Signal Transduction and Apoptose · Transfer of one phosphate group to an organic acceptor (e.g....

Martin Luther University Halle-Wittenberg

Lecture 6:Signal Transduction through

Integrins and Receptor TyrosineKinases

Prof. Thomas Groth

Biomedical Materials Group



Content

• Understanding mechanisms of signal transduction from fixed effectors (ECM molecules) into the cell.

• Understanding also mechanism by which soluble signaling molecules (growth factors) activate cells

• Learning that integrin signaling and growth factor receptors talk with each other

• Effect of material chemistry on integrin-mediated signaling and subsequent behavior of cells

• Role of integrins, cadherins, growth factor receptors and signal transduction in tumor development & progression


Effectors of Signal Transduction

• Adhesive contact with extracellular matrix molecules (fixed, insoluble effector) – e.g. collagen, fibronectin, laminin

• Soluble effector –hormons, cytokines, & growth factors

• Adhesive contact with neighbouring cells – e.g. Cadherins

• Mechanical stress – e.g.. shear stress in blood vessel, mechanical load of bone, etc.


Transduction of Signals– Immobilised Effector

target cellsignal emitting

cell

Signal transfer mediated by trans-membrane proteins (e.g.

cadherins in epithelia)

target cell

extracellular matrix ECM

Signal transfer from ECM molecules to specific cell receptors (e.g. integrins)


Transduction of Signals – Soluble Effectorendocrine gland

blood vessel

target cells

endocrine signaling (e.g.

thyroid hormone)

paracrine signaling (e.g. growth factors)

secreting celltarget cell

autocrine signaling e.g. growth factors


Intracellular signaling from integrinsand growth factor receptors –a detailed view

Note:- Focal adhesion (FA) turnover- Filipodia- Lamellipodia- Stressfibre formation- Cell cycle progressionmitosis(FERM – spezielle Bindungsdomäne)


Phosphorylation Reactions

ATP or GTP (Guanosin triphospate) as phosphate donors

Transfer of one phosphate group to an organic acceptor (e.g. alcohol)

Phosphorylation of aminoacids with hydroxyl groups like serin, tyrosine, threonine

UC Davies ChemWiki


Function of Phosphorylation in Signal Transduction

thermofisher.com


ECM Synthesis

Integrin Functions

Motility Growth Differentiation

Apoptosis

Nucleus

Cytosol

Matrix adhesion


Simplified Model of Focal Adhesion Plaque

Signalling:

FAK – Focal Adhesion Kinase

ILK – Integrin-linked Kinase

Src – Sarcoma-derived Kinase

Linker to cytoskeleton:

α-Act- actinin

Fil – Filamentin

Tal – Talin

Ten – Tensin,,

Pax – Paxillin, Vin - Vinculin

Plasma

membrana a a ab b b b

Extracellular Matrix

Aktin

Ten

a b

Fil Tal

a-Act Pax

ILKFAK

Pax

Vin

Vin

Src


Integrin-Ligand-Binding – Opening of Intracellular Binding Sites by Separation of a & b Subunits

• Integrin transmit signals from outside into the cell Change of conformation upon ligand binding

• exposure of cryptic binding sites for cytoskeletal and signaling proteins

Gupta et al. Blood, 2007 15;109(8):3513-20.


Integrin conformation-function relationships: a model.

Askari J A et al. J Cell Sci 2009;122:165-170

Ligation of Integrins Signal Transduction & Mechanical Tension

• Ligation of integrins to ECM induces signal transduction, cytoskeletal arrangement and mechanical tension

• Mechanical tension affects signal transduction & assembly and dissassembly of cytoskeleton


Signal Transduction via Integrins -Phosphorylation of Focal Adhesion Kinase (FAK)

a b

FN

FAK

Paxillin

Talin

Vinculin

Actin

Src RTKP

Contact integrin -fibronectin

Binding of N-terminus of FAK tointegrin b subunit

conformational change of FAK followed by autophosphorylation

active conformation of FAK withformation of binding site SH2 domain for Src kinase.

Src – Sarcoma

RTK – receptor tyrosine kinase

SH – sarcoma kinase homologous bindingdomains


Integrin Clustering is Prerequiste for Full Catalytic Activity of Kinases

ab

FN

FAKP

Paxillin

Talin

Vinculin

ab

FN

FAKP

Paxillin

Talin

Vinculin

P P

PP

srcsrc

PP

P

P

• Oligomerisation of FAK through clustering of integrins.

• Trans-phosphorylation of FAK.

• Involvment of Src-receptor tyrosine kinases phosphorylation of Tyr-576 and Tyr-577 residues and full catalytic activity of FAK.

• Phosphorylation of Tyr-925 – Formation of binding site for adaptor proteins (growth factor receptor-bound protein 2 (Grb2) und SOS (son of sevenless protein)).

• Paxillin and vinculin become phosphorylated

by Src as well.


Colocalisation of FAK with Integrins in Focal Adhesion Plaques

a b

FN

FAK

FAK Alpha 5

Cell membrane


Tyrosine Phosphorylation of FAK in Focal Adhesion Plaques

Colocalisation of Phosphotyrosine and FAK (orange)

a b

FN

FAK

P

Cell membrane


Hydrophobic Biomaterials Reduce Clustering of Integrins and Signal Transduction

Hydrophobic material

Hydrophilic material

Tyrosin phosphorylationav Integrin

Groth, Altankov (1996) Biomaterials


Tyrosine Phosphorylation Decreased on Hydrophobic Substrata

0

1

2

3

4

5

6

7

8

9

glass APS ODS SINo

rma

lize

d p

ho

sp

ho

tyro

sin

e

ac

tivit

y

Water contact angles

20° 60° 90° 110°Groth, Altankov (1996), Biomaterials


0

20

40

60

80

100

0

0,5

1

1,5

2

2,5

3

CH3 PEG NH2

C11

NH2 C3 COOH OH

Growth index

Observation: Growth and Viability of Fibroblasts Depends on Type of Surfaces

Viability(%)

Cells are seeded and cultured under same conditions -only head groups on substrata are different


Receptor Tyrosine Kinases (RTK) and Signal Transduction

• Ligand (e.g. epidermal growth factor - EGF) binds on RTK domain outside the cell.

• RTKs – transmembrane protein

• RTKs possess intracellular tyrosine kinase domain.

• Cytosolic part of RTK has several tyrosine residues.


Function of Receptor Tyrosin Kinases

• Receptor becomes activated by ligand binding.

• Followed by dimerisation of receptors and trans-phosphorylation.


Growth Factor-Dependent Signal Transduction –Role of Ras I

• EGF – Epidermal growth factor

• Ras – rat sarcoma protein

• Grb2 - growth factor receptor-bound protein 2

• SOS - son of sevenless protein

• SH 2/3 – Src homologous binding domains


Growth Factor-Dependent Signal Transduction –Role of Ras II

• Ras (rat sarcoma) – GTPase switch protein

• Signal transducer for many receptor tyrosine kinases


MAP Kinase Pathway I

• MAP – mitogen-activated proteins

• Active Ras binding + activating Raf

(Rapidly growing fibrosarcoma)


MAP Kinase Pathway II

• MEK – mitogen activated protein kinase

• MAPK – mitogen activated protein kinase


Growth Factor-Dependent Signal Transduction - Overview

P

P

P

P

EGF

Cytosol

Adapter

proteins

Ligand binding induces Dimerisation and trans phosphorylation of receptor tyrosine kinases (RTKs).

Ras

Membrane

MAPK

active

Cascade of kinases:

Raf-MEK-MAPK

Mitogen-Activated-Protein-

Kinase (MAPK) inactiv

Nucleus

P

P

Grb2SOS

MAPK


Cooperation Between Growth Factor and Integrin Dependent Signal Transduction

P

P

P

P

EGF

Cytosol

Adapter-

protein

RasMembrane

MAPK

activ

MAPK

inactiv

Nucleus

P

P

b

FN

aCaveolin

Integrins connected by b subunit & p-FAK via same adaptor proteins like RTKs!


Cooperation Between Growth Factor and Integrin-Dependent Signal Pathways: Caveolin and a-Subunit of Integrins

P

P

P

P

EGF

Cytosol

Adapter-

protein

Ras

Membrane

MAPK

aktiv MAPK

inaktiv

Nucleus

P

P

b

FN

aCaveolin

Fyn/

ShcGrb2/

SOS


Induction of Gene Transcription

• TCF – ternary complex factor

• SRF – serum response factor

• SRE – serum response element of DNA

• RSK - ribosomal s6 kinase c-fos gene – (like c-jun) encoding proteins for progress through cell cycle Mitosis


Survey on Signal Transduction Pathways induced by RTKs


Regulation of RTK Activity

Inhibition of kinase activity by phosphorylation of domains near membrane by protein kinase C (PKC), or dephosphorylation of tyrosine resdiues by protein tyrosinephosphatases (PTP), by endocytosis or by ubiquitinylation and degradation in proteasome


Contact Inhibition (Blocking Mitosis of Cells) by Ligation of Cadherins Related to RTKs

VE–cadherin plays a pivotal role in contact inhibition of cell proliferation

Vascular endothelium (VE)–cadherin (, through its interaction with Dep-1 or other tyrosine phosphatases inhibits vascular endothelial growth factor (VEGF) proliferation signal via mitogen-activated protein kinases (MAPK) activation in three ways:

(i) by reducing VEGFR2 phosphorylation,

(ii) (ii) by inhibiting VEGFR2-associated Shcphosphorylation and

(iii) (iii) by decreasing VEGFR2 internalization and signaling from endosomal compartments.

VE–cadherin complex interaction with activated VEGFR2 also enhances VEGF survival signaling by PI3kinase/Akt pathway.

Biochimica et Biophysica Acta (BBA) – Biomembranes Volume 1778, Issue 3, March 2008, Pages 794–809Apical Junctional Complexes Part I

http://www.sciencedirect.com/science/journal/00052736

http://www.sciencedirect.com/science/journal/00052736/1778/3

http://www.sciencedirect.com/science/journal/00052736

http://www.sciencedirect.com/science/journal/00052736/1778/3


Contact Inhibition by Ligation of CadherinsRelated to RTKs

Okada T et al. J Cell Biol 2005;171:361-371© 2005 Rockefeller University Press

Cadherin-initiated adhesion prevents activation of Pak in contact-inhibited cells and thereby causes accumulation of dephosphorylated Merlin. This closed form of Merlin suppresses integrin-mediated recruitment of Rac, and hence mitogenic signaling. Upon release from contact inhibition, Pak phosphorylates and inactivates Merlin, allowing recruitment of Rac to the membrane and mitogenic signaling.


Role of Receptors and Signal Transduction in Tumor Development and Progression


The Metastatic Cascade

Changes in adhesion duringmetastatic journey

Loss of E-Cadherin mediatedcell-cell adhesions

Migratory phenotype penetratesbasal lamina & invadesinterstitial extracellular matrix

Tumor angiogenesis with entryinto blood stream

Aggregation of tumor cells withplatelets and leukocytes

Occlusion of capillaries in targettissue enables extravasation ofcells


Focal adhesion kinase (FAK) and Src-family kinases (SFKs) integrate pro-migratory signals from integrins and RTKs (receptor tyrosine kinases)

SMAD/AP-1 –transcription factors involved in progression of cell cycle (c-Jun, c-fos genes)

Integrin-RTK-Signaling Induces Cell Migration and Invasion


Integrin-RTKs-Signaling Disrupts Cell-Cell-Adhesion

• Signaling to FAK through integrins activates ILK & SFKs like v-Src

• v-Src phosphorylation & internalisation of E-cadherin

• Signaling through ILK and Src supression of E-Cadherin gen expression

• Activation of Hakai that ubiquitinylates E-Cadherin endocytosis

Decrease of expression & increased endocytosis of E-Cadherin

Loss of cell-cell adhesions

Snail & Slug – Transkription factors blocking E-Cadherin expression


Integrins and Matrix Remodelling

• avb3 integrin recruits metallomatrixprotease – 2 (MMP-2) to cell surface local degradation of extracellular matrix during invasion of cancer cells

• Integrins like avb3 associate with urokinase plasminogen activator (uPA) receptor (uPAR)

• Plasminogen converted to plasmin activates other MMPs precursors increased degradation of ECM


Integrins Promote Oncogenic Signals of Activated RTKs

• Constitutively activated RTKs (mutation or overexpression) induce phosphorylation of b4 subunit of a6b4 integrin (hemisdesmosomes) docking sites for SHCs und PI3K kinases

• Cytoplasmatic domain of b4 signaling adaptor amplifying mitogenic, survival and motogenic signals

• SHC – adaptor protein, PI3K –Phosphatidylinositol-3- kinase


Receptor Cross-Talk in Metastatic Dissemination

• “Resting” (healthy) cell integrins are engaged, cadherin-mediated adhesion is intact and proteinase (MMPs) expression is low.

• “Activated” (cancer - for example, ligand or mutational activation of EGFR signaling, or other mechanisms to stimulate cadherin or integrin-mediated signal transduction Disruption of cadherin-based cell adhesion and/or integrin clustering initiate signaling cascades increased proteinase production and activation Secreted and membrane-associated proteinases disruption of cadherin function through cleavage protein domains and generation of the soluble E-cadherin ectodomain shed E-cadherin ectodomain potentiate junction dissolution through protein-protein interactions with endogenous E-cadherin.

Copyright 2009 Stack Scientific


TK

EGFR Function in Normal Cells

TKP P

Cell Proliferation Antiapoptosis

Angiogenesis

Gene Transcription

Cell Cycle Progression

+

© Dr.M.Jayanthi

Growth factor


Growth Factor-Dependent Signal Transduction – Role of Ras II

• Ras (rat sarcoma) – GTPase switch protein

• Signal transducer for many receptor tyrosine kinases


Constitutively Active Ras/Raf Mutations in Cancer

Nat Rev Cancer 2007;7:295


RTK

Gene transcription

Cell Cycle Progression

Cell Proliferation Metastasis

Anti Apoptosis

Cancer

ATP

Aberant Mutation of EGF Receptor Constitutively Active


Difference between EGFR Variant from Wild Type

EGFR - Variant III EGFR Wild type

No extracellular domain Present

Ligand cannot bind Can bind

RTK constitutively active RTK activated by ligand binding

Cannot dimerize Can dimerize

Not found in normal cells Found in normal cells

More propensity for cancer Up regulation leads to cancer


Normal Cell Cancer Cell

Up Regulation

Mutation

Consequence of Overexpression of EGFR Receptors (Mutants & Wild Type)


Expression of Aberant EGF Receptors in Different Tumors

Tumor EGFR Expression Rate

Breast 14 % - 91 %

Colon 25 % - 77 %

Lung Cancer (Non small cell) 40 % - 80 %

Head & Neck 80 % - 95 %

Ovarian 35 % - 70 %

Pancreatic 30 % - 50 %


Strategies to Inhibit EGFR Signaling

RTK RTK RTK RTK

- - - -

EGFR tyrosine

kinase inhibitorsAnti-EGFR mAbs

Anti-ligand mAbs

BispecificAbs

Imm

un

e effector

cell

ATP


Biopharmaceutical for Treatment of EGF Receptor Malignancies

Biopharmaceutical Effect

Gefitinib Highly selective, potent & reversible EGFR Tyrosine Kinase inhibitor

Erlotinib (see Gefitinib)

Cetuximab Monoclonal Anti-EGF receptorantibody

H 447 Bispecific anti EGF receptro antibodylinked to anti CD 64

MDX 210 (see H 447)

CD 64 – Anti-Fc receptor present on monocytes & macrophages


Take Home Messages

• Signal transduction processes crucial for normal life of cells controlling tissue structure, motility, growth & differentiation

• Cross-talk between receptors for fixed and soluble signals in a cooperative manner

• Cancer development linked to disturbance in signal transduction processes by mutations or overexpression of signaling molecules, loss of inter-cellular adhesions and uncontrolled growth, proteolytic activity and migratory phenotype

• Biopharmaceuticals try to block RTKs activity by inhibiting binding of extra and intracellular ligands


Literature

• Molecular Cell Biology 4th Edition; Lodish, Berk, Zipursky, Matsudaira, Baltimore, Darnell (2002) Freeman & Company

• Giancotti & Ruoslahti Science 285, 1999

• Danen & Yamada Journal of Cellular Physiology 189, 2001

• Hood & Cheresh Nature Reviews Cancer 2, 2002

• Guo & Giancotti, Nature Reviews Molecular Cell Biology 5, 2004

Signal Transduction and Apoptose · Transfer of one phosphate group to an organic acceptor (e.g....

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