Al's Research Overview

8/14/2019 Al's Research Overview

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P-Rex1 and Cool Guanine Exchange Factors

In Human Platelets

Alex Spencer & Daniel Greenberg

Department of Medicine

Hemophilia Treatment Center

Oregon Health Sciences University


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Organization of the Platelet

Cytoskeleton and the RhoGTPase Cycle


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Platelet Adhesion and Spreading

to VWF Under Shear

Tethering Rolling Adhesion Spreading

Immobilized VWF

FLOW


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Platelet Shape Changes after Activation

Quiescent discoid platelet 10 sec: Rounding 30 sec: Filapodia

Focal Adhesions/ Stress Fibers1-2 min: Lamellapodia Spread platelet cytoskeleton

J. G. White 1987


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Elements of the Platelet Cytoskeleton

Microtubule

Outer Ring

Cytoskeleton

Actin Filaments

Transitional Zone

JG White 1998


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Overview of Platelet Cytoskeletal Reorganization

Platelet Activation Signaladhesion, receptor activation

Signal TransductionG-proteins,GEFs, GTPases, ion channels

FilamentReorganization

actin, myosin, filamins


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Small GTP-binding Protein

Superfamily

Small GTPases

Ras Rho Rab Arf Ran

Rho A, B, C, D, E, G, H, Rho 6, 7, 8,

Rac 1, 2, 3, TC10, Cdc42Ral 1


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Shape Changes in Platelets Involving the

Rho Family of Small GTPases

Cdc42

Ridley and Hall (1992)

Nobes and Hall (1995)

Kozma et al(1995)

Filopodia

Rac

Ruffling

Lamellipodia

RhoA

Actin Stress

Fibers and Focal

Adhesion


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Receptor Signaling of Small GTPases:

THE GTPase CYCLE


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Rac and CDC42 Effectors

BISHOPand HALL. Biochem. J. (2000)


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BISHOPand HALL. Biochem. J. (2000)

RhoA Effectors: Stress Fibers & Focal Adhesions


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Biological Activity of the Guanine Nucleotide

Exchange Factors (GEFs)

RhoGDP

RhoGTP

GEF

GDP

GAP

PO4

Protein Phosphorylation

GEF--- GTPase Exchange FactorGAP--- GTPase Activating Protein

GTP


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Guanine Nucleotide Exchange Factors with DH-PH

Domains

Dbl Diffuse B-cell

lymphoma

Vimentin rod

Duet

S/T kinase

P115-RhoGEFCoiled coil

DH1 PH1 DH2 PH2 S/T kinaseSpectrin-likeTrio

VavSH3

SH2Cys richCalponin

homology


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GEFs in Platelets

Vav 1,2,3 Rac1,2 TK

XPLN RhoA,B ?

p115Rho-GEF RhoA GPCR

CalDAG-GEF Rap1 GPCR

GEF GTPase Receptor


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Cell Shape Changes by the Rho/Rho Kinase

Pathway (Ca++ Independent Pathway)

G

PAR

12,13

N

GDP

Low Thrombin

G12,13GTP

Rho

GEF

GDPGTP

RhoA RhoA

MLC-P

Myosin filaments

Cell shape change

(rounding)

MLC (myosin light chain)

GDP

GTP

Pi

Pi

GTP

RhoGAP

Rho kinase

MLC Pase


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G

RhoA-GDPRhoA-GTP

Stress FibersFocal Adhesions

Rac-GTPRac-GDP

Lamellapodia (Rac)Filapodia (CDC 42)

G p115RhoGEF

RhoA and Rac1 Signaling in Platelets

?


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GSH

Sepahrose GSTRac

GSH

Sepharose GST

Rac

Rho-Associated

Protein

GSH

Sepharose GST

Rac+

Rho-Associated

Protein

Add Lysates

-Wash

-Denature

SDS PAGEMass SpecSequence

Homology

Method to Identify Rho-Associated Proteins

Rac-GTP

Rac-GDPRac- Empty

+

Rac1-E

190

120

85

60

Std(kDa)

PlateletLysateprotein

GST-Rac

Rac1-GDP

Rac1-GTP


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Platelet GEF Proteins Identified

by Mass Spectrometry

DH-PH Domain HomologyGTP or Empty Rho Protein CaptureNovel for Platelets

RacE RacGDP RacGTP Protein (Accession number)

77 10 82 IQGAP1 (IQGAP1_Human)

24 3 1 Cool-2 (Alpha PIX)

0 0 23 Vav (VAV_Human)

10 0 0 Cool-1 (Beta PIX)

4 5 8 GEF -H1 (ARHG2_Human)

0 0 15 PREX-1 (PREX1_Human)

0 0 5 Trio (TRIO_Human)

0 0 3 Duet (DUET_Human)


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Guanine Exchange Factor

P-Rex1


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Welch et al., 2002

PREX-1 is 196kDa (1659 amino acids)PH domain contains PIP3 and G/G binding sitesPDZ and DEP domains unknown functionNo demonstrated Inositol 4-phosphatase activityRac1 Specific GEF in Neutrophils

Characteristics of PREX1


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P-Rex1 Specifically Activates Rac

Welch et al., 2002


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P-Rex1 Knock out Mice

Welch et al.,Current Biology 2005.


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Fresh

Pla

telet

s

ARC

Pla

telet

s

Brain

Small

Inte

stin

e

250

150

100

75

50

kD

Western Blots of PREX1 in Tissues

196kD

Monclonal PREX-1 antibody gift of Marcus Theren

Total cell lysate

protein

SDS PAGE

separation

Transfer to Nylon

Membrane

Stain with antibody


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- + +-

Cytosol

Mem

bran

e

Cytosol

Mem

bran

e

250

150

100

75

50

kD

PREX-1 Is Cleaved in Thrombin Activated Platelets

Thrombin(2U/mL) Platelet Lysate

Centrifugation

Supernatent

(Cytosolic Proteins)

Pellet

(Membrane Proteins)


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Western Blot & Silver Gel

of PREX1 Bound to Rac1

Western Silver

Rac

(T17

N)G

DP

Rac

-E

Rac(Q

61L)GTP

Rac

(T17

N)G

DP

Rac

-E

Rac(Q

61L)GTP

Platelet

Lys

ate

Resting Thrombin Activated

Preclea

r

250

150

10075


kD

Pla

tele

tLys

ate

Rac

(T17N)G

DP

Rac

-E

Rac

-E

Rac(Q

61L)GTP

Rac

(T17

N)G

DP

Rac(Q

61L)GTP

Pla

tele

tLys

ate

Resting

Activ

ated

Resting


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Anti-G2 Western Blot with RacAssociated Platelet Lysates


G2

Rac

(T17N)G

DP

Rac(Q

61L)GTP

Rac

E

Rac

(T17N)G

DP

Rac(Q

61L)GTP

Rac

E

Platelet lysates

Incubate with Rac

Consturcts

wash

Western

G2


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Anti-G2

Anti-Rac

Resting

Activate

d

Immunoprecipitation of PlateletLysates with P-REX1 Antibody

Platelet lysates

Immunoprecipitate

With P-REX1 antibody

wash

Western

Rac P-REX1


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Model of PREX1 Signaling


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Conclusions: PREX1 in Platelets

P-Rex1 is relatively abundant in platelets.

P-Rex1 is cleaved and post-translationallymodified (Phophorylated?) in thrombin activatedplatelets.

P-Rex1 interacts with mutant Rac-GDP andRac-GTP.

G2 and Rac1 associate with P-Rex1 after

thrombin activation.

Future Directions Analysis of P-Rex1 deficient mice.

Receptor activation of P-Rex1 activity.

P-Rex1 in Myeloproliferative Disorders.


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Guanine Exchange FactorsCool1(PIX) and Cool2 (PIX)


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Baird et al. Current Biology 2005

Domain structure of Cool family proteins


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Regulation of Cool2 GEF Activity


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GEF Cool-1 Participates in EGFR Degradation

Cerione RA et.al., Nature Cell Biology, 2006.
http://jcs.biologists.org.liboff.ohsu.edu/content/vol119/issue8/images/large/JCS02925F2.jpeg


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G-Protein Coupled Receptor Kinase Interactor (GIT)

Cool Associated Tyrosine Phosphorylated Protein (CAT)

ADP Ribosylation Factor GTPase Activating Protein (ARF-GAP)

Hoefen R., et al. J Cell Science 2006
http://jcs.biologists.org.liboff.ohsu.edu/content/vol119/issue8/images/large/JCS02925F2.jpeghttp://jcs.biologists.org.liboff.ohsu.edu/content/vol119/issue8/images/large/JCS02925F2.jpeg


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Hoefen R., et al. J Cell Science 2006

Summary of GIT Protein Interactions and Functions


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GIT PIX Stacked Complexes


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Anti-Cool-1

Thrombin - + - +

membranecytosol

GEF Cool-1 In Human Platelets

Total Rac1


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Thrombin + - + - + -

CytosolMembrane IP GIT-1

GEF Cool-2 In Human Platelets

Anti-Cool-2

Total Rac1


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IPGIT

-1

Rac1(Q

61L)GTP

Rac1(T1

7N)G

DP

Rac1(T1

7N)G

DP

Rac1(Q

61L)GTP

Thrombin - - + + - - + +

Anti-Cool-2

GEF Cool-2 Associations with Rac1 and GIT

IPGIT

-2

IPGIT

-1

IPGIT

-2

Total Rac1


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Thrombin - - + + - - + +

Rac1(T1

7N)G

DP

Rac1(Q

61L)GTP

Rac1(T1

7N)G

DP

Rac1(Q

61L)GTP

IPGIT

-1

IPGIT

-2

IPGIT

-1

IPGIT

-2

GEF Cool-1 Associations with Rac1 and GIT

Anti-Cool-1

Total Rac1

Silver SDS PAGE of Cool 1 and Cool 2


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Silver SDS-PAGE of Cool-1 and Cool-2

CoIP with Human Platelets

Thrombin - + - +

250

150

100

75

50

37

Cool-1 Cool-2


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GIT ARF6

CoolGIT

Cool

GIT Cool

GIT

GIT

CoolCool

Cool

Focal

Adhesion

P

P

GRacGDP

RacGTP

PAK P

CDCGDP

CDCGTP

Proposed Cool - GIT interactions in Platelets

P

P


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Cool1 and Cool2 Studies

Co-IP gels for Mass Spectroscopy

Confocal microscopy

Do Cools form heterodimers

What receptors activate Cools


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Acknowledgements

Earl Davie

Dominic Chung

Kazuo Fujikawa

Brad McMullen

Jeff Harris

Brian Drucker

Lynn Boshkov

Dave Farrell

Alex Spencer

Owen McCarty

Larry David

University of Washington

Oregon Health Sciences University

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Al's Research Overview