The Cytoplasmic Domain of the Platelet Glycoprotein Ibα Is Phosphorylated at Serine 609

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. 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; PubMed Google 14-3-3 of intracellular with intracellular and signaling molecules D.C. 1994; PubMed Scopus Google Scholar, E. 1994; PubMed Scopus Google Scholar, J.A. Nature. 1994; PubMed Scopus Google Scholar, Cell. Biol. 1996; PubMed Google Scholar, PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar, von T. S. PubMed Scopus Google Scholar, S. T. T. T. PubMed Scopus Google Scholar, J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, D.C. 1994; PubMed Scopus Google and regulates J.A. Nature. 1994; PubMed Scopus Google Scholar, S. J. PubMed Scopus Google Scholar, J. Nature. 1998; PubMed Scopus Google Scholar, J. E. J. S.J. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, E. K.S. 1994; PubMed Scopus Google Scholar). A in and 14-3-3 a phosphorylated residue Cell. 1996; 84: Full Text Full Text PDF PubMed Scopus Google Scholar, S. S.J. S.J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, interaction of intracellular signaling with 14-3-3 is by phosphorylation. We that in the 14-3-3 binding site of GPIbα 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). is not 14-3-3 binding is by phosphorylation of these as 14-3-3 with synthetic nonphosphorylated 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, X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). GPIbα to be a nonphosphorylated protein to phosphorylation of GPIbα B. J. 1986; PubMed Scopus Google Scholar). In this we phosphorylation of GPIbα a We report that the cytoplasmic domain of GPIbα is phosphorylated at Ser609 and that phosphorylation at this site is important for 14-3-3 binding to platelet GPIb-IX. we that GPIbα dephosphorylation at the of spreading platelets, that phosphorylation and dephosphorylation of Ser609 in the cytoplasmic domain of GPIbα is in GPIb-IX platelet adhesion and this we the that GPIbα is phosphorylated and that a phosphorylation site is at phosphorylation of platelet membrane B. J. 1986; PubMed Scopus Google Scholar, J.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The only membrane glycoprotein was GPIbβ B. J. 1986; PubMed Scopus Google Scholar). GPIbβ is phosphorylated at when platelets are with that intracellular J.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Berndt M.C. Fox J.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). a to protein which is of the phosphorylated and is not to that are from dephosphorylation or the In this we an anti-phosphopeptide antibody, anti-pS609, the sequence at the C terminus of GPIbα but not the nonphosphorylated The be to protein phosphorylation or not phosphorylation is and is of GPIbα phosphorylation that not detected by the of the also to phosphorylation at a and are at the C-terminal 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 that the phosphoserine antibody, anti-pS609, specifically to platelet GPIbα and that binding was inhibited by dephosphorylation of GPIb-IX with potato acid In an the phosphorylated sequence not with GPIbα from platelets not These data that the C-terminal domain of GPIbα is phosphorylated at of are by the of protein and In platelet lysates, the of phosphorylated GPIbα in the GPIb-IX is as by the of all GPIb-IX by the anti-phosphopeptide this that under these is phosphorylation of Thus, the of GPIbα is a phosphorylated state. for this phosphorylation is that 14-3-3 may play a the phosphoserine is located in the 14-3-3 binding 14-3-3 to 14-3-3 from dephosphorylation T. PubMed Scopus Google Scholar). The protein that phosphorylation of GPIbα to be protein GPIbα of protein protein and protein C not is that these are not in GPIbα phosphorylation. as the of GPIbα to be a phosphorylated is also that the of these protein is to the that GPIbα is in a phosphorylated and to the of protein at Ser609 of GPIbα is important for GPIb-IX interaction with This is by that the GPIbα C-terminal domain (SIRYSGHpSL), but not the identical nonphosphorylated or inhibited GPIb-IX interaction with 14-3-3 in a manner that interaction between GPIb-IX and 14-3-3 involves a binding site in 14-3-3 that with the GPIbα C-terminal This is with the of (11Andrews R.K. Harris S.J. McNally T. Berndt M.C. Biochemistry. 1998; 37: 638-647Crossref PubMed Scopus (120) Google that a nonphosphorylated GPIbα C-terminal to the binding between 14-3-3 and GPIb-IX. dephosphorylation of GPIb-IX by or with anti-pS609 inhibited 14-3-3 binding and Thus, is that high interaction between the platelet GPIb-IX and 14-3-3 phosphorylation of Ser609 of is to that the 14-3-3 binding site of GPIbα with the of phosphorylated 14-3-3 all an and a at the N-terminal of the phosphorylated Cell. 1996; 84: Full Text Full Text PDF PubMed Scopus Google S. S.J. S.J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). of the are in the of the protein and the in the may a the the 14-3-3 binding site in GPIbα is exposed at the C may not the of a the there are between GPIbα and the The of the region of 14-3-3 X. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google and the data suggest that phosphoserine in the may with in the N-terminal C and region of 14-3-3 S. S.J. S.J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In GPIbα to the region of 14-3-3 X. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google which an J. B. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). synthetic corresponding to C-terminal of GPIbα to 14-3-3 phosphorylation (11Andrews R.K. Harris S.J. McNally T. Berndt M.C. Biochemistry. 1998; 37: 638-647Crossref PubMed Scopus (120) Google Scholar, X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google and the GPIbα cytoplasmic which is not phosphorylated at Ser609 not also to 14-3-3 but with a GPIb-IX from platelets X. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). This that the interaction of 14-3-3 with GPIbα may and binding in platelet Ser609 phosphorylation is for the high binding of of the Ser609 of GPIbα is to play important roles in platelet adhesion and phosphorylation of Ser609 of GPIbα regulates 14-3-3 binding and and we that 14-3-3 binding to GPIb-IX plays an important role in GPIb-IX J. and X. data that a of GPIbα at the of platelets platelet spreading vWF or that the phosphorylation of GPIbα be and that phosphorylation or dephosphorylation of GPIbα may a role platelet spreading. are to phosphorylation of GPIbα may play a role in GPIb-IX is that phosphorylation regulates membrane and regulates the of GPIb-IX. This is by the of J.A. Biochemistry. PubMed Scopus Google that a GPIb-IX the C-terminal acid Ser609 is to the we in that GPIbα is in and that Ser609 phosphorylation not between GPIb-IX and the membrane with this GPIb-IX with the membrane to be mediated by which to the region of the GPIbα cytoplasmic domain from the C terminus (7Andrews R.K. Fox J.E. J. Biol. Chem. 1992; 267: 18605-18611Abstract Full Text PDF PubMed Google and GPIb-IX that the C-terminal domain of GPIbα is associated with filamin and the membrane Fox J.E. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). is that or of GPIb-IX may be by phosphorylation of GPIbα and 14-3-3 binding intracellular signaling 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; PubMed Google Scholar). The 14-3-3 of intracellular with intracellular and signaling molecules D.C. 1994; PubMed Scopus Google Scholar, E. 1994; PubMed Scopus Google Scholar, J.A. Nature. 1994; PubMed Scopus Google Scholar, Cell. Biol. 1996; PubMed Google Scholar, PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar, von T. S. PubMed Scopus Google Scholar, S. T. T. T. PubMed Scopus Google Scholar, J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, D.C. 1994; PubMed Scopus Google and regulates J.A. Nature. 1994; PubMed Scopus Google Scholar, S. J. PubMed Scopus Google Scholar, J. Nature. 1998; PubMed Scopus Google Scholar, J. E. J. S.J. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, E. K.S. 1994; PubMed Scopus Google Scholar). A in and 14-3-3 a phosphorylated residue Cell. 1996; 84: Full Text Full Text PDF PubMed Scopus Google Scholar, S. S.J. S.J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, interaction of intracellular signaling with 14-3-3 is by phosphorylation. We that in the 14-3-3 binding site of GPIbα 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). is not 14-3-3 binding is by phosphorylation of these as 14-3-3 with synthetic nonphosphorylated 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, X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). GPIbα to be a nonphosphorylated protein to phosphorylation of GPIbα B. J. 1986; PubMed Scopus Google Scholar). In this we phosphorylation of GPIbα a We report that the cytoplasmic domain of GPIbα is phosphorylated at Ser609 and that phosphorylation at this site is important for 14-3-3 binding to platelet GPIb-IX. we that GPIbα dephosphorylation at the of spreading platelets, that phosphorylation and dephosphorylation of Ser609 in the cytoplasmic domain of GPIbα is in GPIb-IX platelet adhesion and spreading. this we the that GPIbα is phosphorylated and that a phosphorylation site is at phosphorylation of platelet membrane B. J. 1986; PubMed Scopus Google Scholar, J.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The only membrane glycoprotein was GPIbβ B. J. 1986; PubMed Scopus Google Scholar). GPIbβ is phosphorylated at when platelets are with that intracellular J.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Berndt M.C. Fox J.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). a to protein which is of the phosphorylated and is not to that are from dephosphorylation or the In this we an anti-phosphopeptide antibody, anti-pS609, the sequence at the C terminus of GPIbα but not the nonphosphorylated The be to protein phosphorylation or not phosphorylation is and is of GPIbα phosphorylation that not detected by the of the also to phosphorylation at a and are at the C-terminal 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 that the phosphoserine antibody, anti-pS609, specifically to platelet GPIbα and that binding was inhibited by dephosphorylation of GPIb-IX with potato acid In an the phosphorylated sequence not with GPIbα from platelets not These data that the C-terminal domain of GPIbα is phosphorylated at of are by the of protein and In platelet lysates, the of phosphorylated GPIbα in the GPIb-IX is as by the of all GPIb-IX by the anti-phosphopeptide this that under these is phosphorylation of Thus, the of GPIbα is a phosphorylated state. for this phosphorylation is that 14-3-3 may play a the phosphoserine is located in the 14-3-3 binding 14-3-3 to 14-3-3 from dephosphorylation T. PubMed Scopus Google Scholar). The protein that phosphorylation of GPIbα to be protein GPIbα of protein protein and protein C not is that these are not in GPIbα phosphorylation. as the of GPIbα to be a phosphorylated is also that the of these protein is to the that GPIbα is in a phosphorylated and to the of protein at Ser609 of GPIbα is important for GPIb-IX interaction with This is by that the GPIbα C-terminal domain (SIRYSGHpSL), but not the identical nonphosphorylated or inhibited GPIb-IX interaction with 14-3-3 in a manner that interaction between GPIb-IX and 14-3-3 involves a binding site in 14-3-3 that with the GPIbα C-terminal This is with the of (11Andrews R.K. Harris S.J. McNally T. Berndt M.C. Biochemistry. 1998; 37: 638-647Crossref PubMed Scopus (120) Google that a nonphosphorylated GPIbα C-terminal to the binding between 14-3-3 and GPIb-IX. dephosphorylation of GPIb-IX by or with anti-pS609 inhibited 14-3-3 binding and Thus, is that high interaction between the platelet GPIb-IX and 14-3-3 phosphorylation of Ser609 of is to that the 14-3-3 binding site of GPIbα with the of phosphorylated 14-3-3 all an and a at the N-terminal of the phosphorylated Cell. 1996; 84: Full Text Full Text PDF PubMed Scopus Google S. S.J. S.J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). of the are in the of the protein and the in the may a the the 14-3-3 binding site in GPIbα is exposed at the C may not the of a the there are between GPIbα and the The of the region of 14-3-3 X. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google and the data suggest that phosphoserine in the may with in the N-terminal C and region of 14-3-3 S. S.J. S.J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In GPIbα to the region of 14-3-3 X. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google which an J. B. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). synthetic corresponding to C-terminal of GPIbα to 14-3-3 phosphorylation (11Andrews R.K. Harris S.J. McNally T. Berndt M.C. Biochemistry. 1998; 37: 638-647Crossref PubMed Scopus (120) Google Scholar, X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google and the GPIbα cytoplasmic which is not phosphorylated at Ser609 not also to 14-3-3 but with a GPIb-IX from platelets X. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). This that the interaction of 14-3-3 with GPIbα may and binding in platelet Ser609 phosphorylation is for the high binding of of the Ser609 of GPIbα is to play important roles in platelet adhesion and phosphorylation of Ser609 of GPIbα regulates 14-3-3 binding and and we that 14-3-3 binding to GPIb-IX plays an important role in GPIb-IX J. and X. data that a of GPIbα at the of platelets platelet spreading vWF or that the phosphorylation of GPIbα be and that phosphorylation or dephosphorylation of GPIbα may a role platelet spreading. are to phosphorylation of GPIbα may play a role in GPIb-IX is that phosphorylation regulates membrane and regulates the of GPIb-IX. This is by the of J.A. Biochemistry. PubMed Scopus Google that a GPIb-IX the C-terminal acid Ser609 is to the we in that GPIbα is in and that Ser609 phosphorylation not between GPIb-IX and the membrane with this GPIb-IX with the membrane to be mediated by which to the region of the GPIbα cytoplasmic domain from the C terminus (7Andrews R.K. Fox J.E. J. Biol. Chem. 1992; 267: 18605-18611Abstract Full Text PDF PubMed Google and GPIb-IX that the C-terminal domain of GPIbα is associated with filamin and the membrane Fox J.E. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). is that or of GPIb-IX may be by phosphorylation of GPIbα and 14-3-3 binding intracellular signaling In this we the that GPIbα is phosphorylated and that a phosphorylation site is at phosphorylation of platelet membrane B. J. 1986; PubMed Scopus Google Scholar, J.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The only membrane glycoprotein was GPIbβ B. J. 1986; PubMed Scopus Google Scholar). GPIbβ is phosphorylated at when platelets are with that intracellular J.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Berndt M.C. Fox J.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). a to protein which is of the phosphorylated and is not to that are from dephosphorylation or the In this we an anti-phosphopeptide antibody, anti-pS609, the sequence at the C terminus of GPIbα but not the nonphosphorylated The be to protein phosphorylation or not phosphorylation is and is of GPIbα phosphorylation that not detected by the of the also to phosphorylation at a and are at the C-terminal 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 that the phosphoserine antibody, anti-pS609, specifically to platelet GPIbα and that binding was inhibited by dephosphorylation of GPIb-IX with potato acid In an the phosphorylated sequence not with GPIbα from platelets not These data that the C-terminal domain of GPIbα is phosphorylated at of are by the of protein and In platelet lysates, the of phosphorylated GPIbα in the GPIb-IX is as by the of all GPIb-IX by the anti-phosphopeptide this that under these is phosphorylation of Thus, the of GPIbα is a phosphorylated state. for this phosphorylation is that 14-3-3 may play a the phosphoserine is located in the 14-3-3 binding 14-3-3 to 14-3-3 from dephosphorylation T. PubMed Scopus Google Scholar). The protein that phosphorylation of GPIbα to be protein GPIbα of protein protein and protein C not is that these are not in GPIbα phosphorylation. as the of GPIbα to be a phosphorylated is also that the of these protein is to the that GPIbα is in a phosphorylated and to the of protein at Ser609 of GPIbα is important for GPIb-IX interaction with This is by that the GPIbα C-terminal domain (SIRYSGHpSL), but not the identical nonphosphorylated or inhibited GPIb-IX interaction with 14-3-3 in a manner that interaction between GPIb-IX and 14-3-3 involves a binding site in 14-3-3 that with the GPIbα C-terminal This is with the of (11Andrews R.K. Harris S.J. McNally T. Berndt M.C. Biochemistry. 1998; 37: 638-647Crossref PubMed Scopus (120) Google that a nonphosphorylated GPIbα C-terminal to the binding between 14-3-3 and GPIb-IX. dephosphorylation of GPIb-IX by or with anti-pS609 inhibited 14-3-3 binding and Thus, is that high interaction between the platelet GPIb-IX and 14-3-3 phosphorylation of Ser609 of is to that the 14-3-3 binding site of GPIbα with the of phosphorylated 14-3-3 all an and a at the N-terminal of the phosphorylated Cell. 1996; 84: Full Text Full Text PDF PubMed Scopus Google S. S.J. S.J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). of the are in the of the protein and the in the may a the the 14-3-3 binding site in GPIbα is exposed at the C may not the of a the there are between GPIbα and the The of the region of 14-3-3 X. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google and the data suggest that phosphoserine in the may with in the N-terminal C and region of 14-3-3 S. S.J. S.J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In GPIbα to the region of 14-3-3 X. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google which an J. B. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). synthetic corresponding to C-terminal of GPIbα to 14-3-3 phosphorylation (11Andrews R.K. Harris S.J. McNally T. Berndt M.C. Biochemistry. 1998; 37: 638-647Crossref PubMed Scopus (120) Google Scholar, X. Fox J.E. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google and the GPIbα cytoplasmic which is not phosphorylated at Ser609 not also to 14-3-3 but with a GPIb-IX from platelets X. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). This that the interaction of 14-3-3 with GPIbα may and binding in platelet Ser609 phosphorylation is for the high binding of of the Ser609 of GPIbα is to play important roles in platelet adhesion and phosphorylation of Ser609 of GPIbα regulates 14-3-3 binding and and we that 14-3-3 binding to GPIb-IX plays an important role in GPIb-IX J. and X. data that a of GPIbα at the of platelets platelet spreading vWF or that the phosphorylation of GPIbα be and that phosphorylation or dephosphorylation of GPIbα may a role platelet spreading. are to phosphorylation of GPIbα may play a role in GPIb-IX is that phosphorylation regulates membrane and regulates the of GPIb-IX. This is by the of J.A. Biochemistry. PubMed Scopus Google that a GPIb-IX the C-terminal acid Ser609 is to the we in that GPIbα is in and that Ser609 phosphorylation not between GPIb-IX and the membrane with this GPIb-IX with the membrane to be mediated by which to the region of the GPIbα cytoplasmic domain from the C terminus (7Andrews R.K. Fox J.E. J. Biol. Chem. 1992; 267: 18605-18611Abstract Full Text PDF PubMed Google and GPIb-IX that the C-terminal domain of GPIbα is associated with filamin and the membrane Fox J.E. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). is that or of GPIb-IX may be by phosphorylation of GPIbα and 14-3-3 binding intracellular signaling