The Cytoplasmic Domain of the Platelet Glycoprotein Ibα Is Phosphorylated at Serine 609
1999; Elsevier BV; Volume: 274; Issue: 47 Linguagem: Inglês
10.1074/jbc.274.47.33474
ISSN1083-351X
AutoresRichard J. Bodnar, Minyi Gu, Zhenyu Li, Graham D. Englund, Xiaoping Du,
Tópico(s)14-3-3 protein interactions
ResumoThe α chain of the platelet von Willebrand factor receptor, glycoprotein (GP) Ib, is not known to be phosphorylated. Here, we report that the cytoplasmic domain of GPIbα is phosphorylated at Ser609; this was detected by immunoblotting with an anti-phosphopeptide antibody, anti-pS609, that specifically recognizes the GPIbα C-terminal sequence S606GHSL610 only when Ser609 is phosphorylated. Immunoabsorption with anti-pS609 removed almost all of the GPIbα from platelet lysates, indicating a high proportion of GPIbα phosphorylation. Anti-pS609 inhibited GPIb-IX binding to the intracellular signaling molecule, 14-3-3ζ. Dephosphorylation of GPIb-IX with potato acid phosphatase inhibited anti-pS609 binding and also 14-3-3ζ binding. A synthetic phosphopeptide corresponding to the GPIbα C-terminal sequence (SIRYSGHpSL), but not a nonphosphorylated identical peptide, abolished GPIb-IX binding to 14-3-3ζ. Thus, phosphorylation at Ser609 of GPIbα is important for 14-3-3ζ binding to GPIb-IX. In certain regions of spreading platelets, particularly at the periphery, there was a reduction in GPIbα staining by anti-pS609 as observed under a confocal microscope, indicating that a subpopulation of GPIbα molecules in these regions is dephosphorylated. These data suggest that phosphorylation and dephosphorylation at Ser609 of GPIbα regulates GPIb-IX interaction with 14-3-3 and may play important roles in the process of platelet adhesion and spreading. The α chain of the platelet von Willebrand factor receptor, glycoprotein (GP) Ib, is not known to be phosphorylated. Here, we report that the cytoplasmic domain of GPIbα is phosphorylated at Ser609; this was detected by immunoblotting with an anti-phosphopeptide antibody, anti-pS609, that specifically recognizes the GPIbα C-terminal sequence S606GHSL610 only when Ser609 is phosphorylated. Immunoabsorption with anti-pS609 removed almost all of the GPIbα from platelet lysates, indicating a high proportion of GPIbα phosphorylation. Anti-pS609 inhibited GPIb-IX binding to the intracellular signaling molecule, 14-3-3ζ. Dephosphorylation of GPIb-IX with potato acid phosphatase inhibited anti-pS609 binding and also 14-3-3ζ binding. A synthetic phosphopeptide corresponding to the GPIbα C-terminal sequence (SIRYSGHpSL), but not a nonphosphorylated identical peptide, abolished GPIb-IX binding to 14-3-3ζ. Thus, phosphorylation at Ser609 of GPIbα is important for 14-3-3ζ binding to GPIb-IX. In certain regions of spreading platelets, particularly at the periphery, there was a reduction in GPIbα staining by anti-pS609 as observed under a confocal microscope, indicating that a subpopulation of GPIbα molecules in these regions is dephosphorylated. These data suggest that phosphorylation and dephosphorylation at Ser609 of GPIbα regulates GPIb-IX interaction with 14-3-3 and may play important roles in the process of platelet adhesion and spreading. von Willebrand factor glycoprotein glycoprotein Ib-IX complex polyacrylamide gel electrophoresis maltose-binding protein potato acid phosphatase phosphoserine Platelet adhesion plays a critical role in thrombosis and hemostasis. Platelets in normal circulation are in a resting, nonadherent state. At sites of vascular injury, platelets adhere to the exposed subendothelial matrix. Under the high shear force of blood flow, platelet adhesion involves multiple steps. Initially, platelets adhere in a reversible manner (1Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar). This process is mediated by the interaction between a platelet receptor for von Willebrand factor (vWF),1 the glycoprotein Ib-IX complex (GPIb-IX), and matrix-bound vWF (1Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar, 2Sakariassen K.S. Bolhuis P.A. Sixma J.J. Nature. 1979; 279: 636-638Crossref PubMed Scopus (465) Google Scholar, 3Sakariassen K.S. Nievelstein P.F. Coller B.S. Sixma J.J. Br. J. Haematol. 1986; 63: 681-691Crossref PubMed Scopus (175) Google Scholar). GPIb-IX interaction with vWF mediates signaling leading to activation of integrins that are responsible for platelet spreading and aggregation (1Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar, 4Weiss H.J. Turitto V.T. Baumgartner H.R. Blood. 1986; 67: 322-330Crossref PubMed Google Scholar).GPIb-IX consists of three subunits: GPIbα, GPIbβ, and GPIX. GPIb-IX is loosely associated with glycoprotein V. The N-terminal domain of GPIbα contains binding sites for vWF and thrombin (for reviews see Refs. 5Ware J. Thromb. Haemost. 1998; 79: 466-478Crossref PubMed Scopus (66) Google Scholar and 6Lopez J.A. Blood Coagul. Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar). The cytoplasmic domain of GPIbα contains a binding site (residues 536–568 (7Andrews R.K. Fox J.E. J. Biol. Chem. 1992; 267: 18605-18611Abstract Full Text PDF PubMed Google Scholar)) for filamin (also calledactin-binding protein or ABP-280), which links GPIb-IX to cross-linked actin filamental structures underlying the plasma membrane (the membrane skeleton) (8Fox J.E.B. J. Biol. Chem. 1985; 260: 11970-11977Abstract Full Text PDF PubMed Google Scholar, 9Fox J.E.B. J. Clin. Invest. 1985; 76: 1673-1683Crossref PubMed Scopus (112) Google Scholar). We found that an intracellular signaling molecule, 14-3-3ζ, is associated with GPIb-IX (10Du X. Harris S.J. Tetaz T.J. Ginsberg M.H. Berndt M.C. J. Biol. Chem. 1994; 269: 18287-18290Abstract Full Text PDF PubMed Google Scholar). A binding site for 14-3-3ζ is located in a 15-amino acid residue serine-rich region (residues 595–610) at the C terminus of GPIbα (29Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). 14-3-3 binding also involves an additional 14-3-3 binding site in GPIbβ (11Andrews R.K. Harris S.J. McNally T. Berndt M.C. Biochemistry. 1998; 37: 638-647Crossref PubMed Scopus (120) Google Scholar, 12Calverley D.C. Kavanagh T.J. Roth G.J. Blood. 1998; 91: 1295-1303Crossref PubMed Google Scholar).The 14-3-3 family of highly conserved intracellular proteins interacts with several intracellular serine/threonine kinases and other signaling molecules (13Fu H. Xia K. Pallas D.C. Cui C. Conroy K. Narsimhan R.P. Mamon H. Collier R.J. Roberts T.M. Science. 1994; 266: 126-129Crossref PubMed Scopus (242) Google Scholar, 14Freed E. Symons M. Macdonald S.G. McCormick F. Ruggieri R. Science. 1994; 265: 1713-1716Crossref PubMed Scopus (352) Google Scholar, 15Fanti W.J. Muslin A.J. Kikuchi A. Martin J.A. MacNicol A.M. Gross R.W. Williams L.T. Nature. 1994; 371: 612-614Crossref PubMed Scopus (309) Google Scholar, 16Meller N. Liu Y.C. Collins T.L. Bonnefoy B.N. Baier G. Isakov N. Altman A. Mol. Cell. Biol. 1996; 16: 5782-5791Crossref PubMed Google Scholar, 17Acs P. Szallasi Z. Kazanietz M.G. Blumberg P.M. Biochem. Biophys. Res. Commun. 1995; 216: 103-109Crossref PubMed Scopus (45) Google Scholar, 18Conklin D.S. Galaktionov K. Beach D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7892-7896Crossref PubMed Scopus (245) Google Scholar, 19Bonnefoy B.N. Liu Y.C. von W.M. Sung A. Elly C. Mustelin T. Yoshida H. Ishizaka K. Altman A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10142-10146Crossref PubMed Scopus (133) Google Scholar, 20Furukawa Y. Ikuta N. Omata S. Yamauchi T. Isobe T. Ichimura T. Biochem. Biophys. Res. Commun. 1993; 194: 144-149Crossref PubMed Scopus (87) Google Scholar, 21Liu Y.-C. Elly C. Yoshida H. Bonnefoy-Beard B.N. Altman A. J. Biol. Chem. 1996; 271: 14591-14595Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 22Pallas D.C. Fu H. Haehnel L.C. Weller W. Collier R.J. Roberts T.M. Science. 1994; 265: 535-537Crossref PubMed Scopus (148) Google Scholar) and regulates their functions (15Fanti W.J. Muslin A.J. Kikuchi A. Martin J.A. MacNicol A.M. Gross R.W. Williams L.T. Nature. 1994; 371: 612-614Crossref PubMed Scopus (309) Google Scholar, 23Li S. Janosch P. Tanji M. Rosenfeld G.C. Waymire J.C. Mischak H. Kolch W. Sedivy J.M. EMBO J. 1995; 14: 685-696Crossref PubMed Scopus (154) Google Scholar, 24Tzivion G. Luo Z. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (386) Google Scholar, 25Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2241) Google Scholar, 26Ford J.C. al-Khodairy F. Fotou E. Sheldrick K.S. Griffiths D.J. Carr A.M. Science. 1994; 265: 533-535Crossref PubMed Scopus (298) Google Scholar). A recognition motif, RSXpSXP, has been identified in c-Raf and several other 14-3-3 ligands, requiring a phosphorylated serine residue (27Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar, 28Yaffe M.B. Rittinger K. Volinia S. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar). Thus, interaction of many intracellular signaling proteins with 14-3-3 is regulated by phosphorylation. We hypothesized previously that serine residues in the 14-3-3 binding site of GPIbα might be important for 14-3-3 binding (29Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). However, it is not clear whether 14-3-3 binding is regulated by phosphorylation of these serine residues, as 14-3-3 can interact with synthetic nonphosphorylated peptides corresponding to GPIbα cytoplasmic domain (11Andrews R.K. Harris S.J. McNally T. Berndt M.C. Biochemistry. 1998; 37: 638-647Crossref PubMed Scopus (120) Google Scholar, 29Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Further, GPIbα has been thought to be a nonphosphorylated protein because previous studies failed to show phosphorylation of GPIbα (30Wyler B. Bienz D. Clemetson K.J. Luscher E.F. Biochem. J. 1986; 234: 373-379Crossref PubMed Scopus (24) Google Scholar). In this study, we re-examined phosphorylation states of GPIbα using a phosphoserine-specific anti-GPIbα antibody. We report here that the cytoplasmic domain of GPIbα is phosphorylated at Ser609 and that phosphorylation at this site is important for 14-3-3 binding to intact platelet GPIb-IX. Furthermore, we show that GPIbα dephosphorylation occurs at the edge of spreading platelets, suggesting that phosphorylation and dephosphorylation of Ser609 in the cytoplasmic domain of GPIbα is involved in regulating GPIb-IX functions during platelet adhesion and spreading.DISCUSSIONIn this study, we provide the first evidence that GPIbα is phosphorylated and that a phosphorylation site is at Ser609. Previously, phosphorylation of platelet membrane proteins has been studied (30Wyler B. Bienz D. Clemetson K.J. Luscher E.F. Biochem. J. 1986; 234: 373-379Crossref PubMed Scopus (24) Google Scholar, 36Fox J.E. Reynolds C.C. Johnson M.M. J. Biol. Chem. 1987; 262: 12627-12631Abstract Full Text PDF PubMed Google Scholar). The only serine/threonine-phosphorylated major membrane glycoprotein identified was GPIbβ (30Wyler B. Bienz D. Clemetson K.J. Luscher E.F. Biochem. J. 1986; 234: 373-379Crossref PubMed Scopus (24) Google Scholar). GPIbβ is phosphorylated at Thr166 when platelets are stimulated with agents that enhance intracellular cAMP level (36Fox J.E. Reynolds C.C. Johnson M.M. J. Biol. Chem. 1987; 262: 12627-12631Abstract Full Text PDF PubMed Google Scholar, 37Wardell M.R. Reynolds C.C. Berndt M.C. Wallace R.W. Fox J.E. J. Biol. Chem. 1989; 264: 15656-15661Abstract Full Text PDF PubMed Google Scholar). However, previous studies used a 32P labeling technique to detect protein phosphorylation, which is dependent upon incorporation of exogenous 32P into the phosphorylated proteins and thus is not sensitive to phosphoproteins that are protected from dephosphorylation or rephosphorylation during the procedure. In this study, we used an anti-phosphopeptide antibody, anti-pS609, recognizing the SGHpSL sequence at the C terminus of GPIbα but not the nonphosphorylated peptide. The phosphorylation-dependent antibody can be used to detect protein phosphorylation whether or not phosphorylation is metabolically active and thus is capable of detecting GPIbα phosphorylation that has not been detected by the 32P labeling technique. Use of the phosphorylation-dependent antibody also enabled us to detect phosphorylation at a specific residue. Two serine residues (Ser606 and Ser609) are present at the C-terminal SGHSL region of GPIbα, which is important for 14-3-3 binding (29Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). We showed that the phosphoserine 609-specific antibody, anti-pS609, specifically bound to platelet GPIbα and that its binding was inhibited by dephosphorylation of GPIb-IX with potato acid phosphatase. In addition, an antibody raised against the phosphorylated Ser606-containing sequence (SIRYpSGH) did not react with GPIbα from resting platelets (data not shown). These data indicates that the C-terminal domain of GPIbα is phosphorylated at Ser609.Phosphorylation states of proteins are balanced by the actions of protein kinases and phosphatases. In platelet lysates, the percentage of phosphorylated GPIbα in the whole GPIb-IX population is high, as indicated by the removal of nearly all GPIb-IX by the anti-phosphopeptide antibody (Fig. 3 A); this suggests that balance under these conditions is tilted toward phosphorylation of GPIbα. Thus, it appears that, unlike many other phosphoproteins, the default state of GPIbα is a phosphorylated state. One possible mechanism for this default phosphorylation state is that 14-3-3 may play a protective role, since the phosphoserine 609 is located in the 14-3-3 binding site. 14-3-3 has previously been shown to protect 14-3-3 ligands from dephosphorylation (38Dent P. Jelinek T. Morrison D.K. Weber M.J. Sturgill T.W. Science. 1995; 268: 1902-1906Crossref PubMed Scopus (172) Google Scholar). The protein kinase that catalyzes phosphorylation of GPIbα remains to be identified. Several protein kinase inhibitors had no effect on GPIbα phosphorylation, including inhibitors of protein kinase A, protein kinase G, and protein kinase C (data not shown). It is thus possible that these kinases are not involved in GPIbα phosphorylation. However, as the default state of GPIbα appears to be a phosphorylated form, it is also possible that the ineffectiveness of these protein kinase inhibitors is due to the fact that GPIbα is already in a relatively stable phosphorylated state and thus immune to the effects of protein kinase inhibitors.Phosphorylation at Ser609 of GPIbα is important for GPIb-IX interaction with 14-3-3. This conclusion is supported by our finding that the Ser609-phosphorylated GPIbα C-terminal domain peptide (SIRYSGHpSL), but not the identical nonphosphorylated or Ser606-phosphorylated peptides, inhibited GPIb-IX interaction with 14-3-3 in a concentration-dependent manner (Fig. 6), suggesting that interaction between GPIb-IX and 14-3-3 involves a binding site in 14-3-3 that interacts with the Ser609-phosphorylated GPIbα C-terminal sequence. This result is consistent with the previous result of Andrews et al. (11Andrews R.K. Harris S.J. McNally T. Berndt M.C. Biochemistry. 1998; 37: 638-647Crossref PubMed Scopus (120) Google Scholar) showing that a nonphosphorylated GPIbα C-terminal peptide failed to abolish the binding between 14-3-3 and GPIb-IX. Furthermore, dephosphorylation of GPIb-IX by PAP or preincubation with anti-pS609 inhibited 14-3-3 binding (Figs. 4 and 5). Thus, it is likely that high affinity interaction between the intact platelet GPIb-IX and 14-3-3 requires phosphorylation of Ser609 of GPIbα. It is interesting to note that the 14-3-3 binding site of GPIbα (RYSGHSL) shares similarities with the RSXpSXP-like motifs of other phosphorylated 14-3-3 ligands; they all contain an arginine and a serine at the N-terminal side of the phosphorylated serine (27Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar,28Yaffe M.B. Rittinger K. Volinia S. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar). Most of the identified RSXpSXP motifs are present in the middle of the protein sequence, and the proline in the motif may possibly form a turn exposing the phosphoserine. Because the 14-3-3 binding site in GPIbα is already exposed at the C terminus, it may not require the presence of a proline residue. However, despite the similarities, there are striking differences between GPIbα and the RSXpSXP-like ligands. The prototype RSXpSXP-like ligand of 14-3-3, c-Raf, requires the helix G region of 14-3-3 (33Gu M. Du X. J. Biol. Chem. 1998; 273: 33465-33471Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), and the crystal structure data suggest that phosphoserine in the RSXpSXP motif may interact with residues in the more N-terminal helix C and E region of 14-3-3 (28Yaffe M.B. Rittinger K. Volinia S. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar, 39Zhang L. Wang H. Liu D. Liddington R. Fu H. J. Biol. Chem. 1997; 272: 13717-13724Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). In contrast, GPIbα binds to the helix I region of 14-3-3 (33Gu M. Du X. J. Biol. Chem. 1998; 273: 33465-33471Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), which forms an amphiphilic ligand contact surface (40Petosa C. Masters S.C. Bankston L.A. Pohl J. Wang B. Fu H. Liddington R.C. J. Biol. Chem. 1998; 273: 16305-16310Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Furthermore, synthetic peptides corresponding to C-terminal 15 residues of GPIbα bound to 14-3-3 without requiring phosphorylation (11Andrews R.K. Harris S.J. McNally T. Berndt M.C. Biochemistry. 1998; 37: 638-647Crossref PubMed Scopus (120) Google Scholar, 29Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), and the recombinant GPIbα cytoplasmic domain, which is not phosphorylated at Ser609 (data not shown), also binds to 14-3-3 but with a much lower affinity than GPIb-IX from platelets (33Gu M. Du X. J. Biol. Chem. 1998; 273: 33465-33471Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). This suggests that the interaction of 14-3-3 with GPIbα may involve both phosphorylation-dependent and phosphorylation-independent binding mechanisms. However, in intact platelet GPIb-IX, Ser609 phosphorylation is required for the high affinity binding of 14-3-3.Phosphorylation of the Ser609 of GPIbα is likely to play important roles in GPIb-IX-mediated platelet adhesion and signaling. First, phosphorylation of Ser609 of GPIbα regulates 14-3-3 binding (Figs. 4 and 5), and we have evidence that 14-3-3 binding to GPIb-IX plays an important role in GPIb-IX signaling. 2M. Gu, R. J. Bodnar, Z. Li, G. D. Englund, and X. Du, unpublished data. Furthermore, our data indicate that a population of GPIbα becomes dephosphorylated at the periphery of platelets during platelet spreading on vWF or fibrinogen (Fig. 7), suggesting that the phosphorylation state of GPIbα can be dynamically regulated and that phosphorylation or dephosphorylation of GPIbα may have a functional role during platelet spreading. Although further studies are required to understand how phosphorylation of GPIbα may play a role in GPIb-IX function, one possibility is that phosphorylation regulates GPIb-IX-associated membrane skeleton organization and thus regulates the movement of GPIb-IX. This possibility is supported by the finding of Dong et al. (41Dong J.F. Li C.Q. Sae T.G. Hyun W. Afshar K.V. Lopez J.A. Biochemistry. 1997; 36: 12421-12427Crossref PubMed Scopus (47) Google Scholar) that a GPIb-IX mutant, lacking the C-terminal 4 amino acid residues including Ser609 is more likely to move laterally on the membrane. However, we show in Fig. 3 that Ser609-phosphorylated GPIbα is distributed in both cytoskeleton and non-cytoskeleton fractions, suggesting that Ser609 phosphorylation does not directly regulate association between GPIb-IX and the membrane skeleton. Consistent with this result, GPIb-IX association with the membrane skeleton has been shown to be mediated by filamin, which binds to the central region of the GPIbα cytoplasmic domain distinct from the C terminus (7Andrews R.K. Fox J.E. J. Biol. Chem. 1992; 267: 18605-18611Abstract Full Text PDF PubMed Google Scholar), and mutant GPIb-IX that lacks the C-terminal domain of GPIbα is still associated with filamin and the membrane skeleton (42Cunningham J.G. Meyer S.C. Fox J.E. J. Biol. Chem. 1996; 271: 11581-11587Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). However, it is possible that GPIb-IX-associated cytoskeleton organization or movement of GPIb-IX may be indirectly regulated by phosphorylation of GPIbα and 14-3-3 binding via intracellular signaling pathways. Platelet adhesion plays a critical role in thrombosis and hemostasis. Platelets in normal circulation are in a resting, nonadherent state. At sites of vascular injury, platelets adhere to the exposed subendothelial matrix. Under the high shear force of blood flow, platelet adhesion involves multiple steps. Initially, platelets adhere in a reversible manner (1Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar). This process is mediated by the interaction between a platelet receptor for von Willebrand factor (vWF),1 the glycoprotein Ib-IX complex (GPIb-IX), and matrix-bound vWF (1Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar, 2Sakariassen K.S. Bolhuis P.A. Sixma J.J. Nature. 1979; 279: 636-638Crossref PubMed Scopus (465) Google Scholar, 3Sakariassen K.S. Nievelstein P.F. Coller B.S. Sixma J.J. Br. J. Haematol. 1986; 63: 681-691Crossref PubMed Scopus (175) Google Scholar). GPIb-IX interaction with vWF mediates signaling leading to activation of integrins that are responsible for platelet spreading and aggregation (1Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1003) Google Scholar, 4Weiss H.J. Turitto V.T. Baumgartner H.R. Blood. 1986; 67: 322-330Crossref PubMed Google Scholar). GPIb-IX consists of three subunits: GPIbα, GPIbβ, and GPIX. GPIb-IX is loosely associated with glycoprotein V. The N-terminal domain of GPIbα contains binding sites for vWF and thrombin (for reviews see Refs. 5Ware J. Thromb. Haemost. 1998; 79: 466-478Crossref PubMed Scopus (66) Google Scholar and 6Lopez J.A. Blood Coagul. Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar). The cytoplasmic domain of GPIbα contains a binding site (residues 536–568 (7Andrews R.K. Fox J.E. J. Biol. Chem. 1992; 267: 18605-18611Abstract Full Text PDF PubMed Google Scholar)) for filamin (also calledactin-binding protein or ABP-280), which links GPIb-IX to cross-linked actin filamental structures underlying the plasma membrane (the membrane skeleton) (8Fox J.E.B. J. Biol. Chem. 1985; 260: 11970-11977Abstract Full Text PDF PubMed Google Scholar, 9Fox J.E.B. J. Clin. Invest. 1985; 76: 1673-1683Crossref PubMed Scopus (112) Google Scholar). We found that an intracellular signaling molecule, 14-3-3ζ, is associated with GPIb-IX (10Du X. Harris S.J. Tetaz T.J. Ginsberg M.H. Berndt M.C. J. Biol. Chem. 1994; 269: 18287-18290Abstract Full Text PDF PubMed Google Scholar). A binding site for 14-3-3ζ is located in a 15-amino acid residue serine-rich region (residues 595–610) at the C terminus of GPIbα (29Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). 14-3-3 binding also involves an additional 14-3-3 binding site in GPIbβ (11Andrews R.K. Harris S.J. McNally T. Berndt M.C. Biochemistry. 1998; 37: 638-647Crossref PubMed Scopus (120) Google Scholar, 12Calverley D.C. Kavanagh T.J. Roth G.J. Blood. 1998; 91: 1295-1303Crossref PubMed Google Scholar). The 14-3-3 family of highly conserved intracellular proteins interacts with several intracellular serine/threonine kinases and other signaling molecules (13Fu H. Xia K. Pallas D.C. Cui C. Conroy K. Narsimhan R.P. Mamon H. Collier R.J. Roberts T.M. Science. 1994; 266: 126-129Crossref PubMed Scopus (242) Google Scholar, 14Freed E. Symons M. Macdonald S.G. McCormick F. Ruggieri R. Science. 1994; 265: 1713-1716Crossref PubMed Scopus (352) Google Scholar, 15Fanti W.J. Muslin A.J. Kikuchi A. Martin J.A. MacNicol A.M. Gross R.W. Williams L.T. Nature. 1994; 371: 612-614Crossref PubMed Scopus (309) Google Scholar, 16Meller N. Liu Y.C. Collins T.L. Bonnefoy B.N. Baier G. Isakov N. Altman A. Mol. Cell. Biol. 1996; 16: 5782-5791Crossref PubMed Google Scholar, 17Acs P. Szallasi Z. Kazanietz M.G. Blumberg P.M. Biochem. Biophys. Res. Commun. 1995; 216: 103-109Crossref PubMed Scopus (45) Google Scholar, 18Conklin D.S. Galaktionov K. Beach D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7892-7896Crossref PubMed Scopus (245) Google Scholar, 19Bonnefoy B.N. Liu Y.C. von W.M. Sung A. Elly C. Mustelin T. Yoshida H. Ishizaka K. Altman A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10142-10146Crossref PubMed Scopus (133) Google Scholar, 20Furukawa Y. Ikuta N. Omata S. Yamauchi T. Isobe T. Ichimura T. Biochem. Biophys. Res. Commun. 1993; 194: 144-149Crossref PubMed Scopus (87) Google Scholar, 21Liu Y.-C. Elly C. Yoshida H. Bonnefoy-Beard B.N. Altman A. J. Biol. Chem. 1996; 271: 14591-14595Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 22Pallas D.C. Fu H. Haehnel L.C. Weller W. Collier R.J. Roberts T.M. Science. 1994; 265: 535-537Crossref PubMed Scopus (148) Google Scholar) and regulates their functions (15Fanti W.J. Muslin A.J. Kikuchi A. Martin J.A. MacNicol A.M. Gross R.W. Williams L.T. Nature. 1994; 371: 612-614Crossref PubMed Scopus (309) Google Scholar, 23Li S. Janosch P. Tanji M. Rosenfeld G.C. Waymire J.C. Mischak H. Kolch W. Sedivy J.M. EMBO J. 1995; 14: 685-696Crossref PubMed Scopus (154) Google Scholar, 24Tzivion G. Luo Z. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (386) Google Scholar, 25Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2241) Google Scholar, 26Ford J.C. al-Khodairy F. Fotou E. Sheldrick K.S. Griffiths D.J. Carr A.M. Science. 1994; 265: 533-535Crossref PubMed Scopus (298) Google Scholar). A recognition motif, RSXpSXP, has been identified in c-Raf and several other 14-3-3 ligands, requiring a phosphorylated serine residue (27Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar, 28Yaffe M.B. Rittinger K. Volinia S. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar). Thus, interaction of many intracellular signaling proteins with 14-3-3 is regulated by phosphorylation. We hypothesized previously that serine residues in the 14-3-3 binding site of GPIbα might be important for 14-3-3 binding (29Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). However, it is not clear whether 14-3-3 binding is regulated by phosphorylation of these serine residues, as 14-3-3 can interact with synthetic nonphosphorylated peptides corresponding to GPIbα cytoplasmic domain (11Andrews R.K. Harris S.J. McNally T. Berndt M.C. Biochemistry. 1998; 37: 638-647Crossref PubMed Scopus (120) Google Scholar, 29Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Further, GPIbα has been thought to be a nonphosphorylated protein because previous studies failed to show phosphorylation of GPIbα (30Wyler B. Bienz D. Clemetson K.J. Luscher E.F. Biochem. J. 1986; 234: 373-379Crossref PubMed Scopus (24) Google Scholar). In this study, we re-examined phosphorylation states of GPIbα using a phosphoserine-specific anti-GPIbα antibody. We report here that the cytoplasmic domain of GPIbα is phosphorylated at Ser609 and that phosphorylation at this site is important for 14-3-3 binding to intact platelet GPIb-IX. Furthermore, we show that GPIbα dephosphorylation occurs at the edge of spreading platelets, suggesting that phosphorylation and dephosphorylation of Ser609 in the cytoplasmic domain of GPIbα is involved in regulating GPIb-IX functions during platelet adhesion and spreading. DISCUSSIONIn this study, we provide the first evidence that GPIbα is phosphorylated and that a phosphorylation site is at Ser609. Previously, phosphorylation of platelet membrane proteins has been studied (30Wyler B. Bienz D. Clemetson K.J. Luscher E.F. Biochem. J. 1986; 234: 373-379Crossref PubMed Scopus (24) Google Scholar, 36Fox J.E. Reynolds C.C. Johnson M.M. J. Biol. Chem. 1987; 262: 12627-12631Abstract Full Text PDF PubMed Google Scholar). The only serine/threonine-phosphorylated major membrane glycoprotein identified was GPIbβ (30Wyler B. Bienz D. Clemetson K.J. Luscher E.F. Biochem. J. 1986; 234: 373-379Crossref PubMed Scopus (24) Google Scholar). GPIbβ is phosphorylated at Thr166 when platelets are stimulated with agents that enhance intracellular cAMP level (36Fox J.E. Reynolds C.C. Johnson M.M. J. Biol. Chem. 1987; 262: 12627-12631Abstract Full Text PDF PubMed Google Scholar, 37Wardell M.R. Reynolds C.C. Berndt M.C. Wallace R.W. Fox J.E. J. Biol. Chem. 1989; 264: 15656-15661Abstract Full Text PDF PubMed Google Scholar). However, previous studies used a 32P labeling technique to detect protein phosphorylation, which is dependent upon incorporation of exogenous 32P into the phosphorylated proteins and thus is not sensitive to phosphoproteins that are protected from dephosphorylation or rephosphorylation during the procedure. In this study, we used an anti-phosphopeptide antibody, anti-pS609, recognizing the SGHpSL sequence at the C terminus of GPIbα but not the nonphosphorylated peptide. The phosphorylation-dependent antibody can be used to detect protein phosphorylation whether or not phosphorylation is metabolically active and thus is capable of detecting GPIbα phosphorylation that has not been detected by the 32P labeling technique. Use of the phosphorylation-dependent antibody also enabled us to detect phosphorylation at a specific residue. Two serine residues (Ser606 and Ser609) are present at the C-terminal SGHSL region of GPIbα, which is important for 14-3-3 binding (29Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). We showed that the phosphoserine 609-specific antibody, anti-pS609, specifically bound to platelet GPIbα and that its binding was inhibited by dephosphorylation of GPIb-IX with potato acid phosphatase. In addition, an antibody raised against the phosphorylated Ser606-containing sequence (SIRYpSGH) did not react with GPIbα from resting platelets (data not shown). These data indicates that the C-terminal domain of GPIbα is phosphorylated at Ser609.Phosphorylation states of proteins are balanced by the actions of protein kinases and phosphatases. In platelet lysates, the percentage of phosphorylated GPIbα in the whole GPIb-IX population is high, as indicated by the removal of nearly all GPIb-IX by the anti-phosphopeptide antibody (Fig. 3 A); this suggests that balance under these conditions is tilted toward phosphorylation of GPIbα. Thus, it appears that, unlike many other phosphoproteins, the default state of GPIbα is a phosphorylated state. One possible mechanism for this default phosphorylation state is that 14-3-3 may play a protective role, since the phosphoserine 609 is located in the 14-3-3 binding site. 14-3-3 has previously been shown to protect 14-3-3 ligands from dephosphorylation (38Dent P. Jelinek T. Morrison D.K. Weber M.J. Sturgill T.W. Science. 1995; 268: 1902-1906Crossref PubMed Scopus (172) Google Scholar). The protein kinase that catalyzes phosphorylation of GPIbα remains to be identified. Several protein kinase inhibitors had no effect on GPIbα phosphorylation, including inhibitors of protein kinase A, protein kinase G, and protein kinase C (data not shown). It is thus possible that these kinases are not involved in GPIbα phosphorylation. However, as the default state of GPIbα appears to be a phosphorylated form, it is also possible that the ineffectiveness of these protein kinase inhibitors is due to the fact that GPIbα is already in a relatively stable phosphorylated state and thus immune to the effects of protein kinase inhibitors.Phosphorylation at Ser609 of GPIbα is important for GPIb-IX interaction with 14-3-3. This conclusion is supported by our finding that the Ser609-phosphorylated GPIbα C-terminal domain peptide (SIRYSGHpSL), but not the identical nonphosphorylated or Ser606-phosphorylated peptides, inhibited GPIb-IX interaction with 14-3-3 in a concentration-dependent manner (Fig. 6), suggesting that interaction between GPIb-IX and 14-3-3 involves a binding site in 14-3-3 that interacts with the Ser609-phosphorylated GPIbα C-terminal sequence. This result is consistent with the previous result of Andrews et al. (11Andrews R.K. Harris S.J. McNally T. Berndt M.C. Biochemistry. 1998; 37: 638-647Crossref PubMed Scopus (120) Google Scholar) showing that a nonphosphorylated GPIbα C-terminal peptide failed to abolish the binding between 14-3-3 and GPIb-IX. Furthermore, dephosphorylation of GPIb-IX by PAP or preincubation with anti-pS609 inhibited 14-3-3 binding (Figs. 4 and 5). Thus, it is likely that high affinity interaction between the intact platelet GPIb-IX and 14-3-3 requires phosphorylation of Ser609 of GPIbα. It is interesting to note that the 14-3-3 binding site of GPIbα (RYSGHSL) shares similarities with the RSXpSXP-like motifs of other phosphorylated 14-3-3 ligands; they all contain an arginine and a serine at the N-terminal side of the phosphorylated serine (27Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar,28Yaffe M.B. Rittinger K. Volinia S. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar). Most of the identified RSXpSXP motifs are present in the middle of the protein sequence, and the proline in the motif may possibly form a turn exposing the phosphoserine. Because the 14-3-3 binding site in GPIbα is already exposed at the C terminus, it may not require the presence of a proline residue. However, despite the similarities, there are striking differences between GPIbα and the RSXpSXP-like ligands. The prototype RSXpSXP-like ligand of 14-3-3, c-Raf, requires the helix G region of 14-3-3 (33Gu M. Du X. J. Biol. Chem. 1998; 273: 33465-33471Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), and the crystal structure data suggest that phosphoserine in the RSXpSXP motif may interact with residues in the more N-terminal helix C and E region of 14-3-3 (28Yaffe M.B. Rittinger K. Volinia S. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar, 39Zhang L. Wang H. Liu D. Liddington R. Fu H. J. Biol. Chem. 1997; 272: 13717-13724Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). In contrast, GPIbα binds to the helix I region of 14-3-3 (33Gu M. Du X. J. Biol. Chem. 1998; 273: 33465-33471Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), which forms an amphiphilic ligand contact surface (40Petosa C. Masters S.C. Bankston L.A. Pohl J. Wang B. Fu H. Liddington R.C. J. Biol. Chem. 1998; 273: 16305-16310Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Furthermore, synthetic peptides corresponding to C-terminal 15 residues of GPIbα bound to 14-3-3 without requiring phosphorylation (11Andrews R.K. Harris S.J. McNally T. Berndt M.C. Biochemistry. 1998; 37: 638-647Crossref PubMed Scopus (120) Google Scholar, 29Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), and the recombinant GPIbα cytoplasmic domain, which is not phosphorylated at Ser609 (data not shown), also binds to 14-3-3 but with a much lower affinity than GPIb-IX from platelets (33Gu M. Du X. J. Biol. Chem. 1998; 273: 33465-33471Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). This suggests that the interaction of 14-3-3 with GPIbα may involve both phosphorylation-dependent and phosphorylation-independent binding mechanisms. However, in intact platelet GPIb-IX, Ser609 phosphorylation is required for the high affinity binding of 14-3-3.Phosphorylation of the Ser609 of GPIbα is likely to play important roles in GPIb-IX-mediated platelet adhesion and signaling. First, phosphorylation of Ser609 of GPIbα regulates 14-3-3 binding (Figs. 4 and 5), and we have evidence that 14-3-3 binding to GPIb-IX plays an important role in GPIb-IX signaling. 2M. Gu, R. J. Bodnar, Z. Li, G. D. Englund, and X. Du, unpublished data. Furthermore, our data indicate that a population of GPIbα becomes dephosphorylated at the periphery of platelets during platelet spreading on vWF or fibrinogen (Fig. 7), suggesting that the phosphorylation state of GPIbα can be dynamically regulated and that phosphorylation or dephosphorylation of GPIbα may have a functional role during platelet spreading. Although further studies are required to understand how phosphorylation of GPIbα may play a role in GPIb-IX function, one possibility is that phosphorylation regulates GPIb-IX-associated membrane skeleton organization and thus regulates the movement of GPIb-IX. This possibility is supported by the finding of Dong et al. (41Dong J.F. Li C.Q. Sae T.G. Hyun W. Afshar K.V. Lopez J.A. Biochemistry. 1997; 36: 12421-12427Crossref PubMed Scopus (47) Google Scholar) that a GPIb-IX mutant, lacking the C-terminal 4 amino acid residues including Ser609 is more likely to move laterally on the membrane. However, we show in Fig. 3 that Ser609-phosphorylated GPIbα is distributed in both cytoskeleton and non-cytoskeleton fractions, suggesting that Ser609 phosphorylation does not directly regulate association between GPIb-IX and the membrane skeleton. Consistent with this result, GPIb-IX association with the membrane skeleton has been shown to be mediated by filamin, which binds to the central region of the GPIbα cytoplasmic domain distinct from the C terminus (7Andrews R.K. Fox J.E. J. Biol. Chem. 1992; 267: 18605-18611Abstract Full Text PDF PubMed Google Scholar), and mutant GPIb-IX that lacks the C-terminal domain of GPIbα is still associated with filamin and the membrane skeleton (42Cunningham J.G. Meyer S.C. Fox J.E. J. Biol. Chem. 1996; 271: 11581-11587Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). However, it is possible that GPIb-IX-associated cytoskeleton organization or movement of GPIb-IX may be indirectly regulated by phosphorylation of GPIbα and 14-3-3 binding via intracellular signaling pathways. In this study, we provide the first evidence that GPIbα is phosphorylated and that a phosphorylation site is at Ser609. Previously, phosphorylation of platelet membrane proteins has been studied (30Wyler B. Bienz D. Clemetson K.J. Luscher E.F. Biochem. J. 1986; 234: 373-379Crossref PubMed Scopus (24) Google Scholar, 36Fox J.E. Reynolds C.C. Johnson M.M. J. Biol. Chem. 1987; 262: 12627-12631Abstract Full Text PDF PubMed Google Scholar). The only serine/threonine-phosphorylated major membrane glycoprotein identified was GPIbβ (30Wyler B. Bienz D. Clemetson K.J. Luscher E.F. Biochem. J. 1986; 234: 373-379Crossref PubMed Scopus (24) Google Scholar). GPIbβ is phosphorylated at Thr166 when platelets are stimulated with agents that enhance intracellular cAMP level (36Fox J.E. Reynolds C.C. Johnson M.M. J. Biol. Chem. 1987; 262: 12627-12631Abstract Full Text PDF PubMed Google Scholar, 37Wardell M.R. Reynolds C.C. Berndt M.C. Wallace R.W. Fox J.E. J. Biol. Chem. 1989; 264: 15656-15661Abstract Full Text PDF PubMed Google Scholar). However, previous studies used a 32P labeling technique to detect protein phosphorylation, which is dependent upon incorporation of exogenous 32P into the phosphorylated proteins and thus is not sensitive to phosphoproteins that are protected from dephosphorylation or rephosphorylation during the procedure. In this study, we used an anti-phosphopeptide antibody, anti-pS609, recognizing the SGHpSL sequence at the C terminus of GPIbα but not the nonphosphorylated peptide. The phosphorylation-dependent antibody can be used to detect protein phosphorylation whether or not phosphorylation is metabolically active and thus is capable of detecting GPIbα phosphorylation that has not been detected by the 32P labeling technique. Use of the phosphorylation-dependent antibody also enabled us to detect phosphorylation at a specific residue. Two serine residues (Ser606 and Ser609) are present at the C-terminal SGHSL region of GPIbα, which is important for 14-3-3 binding (29Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). We showed that the phosphoserine 609-specific antibody, anti-pS609, specifically bound to platelet GPIbα and that its binding was inhibited by dephosphorylation of GPIb-IX with potato acid phosphatase. In addition, an antibody raised against the phosphorylated Ser606-containing sequence (SIRYpSGH) did not react with GPIbα from resting platelets (data not shown). These data indicates that the C-terminal domain of GPIbα is phosphorylated at Ser609. Phosphorylation states of proteins are balanced by the actions of protein kinases and phosphatases. In platelet lysates, the percentage of phosphorylated GPIbα in the whole GPIb-IX population is high, as indicated by the removal of nearly all GPIb-IX by the anti-phosphopeptide antibody (Fig. 3 A); this suggests that balance under these conditions is tilted toward phosphorylation of GPIbα. Thus, it appears that, unlike many other phosphoproteins, the default state of GPIbα is a phosphorylated state. One possible mechanism for this default phosphorylation state is that 14-3-3 may play a protective role, since the phosphoserine 609 is located in the 14-3-3 binding site. 14-3-3 has previously been shown to protect 14-3-3 ligands from dephosphorylation (38Dent P. Jelinek T. Morrison D.K. Weber M.J. Sturgill T.W. Science. 1995; 268: 1902-1906Crossref PubMed Scopus (172) Google Scholar). The protein kinase that catalyzes phosphorylation of GPIbα remains to be identified. Several protein kinase inhibitors had no effect on GPIbα phosphorylation, including inhibitors of protein kinase A, protein kinase G, and protein kinase C (data not shown). It is thus possible that these kinases are not involved in GPIbα phosphorylation. However, as the default state of GPIbα appears to be a phosphorylated form, it is also possible that the ineffectiveness of these protein kinase inhibitors is due to the fact that GPIbα is already in a relatively stable phosphorylated state and thus immune to the effects of protein kinase inhibitors. Phosphorylation at Ser609 of GPIbα is important for GPIb-IX interaction with 14-3-3. This conclusion is supported by our finding that the Ser609-phosphorylated GPIbα C-terminal domain peptide (SIRYSGHpSL), but not the identical nonphosphorylated or Ser606-phosphorylated peptides, inhibited GPIb-IX interaction with 14-3-3 in a concentration-dependent manner (Fig. 6), suggesting that interaction between GPIb-IX and 14-3-3 involves a binding site in 14-3-3 that interacts with the Ser609-phosphorylated GPIbα C-terminal sequence. This result is consistent with the previous result of Andrews et al. (11Andrews R.K. Harris S.J. McNally T. Berndt M.C. Biochemistry. 1998; 37: 638-647Crossref PubMed Scopus (120) Google Scholar) showing that a nonphosphorylated GPIbα C-terminal peptide failed to abolish the binding between 14-3-3 and GPIb-IX. Furthermore, dephosphorylation of GPIb-IX by PAP or preincubation with anti-pS609 inhibited 14-3-3 binding (Figs. 4 and 5). Thus, it is likely that high affinity interaction between the intact platelet GPIb-IX and 14-3-3 requires phosphorylation of Ser609 of GPIbα. It is interesting to note that the 14-3-3 binding site of GPIbα (RYSGHSL) shares similarities with the RSXpSXP-like motifs of other phosphorylated 14-3-3 ligands; they all contain an arginine and a serine at the N-terminal side of the phosphorylated serine (27Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar,28Yaffe M.B. Rittinger K. Volinia S. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar). Most of the identified RSXpSXP motifs are present in the middle of the protein sequence, and the proline in the motif may possibly form a turn exposing the phosphoserine. Because the 14-3-3 binding site in GPIbα is already exposed at the C terminus, it may not require the presence of a proline residue. However, despite the similarities, there are striking differences between GPIbα and the RSXpSXP-like ligands. The prototype RSXpSXP-like ligand of 14-3-3, c-Raf, requires the helix G region of 14-3-3 (33Gu M. Du X. J. Biol. Chem. 1998; 273: 33465-33471Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), and the crystal structure data suggest that phosphoserine in the RSXpSXP motif may interact with residues in the more N-terminal helix C and E region of 14-3-3 (28Yaffe M.B. Rittinger K. Volinia S. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar, 39Zhang L. Wang H. Liu D. Liddington R. Fu H. J. Biol. Chem. 1997; 272: 13717-13724Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). In contrast, GPIbα binds to the helix I region of 14-3-3 (33Gu M. Du X. J. Biol. Chem. 1998; 273: 33465-33471Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), which forms an amphiphilic ligand contact surface (40Petosa C. Masters S.C. Bankston L.A. Pohl J. Wang B. Fu H. Liddington R.C. J. Biol. Chem. 1998; 273: 16305-16310Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Furthermore, synthetic peptides corresponding to C-terminal 15 residues of GPIbα bound to 14-3-3 without requiring phosphorylation (11Andrews R.K. Harris S.J. McNally T. Berndt M.C. Biochemistry. 1998; 37: 638-647Crossref PubMed Scopus (120) Google Scholar, 29Du X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), and the recombinant GPIbα cytoplasmic domain, which is not phosphorylated at Ser609 (data not shown), also binds to 14-3-3 but with a much lower affinity than GPIb-IX from platelets (33Gu M. Du X. J. Biol. Chem. 1998; 273: 33465-33471Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). This suggests that the interaction of 14-3-3 with GPIbα may involve both phosphorylation-dependent and phosphorylation-independent binding mechanisms. However, in intact platelet GPIb-IX, Ser609 phosphorylation is required for the high affinity binding of 14-3-3. Phosphorylation of the Ser609 of GPIbα is likely to play important roles in GPIb-IX-mediated platelet adhesion and signaling. First, phosphorylation of Ser609 of GPIbα regulates 14-3-3 binding (Figs. 4 and 5), and we have evidence that 14-3-3 binding to GPIb-IX plays an important role in GPIb-IX signaling. 2M. Gu, R. J. Bodnar, Z. Li, G. D. Englund, and X. Du, unpublished data. Furthermore, our data indicate that a population of GPIbα becomes dephosphorylated at the periphery of platelets during platelet spreading on vWF or fibrinogen (Fig. 7), suggesting that the phosphorylation state of GPIbα can be dynamically regulated and that phosphorylation or dephosphorylation of GPIbα may have a functional role during platelet spreading. Although further studies are required to understand how phosphorylation of GPIbα may play a role in GPIb-IX function, one possibility is that phosphorylation regulates GPIb-IX-associated membrane skeleton organization and thus regulates the movement of GPIb-IX. This possibility is supported by the finding of Dong et al. (41Dong J.F. Li C.Q. Sae T.G. Hyun W. Afshar K.V. Lopez J.A. Biochemistry. 1997; 36: 12421-12427Crossref PubMed Scopus (47) Google Scholar) that a GPIb-IX mutant, lacking the C-terminal 4 amino acid residues including Ser609 is more likely to move laterally on the membrane. However, we show in Fig. 3 that Ser609-phosphorylated GPIbα is distributed in both cytoskeleton and non-cytoskeleton fractions, suggesting that Ser609 phosphorylation does not directly regulate association between GPIb-IX and the membrane skeleton. Consistent with this result, GPIb-IX association with the membrane skeleton has been shown to be mediated by filamin, which binds to the central region of the GPIbα cytoplasmic domain distinct from the C terminus (7Andrews R.K. Fox J.E. J. Biol. Chem. 1992; 267: 18605-18611Abstract Full Text PDF PubMed Google Scholar), and mutant GPIb-IX that lacks the C-terminal domain of GPIbα is still associated with filamin and the membrane skeleton (42Cunningham J.G. Meyer S.C. Fox J.E. J. Biol. Chem. 1996; 271: 11581-11587Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). However, it is possible that GPIb-IX-associated cytoskeleton organization or movement of GPIb-IX may be indirectly regulated by phosphorylation of GPIbα and 14-3-3 binding via intracellular signaling pathways.
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