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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Département d'Hématologie and
Département de Biologie Cellulaire, Institut Cochin, Institut
National de la Santé et de la Recherche Médicale (INSERM),
Centre National de la Recherche Scientifique (CNRS), Université
René Descartes, Laboratoire Central d'Hématologie,
Hôpital Cochin, AP-HP and E9907 INSERM, Faculté Xavier
Bichat, Paris, France.
Platelet activation by thrombin or thrombin receptor-activating
peptide (TRAP) results in extensive actin reorganization that leads to
filopodia emission and lamellae spreading concomitantly with activation
of the Rho family small G proteins, Cdc42 and Rac1. Evidence has been
provided that direct binding of Cdc42-guanosine triphosphate
(GTP) and Rac1-GTP to the N-terminal regulatory domain of the
p21-activated kinase (PAK) stimulates PAK activation and actin
reorganization. In the present study, we have investigated the
relationship between shape change and PAK activation. We show that
thrombin, TRAP, or monoclonal antibody (MoAb) anti-Fc The actin cytoskeleton plays an important role in
defining cell shape and morphology. Actin-rich filopodia and
lamellipodia protrusions are thought to depend on the activation of
small guanosine triphosphatases (GTPases) of the Rho subfamily,
Cdc42 and Rac1, respectively.1,2 In platelets, stimulation
by thrombin induces filopodia extension and lamellae spreading
supported by massive actin polymerization. Subsequently to early shape
change, platelets reorganize adhesion points mainly focused on integrin
The p21-activated kinases (PAKs) are serine/threonine kinases that
have been proposed as downstream effectors of those
GTPases.5,6 At least 4 isoforms have been reported in
mammals. PAKs exhibit a N-terminal regulatory domain with a
GTPase-binding cassette, a proline-rich domain that binds
SH3-containing proteins and a C-terminal kinase domain. The regulatory
mechanisms of PAK activation have been extensively studied and result
both from binding of Cdc42-GTP and Rac-GTP and autophosphorylation of
serine/threonine residues in the regulatory domain that leads to
opening of the molecule, transphosphorylation of threonine 423 in PAK1
or threonine 402 in PAK2, and substrate access to the kinase
domain.7-9 Evidence has been provided that integrin
interaction with extracellular matrix promotes the activation of
Rac1/Cdc42 and PAK in the NIH 3T3 cell line,10 and it has
been hypothesized that PAK also plays an important role in regulating
actin stress fiber disassembly and, thus, the turnover of focal
complexes. These effects are in part mediated through PAK
phosphorylation and inhibition of the myosin light chain
kinase11 and also through an upstream regulation of the
focal adhesion adapter protein paxillin.12 In platelets
aggregated in response to thrombin, PAK2 is rapidly activated.13 This finding suggests that PAK activation may
play a primary role in extensive cytoskeleton reorganization in platelets.
The aim of the present work was to investigate the relationship between
PAK activation and early platelet shape change prior to aggregation.
Filopodia formation and lamellae spreading have been obtained from
platelets stimulated through G-coupled protease-activated receptor 1 (PAR-1) by thrombin or thrombin receptor-activating peptide (TRAP) and
through the low-affinity receptor for immunoglobulin G (IgG)-bearing
immunoreceptor tyrosine-based activation motif (ITAM)
Fc Agonists and antibodies
Platelet isolation and stimulation
Flow cytometry For quantification of actin polymerization, 50 µL of resting or stimulated platelets were fixed with 1.8% paraformaldehyde (PFA), washed in phosphate-buffered saline (PBS) pH 7.4, and then incubated with 10 µM FITC-phalloidin for 30 minutes at room temperature in 0.02% Triton X-100 buffer. After 2 washes, samples were resuspended in 0.5 mL PBS pH 7.4 and analyzed by using an Epics Profile flow cytometer (Beckman Coulter, Miami, FL). Fluorescence signals were set in logarithmic gain. Fluorescence emission was monitored by using a 550-nm (FL1) band pass filter. Results were expressed as relative fluorescence emission intensity (RFI) of stimulated to resting platelets.Confocal microscopy After stimulation, with or without pretreatment with 1µg/mL LT or toxin B, platelets were fixed with 1.8% PFA for 5 minutes at room temperature, washed twice in PBS pH 7.40, and settled on glass slides. Platelets were labeled with 10 µM FITC-phalloidin for 30 minutes in 0.02% Triton X-100 containing PBS, washed twice in PBS, and mounted in Mowiol. Immunolocalization of Rac1, PAK, and cortactin was performed on PFA-fixed platelets settled on glass slides. Platelets were permeabilized in 0.03% saponin-containing PBS (pH 7.4) for 15 minutes at room temperature and then incubated with specific primary antibodies (1-10 µg/mL) for 30 minutes at 4°C. After 2 washes in PBS (pH 7.4), Rac1 and cortactin labeling was revealed by incubation with a goat antimouse-FITC secondary antibody, and PAK labeling was detected by incubation with a goat antirabbit-rhodamine secondary antibody (DAKO, Copenhagen, Denmark) for 30 minutes at room temperature. Purified irrelevant mouse or rabbit IgG (Sigma-Aldrich) was used as negative controls. Fluorescent images (magnification × 100) were taken with a confocal krypton/argon laser scanning microscope (MRC-100, Bio-Rad, Hemel, Hempstead, UK) attached to a diaphot 300 microscope (Nikon, Tokyo, Japan).Production of GST-fused recombinant proteins and purification of recombinant human cortactin The GTPase-binding domain (PBD) of human PAK1 (amino acids 67-150), PAK1 wild type (full length) in fusion with GST cloned into the bacterial expression vector pGEX-4T3 and PAK1 T423E (active mutant) in pCMV6m was a gift from Dr G. Bokoch (Scripps Institute, La Jolla, CA). PAK1 T423E cDNA was inserted into pGEX-4T3. GST-cortactin cloned into PXZ-122 was a gift from Dr X. Zhan (American Red Cross, Rockville, MD). GST-fused proteins were coupled to glutathione-Sepharose beads and stored at 80°C as a 50% suspension in 25 mM Tris
(tris(hydroxymethyl)aminomethane)-HCl, pH 7.4, 0.2 mM DTT
(dichlorodiphenyltrichloroethane), 1 mM MgCl2, and 5%
glycerol. Recombinant human (rHu) cortactin was purified on a
glutathione-Sepharose column and cleaved from GST by thrombin.
Affinity precipitation using GST-fused proteins Precipitation of activated Rac1 or Cdc42 was performed according to Benard et al.21 Platelets were lysed in 25 mM Tris pH 7.4, 5 mM MgCl2, 100 mM NaCl, 1% Nonidet P-40 (NP-40), 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitors. Cytoskeletal and NP-40-soluble fractions were separated by centrifugation at 10 000g for 20 minutes. PBD-GST was used to trap Rac1-GTP or Cdc42-GTP in 100 µL NP-40-soluble fraction. Incubation was performed after addition of 200 µL binding buffer (25 mM Tris-HCl, pH 7.4, 1 mM DTT, 30 mM MgCl2, 40 mM NaCl, 0.5% Nonidet P-40) for 1 hour at 4°C. The bead pellet was then washed 3 times in binding buffer, twice with the same buffer without Nonidet P-40, and finally resuspended in 20 µL Laemmli sample buffer. Proteins were separated by 12% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membrane, and blotted by using either monoclonal anti-Rac1 antibody or polyclonal anti-Cdc42 antibody. In some experiments, cytoskeletal and NP-40-soluble fractions were analyzed for the expression of Cdc42 by direct immunoblotting. As controls, lysates were incubated with 100 µM either nonhydrolysable GTP S or guanosine diphosphate (GDP) before precipitation on PBD-GST beads, and both bead pellets and
supernatants were analyzed. Immunoblots were revealed with the enhanced
chemiluminescence (ECL) kit from Amersham Pharmacia Biotech
(Little Chalfont, United Kingdom).
GST-PAK1WT and GST-PAK1T423E or GST-cortactin
were used to pull down cortactin or PAK, respectively, from platelets
lysed in solubilization buffer (50 mM HEPES, pH 7.4, 150 mM
NaCl, 0.5% Triton X-100, 1 mM sodium fluoride, 10 mM
Immunoprecipitation and immunoblot After stimulation, platelets were lysed in 3× solubilization buffer for 20 minutes on ice. Lysates were clarified by centrifugation at 4°C for 10 minutes at 10 000g and precleared by incubation for 1 hour at 4°C with 20 µL protein G-Sepharose 4B packed beads. In some experiments, lysates were sonicated and incubated for 45 minutes at 37°C with DNase I (Roche Molecular Biochemicals, Mannheim, Germany). Immunoprecipitations were performed for 2 hours in ice with 5 µg/mL polyclonal anti-PAK2 or monoclonal anticortactin antibodies. Immune complexes were collected on 25 µL protein G-Sepharose 4B packed beads during 1 hour at 4°C under agitation. Beads were washed 3 times in 1× solubilization buffer and twice in the same buffer containing 0.1% Triton X-100. Proteins were solubilized in Laemmli buffer, separated by SDS-PAGE, and transferred to nitrocellulose membranes for specific immunoblotting.PAK in vitro kinase assay PAK was immunoprecipitated as described above. Dried beads were recovered in 1 × kinase buffer (50 mM HEPES, pH 7.4, 10 mM MgCl2, 10 mM MnCl2, 0.4 mM DTT). Purified myelin basic protein (MBP; 1 µg) or 5 µg purified rHu cortactin was added, and the reaction was started by the addition of a mixture of 20 µM MgCl2-adenosine triphosphate (ATP) and 0.185 MBq (5 µCi)[32P]-ATP (specific
activity, 111 TBq/mmol) in 40 µL 1× kinase buffer for 20 minutes at 30°C. The reaction was stopped by the addition of
10 µL 5 × Laemmli sample buffer. Samples were boiled for 5 minutes
and analyzed on 12% SDS-PAGE. Proteins were transferred to
nitrocellulose membrane before exposure for 2 hours at 80°C onto
Kodak film. Signals on autoradiographies were quantified by using the
Visio-Mic system (Genomic, Collonges-sous-Saleve, France). After
decrease of radioactivity, PAK was quantified by immunoblot with
anti-PAK1/2 antibody.
Statistical analysis Results for flow cytometry experiments were expressed as mean ± SD and compared by using the Student t test.
Time course of actin polymerization and reorganization in activated platelets Phalloidin binding to polymerized actin during platelet shape change stimulated by optimal concentrations of various activators was quantified by flow cytometry analysis. Platelets were activated through PAR-1 receptor by thrombin (THR) or TRAP, through GpVI by CVX or ITAM-containing receptor FcRIIA by monoclonal IV.3 antibody followed
by stimulation of FcRIIA clustering with antimouse IgG
F(ab')2. In the presence of THR (0.5 UI/mL), TRAP (10 µM), or IV.3 (2 µg/mL), the relative fluorescence emission
intensity (RFI) was already maximal after 0.5 minutes of
stimulation, although it reached its maximum after 1 minute of
stimulation with CVX (250 pmol/L). THR, TRAP, IV.3, and CVX induced a
1.8-, 2.2-, 2.0-, and 1.7-fold increase of the content of polymerized
actin after 1 minute of stimulation, respectively (Figure
1A). Spatial organization of polymerized
actin was visualized by confocal microscopy (Figure 1B). After
stimulation for 1 minute with thrombin or IV.3, platelets emitted few
thin and short filopodia, and actin was rearranged as a cortical ring
under the plasma membrane that spread lamellae. CVX induced extensive
platelet spreading as assessed by the presence of a marked ring of
actin without evidence of filopodia. These results established that CVX
specifically leads platelets to spread that clearly differ from the
physiologic aspect of filopodia and lamellae.
Time course of Cdc42 and Rac1 activation after stimulation with thrombin, TRAP, CVX, or IV.3 Because of the role of Cdc42 and Rac1 in actin reorganization, we studied the level of Cdc42-GTP and Rac1-GTP during stimulation by thrombin, TRAP, CVX, or IV.3. We used the PAK1 binding domain in fusion with GST (PBD-GST) to trap Cdc42 and Rac1 in their GTP-bound form from the detergent-soluble fraction of platelets. In the presence of exogenously added GTPS, full activation of Cdc42 or Rac1 was
obtained. No residual Cdc42 or Rac1 was detected in the supernatants.
In the presence of an excess of GDP, Cdc42 and Rac1, maintained in an
inactive state, were unable to bind to PBD-GST beads and remained in
the supernatants (Figure 2A). These results validate the specificity of PBD-GST beads for the detection of
Cdc42-GTP and Rac1-GTP.
In platelets stimulated by thrombin or TRAP, both Cdc42-GTP and
Rac1-GTP accumulated from 0.2 minutes of stimulation. After Fc Activation of PAK Direct binding of the GTP-bound form of Cdc42 and/or Rac1 to the regulatory domain of PAK resulted in the activation of the kinase.5 In an attempt to compare the amount of activated PAK in stimulated platelets, we used an in vitro phosphorylation assay, allowing the analysis of PAK autophosphorylation and kinase activity toward MBP as an exogenous substrate. As shown in Figure 3A, thrombin (0.5 UI/mL), as well as TRAP (10 µM), CVX (250 pmol/L), or IV.3 (2 µg/mL) induced an efficient autophosphorylation of PAK and an increase in MBP phosphorylation, after 3 minutes of stimulation. Anti-PAK1/2 immunoblot showed that the amounts of immunoprecipitated PAK were equal.
The time course of PAK activation was followed in thrombin-stimulated platelets (Figure 3B, left). Quantification of pMBP to total PAK by densitometry analysis showed that MBP phosphorylation increased until 3 minutes (Figure 3C). The occurrence of PAK autophosphorylation in platelets stimulated by THR for 1 minute was confirmed by direct immunoblot with anti-pPAK1/2 (Thr423/Thr402) antibody (Figure 3B, right). These results demonstrate that platelet agonists induce an activation of PAK that correlates with activation of the GTPases. Inhibition of thrombin-induced activation of Cdc42/Rac1 blocks PAK activation Considering that PAK activation by thrombin was linked to early and sustained activation of Cdc42 and Rac1, we investigated whether small G protein activation would be necessary for PAK activation. To test this hypothesis, we used membrane-permeable clostridial toxins to inactivate Cdc42 or Rac1. Large molecular weight cytotoxins (250-300 kDa) such as toxin B from C difficile and lethal toxin (LT) from C sordellii possess a glucosyltransferase activity, allowing glucosylation and, thus, inactivation of small G proteins. Indeed, toxin B and LT affect different intracellular target proteins. Toxin B has been reported to inactivate Rho, Rac, and Cdc42, whereas the targets of LT, isolated from strain IP-82, are p21Ras, Ral, Rap, and Rac proteins.22,23 Platelets pretreated or not with LT or toxin B were stimulated with thrombin, and, subsequently, the amounts of activated Cdc42, Rac1, and PAK were determined. We observed that the level of Rac1-GTP after a 1-minute stimulation with thrombin was decreased in platelets pretreated with LT compared with untreated cells. Toxin B was also found to efficiently reduce Rac1 activity. The activation of Cdc42 was decreased by a pretreatment with toxin B but not with LT. Phosphorylation of MBP was unaffected by the presence of LT, whereas toxin B did not permit the activation of PAK over the basal level (Figure 4A). A complete blockage of PAK activity results from a decrease of the amount of both Cdc42-GTP and Rac1-GTP. This finding suggests that full activation of both Cdc42 and Rac1 is required for stimulation of PAK activity.
Inhibition of Cdc42 and Rac1 blocks actin polymerization and shape change induced by thrombin The effects of LT or toxin B were tested on the amount of polymerized actin in phalloidin-labeled platelets by flow cytometry. We observed that, whereas LT or toxin B had no significant effect on the content of preexisting polymerized actin in platelets at rest (time 0), they both inhibited early movements of actin after stimulation by thrombin (Figure 4B).The distribution of polymerized actin was studied after treatment with toxin B or LT by confocal microscopy (Figure 4C). In unstimulated platelets, no obvious modification in platelet shape was observed after toxin B or LT treatment. After a 1-minute stimulation with thrombin, LT inhibited the aspect of spread platelets and prevented the formation of the cortical ring of polymerized actin. Toxin B also blocked actin rearrangement. These results indicate that activation of Cdc42 and/or Rac1 is necessary for thrombin-induced shape change. Cortactin is constitutively associated with PAK and dissociated from PAK after thrombin stimulation Cortactin is an ubiquitous cortical-actin binding protein highly present at the cell periphery, within membrane ruffles and lamellipodia.24 In Swiss 3T3 cells, it has been demonstrated that dominant-negative N17Rac1 blocks cortactin translocation to the membrane ruffles and that introduction of an active form of PAK1 stimulated this translocation.25 These observations suggest that translocation of cortactin is controlled by the Rac1/PAK pathway. Thus, we investigated whether cortactin would interact with PAK. In vitro pull-down experiments showed that cortactin associated with GST-PAKWT (full length) and not with the constitutively active mutant GST-PAKT423E in resting platelets. In addition, PAK associated with GST-cortactin (Figure 5A). In vivo coimmunoprecipitation experiments demonstrated that cortactin was constitutively associated with PAK (Figure 5B, left) and, conversely, that PAK coimmunoprecipitated with cortactin in resting platelets (data not shown). Furthermore, cortactin dissociated from PAK after a 1-minute stimulation with thrombin. These results suggest that activated PAK, either the active mutant or the PAK activated by thrombin, loses its ability to bind cortactin. When platelets were incubated with toxin B, PAK autophosphorylation induced by thrombin was inhibited, and the binding of cortactin to PAK was restored (Figure 5C). This result confirms that the interaction of cortactin with PAK is regulated by PAK phosphorylation and that PAK activation causes cortactin dissociation.
In an attempt to determine whether the interaction between PAK and cortactin may depend on the state of actin polymerization, we incubated resting platelet lysates with increasing concentrations of DNase I that depolymerizes F-actin. DNase I forms a very tight 1:1 complex with G-actin, prevents interaction between myosin and G-actin, and blocks the dynamics of filament elongation.26 The level of cortactin that coprecipitated with PAK increased with the enhancement of DNase I concentration (Figure 5B, right). This finding suggests that only a fraction of cortactin is in association with PAK. Furthermore, it could not be excluded that the constitutive association of cortactin with PAK may be indirect because actin has been found to coimmunoprecipitate with these proteins. In vitro phosphorylation of cortactin by PAK In addition to efficient tyrosine phosphorylation by Src, it has been established that cortactin is also phosphorylated on serine or threonine residues. We performed in vitro phosphorylation assays with immunoprecipitated PAK in the presence of bacterially produced and purified recombinant cortactin. As shown in Figure 6, recombinant cortactin was phosphorylated by thrombin-activated PAK, whereas it remained unphosphorylated when PAK was immunoprecipitated from resting platelets. This finding suggests that cortactin is an in vitro substrate for PAK.
Immunolocalization of Rac1, PAK, and cortactin in thrombin-activated platelets Because of the disruption of the interaction between PAK and cortactin after thrombin stimulation, we performed immunofluorescence studies to precise their subcellular localization. Rac1 and cortactin appeared homogeneously distributed in resting platelets. After stimulation, they moved to the submembrane region, forming a ring of fluorescence in the vicinity of lamellae and also invaded filopodia. PAK was sparsely distributed in resting platelets and focused near the membrane after stimulation. Similar results were obtained in TRAP-stimulated platelets. These results confirm that cortactin and PAK are differentially distributed throughout stimulated platelets and that both Rac1 and cortactin relocalize into lamellipodia (Figure 7).
In the present work, we show that lamellae spreading induced by thrombin, TRAP, monoclonal IV.3 antibody, or CVX correlates with the activation of PAK. Furthermore, inhibition of both Cdc42 and Rac1 with clostridial toxin B inhibits PAK activation by thrombin and platelet shape change. In resting platelets, the cortical actin-binding protein, cortactin, is constitutively associated with PAK, and the disruption of cortactin association with PAK is concomitant with the occurrence of lamellae spreading in response to thrombin. Inhibition of PAK activation by toxin B prevents the dissociation of cortactin. Small G protein activation is commonly observed after ligand binding to
heterotrimeric G protein-coupled receptors for bombesin or bradykinin
or to tyrosine kinase receptors such as platelet-derived growth factor
(PDGF), epidermal growth factor (EGF), and insulin receptors.1,2,27 In platelets, Cdc42 and Rac1 activation occurs after stimulation of G-coupled PAR-1 by TRAP or
thrombin.4,28 Rac is also activated in response to
collagen.28 Fc PAK activity is also regulated through heterotrimeric G-coupled
receptors, and, in platelets, PAK2 is activated by
thrombin.6,13 In a recent paper, Suzuki-Inoue et
al32 demonstrate that PAK, concomitantly with Rac, is
activated during platelet spreading on immobilized collagen. This
activation is dependent on integrin For the first time, we show, here, that the cortical actin-binding protein, cortactin, is constitutively associated with PAK in resting platelets and that PAK autophosphorylation by thrombin correlates with the disruption of this interaction. Cortactin is an important regulator of actin filament polymerization. Recent evidence has been provided that cortactin colocalizes with F-actin and the Arp2/3 complex in lamellipodia structures, where it can promote the nucleation of new actin filaments, as demonstrated in proliferating NIH 3T3 fibroblasts and in MDA breast cancer cells with amplification of the gene at chromosome 11q13.34,35 In thrombin-stimulated platelets, cortactin translocates to the cytoskeleton fraction.36 Furthermore, in Swiss 3T3 cells, cortactin is known to translocate from the cytosol to the F-actin at the membrane ruffles under the control of Rac1.25 Thus, cortactin relocalization in platelets could also be under the control of Rac1. Our present data suggest that the subcellular localization of a pool of cortactin is dependent on PAK activation. This suggestion is assessed by the fact that (1) cortactin is unable to associate with an active mutant of PAK in vitro, (2) a pool of cytosolic cortactin dissociates from PAK after activation of PAK by thrombin, and (3) dissociation is inhibited by inhibition of PAK autophosphorylation by toxin B. Furthermore, in the absence of PAK activation in resting platelets, the enhancement of the pool of unpolymerized actin after DNase I treatment also increases the pool of cortactin associated with PAK. Thus, the size of the pool of cytosolic cortactin stably associated with PAK may vary, depending on the degree of actin polymerization. Cortactin is a well-known substrate for the tyrosine kinase Src.24 In a previous paper, we have shown that cortactin translocation to the detergent-insoluble cytoskeleton fraction could not be inhibited by the Src family kinase inhibitor PP2, suggesting that cortactin relocalization in the vicinity of F-actin may be independent of tyrosine phosphorylation.36 In addition, it has been shown that p85 cortactin is heavily charged with phosphate on serine and threonine residues.37 In colon carcinoma cells with cortactin gene amplification, p85 spontaneously relocalizes to the cell periphery at focal contacts with the matrix. Here, we show that bacterially produced recombinant cortactin is phosphorylated in vitro by thrombin-activated PAK and that cortactin colocalizes with Rac1 to the ring of actin filaments in lamellae, in the same conditions of stimulation. Taken together, these data suggest that the determinant for cortactin relocalization is rather a serine/threonine than a tyrosine phosphorylation. As previously published, the Nf2 tumor suppressor gene product, merlin, is phosphorylated by PAK2. Unphosphorylated merlin localizes in the microvilli of the normal kidney epithelial cell line, LLC-PK1, although it relocalizes to larger membrane protrusions when phosphorylated.38 Thus, PAK is able to influence the subcellular localization of its substrates. Tyrosine phosphorylation of cortactin by Src has been demonstrated to down-regulate its ability to bind F-actin in vitro.39 However, the influence of serine/threonine phosphorylation on cortactin F-actin binding activity is not known. Interestingly, it has been recently shown that the intermediate filament protein, vimentin, when phosphorylated by PAK, loses its potential to form 10-nm filaments.40 In contrast, PAK phosphorylates and activates the LIM kinase that inhibits the activity of cofilin, an actin depolymerization factor, thus, facilitating actin polymerization.41 Whether serine/threonine phosphorylation of cortactin by PAK may influence its actin filament growth-promoting activity remains to be determined. In conclusion, we show, in this work, that cortical actin reorganization in lamellae is supported by the Cdc42/Rac1/PAK pathway and is concomitant to the disruption of cortactin binding to PAK.
We thank Dr M. F. Carlier (CNRS, Gif-sur-Yvette, France), Prof E. Cramer (Département d'Hématologie, Institut Cochin, Paris) for helpful discussions, F. Heutte for confocal microscopy, and A.-L. Roupie for assistance in manuscript preparation.
Submitted September 18, 2001; accepted August 14, 2002.
Supported by a fellowship from the Fondation pour la Recherche Médicale (C.V.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Michaëla Fontenay-Roupie, Laboratoire d'Hématologie, Hôpital Cochin, 27, rue du Faubourg Saint-Jacques, F75679 Paris, Cedex 14, France; e-mail: fontenay{at}cochin.inserm.fr.
1. Ridley A, Paterson HF, Johnston C, Diekmann D, Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 1992;70:401-410[CrossRef][Medline] [Order article via Infotrieve]. 2. Nobes CD, Hall A. Rho, Rac and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia and filopodia. Cell. 1995;81:53-62[CrossRef][Medline] [Order article via Infotrieve]. 3. Hartwig J, Bokoch G, Carpenter CL, et al. Thrombin receptor ligation and activated Rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets. Cell. 1995;82:643-653[CrossRef][Medline] [Order article via Infotrieve].
4.
Azim AC, Barkalow K, Chou J, Hartwig JH.
Activation of the small GTPases, rac and cdc42, after ligation of the platelet PAR-1 receptor.
Blood.
2000;95:959-964 5. Manser E, Leung T, Salihuddin H, Zhao ZS, Lim L. A brain serine/threonine protein kinase activated by Cdc42 and Rac1. Nature. 1994;367:40-46[CrossRef][Medline] [Order article via Infotrieve].
6.
Knaus UG, Morris S, Dong H-J, Chernoff J, Bokoch GM.
Regulation of human leucocyte p21-activated kinases through G protein-coupled receptors.
Science.
1995;269:221-223
7.
Knaus UG, Wang Y, Reilly AM, Warnock D, Jackson JH.
Structural requirements for PAK activation by Rac GTPases.
J Biol Chem.
1998;273:21512-21518
8.
Zenke FT, King CC, Bohl BP, Bokoch GM.
Identification of a central phosphorylation site in p21-activated kinase regulating autoinhibition and kinase activity.
J Biol Chem.
1999;274:32565-32573
9.
Chong C, Tan L, Lim L, Manser E.
The mechanism of PAK activation: autophosphorylation events in both regulatory and kinase domains control activity.
J Biol Chem.
2001;276:17347-17353 10. del Poso MA, Price LS, Alderson NB, Ren X-D, Schwartz MA. Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK. EMBO J. 2000;19:2008-2014[CrossRef][Medline] [Order article via Infotrieve].
11.
Sanders LC, Matsumura F, Bokoch GM, de Lanerolle P.
Inhibition of myosin light chain kinase by p21-activated kinase.
Science.
1999;283:2083-2085
12.
Turner CE, Brown MC, Perrotta JA, et al.
Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein. A role in cytoskeletal remodeling.
J Cell Biol.
1999;145:851-863
13.
Teo M, Manser E, Lim L.
Identification and molecular cloning of a p21cdc42/rac1-activated serine/threonine kinase that is rapidly activated by thrombin in platelets.
J Biol Chem.
1995;270:26690-26697 14. Anderson CL, Chacko GW, Osborne JM, Brandt JT. The Fc receptor for immunoglobulin G (Fc gamma RII) on human platelets. Semin Thromb Hemost. 1995;21:1-9[Medline] [Order article via Infotrieve].
15.
Polgar J, Clemetson JM, Kehrel BE, et al.
Platelet activation and signal transduction by convulxin, a C-type lectin from Crotalus durissus terrificus (tropical rattlesnake) venom via the p62/GPVI collagen receptor.
J Biol Chem.
1997;272:13576-13583
16.
Jandrot-Perrus M, Lagrue A-H, Okuma M, Bon C.
Adhesion and activation of human platelets induced by convulxin involve glycoprotein VI and integrin
17.
Jandrot-Perrus M, Busfield S, Lagrue A-H, et al.
Cloning, characterization, and functional studies of human and mouse glycoprotein VI: a platelet-specific collagen receptor from the immunoglobulin superfamily.
Blood.
2000;96:1798-1807
18.
Perelle S, Gilbert M, Boquet P, Popoff M.
Characterization of Clostridium perfringens iota-toxin genes and expression in Escherichia coli.
Infect Immun.
1993;61:5147-5156 19. Perelle S, Gibert M, Boquet P, Popoff MR. Characterization of Clostridium perfringens Iota-toxin genes and expression in Escherichia coli. Infect Immun. 1995;63:4967-4972[Medline] [Order article via Infotrieve]. 20. von Eichel-Streiber C, Harperath U, Bosse D, Hadding U. Purification of two high molecular weight toxins of Clostridium difficile which are antigenically related. Microb Pathog. 1987;2:307-318[CrossRef][Medline] [Order article via Infotrieve].
21.
Benard V, Bohl BP, Bokoch GM.
Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases.
J Biol Chem.
1999;274:13198-13204
22.
Popoff M, Chaves-Olarte E, Lemichez E, et al.
Ras, Rap, and Rac small GTP-binding proteins are targets for the Clostridium sordellii lethal toxin glucosylation.
J Biol Chem.
1996;271:10217-10224 23. Richard J-F, Petit L, Gibert M, Marvaud JC, Bouchaud C, Popoff MR. Bacterial toxins modifying the actin cytoskeleton. Int Microbiol. 1999;2:185-194[Medline] [Order article via Infotrieve].
24.
Wu H, Parsons JT.
Cortactin, an 80/85-kilodalton pp60src substrate, is a filamentous actin-binding protein enriched in the cell cortex.
J Cell Biol.
1993;120:1417-1426 25. Weed SA, Du Y, Parsons JT. Translocation of cortactin to the cell periphery is mediated by the small GTPase Rac1. J Cell Sci. 1998;111:2433-2443[Abstract].
26.
Blanchoin L, Fievez S, Travers F, Carlier M-F, Pantaloni D.
Kinetics of the interaction of myosin subfragment-1 with G-actin. Effects of nucleotides and DNaseI.
J Biol Chem.
1995;270:7125-7133 27. Kozma R, Ahmed S, Best A, Lim L. The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts. Mol Cell Biol. 1995;15:1942-1952[Abstract]. 28. Soulet C, Gendreau S, Missy K, Benard V, Plantavid M, Payrastre B. Characterization of Rac activation in thrombin- and collagen-stimulated human blood platelets. FEBS Lett. 2001;507:253-258[CrossRef][Medline] [Order article via Infotrieve].
29.
Cox D, Chang P, Zhang Q, Reddy PG, Bokoch GM, Greenberg S.
Requirements for both Rac1 and Cdc42 in membrane ruffling and phagocytosis in leucocytes.
J Exp Med.
1997;186:1487-1494
30.
Kim JS, Peng X, De PK, Geahlen RL, Durden DL.
PTEN controls immunoreceptor (immunoreceptor tyrosine-based activation motif) signaling and the activation of Rac.
Blood.
2002;99:694-697
31.
Gratacap M-P, Payrastre B, Viala C, Mauco G, Plantavid M, Chap H.
Phosphatidylinositol 3,4,5-trisphosphate-dependent stimulation of phospholipase C-
32.
Suzuki-Inoue K, Yatomi Y, Asazuma N, et al.
Rac, a small guanosine triphosphate-binding protein, and p21-activate kinase are activated during platelet spreading on collagen-coated surfaces: roles of integrin 33. Daniels RH, Hall PS, Bokock GM. Membrane targeting of p21-activated kinase 1 (PAK1) induces neurite outgrowth from PC12 cells. EMBO J. 1998;17:754-764[CrossRef][Medline] [Order article via Infotrieve]. 34. Kaksonen M, Peng HB, Rauvala H. Association of cortactin with dynamic actin in lamellipodia and endosomal vesicles. J Cell Sci. 2000;113:4421-4426[Abstract]. 35. Urano T, Liu J, Zhang P, et al. Activation of Arp2/3 complex-mediated actin polymerization by cortactin. Nat Cell Biol. 2001;3:259-266[CrossRef][Medline] [Order article via Infotrieve]. 36. Lopez I, Duprez V, Melle J, Dreyfus F, Levy-Toledano S, Fontenay-Roupie M. Thrombopoietin stimulates cortactin translocation to the cytoskeleton independently of tyrosine phosphorylation. Biochem J. 2001;356:875-881[CrossRef][Medline] [Order article via Infotrieve].
37.
van Damme H, Brok H, Schuuring-Scholtes E, Schuuring E.
The redistribution of cortactin into cell-matrix contact sites in human carcinoma cells with 11q13 amplification is associated with both overexpression and post-translational modification.
J Biol Chem.
1997;272:7374-7380
38.
Kissil JL, Johnson KC, Eckman MS, Jacks T.
Merlin phosphorylation by p21-activated kinase 2 and effects of phosphorylation on merlin localization.
J Biol Chem.
2002;277:10394-10399
39.
Huang C, Ni Y, Wang T, et al.
Downregulation of the filamentous actin cross-linking activity of cortactin by src-mediated tyrosine phosphorylation.
J Biol Chem.
1997;272:13911-13915 40. Goto H, Tanabe K, Manser E, Lim L, Yasui Y, Inagaki M. Phosphorylation and reorganization of vimentin by p21-activated kinase (PAK). Gene Cells. 2002;7:91-97. 41. Edwards DC, Sanders LC, Bokoch GM, Gill GN. Activation of LIM-kinase 1 by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics. Nat Cell Biol. 1999;5:253-259.
© 2002 by The American Society of Hematology.
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  D. Pandey, P. Goyal, S. Dwivedi, and W. Siess Unraveling a novel Rac1-mediated signaling pathway that regulates cofilin dephosphorylation and secretion in thrombin-stimulated platelets Blood, July 9, 2009; 114(2): 415 - 424. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Huveneers and E. H. J. Danen Adhesion signaling - crosstalk between integrins, Src and Rho J. Cell Sci., April 15, 2009; 122(8): 1059 - 1069. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. A. Karim, W. Choi, and S. W. Whiteheart Primary Platelet Signaling Cascades and Integrin-mediated Signaling Control ADP-ribosylation Factor (Arf) 6-GTP Levels during Platelet Activation and Aggregation J. Biol. Chem., May 2, 2008; 283(18): 11995 - 12003. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Jia, T. Uekita, and R. Sakai Hyperphosphorylated Cortactin in Cancer Cells Plays an Inhibitory Role in Cell Motility Mol. Cancer Res., April 1, 2008; 6(4): 654 - 662. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Ayala, M. Baldassarre, G. Giacchetti, G. Caldieri, S. Tete, A. Luini, and R. Buccione Multiple regulatory inputs converge on cortactin to control invadopodia biogenesis and extracellular matrix degradation J. Cell Sci., February 1, 2008; 121(3): 369 - 378. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. I. Cosen-Binker and A. Kapus Cortactin: The Gray Eminence of the Cytoskeleton. Physiology, October 1, 2006; 21(5): 352 - 361. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. L. Rothschild, A. H. Shim, A. G. Ammer, L. C. Kelley, K. B. Irby, J. A. Head, L. Chen, M. Varella-Garcia, P. G. Sacks, B. Frederick, et al. Cortactin Overexpression Regulates Actin-Related Protein 2/3 Complex Activity, Motility, and Invasion in Carcinomas with Chromosome 11q13 Amplification Cancer Res., August 15, 2006; 66(16): 8017 - 8025. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. Jacobson, S. M. Dudek, P. A. Singleton, I. A. Kolosova, A. D. Verin, and J. G. N. Garcia Endothelial cell barrier enhancement by ATP is mediated by the small GTPase Rac and cortactin. Am J Physiol Lung Cell Mol Physiol, August 1, 2006; 291(2): L289 - L295. [Abstract] [Full Text] [PDF]
|
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|
|
 |
|
 J. G. Lee and E. P. Kay Cross-talk among Rho GTPases Acting Downstream of PI 3-Kinase Induces Mesenchymal Transformation of Corneal Endothelial Cells Mediated by FGF-2. Invest. Ophthalmol. Vis. Sci., June 1, 2006; 47(6): 2358 - 2368. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Choi, Z. A. Karim, and S. W. Whiteheart Arf6 plays an early role in platelet activation by collagen and convulxin Blood, April 15, 2006; 107(8): 3145 - 3152. [Abstract] [Full Text] [PDF]
|
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|
|
 |
|
 S. Zhou, B. A. Webb, R. Eves, and A. S. Mak Effects of tyrosine phosphorylation of cortactin on podosome formation in A7r5 vascular smooth muscle cells Am J Physiol Cell Physiol, February 1, 2006; 290(2): C463 - C471. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Pandey, P. Goyal, J. R. Bamburg, and W. Siess Regulation of LIM-kinase 1 and cofilin in thrombin-stimulated platelets Blood, January 15, 2006; 107(2): 575 - 583. [Abstract] [Full Text] [PDF]
|
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|
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|
|
B. A. Webb, R. Eves, S. W. Crawley, S. Zhou, G. P. Cote, and A. S. Mak PAK1 induces podosome formation in A7r5 vascular smooth muscle cells in a PAK-interacting exchange factor-dependent manner Am J Physiol Cell Physiol, October 1, 2005; 289(4): C898 - C907. [Abstract] [Full Text] [PDF]
|
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|
H. Kashiwagi, M. Shiraga, H. Kato, T. Kamae, N. Yamamoto, S. Tadokoro, Y. Kurata, Y. Tomiyama, and Y. Kanakura Negative regulation of platelet function by a secreted cell repulsive protein, semaphorin 3A Blood, August 1, 2005; 106(3): 913 - 921. [Abstract] [Full Text] [PDF]
|
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A. Mazharian, S. Roger, P. Maurice, E. Berrou, M. R. Popoff, M. F. Hoylaerts, F. Fauvel-Lafeve, A. Bonnefoy, and M. Bryckaert Differential Involvement of ERK2 and p38 in Platelet Adhesion to Collagen J. Biol. Chem., July 15, 2005; 280(28): 26002 - 26010. [Abstract] [Full Text] [PDF]
|
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A. Oda, H. Miki, I. Wada, H. Yamaguchi, D. Yamazaki, S. Suetsugu, M. Nakajima, A. Nakayama, K. Okawa, H. Miyazaki, et al. WAVE/Scars in platelets Blood, April 15, 2005; 105(8): 3141 - 3148. [Abstract] [Full Text] [PDF]
|
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|
|
|
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S. Sabri, M. Jandrot-Perrus, J. Bertoglio, R. W. Farndale, V. M.-D. Mas, N. Debili, and W. Vainchenker Differential regulation of actin stress fiber assembly and proplatelet formation by {alpha}2{beta}1 integrin and GPVI in human megakaryocytes Blood, November 15, 2004; 104(10): 3117 - 3125. [Abstract] [Full Text] [PDF]
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|
 K. Kawkitinarong, L. Linz-McGillem, K. G. Birukov, and J. G. N. Garcia Differential Regulation of Human Lung Epithelial and Endothelial Barrier Function by Thrombin Am. J. Respir. Cell Mol. Biol., November 1, 2004; 31(5): 517 - 527. [Abstract] [Full Text] [PDF]
|
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|
 R. Nijhara, P. B. van Hennik, M. L Gignac, M. J. Kruhlak, P. L. Hordijk, J. Delon, and S. Shaw Rac1 Mediates Collapse of Microvilli on Chemokine-Activated T Lymphocytes J. Immunol., October 15, 2004; 173(8): 4985 - 4993. [Abstract] [Full Text] [PDF]
|
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 |
|
 N. Martinez-Quiles, H.-Y. H. Ho, M. W. Kirschner, N. Ramesh, and R. S. Geha Erk/Src Phosphorylation of Cortactin Acts as a Switch On-Switch Off Mechanism That Controls Its Ability To Activate N-WASP Mol. Cell. Biol., June 15, 2004; 24(12): 5269 - 5280. [Abstract] [Full Text] [PDF]
|
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S. M. Dudek, J. R. Jacobson, E. T. Chiang, K. G. Birukov, P. Wang, X. Zhan, and J. G. N. Garcia Pulmonary Endothelial Cell Barrier Enhancement by Sphingosine 1-Phosphate: ROLES FOR CORTACTIN AND MYOSIN LIGHT CHAIN KINASE J. Biol. Chem., June 4, 2004; 279(23): 24692 - 24700. [Abstract] [Full Text] [PDF]
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 B. L. Lua and B. C. Low BPGAP1 Interacts with Cortactin and Facilitates Its Translocation to Cell Periphery for Enhanced Cell Migration Mol. Biol. Cell, June 1, 2004; 15(6): 2873 - 2883. [Abstract] [Full Text] [PDF]
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A. G. S. H. van Rossum, J. H. de Graaf, E. Schuuring-Scholtes, P. M. Kluin, Y.-x. Fan, X. Zhan, W. H. Moolenaar, and E. Schuuring Alternative Splicing of the Actin Binding Domain of Human Cortactin Affects Cell Migration J. Biol. Chem., November 14, 2003; 278(46): 45672 - 45679. [Abstract] [Full Text] [PDF]
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K. L. Barkalow, H. Falet, J. E. Italiano Jr., A. van Vugt, C. L. Carpenter, A. D. Schreiber, and J. H. Hartwig Role for phosphoinositide 3-kinase in Fc{gamma}RIIA-induced platelet shape change Am J Physiol Cell Physiol, October 1, 2003; 285(4): C797 - C805. [Abstract] [Full Text] [PDF]
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U. P. Naik and M. U. Naik Association of CIB with GPIIb/IIIa during outside-in signaling is required for platelet spreading on fibrinogen Blood, August 15, 2003; 102(4): 1355 - 1362. [Abstract] [Full Text] [PDF]
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J. M. Dyson, A. D. Munday, A. M. Kong, R. D. Huysmans, M. Matzaris, M. J. Layton, H. H. Nandurkar, M. C. Berndt, and C. A. Mitchell SHIP-2 forms a tetrameric complex with filamin, actin, and GPIb-IX-V: localization of SHIP-2 to the activated platelet actin cytoskeleton Blood, August 1, 2003; 102(3): 940 - 948. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
IIb
3 with emergence of stress fibers
submitted to a regulation by Rho. In an attractive model of
permeabilized platelets, it has been demonstrated that the introduction
of a dominant-negative mutant N17Rac1 prevents exposure of actin
filament barbed ends caused by thrombin, whereas a constitutively
active mutant V12Rac1 promotes exposure of actin ends that precedes
nucleation of new actin filaments.
), 10 µM TRAP (
), 250 pmol/L CVX (
), 2 µg/mL monoclonal IV.3 antibody followed by
clustering of Fc
) or control
immunoglobulin (×), platelets were fixed in 1.8% paraformaldehyde,
labeled with 10 µM FITC-phalloidin in 0.02% Triton X-100 buffer, and
analyzed by flow cytometry. Results are expressed as ratios of mean
fluorescence intensity (RFI) of stimulated to resting
platelets and reported as means (± SD) of RFI from at least 6 experiments. (B) Platelets stimulated for 1 minute were also analyzed
by confocal fluorescence microscopy (× 100). Results are
representative of 3 separate experiments.
