at a

flow rate of 1 ml/min. Unbound proteins were collected in the flow-through and

bound proteins were eluted in 1-ml fractions by applying a 0-100% gradient of 50

mM Tris-HCl (pH 7.4) containing 1 M NaCl over a 15-min period.

7.19 In vitro phosphorylation and identification of potential LIMK2

substrates

10 µl of fractionated extract were incubated with 10 µl activated GST-LIMK2

(immobilised on glutathione Sepharose beads) in 1X phosphorylation buffer (50

mM HEPES [pH 7.4], 2 mM MnCl2, 1 mM DTT, 200 µM ATP) containing 1,000

cpm/pmol [ -32P]ATP at 30 C for 5 min with shaking. The reaction was stopped by

addition of 5 µl 5X Laemmli buffer and incubation at 95 C for 5 min. The beads

were pelleted by centrifugation at 3,000 x g for 2 min and the supernatant was

separated by SDS-PAGE using a 4-20% polyacrylamide gel. Following staining with

Coomassie Blue, autoradiography was performed and differentially

phosphorylated protein bands were excised from the gel for in-gel tryptic

digestion and peptide mass fingerprinting as described in (Suryadinata et al.,

2010). Mass spectrometry data were analysed using Mascot software (Matrix

Science).

7.20 Statistical analysis

Data were analysed using the software Prism Version 5.0d (GraphPad, San Diego,

CA, USA). Statistical analysis was performed using Student’s t test (unpaired, two-

tailed) or one-way ANOVA followed by Bonferroni post hoc test for selected groups

where appropriate. Data obtained from intraperitoneal insulin tolerance test were

analysed using two-way ANOVA followed by Bonferroni post hoc test. P values

<0.05 were considered statistically significant and are denoted by one asterisk (*).

P values <0.01 and <0.005 are denoted by two and three asterisks, respectively.

177

CHAPTER 8 − FUNCTIONAL

CHARACTERISATION OF LIMK2 IN

ADIPOCYTES

Chapter 8 − Functional characterisation of LIMK2 in adipocytes

178


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8.1 Phenotypic characterisation of the LIMK2a knockout mouse

Phenotypic characterisation of the LIMK2a knockout mouse was performed to

obtain an insight into potential roles of LIMK2a in tissue development, cellular

function and whole-body metabolism. The most striking feature of the LIMK2a

knockout mouse was the obese phenotype, which was expressed only in adult

males. These animals had a significantly higher body mass and fat tissue mass

compared to age-matched wildtype littermates (Figure 8-1 A and B). When fed a

high-fat diet, the difference in body mass between LIMK2a knockout and wildtype

mice was even more pronounced (Figure 8-1 C). The obese phenotype was

associated with adipocyte hypertrophy when compared to wildtype mice (Figure

8-1 D).

There are several possible mechanisms whereby the mice can become obese, one

of which is increased caloric intake. However, the food intake of LIMK2a knockout

mice was similar to that of their wildtype littermates (Figure 8-2 A). Also, oxygen

o2

co2 o2 consumed) as determined by indirect

calorimetry were unchanged in the knockout animals, (Figure 8-2 B and C),

indicating a normal metabolic rate and oxidative fuel partitioning, respectively.


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Figure 8-1: Obesity and adipose tissue phenotype of the LIMK2a knockout

mouse.

18-21-week-old male wildtype and LIMK2a knockout mice were culled after being fed a standard chow diet (A, B, D and E) or high-fat diet (C) for 12 weeks. Body mass (A; Wt n=7, KO n=10 or C; Wt n=5, KO n=6) and epididymal fat mass (B; Wt n=7, KO n=10) were obtained by weighing. Epididymal adipose tissue was sectioned and stained with haematoxylin and eosin to assess the size of adipocytes (D). Representative cross-sections are shown for wildtype and LIMK2a knockout mice. Data are expressed as mean ± S.E.M. Asterisk (*) denotes statistical significance (p<0.05).


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Figure 8-2: Metabolic characteristics of the LIMK2a knockout mouse.

Daily food intake (D; Wt n=7, KO n=10) of 18-21-week-old male wildtype and LIMK2a knockout mice was determined by subtracting the amount of food remaining from that given 24 h earlier. Oxygen consumption (B; Wt n=4, KO n=4) and respiratory exchange ratio (C; Wt n=4, KO n=4) were measured by indirect calorimetry. Plasma concentration of free fatty acids (D; Wt n=4, KO n=6) and triacylglycerol (E; Wt n=3, KO n=6), and blood glucose concentration (F; Wt n=9, KO n=12) were determined after a 4-h fasting period. Data are expressed as mean ± S.E.M. Asterisk (*) denotes statistical significance (p<0.05).


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Examination of the lipid metabolism in epididymal adipose tissue from LIMK2a

knockout mice revealed a significant reduction in the rate of both basal and

isoproterenol-stimulated lipolysis (Figure 8-3 A) as well as a trend toward

impaired lipogenesis (Figure 8-3 B; p=0.13). Furthermore, insulin-stimulated

uptake of 2-deoxyglucose was significantly reduced in LIMK2a knockout adipose

tissue (Figure 8-3 C), consistent with decreased clearance of plasma glucose in

response to an insulin load (Figure 8-3 D). However, blood glucose levels after

fasting were similar between wildtype and LIMK2a knockout mice (Figure 8-2 F).

Plasma free fatty acids and plasma triacylglycerol were elevated in LIMK2a

knockout mice (Figure 8-2 D and E), suggesting an impaired lipid clearance. In

contrast, triacylglycerol levels in quadriceps muscle and liver were similar to those

of wildtype mice (Figure 8-3 E and F).

There were no differences in the expression levels of the adipogenic marker genes,

C/EBP , PPAR and aP2 (Figure 8-4 A), or the levels of the lipolytic proteins ATGL,

CGI-58 and perilipin-1, between the adipose tissue of wildtype and LIMK2a

knockout mice (Figure 8-4 B).

Despite apparently normal gene expression, the abnormal morphology of LIMK2a-

deficient adipocytes (adipocyte hypertrophy) may be responsible for the

functional defects (lipolysis) leading to impaired energy homeostasis

(dyslipidaemia, insulin resistance and obesity).


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Figure 8-3: Lipid and glucose metabolism in LIMK2a knockout mice.

The rates of basal and stimulated (1 µM isoproterenol) lipolysis (A; Wt n=11, KO n=11) and lipogenesis (B; Wt n=6, KO n=6) were measured in epididymal adipose tissue from 18-21-week-old wildtype and LIMK2a knockout male mice. The rates of basal and insulin-stimulated (10 nM insulin) 2-deoxyglucose uptake were determined in epididymal adipose tissue of wildtype and LIMK2a knockout mice (C; n=3-6 per group). Plasma glucose levels were measured after administration of 0.5 U/kg insulin (D; Wt n=5, KO n=6). Triacylglycerol levels were determined in quadriceps muscle and liver (E and F, respectively; Wt n=5, KO n=5). Data are expressed as mean ± S.E.M. Asterisk (*) denotes statistical significance (p<0.05).


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Figure 8-4: Expression of adipogenic marker genes and lipolytic proteins.

The transcript abundance for the adipogenic markers genes, C/EBP , PPAR and aP2, in epididymal adipose tissue from wildtype and LIMK2 knockout mice was determined by quantitative real-time PCR (A; Wt n=4, KO n=4). Expression levels of the lipolytic proteins ATGL, CGI-58 and perilipin-1, were determined by Western blotting (B; Wt n=6, KO n=5). Data are expressed as mean ± S.E.M.


185

8.2 LIMK2 mRNA expression in adipose tissue from lean and obese mice

The finding that the LIMK2a knockout mice develop adult-onset obesity raises the

possibility that LIMK2a expression protects from obesity. To study the potential

inverse correlation between the obese phenotype and LIMK2a expression level,

mRNA was prepared from adipose tissue of age-matched male obese ob/ob

(leptin-deficient) and lean (wildtype) mice, and quantitative real-time PCR analysis

of LIMK2 isoform expression was performed. Expression of LIMK2a normalised to

that of the house-keeping gene, ribosomal protein L32, was significantly lower in

ob/ob mice compared to wildtype animals (Figure 8-5 A). LIMK2b expression,

however, was significantly higher in adipose tissue of ob/ob mice (Figure 8-5 B).

The finding of reduced LIMK2a expression in the leptin-deficient obese mice is

consistent with the obese phenotype of the LIMK2a knockout mouse and supports

the notion of a potential link between LIMK2 expression and obesity.


186

Figure 8-5: Expression of LIMK2 isoforms in adipose tissue from obese and

lean mice at mRNA level.

RNA was prepared from epididymal adipose tissue of age-matched, male C57BL/6 wildtype (lean; n=5) and ob/ob mice (obese; n=5). Quantitative real-time PCR analysis was performed using primers specific for the LIMK2a and LIMK2b isoforms and LIMK2 mRNA expression was normalised to the expression of the housekeeping gene, L32. Data are shown as mean ± S.E.M. Asterisk (*) and triple asterisk (***) denote statistical significance (p<0.05 and p<0.005, respectively).


187

8.3 LIMK2 mRNA and protein expression profile during adipogenesis

3T3-L1 pre-adipocytes represent a well-characterised and commonly used model

for adipogenic differentiation. To determine the expression profile of LIMK2

during adipogenesis, quantitative real-time PCR was performed using mRNA

prepared from 3T3-L1 cells before stimulation (day 0) and 2, 4, 6 and 8 days post-

stimulation with the adipogenic differentiation cocktail. The expression of LIMK2a

mRNA was increased 3.5-fold two days post-stimulation when compared to post-

confluent, unstimulated cells on day 0 (Figure 8-6 A). During the later stages of the

differentiation process (day 4 to day 8), LIMK2a levels dropped and were not

significantly different from those on day 0. These data suggest a potential role for

LIMK2a during the early stages of adipogenic differentiation of 3T3-L1 pre-

adipocytes. The mRNA levels of LIMK2b, however, did not change significantly

during the 8-day differentiation period (Figure 8-6 B).

The protein levels of LIMK1, LIMK2 and cofilin (both total and phosphorylated)

were determined in 3T3-L1 cells harvested on day 0, 2, 4, 6 and 8 of the

differentiation time course by Western blotting. Although there were no

statistically significant changes observed in the protein levels of LIMK2, cofilin and

phospho-cofilin, consistent trends were noted in the expression profile of these

proteins based on the analysis of three independent experiments. Similar to the

mRNA level of LIMK2a, total LIMK2 protein levels showed a 1.7-fold increase two

days after induction of 3T3-L1 cell differentiation when compared to day 0 (Figure

8-7). The ratio of phosphorylated cofilin to total cofilin increased 1.7-fold by day 4.

Interestingly, the most significant (and only statistically significant) change in

LIMK protein expression was observed in the levels of LIMK1, which increased

approximately 13-fold over the first two days of differentiation. The similarity in

the overall trend of the expression profiles of LIMK1, LIMK2 and the p-cofilin/total

cofilin ratio suggests a role for the LIMK-cofilin pathway in adipogenic

differentiation, particularly during the early phase of the differentiation program

(days 0 to 4), during which the expression of these proteins was up-regulated.


188

Figure 8-6: Expression of LIMK2 isoforms in 3T3-L1 cells undergoing

adipogenic conversion.

Differentiation of 3T3-L1 cells was induced by incubation with an adipogenic differentiation cocktail. Cells were harvested on alternate days beginning on day 0 (untreated) until 8 days post-treatment and quantitative real-time PCR analysis was performed using primers specific for the LIMK2a and LIMK2b isoforms. Data are shown as mean ± S.E.M of the fold change relative to day 0 (Panels A and B) calculated from three independent experiments. LIMK2 mRNA expression was normalised to the expression of the housekeeping gene, HPRT1. Asterisk (*) denotes statistical significance (p<0.05 versus day 0).


189

Figure 8-7: Expression of LIMK and cofilin in 3T3-L1 cells undergoing

adipogenesis.

3T3-L1 cells stimulated to differentiate into adipocytes were harvested on day 0, 2, 4, 6 and 8 of differentiation. Protein levels of LIMK1, LIMK2, total cofilin and phosphorylated cofilin were determined by Western blot and were normalised to GAPDH. A: Representative Western blot for differentiating 3T3-L1 cells. B: Protein


190

levels are shown as mean ± S.E.M. of three independent experiments. Asterisk (*) and double asterisk (**) denote statistical significance (p<0.05 and p<0.01 versus day 0, respectively).

8.4 Knockdown of LIMK2 expression in 3T3-L1 cells

To determine the effect of LIMK2 knockdown on adipogenic differentiation, a

mixture of four small interfering RNAs (siRNAs) specific for LIMK2 was transfected

into 3T3-L1 cells, which were subsequently trypsinised, plated in 24-well dishes

and stimulated with the differentiation cocktail 2 days post-confluence. As

adipogenic differentiation of 3T3-L1 pre-adipocytes takes a minimum of 6-8 days,

the time window of efficient LIMK2 knockdown was determined.

Cells were transfected on day -6 (day 0 is the day when stimulation with the

differentiation cocktail was performed) and transfection was considered to be

complete on day -5 after overnight incubation with the siRNA mixture. Transfected

cells were harvested on day 0, 2, 4, 6 and 8 and cell lysates were analysed by

Western blotting for LIMK2, phosphorylated cofilin and GAPDH.

Figure 8-8 A shows the protein expression levels of LIMK2, phospho-cofilin and

GAPDH (loading control) in 3T3-L1 cells. The level of LIMK2 in undifferentiated

pre-adipocytes (day 0) transfected with LIMK2 siRNA was reduced by ~90% when

compared to cells transfected with the control siRNA (Figure 8-8 panels A and B),

concomitant with a ~30% reduction in the level of p-cofilin (Figure 8-8 panels A

and C). On day 2 and day 4 of differentiation, LIMK2 levels were reduced by ~70%

and ~30%, respectively. Cofilin phosphorylation was consistently reduced in cells

transfected with LIMK2 siRNA when compared to the controls on day 2 and day 4.

By day 6 of differentiation, the siRNA had lost its effect and LIMK2 protein levels

were similar to those in cells treated with non-targeting siRNA. These preliminary

results indicate that down-regulation of LIMK2 results in a decrease in cofilin

phosphorylation and suggest that an efficient (>70%) knockdown of LIMK2 can

only be maintained until day 2 of differentiation.


191

Figure 8-8: LIMK2 expression and cofilin phosphorylation after transient

LIMK2 knockdown and differentiation of 3T3-L1 pre-adipocytes.

3T3-L1 cells were transfected with non-targeting siRNA (siNT) or LIMK2-specific siRNA (siLK2) and adipogenic differentiation was induced. Cells were harvested on day 0, 2, 4, 6 and 8 of differentiation and were analysed by Western blotting using antibodies specific for LIMK2 (A, B) and phospho-cofilin (A, C). The division of Panel A in two parts indicates that the samples collected on day 0, 2 and 4 were separated on the same gel and those collected on day 6 and 8 were separated on another gel. Protein levels were normalised to GAPDH.


192

3T3-L1 cells transfected with LIMK2 siRNA or control siRNA were stained with the

lysochrome Oil Red O on day 0, 2, 4, 6 and 8 of differentiation. Microscopically,

there was no difference in the staining of cells transfected with control or LIMK2-

specific siRNA (Figure 8-9 A), neither was there any considerable difference in the

absorbance of the eluted Oil Red O dye between sample and control at any time

during the time course (Figure 8-9 B).


193

Figure 8-9: Lipid accumulation in 3T3-L1 cells after transient LIMK2

knockdown.

3T3-L1 cells were transfected in duplicate wells with non-targeting siRNA (siNT) or LIMK2-specific siRNA (siLK2) and adipogenic differentiation was induced. Cells were fixed on day 0, 2, 4, 6 and 8 of differentiation, followed by staining for intracellular neutral lipids with Oil Red O (red colour) and microscopic analysis (A). Subsequently, the dye was eluted with isopropanol and absorbance of the eluate was measured at 530 nm (B).


194

8.5 Protein expression levels of LIMK2, cofilin and phospho-cofilin in MEFs

Similar to 3T3-L1 pre-adipocytes, mouse embryonic fibroblasts (MEFs) represent

another widely used model for adipogenic differentiation. Embryos from the

mating of LIMK2a heterozygous mice were used at E13.5-E16 for the preparation

of fibroblasts. As shown in Figure 8-10, Western blot analysis with an anti-LIMK2

antibody demonstrated high LIMK2 expression in wildtype but not in LIMK2a

knockout MEFs. Longer exposure of the blot revealed a very faint LIMK2 band in

the LIMK2a knockout MEFs, which most likely represents the low levels of LIMK2b

present in the cell lysate. This result suggests that LIMK2a is the dominant LIMK2

isoform in MEFs. To examine whether LIMK1 is up-regulated in the LIMK2a-

deficient MEFs due to a potential compensatory mechanism, immunoblot for

LIMK1 was performed using the cell lysates as above. Only a small increase in

LIMK1 levels was observed in the LIMK2a knockout MEFs when compared to

wildtype MEFs.


195

Figure 8-10: Protein expression of LIMK1 and LIMK2 in mouse embryonic

fibroblasts.

MEFs were prepared from wildtype (WT) and LIMK2a knockout embryos (KO) at E16 and LIMK protein expression in undifferentiated MEFs was determined by Western blotting with anti-LIMK2 (A) and anti-LIMK1 antibodies (B) as well as HRP-conjugated anti-GAPDH antibodies (loading control). Short and long exposures of the LIMK2 immunoblot are shown (A; top and middle panel, respectively).


196

8.6 Differentiation of MEFs extracted from wildtype and LIMK2a knockout

embryos

To investigate the effects of LIMK2a deficiency on adipogenic differentiation in the

mouse embryonic fibroblast model, MEFs were prepared from the LIMK2a

knockout embryos as well as from their heterozygous and wildtype littermates at

E13.5-E14. Differentiation experiments were performed for 6 days to study

differentiation efficiency or were extended until day 13 to determine potential

differences in lipid metabolism (e.g. excessive accumulation of lipids).

Post-confluent MEF cultures were either fixed on day 0 (2 days post-confluence) or

were stimulated with the adipogenic differentiation cocktail and fixed on day 3 and

6 of differentiation, followed by staining with Oil Red O. There were no differences

in the absorbance of dye eluted from wildtype, heterozygous and knockout MEFs

on day 0, day 3 or day 6 (Figure 8-11 A). Comparison of the Oil Red O absorbance

of MEFs to that of 3T3-L1 cells six days post-induction indicated that the MEFs

accumulated considerably less lipids than 3T3-L1 cells. This suggests that the

differentiation of MEFs is much less efficient compared to the committed pre-

adipocyte culture.

To determine whether LIMK2a deficiency affects lipid accumulation, cultures of

knockout and wildtype MEFs were differentiated as described above and fixed

after 0, 3 and 13 days. No difference in Oil Red O absorbance was observed

between knockout and wildtype MEFs (Figure 8-11 B), suggesting that LIMK2a

deficiency did not alter lipid accumulation in fibroblasts differentiating into

adipocytes. Similarly, there was no difference in the morphology of lipid-laden

wildtype and LIMK2a knockout MEF-derived adipocytes (Figure 8-12).


197

Figure 8-11: Lipid accumulation in differentiating LIMK2a knockout mouse

embryonic fibroblasts.

MEFs were prepared from WT, HET and LIMK2a KO littermate embryos at E13.5 (A) and E14 (B). Post-confluent MEF cultures were either fixed on day 0 or stimulated with adipogenic differentiation cocktail and fixed on day 3 and 6 (A; short-term study) or on day 3 and 13 of differentiation (B; long-term study). 3T3-L1 cells (L1) were used as a positive control for adipogenic differentiation. Intracellular neutral lipids were stained with Oil Red O, followed by elution of the dye and absorbance measurement at 530 nm. Each data point represents one MEF culture from one embryo.


198

Figure 8-12: Lipid staining in differentiating LIMK2a knockout mouse


Mouse embryonic fibroblasts from wildtype and LIMK2a knockout MEFs were differentiated into lipid-laden adipocytes using an adipogenic differentiation cocktail. 3T3-L1 cells were used as a positive control for adipogenic differentiation. Cells were fixed on day 0 and 6 of differentiation prior to staining with Oil Red O, and were analysed using a microscope with a 10X and 40X objective lens. Representative images are shown for stained wildtype and LIMK2a knockout MEFs.


199

8.7 Differentiation of LIMK2 knockout MEFs

In addition to MEFs prepared from the LIMK2a-deficient embryos, further

differentiation experiments were performed with MEFs prepared from mice

deficient in the expression of both LIMK2a and LIMK2b. Post-confluent cultures

were either fixed on day 0 or stimulated with the differentiation cocktail and fixed

after 6 and 10 days of differentiation. The mean absorbance of dye eluted from

LIMK2 knockout MEFs was similar between wildtype or heterozygous MEFs on

day 6 or day 10 (Figure 8-13). However, one of the LIMK2 knockout MEF cultures

(top closed square) showed a markedly higher Oil Red O absorbance than the

remaining LIMK2 knockout MEFs.


200

Figure 8-13: Lipid accumulation in differentiating LIMK2 knockout mouse


MEFs were prepared from WT, HET and LIMK2 KO littermate embryos at E14. Post-confluent MEF cultures were either fixed on day 0 or stimulated with adipogenic differentiation cocktail and fixed on day 6 or 10 of differentiation. Intracellular neutral lipids were stained with Oil Red O, followed by elution of the dye and absorbance measurement at 490 nm. Each data point represents one MEF culture from one embryo.


201

8.8 Identification of novel LIMK2 substrates

Due to the accumulating data suggesting that LIMK1 and LIMK2 have non-

overlapping functions within the cell or in different tissues, it is likely that there

are yet unidentified LIMK2-specific substrates other than cofilin. One strategy to

identify novel protein kinase substrates involves incubation of cell extracts with

the kinase of interest in the presence of 32P-labelled ATP, followed by analysis of

phosphorylated proteins by mass spectrometry. In order to reduce the expected

high background in the phosphorylation reaction, it has been suggested to use

short incubation times, high concentrations of the added kinase and ATP of high

specific radioactivity (Cohen & Knebel, 2006). In addition, manganese (Mn2+)

rather than magnesium (Mg2+) can be used as a counter-ion for ATP to further

reduce the background signal. Fractionation of cell extract by ion-exchange

chromatography has been used previously to concentrate potential substrates and

separate them from their endogenous kinases (Knebel et al., 2001).

To produce sufficient amounts of kinase for the in vitro phosphorylation assay,

GST-tagged LIMK2 was expressed in High-Five insect cells and purified by

ammonium sulphate precipitation and glutathione affinity chromatography.

Immobilised GST-LIMK2 was incubated with Flag-tagged ROCK1 in the presence of

ATP. Analysis of GST-LIMK2 by gel electrophoresis and Coomassie staining

revealed extensive degradation of the ~100 kDa fusion protein (Figure 8-13 lane

1). The most abundant degradation product was represented by a strongly stained

protein band at ~55 kDa, which was present consistently in different batches of

purified GST-LIMK2. In addition to the activated LIMK2 protein, a small aliquot of

non-activated LIMK2 was kept as a negative control for the subsequently

performed in vitro kinase assay using cofilin as a substrate.

In the absence of LIMK2, cofilin was not phosphorylated (Figure 8-14). However,

cofilin was highly phosphorylated in the presence of activated GST-LIMK2, while p-

cofilin levels were very low when LIMK2 was not activated by ROCK. It was

concluded that the purified and activated GST-LIMK2 could be used for

biochemical assays despite its relatively high degree of degradation.


202

Figure 8-14: In vitro phosphorylation of cofilin by activated and non-

activated LIMK2.

GST-tagged LIMK2 was purified from baculovirus-infected insect cells and activated with Flag-tagged ROCK1. GST-cofilin was incubated with [ -32P]ATP in the presence or absence of ROCK-treated and untreated GST-LIMK2. Arrows in the Coomassie-stained polyacrylamide gel (top panel) or in the autoradiograph (bottom panel) indicate GST-cofilin.


203

As a source of potential LIMK2 substrates, lipid-depleted adipose tissue extract

was prepared, desalted and subsequently fractionated using a Q Sepharose ion-

exchange chromatography column. The elution profile showed that approximately

60% of the total protein in the tissue extract did not bind to the column in the

absence of sodium chloride. Only about 40% of the total protein was able to bind

to the positively charged Q Sepharose column, and was eluted using a salt gradient.

The elution profile showed that the majority of proteins in the tissue extract did

not bind to the column in the absence of sodium chloride. Only a small proportion

of the total protein was able to bind to the positively charged Q Sepharose column,

and was eluted using a salt gradient. The protein content of the tissue extract

fractions was assessed by SDS-PAGE and staining with Coomassie Blue (Figure 8-

15 A). Subsequently, fractions 16, 18, 20 and 22 were incubated with [ -32P]ATP in

the presence or absence of activated LIMK2, followed by separation of the samples

on a 4-20% SDS-polyacrylamide gel and autoradiography. Phospho-bands at an

apparent molecular weight of ~40 kDa were observed in tissue extract fractions 16

and 18 only in the presence of LIMK2, but not in its absence (Figure 8-15 B).

Another potential hit was found in tissue extract fractions 16 and 18 at an

apparent molecular weight of ~18 kDa. These bands were excised from the gel and

were subjected to analysis by mass spectrometry. The vast majority of the

identified peptides were assigned to keratin, a common and highly abundant

contaminant whose presence can be detrimental to the identification of less

abundant proteins in the sample.

For the purpose of assay optimisation, cell extracts were prepared from NIH3T3

cells, followed by fractionation using a negatively charged (SP Sepharose) ion-

exchange chromatography column. Figure 8-16 A shows the protein content of the

cell extract before and after fractionation.


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Figure 8-15: In vitro phosphorylation of fractionated adipose tissue extract

by activated and non-activated LIMK2.

A: Protein content of ion-exchange chromatography fractions 16, 18, 20 and 22 of lipid-depleted adipose tissue extract was analysed by SDS-PAGE and Coomassie staining. B: Fractions of adipose tissue extract were incubated with activated GST-LIMK2 in the presence of [ -32P]ATP. Samples were separated by SDS-PAGE, followed by autoradiography (top panel: long exposure; bottom panel: shorter exposure). Differentially phosphorylated protein bands (highlighted by red boxes) were excised from the gel and analysed by mass spectrometry.


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Figure 8-16: In vitro phosphorylation of fractionated NIH3T3 cell extract by

activated LIMK2.

A: The protein content of NIH3T3 whole cell lysate (WCL) and of ion-exchange chromatography fractions 4, 6, 8 and 10 was analysed by SDS-PAGE and Coomassie staining. B: Fractions of cell extract were incubated with activated GST-LIMK2 in the presence of [ -32P]ATP. Samples were separated by SDS-PAGE, followed by autoradiography (top panel: short exposure; bottom panel: longer exposure). Differentially phosphorylated protein bands (highlighted by numbered red boxes) were excised from the gel for analysis by mass spectrometry.


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Cell extract fractions 4, 6, 8 and 10 were incubated with [ -32P]ATP in the presence

and absence of ROCK-activated LIMK2, and were separated by gel electrophoresis.

The autoradiograph showed differentially phosphorylated protein bands in extract

fractions 6 and 8 (Figure 8-16 B). Mass spectrometry analysis identified several

proteins in the excised phospho-bands, including nucleolin (band 1), DNA

replication licensing factor MCM3 (band 3), vimentin and Myc box-dependent-

interacting protein 1 (band 4). The identified peptides for each protein are listed in

Table 8-1 together with the specific type of modification (e.g. phosphorylation,

oxidation) as well as their experimental and calculated molecular mass. For

nucleolin, MCM3 and vimentin, no phosphorylated peptides were found. In

contrast, one phospho-peptide of Myc box-dependent-interacting protein 1 was

identified. However, this peptide was not unique to the sample incubated with

activated LIMK2. In bands 2 and 5, no proteins were found that met the criteria for

positive identification, i.e. at least one detected peptide with a confidence interval

for identity or extensive homology of 95%. As relatively large amounts of activated

LIMK2 were added to the fractionated cell extract, numerous LIMK2-specific

peptides, and more interestingly, LIMK2-specific phospho-peptides were detected.

Potential phosphorylation sites found in LIMK2 included serine residues 289, 293

and 298. However, none of these residues were detected in site-determining ions,

i.e. in peptides containing only one phosphorylatable residue. Phosphorylation of

these sites is likely to be the result of trans-phosphorylation induced by ROCK-

mediated LIMK2 activation.


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Table 8-1: Peptides identified in differentially phosphorylated protein bands.

Differentially phosphorylated protein bands as shown in Figure 8-16 B were analysed by mass spectrometry and detected peptides were assigned to individual proteins in each band using the software MASCOT. Addition and omission of activated LIMK2 is indicated by a plus (+) and minus (-) symbol, respectively. Modified residues in the peptide sequence are highlighted in bold. Phosphorylation, oxidation and deamination are indicated by addition of +P, +Ox and +Deam, respectively. The experimental (expt) and calculated (calc) molecular mass (Mr) as well as the ion score is shown for each peptide.

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CHAPTER 9 − DISCUSSION


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9.1 Obesity phenotype

The members of the LIMK family of serine/threonine kinases play an important

role in the regulation of actin dynamics (Vardouli et al., 2005; Yang et al., 1998)

and have been implicated in the pathogenesis of diseases such as cancer and ocular

hypertension (Harrison et al., 2009; Scott et al., 2010). This role is tightly linked to

their ability to modulate actin dynamics through phosphorylation of the actin-

depolymerising protein, cofilin. However, to date there have been no published

reports relating to the involvement of the LIMK family members in the

development of obesity and its associated complications. The surprising finding

that mice deficient in LIMK2a expression exhibit adult-onset obesity and signs of

insulin resistance raised the question of how LIMK2a is involved in the

development of the obese phenotype.

The obese phenotype was observed exclusively in adult LIMK2a-deficient males.

These mice had a significantly increased body mass compared to their wildtype

littermates at the age of 18-21 weeks. The increased body mass was attributed

mainly to an increase in fat mass relative to body mass. The genotype-specific

differences in body and fat mass were even more pronounced when the mice were

fed a high-fat diet. The lack of considerable weight gain in the wildtype mice after

12 weeks on a high-fat diet may be attributed to the relatively low propensity of

C57BL/6 mice for diet-induced weight gain compared to other inbred mouse

strains (Andrikopoulos et al., 2005).

The epididymal adipocytes of LIMK2a knockout mice appeared clearly

hypertrophic. These features were reminiscent of the adult-onset obesity

phenotype observed in many other knockout mouse models, such as the

aquaporin-7 knockout mouse (Hara-Chikuma et al., 2005; Hibuse et al., 2005).

Disruption of the Aqp7 gene resulted in increased adipocyte diameter (adipocyte

hypertrophy) due to impaired glycerol efflux caused by the absence of this

normally highly expressed water/glycerol transporter in adipocytes (Hibuse et al.,

2006).

When studying body mass and body composition, three contributing processes

must be taken into consideration: energy intake, energy storage and energy


212

expenditure (Argmann et al., 2006). The lack of significant differences in food

intake, oxygen consumption or respiratory exchange ratio indicate that the

increased body and fat mass of the LIMK2a-deficient mice was not a consequence

of dramatically altered behaviour (relating to feeding or physical activity), changes

in overall metabolic rate or fuel utilisation. Therefore, it is likely that the obesity

phenotype was caused by tissue-specific defects in adipose tissue. It should be

noted that the subtle differences in energy balance between knockout and

wildtype mice that were expected based on the obese phenotype of the LIMK2a

knockout mouse may fall beyond the detection limits of the analytical methods

used. Future studies should include the measurement of fecal energy loss to

determine potential differences in the energy excreted. In addition, a larger sample

size and a longer observation period may be required to detect small differences in

energy balance. Future studies aiming to determine the energy expenditure of

LIMK2a knockout mice should include the measurement of lean body mass by

dual-energy x-ray absorptiometry. The relevance of this data becomes apparent

when one acknowledges the fact that the higher body mass of obese mice is mostly

due to an expansion of white adipose tissue, which is known to have a much lower

metabolic activity than many non-adipose tissues (Wang et al., 2010). Using this

additional data, the effect of the genotype on energy expenditure could be

determined while controlling for the effect of body composition through analysis

of co-variance (ANCOVA) (Tschop et al., 2012).

The observation that the adipocytes in epididymal fat pads of LIMK2a knockout

mice were hypertrophic supports the notion of altered energy storage. Normal

triacylglyceride levels in muscle and liver suggest that ectopic fat deposition did

not occur in the LIMK2-deficient mice. Based on the theory of limited adipose

tissue expandability (Gray & Vidal-Puig, 2007), the lack of ectopic lipid

accumulation suggests that the potential for adipose tissue expansion through

adipocyte hypertrophy and adipose tissue hyperplasia had not been exhausted in

mice at the age of 18-21 weeks, or that the expansion limit had not been reached

for long enough to evoke measurable “lipid overflow” into non-adipose tissues.

Consistent with this notion is the observation that mice can reach a maximum


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body mass in excess of 60 g and accumulate significantly larger amounts of fat

(Coleman & Hummel, 1973).

In contrast to the obese phenotype of the LIMK2a knockout mice, the previously

generated mouse model of LIMK2-deficiency have not been reported to exhibit an

adipose tissue phenotype (Takahashi et al., 2002). This is not surprising because

these LIMK2 knockout mice (more specifically, LIMK2a, LIMK2b and LIMK2t

knockout mice) were generated by Cre-mediated excision of exons 3-5 (encoding

the second LIM domain and part of the PDZ domain in LIMK2a and LIMK2b) as

well as of exon 1t (initiation exon for LIMK2t). Therefore, active LIMK2 protein

may still be expressed as a truncated form containing a functional kinase domain.

This assumption is based on the finding that the similarly generated LIMK1

knockout mice express a truncated protein comprised of one and a half LIM

domains spliced into the kinase domain (Acevedo et al., 2006). The catalytic

activity of the immunoprecipitated protein has previously been demonstrated by

in vitro kinase assay (unpublished data).

Interestingly, there was also no indication for an obesity phenotype in the LIMK2-

deficient mouse model generated by Lexicon Pharmaceuticals by truncation of the

kinase domain (Rice et al., 2012). In this model, the expression of both LIMK2a and

LIMK2b is disrupted. Therefore, imbalance between the two isoforms (rather than

lack thereof) may be required for development of the obesity phenotype.

9.2 Insulin-stimulated glucose uptake

Responsiveness to insulin is a critical feature of metabolically active tissues such as

adipose tissue, muscle and liver. Isolated epididymal adipose tissue and

quadriceps muscle from LIMK2a knockout mice showed similar basal 2-

deoxyglucose uptake to the corresponding wildtype tissues. However, when

stimulated with insulin, only LIMK2a-deficient adipose tissue, but not muscle,

showed a reduction in glucose uptake relative to the wildtype. The results of the

insulin tolerance tests are suggestive of whole-body insulin resistance, which may

be due to an adipose tissue-specific defect in the response to insulin. Future


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studies should also include the measurement of plasma insulin and glucose

tolerance tests to further explore potential alterations of insulin sensitivity.

Similar studies using human adipose tissue explants have demonstrated that

overweight and obesity are associated with normal basal, but decreased insulin-

stimulated glucose uptake when compared to lean subjects (Stolic et al., 2002).

Basal glucose uptake is mediated by the GLUT1 glucose transporter in a largely

insulin-independent fashion (Harrison et al., 1990). The reduction in insulin

responsiveness, however, indicates a defect in insulin signalling that is specific to

adipocytes. Elevated levels of the inflammatory cytokine TNF are commonly

found in adipose tissue of obese individuals and are thought to contribute to

obesity-related insulin resistance by down-regulating the expression of GLUT4 and

several insulin signalling proteins including the insulin receptor, IRS-1 and Akt

(Hotamisligil et al., 1995; Ruan et al., 2002). Insulin-stimulated glucose uptake

requires the translocation of the GLUT4 hexose transporter from intracellular

storage vesicles to the plasma membrane (Cushman & Wardzala, 1980; Suzuki &

Kono, 1980). Remodelling of cortical actin has been demonstrated to be essential

for the final exocytotic step in the distal GLUT4 trafficking events (Kanzaki &

Pessin, 2001; Lopez et al., 2009). ROCK1-dependent phosphorylation of cofilin has

been implicated in the regulation of insulin-induced GLUT4 translocation in

adipocytes, suggesting the involvement of LIMK as a mediator between ROCK and

cofilin (Chun et al., 2012). Therefore, it seems plausible that LIMK2a deficiency

may cause impairment of insulin-induced glucose uptake in adipocytes under

inflammatory stress.

9.3 Lipid metabolism

The storage and release of lipids from adipose tissue are critical steps in energy

homeostasis as they connect localised adipose tissue function with systemic

energy supply. Basal and stimulated lipolysis were significantly decreased in

adipose tissue of LIMK2a knockout mice when compared to that of wildtype mice.

This is consistent with the concept that reduced lipolytic activity may contribute to

lipid accumulation in adipose tissue, and therefore, obesity (Jaworski et al., 2007).


215

Considering the inhibitory effect of insulin on lipolysis (Morimoto et al., 1998;

Richter et al., 1990) and the reduced insulin sensitivity of adipose tissue in obesity

(Xu et al., 2003), it appears paradoxical to find decreased lipolytic activity in

adipose tissue from obese LIMK2a knockout mice. Despite the reduction in

lipolytic activity per mass unit of LIMK2a knockout adipose tissue, these animals

showed higher levels of circulating free fatty acids than their wildtype littermates.

Plasma free fatty acids correlate positively with visceral adiposity (Baltzell et al.,

1991; Jensen, 2006). Therefore, elevated plasma free fatty acids may be attributed

to the higher adipose tissue mass in the obese mice. It is also possible that the

uptake of free fatty acids is impaired specifically in adipose tissue of LIMK2a-

deficient mice, consistent with the normal triacylglycerol levels found in muscle

and liver.

The fact that the levels of some of the major lipolytic proteins, such as ATGL and

perilipin, were unchanged indicates that the decreased lipolytic activity is not a

consequence of reduced expression of proteins involved in the hydrolysis of

triacylglycerols. It suggests that the expression of genes characteristic for terminal

differentiation of adipocytes is not impaired in LIMK2a-deficient mice, which is

supported by the normal expression of C/EBP and PPAR , the master

transcriptional regulators of adipogenesis (Shao & Lazar, 1997). With no changes

in adipocyte-specific gene and protein expression being apparent in mature

LIMK2a knockout adipocytes, there was a possibility that LIMK2a might be

involved in the commitment and/or differentiation of adipocyte progenitors or

pre-adipocytes.

9.4 LIMK2 expression in adipose tissue

Determination of the expression levels of LIMK2a and LIMK2b in epididymal

adipose tissue from obese (ob/ob) mice and lean (wildtype) mice provided further

evidence for the intriguing correlation between LIMK2 expression and the obese

phenotype. LIMK2a mRNA was less abundant in epididymal adipose tissue from

obese (ob/ob) mice when compared to lean (wildtype) mice. This significant

difference was in agreement with the obese phenotype of LIMK2a-deficient mice.


216

Interestingly, LIMK2b mRNA levels were higher in adipose tissue from obese mice,

indicating a potential regulatory mechanism that coordinates the transcription of

the LIMK2a and LIMK2b isoforms in adipose tissue.

Even though the epididymal adipose tissue was harvested from obese and lean

mice of the same genetic background (C57BL/6), it remains uncertain whether the

observed down-regulation of LIMK2a and up-regulation of LIMK2b expression was

a direct consequence of obesity or merely a secondary effect caused by leptin-

deficiency. This possibility should be addressed in future studies using a mouse

model of diet-induced obesity (Moraes et al., 2003).

Despite the lack of published reports concerning the abundance of LIMK2

transcripts specifically in adipose tissue, LIMK2a and LIMK2b mRNA was reported

to be differentially expressed in a tissue-specific manner (Honma et al., 2006;

Osada et al., 1996). Osada et al. demonstrated that LIMK2a was the dominant

isoform is human liver, colon, stomach and spleen, while LIMK2b was predominant

in human brain, kidney and placenta. In human lung tissue, the two isoforms were

expressed at approximately equal levels. Interestingly, the isoform ratios observed

in normal tissues were altered in some cancer cell lines. It was speculated that

LIMK2b protein might competitively and/or negatively regulate the function of

LIMK2a, suggesting that the balance between the two isoforms may be of

functional importance (Osada et al., 1996).

Further complexity is added when LIMK2 expression is considered in a

developmental context. Northern blot and RT-PCR analysis of the LIMK2 isoforms

in adult mouse tissues showed ubiquitous expression of LIMK2a, while LIMK2b

was expressed more selectively (Ikebe et al., 1997; Koshimizu et al., 1997).

However, there was no specific reference to LIMK2 expression in adipose tissue.

Interestingly, during embryogenesis, both isoforms were detected at high levels

between E10 and E12 (Koshimizu et al., 1997), suggesting a potential role at this

stage of mouse development. It is uncertain whether the increased expression of

the LIMK2 isoforms in E10-12 embryos is of relevance for the development of

adipose tissue in mice, as there is no macroscopically detectable white adipose

tissue during embryonic life and at birth in rodents (Ailhaud et al., 1992). In tissues

of E14 embryos and adult mice, it has been demonstrated by Western blotting and


217

immunohistochemistry that LIMK2 protein is widely expressed in all tissues

(Acevedo et al., 2006). However, adipose tissue was not examined. Due to the lack

of reliable isoform-specific antibodies, the expression patterns of LIMK2a and

LIMK2b protein remain elusive. Detailed immunohistochemical analysis focussing

on the spatial and temporal aspects of LIMK2 expression may provide further

insights into the role of LIMK2 in the development of mouse tissues, as it has done

in the case of LIMK1, revealing a temporally dynamic and tissue-specific

expression pattern during mouse embryogenesis (Lindstrom et al., 2011). All

currently available data on LIMK2 expression suggest a very similar tissue

distribution between LIMK2 and LIMK1 in the adult mouse (Acevedo et al., 2006;

Foletta et al., 2004).

Deletion of LIMK2a specifically in adipose tissue by conditional knockout

represents a promising approach for future studies aiming to directly determine

the role of LIMK2a in regulating the size as well as the diverse functions of

adipocytes. In addition, this model would be useful to study the effects of specific

adipose tissue defects on the whole-body metabolism in the absence of potentially

confounding defects in other tissues.

9.5 LIMK2 expression and function during adipogenesis

In 3T3-L1 cells stimulated with an adipogenic differentiation cocktail, a spike in

LIMK2a mRNA expression was observed after two days of incubation. The

abundance of LIMK2b transcript in response to adipogenic stimuli, however,

remained unchanged compared to the basal level. LIMK2 protein levels were also

increased on day 2 of differentiation, consistent with the increase in LIMK2a

mRNA. The observed increase in LIMK2a expression on day 2 of differentiation

suggests a role for LIMK2a during early adipogenesis, which may relate to the

morphological changes that are characteristic for the initial phase of

differentiation. Reorganisation of the actin cytoskeleton as induced by the

metalloprotease, ADAM12, has been associated with the coordination of

morphological changes in adipogenic differentiation (Kawaguchi et al., 2002). In

committed pre-adipocytes, ADAM12 is highly expressed during early adipogenesis,


218

thus pinpointing a critical stage of permissiveness for differentiation (Kawaguchi

et al., 2003).

However, cell shape changes have not only been implicated in the regulation of

differentiation of committed pre-adipoctyes to mature adipocytes, but also in the

commitment of pluripotent stem cells to the adipogenic lineage. Significant

changes in the expression of -actin and several key actin-binding proteins

(including tropomyosin-1 and profilin) were observed during the early stage of

adipogenic induction of umbilical mesenchymal stem cells (Peng & Liou, 2012).

Several other cytoskeleton-associated proteins (lysyl oxidase (LOX), translationally

controlled tumor protein 1 (TPT1) and αB-crystallin) are involved in the

regulation of cell shape-dependent stem cell commitment (Huang et al., 2011).

Consistent with the increase in LIMK2a expression on day 2 of differentiation, the

level of phospho-cofilin was also elevated in differentiating 3T3-L1 cells between

day 2 and day 4, while the expression of total cofilin decreased continuously from

day 0 to day 8. Apart from LIMK2, which regulates actin dynamics through

phosphorylation of cofilin in fibroblasts (Vardouli et al., 2005), cofilin

phosphorylation could also be mediated by LIMK1, whose levels were increased

13-fold two days post-induction with insulin containing differentiation medium.

Insulin has been reported to increase LIMK1 activity via activation of Rac in HeLa

cells (Yang et al., 1998), which may result in LIMK1 auto- and trans-

phosphorylation, thereby leading to increased protein stability (Li et al., 2006).

Knockdown of LIMK2 in 3T3-L1 cells resulted in transiently reduced levels of

phospho-cofilin when compared to cells transfected with non-targeting siRNA,

indicating that cofilin is a LIMK2 substrate in differentiating 3T3-L1 cells. It further

indicates that phospho-cofilin levels are notably affected by lack of LIMK2 despite

a dramatic increase of LIMK1 levels during early adipogenesis. The siRNA-

mediated approach enabled efficient knockdown of LIMK2 protein levels that was

maintained until at least day 2 of differentiation. On day 4, LIMK2 and phospho-

cofilin levels were only slightly reduced in cells transfected with LIMK2 siRNA

relative to the controls.

However, it was noted that the expression profiles of LIMK2 and phospho-cofilin

for the first 4 days post-stimulation were different from those of non-transfected


219

3T3-L1 cells (Figures 8-6 and 8-7). LIMK2 levels were not increased on day 2 and 4

of differentiation as observed in non-transfected cells, and there was no increase in

phospho-cofilin relative to day 0. In contrast, both LIMK2 and phospho-cofilin

levels were decreased on day 2 and 4, which may be due to non-specific effects of

the transfection reagent, Lipofectamine 2000. Nonetheless, the transfected cells

differentiated efficiently into lipid-laden adipocytes as determined by Oil Red O

staining, suggesting that elevated protein levels of LIMK2 and phosphorylated

cofilin (compared to basal levels) are not essential for adipogenic differentiation.

Knockdown of LIMK2 expression during the early phase of adipogenesis did not

result in altered lipid accumulation, suggesting that long-term reduction or a more

efficient down-modulation of LIMK2 levels may be required to elicit an effect on

adipogenesis. In contrast, pharmacological inhibition of ROCK during the first four

days of differentiation has been shown to be almost exclusively responsible for the

observed enhancement of adipogenesis in 3T3-L1 cells (Noguchi et al., 2007). The

major limitation of the siRNA-mediated approach of LIMK2 knockdown lies in its

transient nature, which appears to be incompatible with the lengthy experimental

protocol involving transfection, cell growth to full confluence and induction of

differentiation on day 2 post-confluence, followed by an at least 6-day period after

which differentiation efficiency can be assessed.

As an alternative approach, mouse embryonic fibroblasts were prepared from

LIMK2-deficient and LIMK2a-deficient embryos and their heterozygous and

wildtype littermates to compare their potential for adipogenic differentiation.

However, no differences in adipogenesis were found in either short-term (6 days)

or long-term studies (10-13 days) focusing on initiation of the adipogenic

differentiation program and lipid metabolism (net lipid accumulation),

respectively. The minor differences in lipid accumulation observed between MEFs

from different embryos are most likely due to unrelated genetically and

environmentally caused differences between individuals. In this context, it should

be noted that not all embryos found in the uterus at the time of embryo collection

would survive until birth if undisturbed. During the extraction of embryos from the

uterus it became obvious that there were small but noticeable differences in the

size of the embryos as well as in their developmental stage as judged by the


220

development of their internal organs. The percentage of cells that differentiated

into lipid-laden adipocytes upon stimulation with the adipogenic cocktail

(approximately 10-15%) was consistent with the expected proportion of cells

within the MEF population that are committed to the adipogenic lineage (Tang et

al., 2003). Although the MEF model of differentiation is one step closer to the in

vivo situation compared to 3T3-L1 pre-adipocytes, the model is limited by the

relatively low percentage of committed cells, which may obscure any potential

effects of LIMK2 or LIMK2a deficiency on adipogenic differentiation. Despite the

limitations of the mouse embryonic fibroblast model, significant enhancement of

adipogenesis was observed in ROCK-II-deficient MEFs (Noguchi et al., 2007),

indicating that it is suitable for studying the effect of “positive” regulators of

adipogenesis. The adipogenesis-enhancing effect of ROCK-II deficiency has been

attributed to the anti-adipogenic and pro-myogenic effect of Rho/ROCK signalling

(Sordella et al., 2003). Thus, inactivation of this critical switch in the adipogenesis-

myogenesis cell fate decision results in an increased number of committed pre-

adipocytes in the total population of mouse embryonic fibroblasts. In contrast, lack

of LIMK2 or LIMK2a does not appear to impact on the number of committed pre-

adipocytes in MEF cultures. In light of these results, future studies should

investigate permanent LIMK2 knockdown in 3T3-L1 pre-adipocytes by means of

isoform-specific shRNA. This may help establish a role for LIMK2a in regulating the

differentiation of pure pre-adipocyte cultures.

9.6 Identification of novel LIMK2 substrates

The identification of novel substrates for LIMK2 represents a major challenge in

understanding its biological functions in addition to those shared by LIMK1.

Incubation of fractionated tissue and cell extracts with radioactively labelled ATP

in the presence or absence of activated LIMK2 resulted in differential

phosphorylation patterns, indicating that LIMK2 can phosphorylate several

proteins in vitro. Peptide mass fingerprinting performed on selected phospho-

bands revealed the identity of the most abundant proteins, which could be

potential LIMK2-specific substrates. Proteins identified in the excised phospho-


221

bands included vimentin and nucleolin. However, the peptides derived from these

proteins were not phosphorylated, indicating that they were either not

phosphorylated by LIMK2 or the abundance of their phospho-peptides was too low

to be detected by mass spectrometry. On the other hand, several phospho-peptides

were assigned to Myc box-dependent-interacting protein 1 and DNA replication

licensing factor MCM3, however, none of them were unique to the sample

incubated with activated LIMK2.

Keratin contamination was more pronounced in fractionated adipose tissue extract

compared to extracts of cultured cells, which is most likely due to contamination of

the tissue sample with mouse hair at harvest. The remaining keratin that was

detected by mass spectrometry could be of human origin, introduced into the

sample through contaminated buffers or SDS polyacrylamide gels.

Several phospho-peptides originating from LIMK2 were identified by peptide mass

fingerprinting, indicating that this method is more efficient with highly abundant

proteins. Data analysis using the software MASCOT revealed serine residues 289,

293 and 298 as likely phosphorylation sites. Phosphorylation of these residues in

mouse and human LIMK2 has been found in several proteomics studies (Daub et

al., 2008; Dephoure et al., 2008; Huttlin et al., 2010; Yu et al., 2011). Notably, the

majority of these potential phosphorylation sites appears to be clustered and is

most likely the result of auto- and trans-phosphorylation of activated LIMK2

molecules. Despite all the measures taken to favour LIMK2-specific

phosphorylation of previously unknown protein substrates (in particular, short

incubation time, high specific radioactivity, high concentration of added kinase as

well as substitution of magnesium by manganese as counter-ion for ATP), further

optimisation of the assay is required to enable the detection of a larger number of

phosphorylated substrate peptides. One important limiting factor is probably the

low abundance of potential substrate molecules. In addition, separation of the in

vitro phosphorylation reaction by two-dimensional gel electrophoresis is likely to

decrease the number of proteins contained in one phospho-band, thereby

facilitating identification of the proteins specifically phosphorylated by LIMK2.

223

CHAPTER 10 − CONCLUSION


224


225

Since its discovery in the 1990s, the LIMK family of protein kinases has been

established as important regulators of cell motility that act through regulation of

cofilin-mediated actin reorganisation (Arber et al., 1998; Sumi et al., 1999; Yang et

al., 1998). Although several mouse models of LIMK deficiency have been

generated, their analysis revealed only minor phenotypic abnormalities in neurons

and testes, most probably due to the incomplete deletion of these genes (Meng et

al., 2002; Meng et al., 2004; Takahashi et al., 2002). In this work, the effect of the

knockout of the LIMK2a isoform on body composition, energy homeostasis and

adipogenesis has been characterised, implicating LIMK2a in the pathogenesis of

obesity and its associated metabolic complications.

In contrast to the previously generated LIMK2 knockout mouse (Takahashi et al.,

2002), LIMK2a-deficient mice showed an obese phenotype, which was exclusive to

adult males. When exposed to a high-fat diet, LIMK2a knockout animals displayed

an even higher susceptibility to developing obesity than their wildtype littermates.

The phenotype was attributed to aberrant energy storage, as energy intake and

energy expenditure were similar to those in wildtype mice. One striking

characteristic of LIMK2a knockout adipose tissue was adipocyte hypertrophy,

which was hypothesised to be the result of dysregulation of actin dynamics due to

the lack of LIMK2a. Defects in actin re-organisation, in turn, have a potential to

affect adipocyte function as well as adipogenesis.

The LIMK2a knockout mice showed reduced blood glucose clearance in response

to insulin, which was attributed to insulin resistance in adipose tissue, but not in

isolated muscle. Although skeletal muscle is responsible for ~85% of insulin-

stimulated glucose uptake (Peppa et al., 2010), considerable cross-talk takes place

between muscle and adipose tissue (e.g. through circulating free fatty acids and

adipokines), which can directly affect skeletal muscle insulin sensitivity (Havekes

& Sauerwein, 2010). There is accumulating evidence suggesting that systemic

insulin resistance has its origin in the loss of insulin sensitivity in adipose tissue

(Iozzo, 2009). A recent study on obese human subjects found that systemic,

skeletal muscle and hepatic insulin resistance as well as dyslipidaemia and

hyperglycaemia can be reversed by weight loss, while adipose tissue insulin

resistance remained unchanged (Viljanen et al., 2009). Therefore, LIMK2a appears


226

to act at a critical junction in adipose tissue development leading to either

physiologically normal adipose tissue or potentially permanent adipose tissue

dysfunction. This notion is supported by the finding that LIMK2a expression was

down-regulated in adipose tissue of obese ob/ob mice when compared to lean

wildtype mice.

Rho and ROCK, the upstream regulators of LIMK2, have previously been implicated

in the regulation of adipogenesis (Noguchi et al., 2007; Sordella et al., 2003).

Increased expression of LIMK2 and phosphorylation of its substrate cofilin in

differentiating 3T3-L1 cells suggests that LIMK2 activity may be important for the

early stages of differentiation of pre-adipocytes into mature adipocytes. Transient

knockdown of LIMK2 in 3T3-L1 cells supported the notion of LIMK2-dependent

cofilin phosphorylation during adipogenesis. However, differentiation as

determined by lipid accumulation was unaffected by transient LIMK2 down-

regulation. Further cell culture studies employing permanent knockdown of

LIMK2a are needed to determine the role of LIMK2a in the differentiation of

committed 3T3-L1 pre-adipocytes. In contrast to ROCK-II-deficient MEF cultures,

lack of expression of LIMK2 or LIMK2a in mouse embryonic fibroblasts did not

enhance adipogenic differentiation. This finding indicates that LIMK2, unlike

ROCK-II, is not involved in the commitment of progenitor cells to the adipogenic

lineage.

The gender-specific expression of the adipose tissue phenotype in LIMK2a

knockout mice suggests the involvement of male and female gonadal hormones in

adipose tissue development and function. In two recent studies, the female

hormone oestrogen was shown to protect female mice from adipocyte

hypertrophy, adipose tissue inflammation and insulin resistance (Stubbins et al.,

2011; Stubbins et al., 2012). Microarray analysis of adipose tissue from diet-

induced obese mice has provided first mechanistic insights into the sexual

dimorphism by demonstrating significant gender-specific differences in the

expression of genes associated with inflammation and insulin signalling (Grove et

al., 2010). Elucidation of the underlying molecular mechanisms may provide new

avenues for the treatment and prevention of obesity-induced insulin resistance

and adipose tissue dysfunction.


227

Future studies aiming at understanding the role of LIMK2 in adipose tissue should

explore specifically the functional importance of the first one and a half LIM

domains at the protein’s N-terminus, which are part of the LIMK2a, LIMK2c and

LIMK2d isoforms, but are missing in LIMK2b. LIM domains are known to mediate

protein-protein interactions and may therefore determine the sets of proteins with

which the individual isoforms of LIMK2 interact. These proteins may also include

other LIMK2 isoforms, which could have implications for the reciprocal regulation

of their activity as well as for their potentially different functions. As demonstrated

in neurons for the isoform LIMK2d, which is devoid of the kinase domain, these

functions can be independent of catalytic activity (Tastet et al., 2012).

In conclusion, LIMK2a was identified as a novel genetic factor that determines the

susceptibility to developing obesity in males. LIMK2a deficiency results in

adipocyte hypertrophy and insulin resistance in mouse adipose tissue, thereby

affecting many of its basic metabolic functions.


228

229

CHAPTER 11 - FINAL CONCLUSION

Chapter 11 - Final Conclusion

230


231

The LIMK family of protein kinases consists of two important regulators of actin

dynamics, which have been implicated in diverse pathologies associated with the

actin cytoskeleton. Similar to other protein kinases associated with disease, small-

molecule inhibitors have been identified that target the kinase domain and prevent

substrate phosphorylation. In pursuit of the primary aim of solving the atomic structure of the LIMK kinase

domain to enable rational optimisation of specific inhibitor compounds, an

effective procedure was developed for the expression and purification of

recombinant LIMK1 kinase domain. The use of the baculovirus/insect cell systems

satisfied the need for large protein quantities for X-ray crystallographic studies,

while protein purity was continuously improved by employing a multi-step

purification process. Refinement of the kinase domain boundaries increased

protein stability while retaining catalytic activity. The low solubility of the LIMK1

kinase domain in the absence of the GST tag was enhanced by addition of the

carbohydrate-based solubilising agent, NV10. However, the concentrated protein

sample did not yield crystals in crystallisation trials. Using a considerably more

concentrated sample stabilised by the addition of L-arginine and L-glutamate,

members of the Structural Genomics Consortium successfully crystallised the

LIMK1 kinase domain in complex with staurosporine. The deposited structure

shows a typical two-lobed fold of the kinase domain with the inhibitor occupying

the ATP binding pocket and stabilising the active conformation. However, the

activation loop of the LIMK1 kinase domain was not resolved in this structure,

suggesting that it did not form significant interactions with the inhibitor.

The structure of the LIMK1 kinase domain/staurosporine complex represents a

promising tool for the design of more specific inhibitor compounds. However, to

obtain a more reliable model of the LIMK1 kinase domain that reflects its

conformational flexibility, further structures are required that show the protein’s

conformation in the presence and absence of more specific inhibitors. Using the

knowledge obtained from the purification and crystallisation of the LIMK1 kinase

domain to determine the structure of the closely related kinase LIMK2 will

facilitate the elucidation of structural differences between the kinase domains,

which may provide valuable clues to explain their non-overlapping functions. In


232

addition, these structural differences may enable the specific targeting of only one

member of the LIMK family.

The identification of LIMK2a as a novel genetic factor determining the

susceptibility to develop obesity highlights the importance of proper

characterisation of the often overlooked adipose tissue. Extensive gene and protein

expression profiles have been established for a wide range of tissues. However, it

appears that adipose tissue is often excluded from detailed analysis, presumably

due to a lack of appreciation of its structural and functional complexity. One

adipocyte-specific feature that remains poorly understood is the cortical actin

network, which gives this cell type its characteristic spherical shape and has been

associated with the expression of inflammatory cytokines (Nakayama et al., 2009)

and with insulin-response pathways (Hagan et al., 2008; Lopez et al., 2009).

Studying the adipogenic potential of LIMK2a-deficient mouse embryonic

fibroblasts, no evidence was found supporting the notion that LIMK2a is essential

for the regulation of adipogenesis or adipocyte morphology. Although mouse

embryonic fibroblasts represent a commonly used and well-established model of

adipogenic differentiation, they can only serve to investigate the later steps in

adipocyte development. Future studies should consider mesenchymal stem cells as

a model to determine the role of LIMK2a in the organisation of the actin

cytoskeleton prior to commitment to the adipogenic lineage. Combined with recent

evidence for the involvement of several cytoskeleton-associated proteins (Huang

et al., 2011), this will further our understanding of the molecular pathways

regulating cell shape-dependent cell fate decisions.

Considering the central dogma that the structure of a protein determines its

function, the recent progress in LIMK structure determination promises to open

new avenues to understanding the non-overlapping functions of LIMK1 and LIMK2

in different cell types and tissues.

233

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Minerva Access is the Institutional Repository of The University of Melbourne

Author/s:

Mittelstaedt, Kevin Yves Mark Knut

Title:

Structural and functional characterisation of LIM kinases

Date:

2012

Citation:

Mittelstaedt, K. Y. M. K. (2012). Structural and functional characterisation of LIM kinases.

PhD thesis, Department of Medicine, St. Vincent’s Hospital, St. Vincent’s Institute of Medical

Research, The University of Melbourne.

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http://hdl.handle.net/11343/38130

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Structural and functional characterisation of LIM kinases

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Structural and Functional Characterisation of LIM kinases

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