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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
Resting Thrombin Activated
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
Resting Thrombin Activated
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.jpeg8/14/2019 Al's Research Overview
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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.jpeg8/14/2019 Al's Research Overview
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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