The Role of Focal Adhesion Kinase in Vascular Smooth Muscle Cell
Migration
Lee MangianteMasters Thesis Defense
Cellular and Molecular PathologyJoan M. Taylor, PhD
Outline
Background: Vascular SMCs and FAKResults: FAK mediates SMC migration to PDGF Moving forward: Dia2 and cortactin
Appendix: Knockdown of leupaxin in human aortic SMCs
Background
Vascular SMCs and FAK
Vascular Smooth Muscle Cells (SMCs)
Comprise the medial layer of all arteriesRegulate blood pressure by modifying vessel toneProper SMC migration is critical for vasculogenesis & wound repair But, can also contribute to vascular pathogenesis
http://www.lab.anhb.uwa.edu
SMCs in Atherosclerosis
Injury to the vessel wall triggers the inflammatory responseInflammatory cells and damaged endothelium release SMC chemoattractants including platelet-derived growth factor (PDGF)SMCs invade the intima, occluding the arterial lumen
www.siumed.edu/ ~dking2/crr/CR026b.htm
PDGF-BB homodimer
Isoforms: A, B, C, and DReceptors: PDGFRA, PDGFRBBB is a potent chemoattractant/mitogen for SMCsIn culture, stimulates formation of ring-shaped actin structures called dorsal rufflesImportant for SMC differentiation during developmentGermline deletion is embryonic lethal, with failed recruitment of SMC precursors to the vasculatureStimulates growth and migration of SMCs during vascular injury response
Focal Adhesion Kinase (FAK)
Nonreceptor tyrosine kinase found at focal adhesionsTransduces adhesion signals from the extracellular matrix; can also cooperate in growth factor & contractile agonist signalingActivates myriad pathways important in numerous biological processes
FATFATKinaseKinase
PaxillinPaxillin
GRAFGRAF GRAFGRAF
CASCASCASCAS
Site ISite ISite ISite ISite IISite IISite IISite II
YY397397YY397397
SRCSRCSRCSRC
SH2SH2SH2SH2
SI SIISI SII
PPPP1111
ASAP ASAP ASAP ASAP Pi3KPi3KPi3KPi3K
SH2SH2SH2SH2
Integrin Binding
A Unique Role for FAK in SMC Biology
Germline deletion of FAK is lethal at E 8.5 – 10; embryos exhibit “leaky vasculature”
FRNK, an endogenous dominant negative for FAK, is expressed exclusively in SMCs during
development and injury response
This suggests that FAK activity requires tight control in SMCs and may play a special role in
this cell type
FAK in Migration: Extant Questions
Known:FAK depletion Impaired wound closure, transwell migration to fibronectin, spreading, in fibroblasts, endothelials, keratinocytes…FAK overexpression increased motility/invasiveness
Unknown:
Structural specifics ?Signaling mechanisms?Smooth muscle cells?
PDGF?
The Migration Cycle: Where is FAK Involved?
Leading Edge Protrusion
pola
rizatio
n
Trailing EdgeRetraction
FA Disassembly/assembly
FAK/Rho?
FAK/ERK?
Fak/Rac?
Overall Research Goal:
Determine the role of FAK in aortic SMC migration toward platelet-derived growth factor-BB (PDGF).
Identify the biomechanical events controlled by FAK
Determine the spatiotemporal signaling events by which this structural regulation is
accomplished
Results
FAK mediates SMC migration to PDGF
LacZ Cre
= FAK
= phalloidin (F-actin)
Deletion of fak in VSMCs: the fak flox/flox mouse
ATP-Binding Dom.
loxP Exon 18
loxP
Cre recombinase
No FAK produced
72 Hours Post-Virus
FAK
ERK
Lac Z Cre
FAK is required for 3D migration to PDGF
FAK- SMCs are spread, focal adhesions appear normalFAK depletion blocks three-dimensional migration toward PDGF, but not 10% serumMigration is rescued by overexpression of wild-type FAK, but not FAK Y397F
Stain: vinculin/phalloidin
Transwell migration assay
LacZ Cre
What are the cytoskeletal characteristics of FAK-depleted SMCs treated with PDGF?
Immunofluorescent staining:
Cortactin: localizes to dorsal ruffles, lamellipodia Phalloidin: binds F-actin stress fibers
PDGF-induced dorsal ruffling is FAK-independent
Lac Z (FAK+) Cre (FAK-)
2.5 min 20x
= cortactin
= phalloidinPeripheral ruffles
Dorsal ruffles
PDGF-induced cell polarization is FAK-dependent
LacZ (FAK+) Cre (FAK-)
Dorsal Ruffling in FFSMCs
0
20
40
60
80
100
0 2.5 5 7.5 15
Minutes Post-PDGF
% C
ells W
ith
Dor
sal
Ruffl
es LacZ
Cre
Cell Polarization in FFSMCs
0
10
20
30
40
50
0 2.5 5 7.5 15
Minutes Post-PDGF
% C
ells P
olar
ized
LacZ
Cre
What molecular mechanisms explain the polarization defect of
FAK-depleted SMCs?
Activity of the Rho subfamily GTPases
Ridley, AJ. J Cell Sci. 2001 Aug;114(Pt 15):2713-22.
Rac = PUSH Rho = PULL
Rho GTPase Signaling Pathways
Rho, Rac, and Cdc42
Dynamic cycle of activation/inhibition during cell migration
Rac facilitates membrane protrusion
Rho controls cell contraction and focal adhesion dynamics
Rac-PI3K Signaling is unperturbed by FAK depletion
AKT, WAVE1/2
PI3K
GTP- Rac
Arp2/3
Membrane protrusion Leading edge formation
Pulldown: GTP-Rac1
IB: pAKT
Live cell: GFP-WAVE2
Myosin activation, but not global RhoA activity, is attenuated by FAK depletion
ROCK
GTP- RhoA
pMLC
contractility
MLC phosphatase
Pulldown: GTP-RhoA
IB: pMLC
Dia2 localizes to focal adhesions dependently of FAK
In LacZ-infected SMCs, Dia2 commonly targets to peripheral streaks after PDGF treatment (85% of 26 movies)
This pattern is absent or less dramatic in Cre-infected SMCs (18% of 22 movies)
GFP-Dia2 colocalizes with mCherry-paxillin after PDGF treatment, suggesting that these “streaks” are indeed focal adhesions
Dia2 is enriched at peripheral/dorsal ruffles following PDGF treatment, regardless of FAK content
Dia2 also colocalizes with cortactin in serum-maintained fixed cells
Dia2 localizes to ruffles independently of FAK
Stain: cortactin/GFP-Dia2
Live cells: GFP-Dia2
What is the biological significance of Dia2 at membrane ruffles vs. focal
adhesions?
What is Dia2 doing at each location?What signaling events drive Dia2 to each
location?Why is FAK required for one, but not the
other?
FAKDia2Stable Microtubules? No
• Palazzo, et al. Science (2004): FRNK overexpression abolishes stable MT’s, and can be rescued by constitutively active mDia1
1. PDGF does not alter levels of glu-tubulin (stable MT’s)
2. FAK depletion does not abolish glu-tubulin staining
Chan et al. (1996) Identified cortactin as a formin binding protein by screening a mouse limb expression library with a formin probeThey proposed that the SH3 domain of cortactin bound to the proline-rich portion of the formin probe
Can we detect such an interaction between cortactin and mDia2 in vitro?
Might this interaction regulate the “switch” between ruffle localization and FA localization?
Cortactin: a structurally distinct Arp2/3 activator
A = acidic region; facilitates Arp2/3 binding
P = proline-rich domain
SH3 = Src homology; binds proline-rich motifs
Repeat domain: binds F-actin (20 fold higher than Arp2/3)
W = WASP homology; binds G-actin
C = central region; binds/activates Arp2/3
GB = GTPase binding domain (Cdc42, Rac)
B = basic region
Sufficient to activate Arp2/3
CTN
WASP, WAVE
WASP
CTN, WASP, WAVE
Daly, RJ. 2004
Structure and Regulation of Dia2
GBD = GTPase Binding DomainDID = Diaphanous Inhibitory DomainFH1, FH2 = Formin Homology 1, 2FH3 = Formin Homology 3
DAD = Diaphanous Autoinhibitory Domain
Dia2 and cortactin interact independently of F-actin
I. GST pulldown (SMC lysates)
II. Co-IP (COS-7 lysates)
FH1 = proline rich
FH2 = no prolines; binds G-actin
Moving Forward
Dia2 and Cortactin
Main Questions
What regulates Dia2-cortactin binding?Extracellular cues?Upstream signaling?Post-translational modifications?Conformation of Dia2?
What is the biological function of this interaction?
Actively cooperating in actin polymerization? How and for what purpose?Sequestering Dia2?
Why would Dia2 and cortactin associate?
Formins and Arp2/3 are traditionally seen as two separate actin nucleatorsArp2/3 controls protrusive machinery (lamellipodia); formins control contractile machinery (stress fibers, focal adhesions, contractile ring in yeast)Arp2/3 nucleates branches from existing filaments Formins generate filaments from monomeric actin
Goode et al. Ann Rev Biochem. (2007)
Shifting the actin paradigm:New evidence suggests that DRFs may interact with the WANP complex
WAVE Abi1 Nap1PIR121
Arp2/3
Dia2/WANP interactions
Beli et al. Nat Cell Biol. (2008): Dia2 N-term/C-term bind to the Scar homology domain/proline-rich domain of WAVE2
Yang et al. PLoS Biol. (2007): N-term of Dia2 interacts with Abi1 C-term (no interaction with WAVE)
* Neither report detected an interaction with the FH1 or FH2 domains of Dia2
Is Dia2 passive or active?
Passive: WAVE2 sequesters Dia2 to prevent filipodia formation Active: Dia2 provides “mother filaments” for Arp2/3; bundles branched filaments into filipodia, as shown below
Can Dia2 interact with cortactin in its “active” (open) conformation?
Yang, et al. PLoS Biol. (2007)
“Active” (open) mutants of Dia2
GBD
GBD
ID
DID
DID
FH1
FH1
FH2
FH2
DAD
DAD
AD
ΔGBD
A272D
All kept in the “open” conformation by disrupting the DID-DAD interaction
Cortactin colocalizes intensely with Dia2 A272D
WT
FL
A27
2D
GFP cortactin merge phalloidin
Does A272D associate more strongly with cortactin than WT Dia2?
Could Src regulate Dia2-cortactin binding?
Three putative Src phosphorylation sites within the FH2 domainOverexpression of constitutively active Src induces tyrosine phosphorylation of Flag-Dia2
Does tyrosine phosphorylation of the FH2 domain by Src modify the association of cortactin and Dia2?
Dia2 in PDGF-Stimulated Migration
“Protrusive Dia”
pola
rizatio
n
“Retractile Dia”
FA Disassembly/assembly
Dia2
Dia2
FAKFAK coordinates these two activities to enable fluid forward movement of the SMC
Dia2
Dia2 cortactin
cortactin
Src
P?
Src
P?
How might Dia2 promote contractility?Direct mechanisms:
Localized actin polymerization events can promote SMC contractility independently of MLCDia1 can regulate myosin-mediated contractility by targeting microtubules to focal adhesions
Indirect mechanisms:Potential crosstalk between Dia2 and ROCKIn endothelial cells, cortactin and myosin light chain kinase (MLCK) interact to form a contractile apparatus at the cell periphery. Does this occur in SMCs?
cortactin EC MLCK Merge
Dudek, et al. J. Biol Chem. (2004)
Future Experiments
Further map the interaction sites on Dia2 and cortactinDetermine whether cortactin and Dia2 associate directly or indirectlyDetermine if cortactin binds only to Dia2, or also to Dia1Use in vitro assays to determine if Dia2-cortactin binding changes the actin polymerizing activities of either proteinAssess the impact of FAK depletion/inactivation on Dia2-cortactin bindingElucidate the signaling events that regulate binding
Appendix
Knockdown of leupaxin in human aortic SMCs
Leupaxin: Structure
Member of the paxillin family of LIM proteinsContains four LIM domains (zinc finger motifs; target paxillin to focal adhesions)Contains three LD motifs (bind to c-Src, Lyn, and FAK)
Turner, CE. Nat Cell Biol. 2000
Leupaxin: Putative FunctionsFirst identified in leukocytes (JBC, 1998)Mostly studied in hematopoetic cells (macrophages, B-cells, osteoclasts)Also enriched in prostate cancer cells and vascular SMCs
Binding Partner Cell Type Putative Biological Function
PYK2Macrophages116, osteoclasts127, prostate cancer cells120
Focal adhesion adapter protein
PTP-PESTSpleen128, osteoclasts126, prostate cancer cells120
Regulation of antigen receptor signaling; podosomal remodeling
c-Src Osteoclasts126, protstate cancer cells120 Podosomal complex signaling, osteoclast activation
p95 PKL Osteoclasts127 Podosomal remodeling
FAK Vascular SMCs123, osteoclasts127 Podosomal complex signaling
Lyn B-cell lymphoma121 Regulation of B-cell receptor signaling
SRF Vascular SMCs123 Promotion of SMC differentiation
Leupaxin in SMCs Enriched in arterial and visceral SMCs Binds to FAK via its LD3 motifGFP-leupaxin shuttles in and out of the nucleusGFP-leupaxin binds directly to serum response factor (SRF) and activates SMC gene transcriptionFAK activity modulates leupaxin localization and function
Sundberg-Smith, et al. Circ Res. 2008
How does endogenous leupaxin knockdown impact SMC biology?
Differentiation?Migration?Proliferation?Apoptosis?
Leupaxin Knockdown in Human ASMCs
siRNA designed to 3’ UTR of human leupaxin
No effect on localization/expression of paxillin or Hic-5
Control knockdown
leup
axin
Hic
-5pa
xilli
n
phalloidin
Leupaxin in Migration: 3D vs. 2D
Transwell assay: leupaxin knockdown SMCs cannot migrate three-dimensionally to serum
Wounding assay: SMCs do not require leupaxin to close a wound on uncoated plastic
2D Motility in Sparsely Plated SMCs
In serum media, leupaxin knockdown cells display slower random motility Cell paths are more confined in knockdown cellsDisplacement from origin is reduced approx. 55%
Aberrant PDGF-induced membrane ruffles
Stain: cortactin/phalloidin
Leupaxin knockdown SMCs lack smooth, continuous areas of ruffling
Ruffles appear spiky, disconnected
Tre
atm
ent:
5’
PD
GF
Preliminary Conclusions
Leupaxin is required for 3D migration to serum, but not 2D wound healingLeupaxin knockdown cells show reduced velocity and displacement under sparsely plated conditionsLeupaxin knockdown cells form spiky, ragged membrane ruffles in response to PDGFLeupaxin silencing impairs cell proliferation (*not quantified)
Future Leupaxin Studies
Elucidate the mechanisms by which leupaxin facilitates motility in different contexts (2D? 3D? Serum? PDGF?)Clarify our understanding of leupaxin in SMC proliferation and differentiationExamine leupaxin expression in vivo in the developing mouse (SMC lineages)Create a leupaxin knockout animal modelExplore the role of leupaxin in vascular disease states (mouse, human)
Committee Members
William B. Coleman
Adrienne Cox
Financial Support
Robert H. Wagner Scholarship
Joseph E. Pogue Fellowship
Joan Taylor
Laura DiMichele
Jason Doherty
Lisa Galante
Zeenat Hakim
Rebecca Sayers
Liisa Smith
Chris Mack
Alicia Blaker
Jeremiah Hinson
Kashelle Lockman
Matt Medlin
Dean Staus
Jim Bear
Liang CaiTom Marshall
Microscopy Services Lab
Bob Bagnell
Elena Davis
Vicki Madden
Acknowledgments
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