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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
Organization of the Platelet
Cytoskeleton and the Rho
GTPase Cycle
Platelet Adhesion and Spreading
to VWF Under Shear

Tethering Rolling Adhesion Spreading

Immobilized VWF

FLOW
Platelet Shape Changes after Activation

Quiescent discoid platelet 10 sec: Rounding 30 sec: Filapodia

1-2 min: Lamellapodia Focal Adhesions/ Stress Fibers Spread platelet cytoskeleton

J. G. White 1987
Elements of the Platelet Cytoskeleton

Microtubule
Outer Ring

Cytoskeleton
Actin Filaments

Transitional Zone

JG White 1998
Overview of Platelet Cytoskeletal Reorganization

Platelet Activation Signal


adhesion, receptor activation

Signal Transduction
G-proteins,GEFs, GTPases, ion channels

Filament Reorganization
actin, myosin, filamins
Small GTP-binding Protein
Superfamily
Small GTPases

Ras Rho Rab Arf Ran

Ral 1 Rho A, B, C, D, E, G, H, Rho 6, 7, 8,


Rac 1, 2, 3, TC10, Cdc42
Shape Changes in Platelets Involving the
Rho Family of Small GTPases

Rac

RhoA Cdc42

Ruffling
Actin Stress Lamellipodia Filopodia
Fibers and Focal
Adhesion

Nobes and Hall (1995)


Kozma et al (1995)
Ridley and Hall (1992)
Receptor Signaling of Small GTPases:
THE GTPase CYCLE
Rac and CDC42 Effectors

BISHOP and HALL. Biochem. J. (2000)


RhoA Effectors: Stress Fibers & Focal Adhesions

BISHOP and HALL. Biochem. J. (2000)


Biological Activity of the Guanine Nucleotide
Exchange Factors (GEFs)
Rho
GDP
GEF
PO4
GDP

GTP
GAP
Rho
GTP

GEF--- GTPase Exchange Factor


GAP--- GTPase Activating Protein

Protein Phosphorylation
Guanine Nucleotide Exchange Factors with DH-PH
Domains

Vimentin rod
Dbl Diffuse B-cell
lymphoma
S/T kinase
Duet

SH3
Vav
Calponin Cys rich SH2
homology
Coiled coil
P115-RhoGEF

Spectrin-like DH1 PH1 DH2 PH2 S/T kinase


Trio
GEFs in Platelets

GEF GTPase Receptor


• Vav 1,2,3 Rac1,2 TK

• XPLN RhoA,B ?

• p115Rho-GEF RhoA GPCR

• CalDAG-GEF Rap1 GPCR


Cell Shape Changes by the Rho/Rho Kinase
Pathway (Ca++ Independent Pathway)
Low Thrombin
N

PAR
Pi
RhoGAP

γ Gαβγ
β RhoA
GDP Pi RhoA
GTP
α12,13 GDP
Rho kinase

GTP MLC (myosin light chain)


GTP GTP
Rho MLC Pase
Gα12,13 MLC-P
GDP GEF

Myosin filaments

Cell shape change


(rounding)
RhoA and Rac1 Signaling in Platelets

Gα β β
Gα γ
γ

p115RhoGEF
?

RhoA-GDP RhoA-GTP Rac-GTP Rac-GDP

•Stress Fibers •Lamellapodia (Rac)


•Focal Adhesions •Filapodia (CDC 42)
Method to Identify Rho-Associated Proteins

Rac1-GDP
Rac1-GTP
Std (kDa)

Rac1-E
•Rac-GTP
GSH
Sepahrose GST
•Rac-GDP
Rac •Rac- Empty

Platelet Lysate protein


Add Lysates 190

120
Rho-Associated
Protein
GSH
Sepharose GST 85
Rac
60 GST-Rac

-Wash
-Denature

Rho-Associated •SDS PAGE


Protein
GSH •Mass Spec
Sepharose + GST
Rac
+ •Sequence
Homology
Platelet GEF Proteins Identified
by Mass Spectrometry

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)

•DH-PH Domain Homology


•GTP or Empty Rho Protein Capture
•Novel for Platelets
Guanine Exchange Factor
P-Rex1
Characteristics of PREX1

PREX-1 is 196kDa (1659 amino acids)


PH domain contains PIP3 and Gβ/Gγ binding sites
PDZ and DEP domains unknown function
No demonstrated Inositol 4-phosphatase activity
Rac1 Specific GEF in Neutrophils

Welch et al., 2002


P-Rex1 Specifically Activates Rac

Welch et al., 2002


P-Rex1 Knock out Mice

Welch et al.,Current Biology 2005.


Western Blots of PREX1 in Tissues

kD Total cell lysate


protein
250
196kD
150

100

75 SDS PAGE
separation
50

Transfer to Nylon
Membrane
ne
ain
ets
s t

sti
ele

Br
tel

nte
lat

Pla
P

I
all
C
sh

AR

Sm
Fre

Stain with antibody

Monclonal PREX-1 antibody gift of Marcus Theren


PREX-1 Is Cleaved in Thrombin Activated Platelets

Thrombin (2U/mL)
- - + + Platelet Lysate
250
150
100 Centrifugation

kD
75

50 Supernatent
(Cytosolic Proteins)

Pellet
e

e
l

(Membrane Proteins)
o
ol

an

an
os
os

br

br
yt
yt

em

em
C
C

M
Ra
c

75
kD

150
100
250
(T
17
N)
GD
P
Ra
Western
c-
Ra E
c(
Resting
Q6
1L
Ra )G
c TP
(T
17
N)
GD
P
Ra
c-
Ra E
c(
Q6
1L
)G
Pl TP
at
Thrombin Activated

el
et
Ly
sa Re
te sti
Pl
ate n g
let
Pl Ly
sa Re
Pratel
ec et te st i
lea Ly ng
Ra r sa
c( te Ac
T1 t
7N iva
)G te
DP d
Ra
Ra c-
c( E
Q6
1
Resting

Ra L)
c GT
(T P
Western Blot & Silver Gel
of PREX1 Bound to Rac1

17
N)
GD
P
Ra
Ra c-
c( E
Q6
1L
)G
TP
Silver
Thrombin Activated
Anti-Gγ2 Western Blot with Rac
Associated Platelet Lysates

Resting
Platelet lysates
Thrombin Activated

Incubate with Rac


Consturcts
Gγ 2

wash

TP
E
P
P
P

GD
GT
GD

c
c

G
Ra
Ra

L)
L)

N)
N)

61
61

17
17

Q
Q

(T
(T

Western

c(
c(

c
c

Ra
Ra

Ra
Ra

Gγ2
Immunoprecipitation of Platelet
Lysates with P-REX1 Antibody
Platelet lysates

ed
g
in

at
st

tiv
Re

Ac
Immunoprecipitate
Anti-Rac With P-REX1 antibody

wash

Western

Anti-Gγ2

Rac P-REX1
Model of PREX1 Signaling
Conclusions: PREX1 in Platelets
• P-Rex1 is relatively abundant in platelets.
• P-Rex1 is cleaved and post-translationally
modified (Phophorylated?) in thrombin activated
platelets.
• P-Rex1 interacts with mutant Rac-GDP and
Rac-GTP.
• Gγ2 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.
Guanine Exchange Factors
Cool1(βPIX) and Cool2 (αPIX)
Domain structure of Cool family proteins

Baird et al. Current Biology 2005


Regulation of Cool2 GEF Activity
GEF Cool-1 Participates in EGFR Degradation

Cerione RA et.al., Nature Cell Biology, 2006.


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


Summary of GIT Protein Interactions and Functions

Hoefen R., et al. J Cell Science 2006


GIT – PIX ‘Stacked’ Complexes
GEF Cool-1 In Human Platelets
cytosol membrane

Thrombin - + - +

Anti-Cool-1

Total Rac1
GEF Cool-2 In Human Platelets

Membrane Cytosol IP GIT-1

Thrombin + - + - + -

Anti-Cool-2

Total Rac1
Total Rac1
Ra
Thrombin
c

Anti-Cool-2
1(T
1 7N
Ra )G
-
c1 DP
( Q6
1L
)G
T
-

Ra
c1 P
(T 17
N)
Ra G
+

c1 DP
( Q6
1L
)G
+

T P

IP
GI
-

T-
1
IP
GI
-

T-
2
IP
+

GI
T-
1
IP
GI
+

T-
2
GEF Cool-2 Associations with Rac1 and GIT
Ra
c1

Anti-Cool-1
(T

Total Rac1
17

Thrombin
N)
Ra GD
c1 P

-
(Q
6 1L
)GT
Ra
c P -
1(T
1 7N
Ra )G
c1 DP
(
+

Q6
1 L)
GT
P
+

IP
GI
T-
1
-

IP
GI
T-
2
-

IP
GI
T-
1
+

IP
GI
T-
2
+
GEF Cool-1 Associations with Rac1 and GIT
Silver SDS-PAGE of Cool-1 and Cool-2
CoIP with Human Platelets
Cool-1 Cool-2

250
150

100

75

50

37

Thrombin - + - +
Proposed Cool - GIT interactions in Platelets

GIT ARF6

RacGDP PAK P Gβγ


P Cool
GIT
Cool
RacGTP

CDCGDP

P GIT Cool
CDCGTP

P GIT Cool
Focal
Cool
Adhesion
P GIT Cool
Cool1 and Cool2 Studies

•Co-IP gels for Mass Spectroscopy

•Confocal microscopy

•Do Cools form heterodimers

•What receptors activate Cools


Acknowledgements
Earl Davie
Dominic Chung
Kazuo Fujikawa University of Washington
Brad McMullen
Jeff Harris

Brian Drucker
Lynn Boshkov
Dave Farrell Oregon Health Sciences University
Alex Spencer
Owen McCarty
Larry David