Masters Defense - [PPT Powerpoint] (2024)

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 hom*odimer

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

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

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 hom*ology; binds proline-rich motifs

Repeat domain: binds F-actin (20 fold higher than Arp2/3)

W = WASP hom*ology; 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 hom*ology 1, 2FH3 = Formin hom*ology 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 hom*ology 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