Regulation of Outside-in Signaling in Platelets by Integrin-associated Protein Kinase Cβ
2004; Elsevier BV; Volume: 280; Issue: 1 Linguagem: Inglês
10.1074/jbc.m410229200
ISSN1083-351X
AutoresCharito S. Buensuceso, Achim Obergfell, Alessandra Soriani, Koji Eto, William B. Kiosses, Elena G. Arias‐Salgado, Toshiaki Kawakami, Sanford J. Shattil,
Tópico(s)Antiplatelet Therapy and Cardiovascular Diseases
ResumoStudies with inhibitors have implicated protein kinase C (PKC) in the adhesive functions of integrin αIIbβ3 in platelets, but the responsible PKC isoforms and mechanisms are unknown. αIIbβ3 interacts directly with tyrosine kinases c-Src and Syk. Therefore, we asked whether αIIbβ3 might also interact with PKC. Of the several PKC isoforms expressed in platelets, only PKCβ co-immunoprecipitated with αIIbβ3 in response to the interaction of platelets with soluble or immobilized fibrinogen. PKCβ recruitment to αIIbβ3 was accompanied by a 9-fold increase in PKC activity in αIIbβ3 immunoprecipitates. RACK1, an intracellular adapter for activated PKCβ, also co-immunoprecipitated with αIIbβ3, but in this case, the interaction was constitutive. Broad spectrum PKC inhibitors blocked both PKCβ recruitment to αIIbβ3 and the spread of platelets on fibrinogen. Similarly, mouse platelets that are genetically deficient in PKCβ spread poorly on fibrinogen, despite normal agonist-induced fibrinogen binding. In a Chinese hamster ovary cell model system, adhesion to fibrinogen caused green fluorescent protein-PKCβI to associate with αIIbβ3 and to co-localize with it at lamellipodial edges. These responses, as well as Chinese hamster ovary cell migration on fibrinogen, were blocked by the deletion of the β3 cytoplasmic tail or by co-expression of a RACK1 mutant incapable of binding to β3. These studies demonstrate that the interaction of αIIbβ3 with activated PKCβ is regulated by integrin occupancy and can be mediated by RACK1 and that the interaction is required for platelet spreading triggered through αIIbβ3. Furthermore, the studies extend the concept of αIIbβ3 as a scaffold for multiple protein kinases that regulate the platelet actin cytoskeleton. Studies with inhibitors have implicated protein kinase C (PKC) in the adhesive functions of integrin αIIbβ3 in platelets, but the responsible PKC isoforms and mechanisms are unknown. αIIbβ3 interacts directly with tyrosine kinases c-Src and Syk. Therefore, we asked whether αIIbβ3 might also interact with PKC. Of the several PKC isoforms expressed in platelets, only PKCβ co-immunoprecipitated with αIIbβ3 in response to the interaction of platelets with soluble or immobilized fibrinogen. PKCβ recruitment to αIIbβ3 was accompanied by a 9-fold increase in PKC activity in αIIbβ3 immunoprecipitates. RACK1, an intracellular adapter for activated PKCβ, also co-immunoprecipitated with αIIbβ3, but in this case, the interaction was constitutive. Broad spectrum PKC inhibitors blocked both PKCβ recruitment to αIIbβ3 and the spread of platelets on fibrinogen. Similarly, mouse platelets that are genetically deficient in PKCβ spread poorly on fibrinogen, despite normal agonist-induced fibrinogen binding. In a Chinese hamster ovary cell model system, adhesion to fibrinogen caused green fluorescent protein-PKCβI to associate with αIIbβ3 and to co-localize with it at lamellipodial edges. These responses, as well as Chinese hamster ovary cell migration on fibrinogen, were blocked by the deletion of the β3 cytoplasmic tail or by co-expression of a RACK1 mutant incapable of binding to β3. These studies demonstrate that the interaction of αIIbβ3 with activated PKCβ is regulated by integrin occupancy and can be mediated by RACK1 and that the interaction is required for platelet spreading triggered through αIIbβ3. Furthermore, the studies extend the concept of αIIbβ3 as a scaffold for multiple protein kinases that regulate the platelet actin cytoskeleton. In addition to their roles in cell adhesion, integrins transmit signals in both directions across the plasma membrane to regulate cytoskeletal organization, motility, and other anchorage-dependent cellular responses (1Hynes R. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6687) Google Scholar). In platelets, for example, αIIbβ3 responds to "inside-out" signals with an increase in affinity for cognate ligands such as fibrinogen that bridge platelets to each other and mediate platelet adhesion to sites of vascular damage. In turn, ligand binding to αIIbβ3 triggers outside-in signals that promote cytoskeletal changes necessary for full platelet aggregation and spreading (2Hartwig J.H. Barkalow K. Azim A. Italiano J. Thromb. Haemostasis. 1999; 82: 392-398Crossref PubMed Scopus (83) Google Scholar, 3Shattil S.J. Newman P.J. Blood. 2004; 104: 1606-1615Crossref PubMed Scopus (433) Google Scholar). Bidirectional αIIbβ3 signaling is controlled, in part, by specific intracellular proteins that interact with the relatively short cytoplasmic tails of αIIb or β3. For example, binding of talin to β3 is a final common step in the cellular modulation of αIIbβ3 affinity (4Tadokoro S. Shattil S.J. Eto K. Tai V. Liddington R.C. de Pereda J.M. Ginsberg M.H. Calderwood D.A. Science. 2003; 302: 103-106Crossref PubMed Scopus (953) Google Scholar), and binding of c-Src and Syk protein-tyrosine kinases to β3 is required for platelet spreading on fibrinogen (5Arias-Salgado E.G. Lizano S. Sarker S. Brugge J.S. Ginsberg M.H. Shattil S.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13298-13302Crossref PubMed Scopus (441) Google Scholar, 6Obergfell A. Eto K. Mocsai A. Buensuceso C. Moores S.L. Brugge J.S. Lowell C.A. Shattil S.J. J. Cell Biol. 2002; 157: 265-275Crossref PubMed Scopus (344) Google Scholar). Several other intracellular proteins, for example, CIB and β3-endonexin, can also bind to αIIb or β3 tails, respectively, and may influence αIIbβ3 functions (7Naik U.P. Patel P.M. Parise L.V. J. Biol. Chem. 1997; 272: 4651-4654Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 8Naik U.P. Naik M.U. Blood. 2003; 102: 1355-1362Crossref PubMed Scopus (61) Google Scholar, 9Kashiwagi H. Schwartz M.A. Eigenthaler M.A. Davis K.A. Ginsberg M.H. Shattil S.J. J. Cell Biol. 1997; 137: 1433-1443Crossref PubMed Scopus (113) Google Scholar). However, the full complement of intracellular proteins that are capable of interacting directly or indirectly with αIIbβ3 is unknown. The protein kinase C (PKC) 1The abbreviations used are: PKC, protein kinase C; RACK, receptor for activated PKC; CHO, Chinese hamster ovary; GFP, green fluorescent protein; TRITC, tetramethylrhodamine isothiocyanate; BSA, bovine serum albumin; PMA, phorbol 12-myristate 13-acetate. 1The abbreviations used are: PKC, protein kinase C; RACK, receptor for activated PKC; CHO, Chinese hamster ovary; GFP, green fluorescent protein; TRITC, tetramethylrhodamine isothiocyanate; BSA, bovine serum albumin; PMA, phorbol 12-myristate 13-acetate. subfamily of AGC serine/threonine kinases has been implicated in integrin function or dynamics in many cell types (10Ivaska J. Kermorgant S. Whelan R. Parsons M. Ng T. Parker P.J. Biochem. Soc. Trans. 2003; 31: 90-93Crossref PubMed Google Scholar). In platelets, PKC is thought to regulate αIIbβ3 affinity, based on the stimulatory effects of phorbol esters, which bind to PKC C1 domains, and the blocking effects of broad spectrum PKC inhibitors (3Shattil S.J. Newman P.J. Blood. 2004; 104: 1606-1615Crossref PubMed Scopus (433) Google Scholar). However, the lack of specificity of these compounds limits data interpretation and does not permit conclusions about the roles of specific PKC isoforms (11Brose N. Rosenmund C. J. Cell Sci. 2002; 115: 4399-4411Crossref PubMed Scopus (303) Google Scholar). PKCs have been categorized as classical (diacylglycerol- and Ca2+-regulated through C1 and C2 domains, respectively), novel (Ca2+-independent but diacylglycerol-regulated), or atypical (Ca2+- and diacylglycerol-independent) (12Newton A.C. Johnson J.E. Biochim. Biophys. Acta. 1998; 1376: 155-172Crossref PubMed Scopus (242) Google Scholar, 13Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Crossref PubMed Scopus (1345) Google Scholar). Platelets are reported to contain members of all three classes of PKC isozymes (14Baldassare J.J. Henderson P.A. Burns D. Loomis C. Fisher G.J. J. Biol. Chem. 1992; 267: 15585-15590Abstract Full Text PDF PubMed Google Scholar, 15Grabarek J. Raychowdhury M. Ravid K. Kent K.C. Newman P.J. Ware J.A. J. Biol. Chem. 1992; 267: 10011-10017Abstract Full Text PDF PubMed Google Scholar, 16Khan W.A. Blobe G. Halpern A. Taylor W. Wetsel W.C. Burns D. Loomis C. Hannun Y.A. J. Biol. Chem. 1993; 268: 5063-5068Abstract Full Text PDF PubMed Google Scholar, 17Wang F. Naik U.P. Ehrlich Y.H. Osada S. Ohno S. Kornecki E. Biochem. J. 1995; 311: 401-406Crossref PubMed Scopus (22) Google Scholar, 18Hillen T.J. Aroor A.R. Shukla S.D. Biochem. Biophys. Res. Commun. 2001; 280: 259-264Crossref PubMed Scopus (9) Google Scholar, 19Yoshioka A. Shirakawa R. Nishioka H. Tabuchi A. Higashi T. Ozaki H. Yamamoto A. Kita T. Horiuchi H. J. Biol. Chem. 2001; 276: 39379-39385Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 20Crosby D. Poole A.W. J. Biol. Chem. 2002; 277: 9958-9965Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 21Crosby D. Poole A.W. J. Biol. Chem. 2003; 278: 24533-24541Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), as well as the related protein kinase D (22Stafford M.J. Watson S.P. Pears C.J. Blood. 2003; 101: 1392-1399Crossref PubMed Scopus (22) Google Scholar). Recently, experimental tools have become available to study specific PKC isoforms in cells, including overexpression, gene targeting and gene knock-down strategies, and molecular imaging (23Irie N. Sakai N. Ueyama T. Kajimoto T. Shirai Y. Saito N. Biochem. Biophys. Res. Commun. 2002; 298: 738-743Crossref PubMed Scopus (47) Google Scholar, 24Toker A. Newton A.C. Protein Kinase C Protocols. 233. Humana Press, Inc., Totowa, NJ2003: 475-489Google Scholar, 25Saito N. Newton A.C. Protein Kinase C Protocols. 233. Humana Press, Inc., Totowa, NJ2003: 93-103Google Scholar). Some of these tools are potentially relevant to platelets. PKC function depends on the maturation of catalytic activity of the enzyme through phosphorylation and PKC binding to membranes or specific proteins. The latter interactions place PKC in proximity to substrates and relieve autoinhibitory restraints imposed by the binding of a pseudosubstrate sequence to the active site (12Newton A.C. Johnson J.E. Biochim. Biophys. Acta. 1998; 1376: 155-172Crossref PubMed Scopus (242) Google Scholar, 13Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Crossref PubMed Scopus (1345) Google Scholar, 26Newton A.C. Biochem. J. 2003; 370: 361-371Crossref PubMed Scopus (654) Google Scholar). One group of PKC targeting proteins has been termed RACK, which binds selectively to activated PKCs (27Schechtman D. Mochly-Rosen D. Oncogene. 2001; 20: 6339-6347Crossref PubMed Scopus (282) Google Scholar). The best characterized protein of this group is RACK1, a 36-kDa protein composed of seven WD40 repeats. RACK1 was originally identified based on its interaction with activated PKCβ and subsequently shown to interact with certain other PKC isoforms and with several other proteins, most notably integrin β cytoplasmic tails and c-Src (see Fig. 1A) (28Liliental J. Chang D.D. J. Biol. Chem. 1998; 273: 2379-2383Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 29Chang B.Y. Chiang M.L. Cartwright C.A. J. Biol. Chem. 2001; 276: 20346-20356Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 30McCahill A. Warwicker J. Bolger G.B. Houslay M.D. Yarwood S.J. Mol. Pharmacol. 2002; 62: 1261-1273Crossref PubMed Scopus (327) Google Scholar). Given the potential for the cytoplasmic tails of αIIbβ3 to serve as binding sites for signaling molecules and the apparent functional relationships between PKC and αIIbβ3, the present studies were carried out to determine whether specific PKCs associate with αIIbβ3 and if so to determine what the functional relevance of the association is. By using human and mouse platelets and a CHO cell model system, the results show that one particular PKC isoform, PKCβ, inducibly associates with αIIbβ3 in response to fibrinogen binding to cells. The PKCβ/αIIbβ3 interaction appears to be mediated by RACK1 and is required for cytoskeletal reorganization and platelet spreading on fibrinogen, but it is dispensable for the affinity modulation of αIIbβ3. Reagents—Monoclonal antibodies to PKCα, -β, -δ, -τ, -ϵ, -λ, and -ι were from Transduction Laboratories (Lexington, KY), and antibodies specific for PKCβI, PKCβII, or cortactin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The phospho-specific antibody to c-Src, tyrosine 418, was from BIOSOURCE, and anti-phosphotyrosine antibodies 4G10 and PY20 were from Upstate Biotechnology (Lake Placid, NY) and Transduction Laboratories, respectively. Monoclonal antibody 327 to c-Src was from Dr. Joan Brugge (Harvard Medical School), and monoclonal antibody D57 (specific for αIIbβ3) and polyclonal antibody Rb8053 (specific for integrin β3) were from Dr. Mark H. Ginsberg (University of California San Diego, La Jolla, CA). Horseradish peroxidase-conjugated secondary antibodies were from Bio-Rad, and Cy-5- and TRITC-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Rhodamine-phalloidin was from Molecular Probes (Eugene, OR), purified human fibrinogen was from Enzyme Research Laboratories, Inc. (South Bend, IN), and protein A-Sepharose was from Amersham Biosciences. Cell-permeable PKC antagonist peptide p99 was purchased from Stanford University (31Schechtman D. Mochly-Rosen D. Methods Enzymol. 2002; 345: 470-489Crossref PubMed Scopus (46) Google Scholar). Src kinase inhibitor SU6656 was from SUGEN, Inc. (South San Francisco, CA). Platelet agonists were from Sigma, except for convulxin, which was a gift from Dr. Steve Watson (University of Birmingham, Birmingham, UK). Cell Culture, Plasmids, and Transfections—A5 CHO cells stably expressing αIIbβ3 were maintained in culture as described previously (32O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (573) Google Scholar). Transfections were performed at 70-80% confluency using Lipofectamine (Invitrogen) according to the manufacturer's instructions. Twenty-four hours later, the concentration of fetal calf serum was decreased from 10 to 0.5%, and cells were cultured for another 24 h before being used in functional assays. Mammalian expression plasmids included pEGFP-PKCβI (a chimera containing GFP fused to the N terminus of full-length PKCβI (a gift from Dr. Stephen S. G. Ferguson, Robarts Research Institute, Ontario, Canada)), pRC-CMV/Src (encoding wild-type non-neuronal murine c-Src), and pcDNA/RACK1/WD6/7/HA (a hemagglutinin-tagged chimera containing RACK1 WD repeats 6 and 7). To construct the latter, pTarget/WD6/7 (a gift from Dr. Arnaud Besson, Fred Hutchinson Cancer Research Center, Seattle, WA) was subjected to PCR using Platinum Pfx polymerase to introduce a5′-KpnI site and a hemagglutinin tag-3′-XhoI site. After digestion and ligation into pcDNA3.1, transformed colonies were screened by colony PCR, and coding sequences were verified by DNA sequencing. Plasmids were amplified and purified before use (QIAfilter plasmid Maxi kit, Qiagen, Inc., Chatsworth, CA). Interaction of Cells with Fibrinogen—Human platelets or mouse platelets from PKCβ-/-, PKCβ+/-, and PKCβ+/+ littermates (33Leitges M. Schmedt C. Guinamard R. Davoust J. Schaal S. Stabel S. Tarakhovsky A. Science. 1996; 273: 788-791Crossref PubMed Scopus (412) Google Scholar) were obtained from fresh, anticoagulated whole blood and were washed and resuspended to 3 × 108 cells/ml in a platelet incubation buffer (34Leng L. Kashiwagi H. Ren X-D. Shattil S.J. Blood. 1998; 91: 4206-4215Crossref PubMed Google Scholar, 35Law D.A. Nannizzi-Alaimo L. Ministri K. Hughes P. Forsyth J. Turner M. Shattil S.J. Ginsberg M.H. Tybulewicz V. Phillips D.R. Blood. 1999; 93: 2645-2652Crossref PubMed Google Scholar). The PKCβ status of mouse platelets was established by genotyping the animals and by Western blotting of platelet lysates. CHO cells were harvested using 0.5 mm EDTA, washed once in Dulbecco's modified Eagle's medium, resuspended to 3 × 106 cells/ml, and incubated for 45 min in the presence 20 μm cycloheximide. Binding of fluorescein isothiocyanate-fibrinogen to mouse platelets was quantified by flow cytometry (35Law D.A. Nannizzi-Alaimo L. Ministri K. Hughes P. Forsyth J. Turner M. Shattil S.J. Ginsberg M.H. Tybulewicz V. Phillips D.R. Blood. 1999; 93: 2645-2652Crossref PubMed Google Scholar). To test the effects of soluble fibrinogen binding on the interaction of intracellular proteins with αIIbβ3, platelets or CHO cells were incubated for 20 or 30 min, respectively, with 250 μg/ml fibrinogen in the presence or absence of 0.5 mm MnCl2 (to activate αIIbβ3) (36Litvinov R.I. Nagaswami C. Vilaire G. Shuman H. Bennett J.S. Weisel J.W. Blood. 2004; (10.1182/blood-2004-04-1411, in press)PubMed Google Scholar) and in some cases with 2 mm RGDS (to block fibrinogen binding). Cells were collected by centrifugation, washed once with phosphate-buffered saline, and solubilized for 10 min on ice in a lysis buffer containing 0.5% Nonidet P-40, 50 mm NaCl, 50 mm Tris, pH 7.4, and inhibitors (1 mm sodium vanadate, 0.5 mm sodium fluoride, and 1× Complete protease inhibitor (Roche Applied Science)). Lysates were clarified at 4 °C by sedimentation at 10,000 rpm in a microcentrifuge and subjected to immunoprecipitation and Western blotting. To test the effects of cell adhesion to fibrinogen, 100-mm bacterial culture dishes were precoated with 5 mg/ml bovine serum albumin (BSA) or 100 μg/ml fibrinogen. After blocking with heat-denatured BSA, 4.5 × 108 platelets in 1.5 ml or 3 × 106 CHO cells in 2 ml were added to each dish, and incubations were carried out in a CO2 incubator for the indicated periods of time at 37 °C. Non-adherent cells from the BSA plates were sedimented and lysed immediately. Cells adherent to fibrinogen were gently washed twice with phosphate-buffered saline, lysed on the plates, and subjected to immunoprecipitation and Western blotting. Immunoprecipitation and Western Blotting—Five hundred-microliter aliquots of lysate containing equal amounts of protein (ranging from 500 to 800 μg, depending on the experiment) were incubated with relevant antibodies for 2 h or overnight at 4 °C with gentle agitation. Then, 50 μl of protein A-Sepharose (50% v/v) were added for 2 h at 4 °C. Immune complexes were washed twice with phosphate-buffered saline, SDS-PAGE sample buffer was added, and samples were electrophoresed in 7.5 or 10% SDS-polyacrylamide gels. After electrotransfer to nitrocellulose, Western blotting was carried out using enhanced chemiluminescence for detection (Supersignal West Pico substrate, Pierce). PKC Activity Measurements—αIIbβ3 was immunoprecipitated from platelet lysates with antibody Rb8053 and protein A-Sepharose. Beads were washed twice with lysis buffer and resuspended in kinase buffer and [32P]ATP, and PKC activity was measured using a substrate peptide (QKRPSQRSKYL) according to the manufacturer's instructions (PKC assay kit, Upstate Biotechnology, Inc., Lake Placid, NY). Cell Spreading and Migration Assays—Glass coverslips were coated with 100 μg/ml fibrinogen, washed cells were allowed to adhere for 45 min at room temperature, and cell morphology and spreading were assessed by confocal microscopy (6Obergfell A. Eto K. Mocsai A. Buensuceso C. Moores S.L. Brugge J.S. Lowell C.A. Shattil S.J. J. Cell Biol. 2002; 157: 265-275Crossref PubMed Scopus (344) Google Scholar, 34Leng L. Kashiwagi H. Ren X-D. Shattil S.J. Blood. 1998; 91: 4206-4215Crossref PubMed Google Scholar). To study CHO cell migration on fibrinogen, cells were serum-starved overnight in Dulbecco's modified Eagle's medium containing 0.5% fetal calf serum and resuspended in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, and 1-ml aliquots were seeded at 30,000 cells/ml onto fibrinogen-coated coverslips. After 1 h, cells were microinjected with a 50 μg/ml solution of RACK1 WD6/7 and/or GFP-PKCβI or GFP. Microinjected cells were monitored in real time in a temperature-controlled environment chamber using a Nikon TE2000U microscope. Images were acquired every 10 min for a 6-h period with a CoolSnap HQ charge-coupled device camera (Roper Scientific, Tucson, AZ) running on a Linux work station using ISee software (ISee Imaging Systems, Raleigh, NC). At the end of the filming period, cells were stained with an antibody to hemagglutinin tag to detect those cells expressing RACK1 WD6/7. Interactions between αIIbβ3and RACK1 in Platelets—The RACK1 adapter molecule contains binding sites for c-Src, certain isoforms of activated PKC, and integrin β cytoplasmic tails (Fig. 1A) (27Schechtman D. Mochly-Rosen D. Oncogene. 2001; 20: 6339-6347Crossref PubMed Scopus (282) Google Scholar, 29Chang B.Y. Chiang M.L. Cartwright C.A. J. Biol. Chem. 2001; 276: 20346-20356Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 30McCahill A. Warwicker J. Bolger G.B. Houslay M.D. Yarwood S.J. Mol. Pharmacol. 2002; 62: 1261-1273Crossref PubMed Scopus (327) Google Scholar). In platelets, αIIbβ3 is constitutively associated with c-Src, and fibrinogen binding leads to c-Src activation (5Arias-Salgado E.G. Lizano S. Sarker S. Brugge J.S. Ginsberg M.H. Shattil S.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13298-13302Crossref PubMed Scopus (441) Google Scholar). Therefore, we considered whether RACK1 might also be associated with αIIbβ3. Washed human platelets were allowed to adhere for 30 min to immobilized fibrinogen or incubated in suspension over BSA. During this time, adherent platelets reorganize their actin cytoskeletons and spread to varying degrees (34Leng L. Kashiwagi H. Ren X-D. Shattil S.J. Blood. 1998; 91: 4206-4215Crossref PubMed Google Scholar). After washing and cell lysis in buffer containing Nonidet P-40 detergent, αIIbβ3 immunoprecipitates were probed for the presence of RACK1 by Western blotting. αIIbβ3 associated with RACK1 whether platelets were in suspension or adherent to fibrinogen (Fig. 1B). Therefore, potential relationships among αIIbβ3, RACK1, and PKC were assessed. Interactions between αIIbβ3and PKC in Platelets—As reported previously (14Baldassare J.J. Henderson P.A. Burns D. Loomis C. Fisher G.J. J. Biol. Chem. 1992; 267: 15585-15590Abstract Full Text PDF PubMed Google Scholar, 15Grabarek J. Raychowdhury M. Ravid K. Kent K.C. Newman P.J. Ware J.A. J. Biol. Chem. 1992; 267: 10011-10017Abstract Full Text PDF PubMed Google Scholar, 16Khan W.A. Blobe G. Halpern A. Taylor W. Wetsel W.C. Burns D. Loomis C. Hannun Y.A. J. Biol. Chem. 1993; 268: 5063-5068Abstract Full Text PDF PubMed Google Scholar, 17Wang F. Naik U.P. Ehrlich Y.H. Osada S. Ohno S. Kornecki E. Biochem. J. 1995; 311: 401-406Crossref PubMed Scopus (22) Google Scholar, 18Hillen T.J. Aroor A.R. Shukla S.D. Biochem. Biophys. Res. Commun. 2001; 280: 259-264Crossref PubMed Scopus (9) Google Scholar, 19Yoshioka A. Shirakawa R. Nishioka H. Tabuchi A. Higashi T. Ozaki H. Yamamoto A. Kita T. Horiuchi H. J. Biol. Chem. 2001; 276: 39379-39385Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 20Crosby D. Poole A.W. J. Biol. Chem. 2002; 277: 9958-9965Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 21Crosby D. Poole A.W. J. Biol. Chem. 2003; 278: 24533-24541Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), two classical PKC isoforms (PKCα and PKCβ) and two novel isoforms (PKCδ and PKCθ) were detected readily in human platelets, whereas other isoforms were less prominent or undetectable with the antibodies used (Fig. 2A). Of the PKC isoforms detected, only PCKβ was inducibly associated with αIIbβ3 in response to platelet adhesion to fibrinogen (Fig. 2B). In addition, when platelets were incubated in suspension for 10 min with 200 nm phorbol 12-myristate 13-acetate (PMA) to activate PKC, PKCβ became associated with αIIbβ3 (Fig. 2B). Similar results were obtained if platelets were stimulated instead with 50 μm PAR1 thrombin receptor-activating peptide (SSFLRN) or if murine platelets were used instead of human platelets (data not shown). There are two splice variants of PKCβ, PCKβI and PKCβII, in which the sequences differ in the C-terminal V5 region (37Kawakami T. Kawakami Y. Kitaura J. J. Biochem. (Tokyo). 2002; 132: 677-682Crossref PubMed Scopus (57) Google Scholar). Human platelets are reported to contain both variants (16Khan W.A. Blobe G. Halpern A. Taylor W. Wetsel W.C. Burns D. Loomis C. Hannun Y.A. J. Biol. Chem. 1993; 268: 5063-5068Abstract Full Text PDF PubMed Google Scholar), and we could detect both in human and mouse platelets with variant-specific antibodies (data not shown). The platelet studies described below used an antibody that recognizes both variants of PKCβ. The inducible association of PKCβ with αIIbβ3 could be a direct consequence of fibrinogen binding and αIIbβ3 clustering (38Buensuceso C. de Virgilio M. Shattil S.J. J. Biol. Chem. 2003; 278: 15217-15224Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) or the result of additional signaling events stimulated during platelet spreading caused by co-stimulation with endogenous ADP (3Shattil S.J. Newman P.J. Blood. 2004; 104: 1606-1615Crossref PubMed Scopus (433) Google Scholar). However, the association of PKCβ with αIIbβ3 that was induced by platelet adhesion to fibrinogen was not affected by the presence of 2 μm apyrase, an amount of enzyme sufficient to remove any ADP that might be released from the platelets (Fig. 2B). In addition, soluble fibrinogen binding to platelets in suspension was induced with 0.5 mm MnCl2, which activates αIIbβ3 directly. Under these conditions, PKCβ became associated with αIIbβ3 as early as 30 s after fibrinogen binding, the earliest time point tested, and the association was stable for up to 20 min (Fig. 2C). This association was maintained even if platelets were pretreated with 10 μm latrunculin A to block actin polymerization (data not shown). However, the interaction was blocked by 2 mm RGDS, a competitive inhibitor peptide of fibrinogen binding to αIIbβ3 (Fig. 2C), and it was not observed whether fibrinogen was omitted from the incubation mixture. Thus, the recruitment of PKCβ to αIIbβ3 is caused by fibrinogen binding and is not the result of additional signaling events induced by ADP co-stimulation. Factors Regulating PKCβ Association with αIIbβ3—Recruitment of PKC to plasma membranes often coincides with PKC activation (12Newton A.C. Johnson J.E. Biochim. Biophys. Acta. 1998; 1376: 155-172Crossref PubMed Scopus (242) Google Scholar, 13Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Crossref PubMed Scopus (1345) Google Scholar). To test PKC activity associated with αIIbβ3, platelets were incubated with MnCl2 with or without fibrinogen for up to 20 min, and PKC activity was quantified in αIIbβ3 immunoprecipitates. Immunoprecipitates from control platelets incubated with MnCl2 or fibrinogen alone displayed relatively low PKC activity. However, αIIbβ3 immunoprecipitates from platelets incubated with MnCl2 and fibrinogen displayed a significant increase in PKC activity, even at 30 s (p ≤ 0.001). At 20 min, this increase was 9-fold over baseline, which was 25-30% of the response observed with maximal PKC activation by PMA, and it could be blocked by RGDS (Fig. 3A). In addition, the fibrinogen-dependent increase in the association of PKCβ with αIIbβ3 was blocked partially by p99, a cell-permeable pseudosubstrate peptide inhibitor of classical and novel PKCs (31Schechtman D. Mochly-Rosen D. Methods Enzymol. 2002; 345: 470-489Crossref PubMed Scopus (46) Google Scholar) and by bisindolylmaleimide I, a general PKC inhibitor (Fig. 3B). These results indicated that fibrinogen binding stimulates an increase in PKC activity associated with αIIbβ3 and suggest that the association of PKCβ with αIIbβ3 requires catalytically active PKCβ. The activity and subcellular localization of classical PKCs are influenced by phosphorylation and products of phospholipid hydrolysis, e.g. Ca2+ and diacylglycerol (12Newton A.C. Johnson J.E. Biochim. Biophys. Acta. 1998; 1376: 155-172Crossref PubMed Scopus (242) Google Scholar, 13Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Crossref PubMed Scopus (1345) Google Scholar). Because fibrinogen binding to platelets leads to both the activation of integrin-associated c-Src (5Arias-Salgado E.G. Lizano S. Sarker S. Brugge J.S. Ginsberg M.H. Shattil S.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13298-13302Crossref PubMed Scopus (441) Google Scholar) and the association of active PKCβ with αIIbβ3, functional relationships between PKC and c-Src were examined. In fibrinogen-adherent platelets, 2 μm SU6656, a selective inhibitor of Src family kinases, failed to block the interaction of PKCβ with αIIbβ3 (Fig. 4A), despite the fact that it adequately blocked c-Src activation, as assessed by tyrosine phosphorylation of c-Src activation loop Tyr-418 (Fig. 4B). Furthermore, the inhibition of PKC by 12 μm bisindolylmaleimide I failed to block the activation of integrin-associated c-Src (data not shown). Platelet and αIIbβ3Responses Dependent on PKCβ—Prominent responses following platelet adhesion to fibrinogen include reorganization of the actin cytoskeleton and spreading (34Leng L. Kashiwagi H. Ren X-D. Shattil S.J. Blood. 1998; 91: 4206-4215Crossref PubMed Google Scholar). Forty-five minutes after plating on fibrinogen, normal human platelets were generally well spread and exhibited prominent F-actin cables and a peripheral distribution of phosphotyrosine-containing proteins (Fig. 5B). In contrast, platelets incubated with the cell-permeable p99 peptide to inhibit classical and novel PKCs attached to fibrinogen and displayed filopodia but largely failed to reorganize their cytoskeletons, develop a peripheral distributi
Referência(s)