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• W2025827931 abstract "Collagen-related peptide (CRP), a collagen homologue, induces platelet activation through a tyrosine kinase-dependent pathway, leading to sequential tyrosine phosphorylation of Fc receptor (FcR) γ-chain, Syk, and phospholipase C-γ2. Here we report that CRP and the platelet low affinity immune receptor FcγRIIA stimulate tyrosine phosphorylation of the T cell adapter SLP-76, whereas the G protein-coupled receptor agonist thrombin induces only minor tyrosine phosphorylation. This suggests that SLP-76 has a specific role downstream of receptors that signal via an immunoreceptor tyrosine-based activation motif. Immunoprecipitation studies demonstrate association of SLP-76 with SLAP-130, Vav, Fyn, Lyn, and the FcR γ-chain in CRP-stimulated platelets. Several of these proteins, including SLP-76, undergo tyrosine phosphorylation in in vitro kinase assays performed on SLP-76 immunoprecipitates. Tyrosine phosphorylation of all of these proteins in the in vitro kinase assay was abrogated by the Src family kinase inhibitor PP1, suggesting that it is mediated by either Fyn or Lyn. The physiological significance of this is uncertain, however, since tyrosine phosphorylation of SLP-76in vivo is not altered in either Fyn- or Lyn-deficient platelets. CRP stimulation of Syk-deficient platelets demonstrated thatin vivo tyrosine phosphorylation of SLP-76 is downstream of Syk. The absence of Syk in the SLP-76 immunoprecipitates raises the possibility that another protein is responsible for bringing SLP-76 to Syk. Candidates for this include those proteins that co-immunoprecipitate with SLP-76, including the FcR γ-chain. Tyrosine phosphorylation of PLC-γ2 and Ca2+mobilization is markedly attenuated in SLP-76-deficient platelets following CRP stimulation, suggesting that the adapter plays a critical role in the regulation of the phospholipase. The increase in tyrosine phosphorylation of SLAP-130 in response to CRP is also inhibited in SLP-76-deficient platelets, placing it downstream of SLP-76. This work identifies SLP-76 as an important adapter molecule that is regulated by Syk and lies upstream of SLAP-130 and PLC-γ2 in CRP-stimulated platelets. Collagen-related peptide (CRP), a collagen homologue, induces platelet activation through a tyrosine kinase-dependent pathway, leading to sequential tyrosine phosphorylation of Fc receptor (FcR) γ-chain, Syk, and phospholipase C-γ2. Here we report that CRP and the platelet low affinity immune receptor FcγRIIA stimulate tyrosine phosphorylation of the T cell adapter SLP-76, whereas the G protein-coupled receptor agonist thrombin induces only minor tyrosine phosphorylation. This suggests that SLP-76 has a specific role downstream of receptors that signal via an immunoreceptor tyrosine-based activation motif. Immunoprecipitation studies demonstrate association of SLP-76 with SLAP-130, Vav, Fyn, Lyn, and the FcR γ-chain in CRP-stimulated platelets. Several of these proteins, including SLP-76, undergo tyrosine phosphorylation in in vitro kinase assays performed on SLP-76 immunoprecipitates. Tyrosine phosphorylation of all of these proteins in the in vitro kinase assay was abrogated by the Src family kinase inhibitor PP1, suggesting that it is mediated by either Fyn or Lyn. The physiological significance of this is uncertain, however, since tyrosine phosphorylation of SLP-76in vivo is not altered in either Fyn- or Lyn-deficient platelets. CRP stimulation of Syk-deficient platelets demonstrated thatin vivo tyrosine phosphorylation of SLP-76 is downstream of Syk. The absence of Syk in the SLP-76 immunoprecipitates raises the possibility that another protein is responsible for bringing SLP-76 to Syk. Candidates for this include those proteins that co-immunoprecipitate with SLP-76, including the FcR γ-chain. Tyrosine phosphorylation of PLC-γ2 and Ca2+mobilization is markedly attenuated in SLP-76-deficient platelets following CRP stimulation, suggesting that the adapter plays a critical role in the regulation of the phospholipase. The increase in tyrosine phosphorylation of SLAP-130 in response to CRP is also inhibited in SLP-76-deficient platelets, placing it downstream of SLP-76. This work identifies SLP-76 as an important adapter molecule that is regulated by Syk and lies upstream of SLAP-130 and PLC-γ2 in CRP-stimulated platelets. Src homology 2 collagen-related peptide gluthationeS-transferase Fc receptor phospholipase C-γ T cell receptor polyacrylamide gel electrophoresis monoclonal antibody polyvinylidene difluoride glycoprotein VI SLP-76 (SH21domain-containing leukocyte protein of76 kDa) was identified by association with the SH3 domain of the Grb2 adapter protein in T cells and becomes tyrosine-phosphorylated upon T cell receptor (TCR) stimulation (1Jackman J.K. Motto D.G. Sun Q. Tanemoto M. Turck C.W. Peltz G.A. Koretzky G.A. Findell P.R. J. Biol. Chem. 1995; 270: 7029-7032Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). SLP-76 has three potential tyrosine phosphorylation sites within its amino terminus region: Tyr113, Tyr128, and Tyr145. Tyr113 and Tyr128 have a consensus binding site for the SH2 domain of Vav (DYESP) (2Onodera H. Motto D.G. Koretzky G.A. Rothstein D.M. J. Biol. Chem. 1996; 271: 22225-22230Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 3Tuosto L. Michel F. Acuto O. J. Exp. Med. 1996; 184: 1161-1166Crossref PubMed Scopus (173) Google Scholar, 4Wu J. Motto D.G. Koretzky G.A. Weiss A. Immunity. 1996; 4: 593-602Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 5Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 155-164Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) and are heavily tyrosine-phosphorylated following TCR engagement, whereas Tyr145, which falls in the sequence DYEPP, is phosphorylated to a lesser extent (6Fang N. Motto D.G. Ross S.E. Koretzky G.A. J. Immunol. 1996; 157: 3769-3773PubMed Google Scholar). SLP-76 also contains a central proline-rich region that mediates the association with Grb2 (7Motto D.G. Ross S.E. Wu J. Hendricks-Taylor L.R. Koretzky G.A. J. Exp. Med. 1996; 183: 1937-1943Crossref PubMed Scopus (181) Google Scholar) and a carboxyl-terminal SH2 domain that binds to at least two tyrosine-phosphorylated proteins, SLAP-130 (SLP-76-associated phosphoprotein of 130 kDa) (8Musci M.A. Hendricks-Taylor L.R. Motto D.G. Paskind M. Kamens J. Turck C.W. Koretzky G.A. J. Biol. Chem. 1997; 272: 11674-11677Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), a 62-kDa protein, and an uncharacterized serine/threonine kinase after TCR engagement (7Motto D.G. Ross S.E. Wu J. Hendricks-Taylor L.R. Koretzky G.A. J. Exp. Med. 1996; 183: 1937-1943Crossref PubMed Scopus (181) Google Scholar). SLP-76 is believed to be an essential adapter protein in T cells. Overexpression of SLP-76 results in an enhancement of TCR-mediated induction of nuclear factor of activated T cell and interleukin-2 promoter activity (3Tuosto L. Michel F. Acuto O. J. Exp. Med. 1996; 184: 1161-1166Crossref PubMed Scopus (173) Google Scholar, 5Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 155-164Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 6Fang N. Motto D.G. Ross S.E. Koretzky G.A. J. Immunol. 1996; 157: 3769-3773PubMed Google Scholar, 7Motto D.G. Ross S.E. Wu J. Hendricks-Taylor L.R. Koretzky G.A. J. Exp. Med. 1996; 183: 1937-1943Crossref PubMed Scopus (181) Google Scholar, 9Wardenburg J.B. Fu C. Jackman J.K. Flotow H. Wilkinson S.E. Williams D.H. Johnson R. Kong G. Chan A.C. Findell P.R. J. Biol. Chem. 1996; 271: 19641-19644Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar,10Musci M.A. Motto D.G. Ross S.E. Fang N. Koretzky G.A. J. Immunol. 1997; 159: 1639-1647PubMed Google Scholar). More recently, lack of expression of SLP-76 in Jurkat cells demonstrated that SLP-76 is necessary for tyrosine phosphorylation of phospholipase C-γ1 (PLC-γ1) and activation of the Ras pathway (11Yablonski D. Kuhne M.R. Kadlecek T. Weiss A. Science. 1998; 281: 413-416Crossref PubMed Scopus (357) Google Scholar). Moreover, SLP-76 is required for normal thymocyte development, since SLP-76 knock-out mice lack peripheral T cells (12Clements J.L. Yang B. Ross-Barta S.E. Eliason S.L. Hrstka R.F. Williamson R.A. Koretzky G.A. Science. 1998; 281: 416-419Crossref PubMed Scopus (363) Google Scholar, 13Pivniouk V. Tsitsikov E. Swinton P. Rathburn G. Alt F.W. Geha R.S. Cell. 1998; 94: 229-238Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). The three tyrosine phosphorylation sites, the proline-rich region, and the SH2 domain of SLP-76 have all been shown to be important for the regulation of T cell interleukin 2 production (10Musci M.A. Motto D.G. Ross S.E. Fang N. Koretzky G.A. J. Immunol. 1997; 159: 1639-1647PubMed Google Scholar). The inducible tyrosine phosphorylation of SLP-76 is mediated by ZAP-70 or Syk in COS cells (9Wardenburg J.B. Fu C. Jackman J.K. Flotow H. Wilkinson S.E. Williams D.H. Johnson R. Kong G. Chan A.C. Findell P.R. J. Biol. Chem. 1996; 271: 19641-19644Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar) and rat basophilic leukemia cells (14Hendricks-Taylor L.R. Motto D.G. Zhang J. Siraganian R.P. Koretzky G.A. J. Biol. Chem. 1997; 272: 1363-1367Crossref PubMed Scopus (65) Google Scholar), respectively. The mechanism by which SLP-76 is phosphorylated by ZAP-70 or Syk is not known. We have previously reported the association of tyrosine-phosphorylated SLP-76 with the SH3 domain of Grb2 in platelets in response to stimulation of the low affinity IgG immunoreceptor FcγRIIA (15Robinson A. Gibbins J. Rodrı́guez-Liñares B. Finan P.M. Wilson L. Kellie S. Findell P. Watson S.P. Blood. 1996; 88: 522-530Crossref PubMed Google Scholar). Increasing evidence suggests that the collagen receptor underlying the major increase in tyrosine phosphorylation in platelets also signals like an immune receptor. The collagen receptor is believed to comprise a multimeric structure, containing the glycoprotein VI (GPVI), and the Fc receptor (FcR) γ-chain (16Gibbins J. Asselin J. Farndale R. Barnes M. Law C.-L. Watson S. J. Biol. Chem. 1996; 271: 18095-18099Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 17Polgár J. Clemetson J.M. Kehrel B.E. Wiedemann M. Magnenat E.M. Wells T.N.C. Clemetson K.J. J. Biol. Chem. 1997; 272: 13576-13583Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 18Gibbins J.M. Okuma M. Farndale R. Barnes M. Watson S.P. FEBS Lett. 1997; 413: 255-259Crossref PubMed Scopus (261) Google Scholar, 19Francischetti I.M.B. Ghazaleh F.A. Reis R.A.M. Carlini C.R. Guimaraes J.A. Arch. Biochem. Biophys. 1998; 353: 239-250Crossref PubMed Scopus (26) Google Scholar). Binding of collagen to GPVI induces tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motif in the cytoplasmic tail of FcR γ-chain (18Gibbins J.M. Okuma M. Farndale R. Barnes M. Watson S.P. FEBS Lett. 1997; 413: 255-259Crossref PubMed Scopus (261) Google Scholar), leading to tyrosine phosphorylation of Syk and PLC-γ2(20Poole A. Gibbins J.M. Turner M. van Vugt M. van de Winkel J.G.J. Saito T. Tybulewicz V.L.J. Watson S.P. EMBO J. 1997; 16: 2333-2341Crossref PubMed Scopus (398) Google Scholar). SLP-76 was recently reported to be a crucial adapter protein in collagen-stimulated platelets, since aggregation and tyrosine phosphorylation of PLC-γ2 in response to collagen is abolished in SLP-76-deficient platelets. 2Clements, J. L., Lee, J. Ran, Gross, B., Yang, B., Olson, J. D., Sandra, A., Watson, S., Lentz, S. R., and Koretzky, G. A., (1999) J. Clin. Invest. 103,19–25 In this study, we have investigated the mechanism of tyrosine phosphorylation of SLP-76 and the function of SLP-76 in platelets following stimulation by a collagen-related peptide (CRP), through the specific binding to GPVI (22Kehrel B. Wierwille S. Clemetson K.J. Anders O. Steiner M. Knight C.G. Farndale R.W. Okuma M. Barnes M.J. Blood. 1998; 91: 491-499Crossref PubMed Google Scholar). CRP is a synthetic, triple helical peptide composed of Gly-Pro-hydroxyproline repeats, cross-linked by cysteines at the N and C termini (23Morton L.F. Hargreave P.G. Farndale R.W. Young R.D. Barnes M.J. Biochem. J. 1995; 306: 337-344Crossref PubMed Scopus (285) Google Scholar). CRP activates platelets through the GPVI but, in contrast to collagen, is unable to bind the integrin α2β1 (23Morton L.F. Hargreave P.G. Farndale R.W. Young R.D. Barnes M.J. Biochem. J. 1995; 306: 337-344Crossref PubMed Scopus (285) Google Scholar, 24Asselin J. Gibbins J. Achison M. Lee Y.H. Morton L.F. Farndale R.W. Barnes M.J. Watson S.P. Blood. 1997; 89: 1235-1242Crossref PubMed Google Scholar). It is a more powerful agonist than collagen and exhibits less variation in response between individuals. A CRP (GCP*(GPP)10GCP*G; single amino acid code P* represents hydroxyproline) was cross-linked via cysteine residues as described previously (23Morton L.F. Hargreave P.G. Farndale R.W. Young R.D. Barnes M.J. Biochem. J. 1995; 306: 337-344Crossref PubMed Scopus (285) Google Scholar); CRP was kindly donated by Dr. M. Barnes (Cambridge, UK). Collagen (native collagen fibrils from equine tendons) was from Nycomed (Munich, Germany). FcγRIIA-specific monoclonal antibody (mAb) was purchased from Madarex Inc (Annandale, NJ). Sheep F(ab′)2 raised against mouse IgG (M-1522) and thrombin were purchased from Sigma (Poole, UK). Fura-2/AM was from Molecular Probes, Inc. (Eugene, OR). Anti-phosphotyrosine mAb 4G10 was purchased from Upstate Biotechnology, Inc. (TCS Biologicals Ltd., Botolph Claydon, UK); polyclonal anti-Lyn antibody, Lyn(44), and polyclonal anti-Fyn antibody, Fyn(FYN3), were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-SLP-76 mAb and the GST-SLP-SH2 were described previously (1Jackman J.K. Motto D.G. Sun Q. Tanemoto M. Turck C.W. Peltz G.A. Koretzky G.A. Findell P.R. J. Biol. Chem. 1995; 270: 7029-7032Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar), and polyclonal anti-SLAP-130 rabbit antiserum was described previously (8Musci M.A. Hendricks-Taylor L.R. Motto D.G. Paskind M. Kamens J. Turck C.W. Koretzky G.A. J. Biol. Chem. 1997; 272: 11674-11677Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Polyclonal anti-FcR γ-chain rabbit antiserum was a gift from Dr. J. P. Kinet (Beth Israel Hospital, Boston, MA). PP1 (CP118,556) was kindly donated by Dr. J. Hanke (Pfizer Central Research, CT). The Syk-deficient mice and murine anti-Syk antiserum were described previously (25Turner M. Mee P.J. Costello P.S. Williams O. Price A.A. Dubby L.P. Furlong M.T. Geahlen R.L. Tybulewicz V.L.T. Nature. 1997; 378: 298-302Crossref Scopus (650) Google Scholar). The SLP-76 knock-out mice were previously described (12Clements J.L. Yang B. Ross-Barta S.E. Eliason S.L. Hrstka R.F. Williamson R.A. Koretzky G.A. Science. 1998; 281: 416-419Crossref PubMed Scopus (363) Google Scholar). The Lyn knock-out mice were a gift from Dr. A. Dunn (Ludwig Institute, Melbourne, Australia) (26Hibbs M. Tarlinton D.M. Armes J. Grail D. Hodgson G. Maglitto R. Stacker S.A. Dunn A.R. Cell. 1995; 83: 301-311Abstract Full Text PDF PubMed Scopus (631) Google Scholar). The Fyn knock-out mice were purchased from Jackson Laboratories (Bar Harbor, ME). Human platelets were isolated from blood taken on the day of the experiment as described previously (27Blake R.A. Walker T.R. Watson S.P. Biochem. J. 1993; 290: 471-475Crossref PubMed Scopus (61) Google Scholar). Mouse platelets were prepared as described previously (20Poole A. Gibbins J.M. Turner M. van Vugt M. van de Winkel J.G.J. Saito T. Tybulewicz V.L.J. Watson S.P. EMBO J. 1997; 16: 2333-2341Crossref PubMed Scopus (398) Google Scholar). Stimulations were performed at 37 °C in the presence of 1 mm EGTA and 10 μm indomethacin with continuous stirring at 1,200 rpm. Platelets were stimulated with 3 μg/ml CRP, 30 μg/ml collagen, or 1 unit/ml thrombin for 90 s. Platelets were stimulated via FcγRIIA using mAb IV.3 (1 μg/ml) for 1 min and then the cross-linker F(ab′)2 (30 μg/ml) for 90 s. Platelets were lysed with an equal volume of lysis buffer (2% Nonidet P-40, 300 mm NaCl, 20 mmTris, 10 mm EDTA containing 2 mmNa3VO4, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 μg/ml pepstatin A, pH 7.3). Insoluble cell debris was removed by centrifugation. Cell lysates were precleared with glutathione-agarose or Protein A-Sepharose for GST precipitation and immunoprecipitation, respectively. For some experiments, antibodies were covalently linked to Protein A-Sepharose as described previously (28Schneider C. Newman R.A. Sutherland D.R. Asser U. Greaves M.F. J. Biol. Chem. 1982; 257: 10766-10769Abstract Full Text PDF PubMed Google Scholar). For GST precipitation, lysates were incubated with 5 μg of fusion protein immobilized on agarose. Endogenous SLP-76 was immunoprecipitated using 4 μg of anti-SLP-76 mAb. The resulting protein complexes and immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. Immunoblotting was carried out as described previously (24Asselin J. Gibbins J. Achison M. Lee Y.H. Morton L.F. Farndale R.W. Barnes M.J. Watson S.P. Blood. 1997; 89: 1235-1242Crossref PubMed Google Scholar) with protein detection by enhanced chemiluminescence. Sequential immunoprecipitation was performed following kinase assay as described previously (29Mizuno K. Katagiri T. Hasegawa K. Ogimoto M. Yakura H. J. Exp. Med. 1996; 184: 457-463Crossref PubMed Scopus (41) Google Scholar). Protein immunoprecipitations or precipitations were submitted to kinase assay as described (16Gibbins J. Asselin J. Farndale R. Barnes M. Law C.-L. Watson S. J. Biol. Chem. 1996; 271: 18095-18099Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Proteins were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were treated with 1 m KOH for 1 h at 55 °C to cleave serine/threonine phosphorylation and then subjected to autoradiography and immunoblotting. Wild type SLP-76 (1Jackman J.K. Motto D.G. Sun Q. Tanemoto M. Turck C.W. Peltz G.A. Koretzky G.A. Findell P.R. J. Biol. Chem. 1995; 270: 7029-7032Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar) and the triple mutant SLP-76 YYY/FFF (5Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 155-164Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) were used as template for polymerase chain reaction using the following primers: SLP-TyrF (5′-ATT GGA TCC GGG GGT TGG TCG TCC TTT GAA-3′) and SLP-TyrR (5′-ATTCCC GGG GCT TCC TCG TCA TTG GAG GG-3′). The polymerase chain reaction products were expressed in pGEX-2T (Amersham Pharmacia Biotech, St. Albans, UK) as described previously (30Frangioni J.V. Neel B.G. Anal. Biochem. 1993; 210: 179-187Crossref PubMed Scopus (833) Google Scholar). Mouse platelets (108 cells/ml) were loaded with 1 μm Fura-2/AM for 30 min at 37 °C in RPMI 1640 medium containing 1% fetal calf serum and resuspended at 2 × 108 cells/ml in Hepes-buffered salt solution (135 mm NaCl, 5 mm KCl, 10 mm Hepes, 1.2 mm CaCl2, 1.2 mm MgCl2) containing 1% fetal calf serum. Stimulation and measurements were performed at 37 °C. Cytosolic Ca2+ levels were measured as described previously (31Rodrı́guez-Liñares B. Watson S.P. Biochem. J. 1996; 316: 93-98Crossref PubMed Scopus (61) Google Scholar). The intracellular Ca2+concentration was estimated using the equation described by Grynkiewiczet al. (32Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar): (R −R min)/(R max −R) × K, where K represents the valueK d × Sf2/Sb2. The experimentally determined value of K used in this study is 3.6 μm. We have previously reported that FcγRIIA cross-linking stimulates marked tyrosine phosphorylation of SLP-76 in human platelets (15Robinson A. Gibbins J. Rodrı́guez-Liñares B. Finan P.M. Wilson L. Kellie S. Findell P. Watson S.P. Blood. 1996; 88: 522-530Crossref PubMed Google Scholar), a result that is confirmed in the present study (Fig.1 A). Collagen was also observed to stimulate marked tyrosine phosphorylation of SLP-76, in agreement with the observation in mouse platelets,2 whereas the G protein-coupled receptor agonist thrombin induced only a low level of tyrosine phosphorylation of SLP-76 (Fig. 1 A). CRP, a synthetic peptide based on the triple-helical structure of collagen, also stimulated tyrosine phosphorylation of SLP-76 (Fig.1 A). Reprobing the membrane for SLP-76 to check loading revealed that the anti-SLP-76 antibody has a better affinity for the unphosphorylated form of SLP-76 (Fig. 1 A). Further studies were performed with CRP, since this is a powerful agonist for GPVI but is unable to bind to α2β1 (23Morton L.F. Hargreave P.G. Farndale R.W. Young R.D. Barnes M.J. Biochem. J. 1995; 306: 337-344Crossref PubMed Scopus (285) Google Scholar, 24Asselin J. Gibbins J. Achison M. Lee Y.H. Morton L.F. Farndale R.W. Barnes M.J. Watson S.P. Blood. 1997; 89: 1235-1242Crossref PubMed Google Scholar). Tyrosine phosphorylation of SLP-76 by CRP occurred within 10 s and reached a maximum at 60 s, being sustained for up to 10 min (Fig. 1 B). The time course showing tyrosine phosphorylation of total platelet protein indicated that a major band of 75 kDa displayed the same tyrosine phosphorylation pattern as SLP-76 (not shown). SLP-76 was identified as a component of this band. We have previously shown that Syk is also a component of this band. Pretreatment of platelets with the Ca2+ chelator bis(O-aminophenoxy)-N,N,N′,N′-tetraacetic acid and the protein kinase C antagonist Ro 31-8220 to inhibit the action of the second messengers produced by PLC-γ2indicated that tyrosine phosphorylation of SLP-76 is independent of PLC-γ2 activation (Fig. 1 A). SLP-76 co-immunoprecipitated with a tyrosine phosphoprotein of 130 kDa following stimulation by CRP, collagen, and thrombin (Fig. 1 A). Cross-linking of FcγRIIA only induces a small increase in phosphorylation of this 130-kDa phosphoprotein. SLAP-130, which associates with the SH2 domain of SLP-76 following TCR engagement (8Musci M.A. Hendricks-Taylor L.R. Motto D.G. Paskind M. Kamens J. Turck C.W. Koretzky G.A. J. Biol. Chem. 1997; 272: 11674-11677Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), and PLC-γ2, the only PLC-γ isoform to be tyrosine-phosphorylated in CRP and collagen-stimulated platelets (24Asselin J. Gibbins J. Achison M. Lee Y.H. Morton L.F. Farndale R.W. Barnes M.J. Watson S.P. Blood. 1997; 89: 1235-1242Crossref PubMed Google Scholar, 33Blake R.A. Schieven G.L. Watson S.P. FEBS Lett. 1994; 353: 212-216Crossref PubMed Scopus (127) Google Scholar), have a similar electrophoretic mobility to this tyrosine-phosphorylated protein of 130 kDa. However, subsequent immunoblotting with SLAP-130- or PLC-γ2-specific antibodies failed to identify conclusively the 130-kDa co-precipitated protein, possibly because the level of protein was below that of the sensitivity of detection. The presence of SLAP-130 in the 130-kDa band was subsequently shown by sequential immunoprecipitations as described below. Immunoprecipitation of SLP-76 using an antibody covalently linked to Protein A-Sepharose revealed the presence of a tyrosine-phosphorylated band of approximately 55 kDa (Fig. 1 C) that was sometimes resolved as a doublet. This band was hidden by the IgG heavy chain band under standard immunoprecipitation conditions. The 55-kDa band was present in resting platelets, and its level of tyrosine phosphorylation increased with stimulation of platelets by CRP. It was identified as the tyrosine kinase Lyn by immunoblotting (Fig. 1 C). There was a small increase in association with SLP-76 following CRP stimulation, although this was less than the increase in tyrosine phosphorylation. Immunoblotting for Grb2 revealed the presence of a similar level of the adapter protein co-immunoprecipitating with SLP-76 in resting and CRP-stimulated platelets (not shown). This is likely to be mediated through the SH3 domain of Grb2 as previously shown in FcγRIIA-stimulated platelets (15Robinson A. Gibbins J. Rodrı́guez-Liñares B. Finan P.M. Wilson L. Kellie S. Findell P. Watson S.P. Blood. 1996; 88: 522-530Crossref PubMed Google Scholar). We used a fusion protein containing the SH2 domain of SLP-76, GST-SLP-SH2, to locate the binding site of Lyn and the component(s) of the 130-kDa protein. GST alone, used as control, did not bind any tyrosine-phosphorylated proteins (Fig.2). GST-SLP-SH2 precipitates two major tyrosine-phosphorylated proteins of 55 and 130 kDa (Fig. 2) in resting platelets. Stimulation of platelets by CRP strongly increased the degree of tyrosine phosphorylation of 130-kDa protein that is precipitated by GST-SLP-SH2, whereas the level of phosphorylation of the 55-kDa protein underwent a small increase. Reprobing revealed Lyn and SLAP-130 as components of these bands, respectively (Fig. 2). There was a small increase in the association of Lyn and SLAP-130 in CRP-stimulated lanes (Fig. 2). Association of SLAP-130 to SLP-76 under basal conditions was also reported in T cells (8Musci M.A. Hendricks-Taylor L.R. Motto D.G. Paskind M. Kamens J. Turck C.W. Koretzky G.A. J. Biol. Chem. 1997; 272: 11674-11677Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Minor tyrosine-phosphorylated proteins of 90 and 75 kDa were also associated with GST-SLP-SH2 in CRP-stimulated platelets. The 75-kDa band was not identified as Syk or Btk by immunoblotting, although either or both may be below the detection sensitivity of the antibodies. Comparison of autoradiographs of kinase assays of immunoprecipitated SLP-76 before and after KOH treatment indicated the absence of serine/threonine kinase co-immunoprecipitating with SLP-76 in resting and CRP-stimulated platelets (not shown). Three major bands of 130, 75, and 55 were phosphorylated in SLP-76 immunoprecipitates from resting platelets (Fig.3 A). The three bands of 130, 75, and 55 kDa correspond to the major tyrosine-phosphorylated bands observed in SLP-76 immunoprecipitates when immunoblotted for phosphotyrosine (Fig. 3 A). The level of tyrosine phosphorylation of the 130-, 75-, and 55-kDa bands increased dramatically in SLP-76 immunoprecipitates from CRP-stimulated platelets. CRP stimulation also induced the appearance of three other minor tyrosine-phosphorylated bands of 90, 60, and 38 kDa following kinase assay. On a longer exposure, a doublet of 13/11.5 kDa could also be seen in CRP-stimulated samples (not shown). The 90-kDa band was also seen by anti-phosphotyrosine immunoblotting in Fig. 3 A, while the 60-kDa band could be seen in a longer exposure. The 75-kDa band was identified as SLP-76 by immunoblotting. The remaining proteins were identified through sequential immunoprecipitation. Followingin vitro kinase assay, the proteins co-immunoprecipitating with SLP-76 were dissociated by boiling in the presence of 2% SDS. Supernatant was diluted down to 0.1% SDS, and proteins from the supernatant were immunoprecipitated with specific antibodies. Components of the 13/11.5-, 55-, 60-, 90-, and 130-kDa radiolabeled bands were identified as FcR γ-chain (Fig.4 A), Lyn (Fig. 4 B), Fyn (Fig. 4 C), Vav (Fig. 4 D), and SLAP-130 (Fig.4 E), respectively. A more prominent association with Fyn was seen in some studies as illustrated in Fig.5. The weak band corresponding to Vav is likely to reflect both a low level of binding and the fact that it serves as a poor substrate in the in vitro kinase assay. Neither Syk nor PLC-γ2 were detected in these studies, suggesting that they do not associate with SLP-76 or that PLC-γ2 is not a substrate in the in vitrokinase assay.Figure 4FcR γ-chain, Lyn, Fyn, Vav, and SLAP-130 co-immunoprecipitate with SLP-76 following CRP stimulation. SLP-76 was immunoprecipitated as described in the legend of Fig. 3. Following an in vitro kinase assay, immunoprecipitated proteins were parted in lysis buffer containing 2% SDS. Proteins were heated, and subsequently the SDS concentration of the supernatant was reduced to 0.1% by the addition of Nonidet P-40 lysis buffer. Lysates were used for immunoprecipitation of FcR γ-chain (A), Lyn (B), Fyn (C), Vav (D), and SLAP-130 (E). Proteins were analyzed on 10% SDS-PAGE, expect for FcR γ-chain immunoprecipitation, which was resolved on 10–18% gradient SDS-PAGE. SDS-polyacrylamide gels were dried and exposed to autoradiography.View Large Image Figure ViewerDownload (PPT)Figure 5Effect of PP1 on in vitrokinase assay performed on SLP-76 immunoprecipitated from resting or CRP-stimulated platelets lysed in Nonidet P-40- or Brij 96-containing buffer. Stimulation of platelets with Tyrode-Hepes buffer or 3 μg/ml CRP was stopped after 90 s by the addition of Nonidet P-40 lysis buffer or Brij 96 lysis buffer. SLP-76 was immunoprecipitated and submitted to an in vitro kinase assay. When indicated, kinase assay was performed in the presence of 10 μm PP1. Proteins were resolved on 10–18% gradient SDS-PAGE and transferred to PVDF membranes. Membranes were treated with 1 m KOH for 1 h at 55 °C and submitted to autoradiography (upper panel). Equal loading was checked by immunoblotting the membranes using the anti-SLP-76 mAb (bottom panel).View Large Image Figure ViewerDownload (PPT) A kinase assay was also performed on proteins precipitated with GST-SLP-SH2. This showed a similar profile of kinase activity as SLP-76 immunoprecipitates, except for the presence of a 38-kDa protein, an uncharacterized doublet of 75 kDa and the lack of SLP-76 (Fig.3 B). The tyrosine-phosphorylated band of 38" @default.
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• W2025827931 date "1999-02-01" @default.
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• W2025827931 title "Tyrosine Phosphorylation of SLP-76 Is Downstream of Syk following Stimulation of the Collagen Receptor in Platelets" @default.
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