The cytoplasmic domain of the platelet glycoprotein Ibα is phosphorylated at Serine 609

The a 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 GPIb alpha is phosphorylated at Ser(609); this was detected by immunoblotting with an anti-phosphopeptide antibody, anti-pS609, that specifically recognizes the GPIb alpha C-terminal sequence S(606)GHSL(610) only when Ser(609) is phosphorylated. Immunoabsorption with anti-pS(609) removed almost all of the GPIb alpha from platelet lysates, indicating a high proportion of GPIb alpha phosphorylation. Anti-pS609 inhibited GPIb-IX binding to the intracellular signaling molecule, 14-3-3 zeta. Dephosphorylation of GPIb-IX with potato acid phosphatase inhibited anti-pS609 binding and also 14-3-3 zeta binding. A synthetic phosphopeptide corresponding to the GPIb alpha C-terminal sequence (SIRYSGHpSL), but not a nonphosphorylated identical peptide, abolished GPIb-IX binding to 14-3-3 zeta. Thus, phosphorylation at Ser(609) of GPIb alpha is important for 14-3-3 zeta binding to GPIb-IX. In certain regions of spreading platelets, particularly at the periphery, there was a reduction in GPIb alpha staining by anti-pS609 as observed under a confocal microscope, indicating that a subpopulation of GPIb alpha molecules in these regions is dephosphorylated. These data suggest that phosphorylation and dephosphorylation at Ser(609) of GPIb alpha regulates GPIb-IX interaction with 14-3-3 and may play important roles in the process of platelet adhesion and spreading.