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 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 GPIba 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.