Ser⁵⁵⁹ of Glycoprotein Ibα Is Phosphorylated by Protein Kinase A Which Plays Key Roles In Regulating Von Willebrand Factor Binding, Surface Expression and Signaling of Glycoprotein Ib-IX

Abstract 3192

The interaction of glycoprotein (GP) Iba with von Willebrand factor (VWF) is regulated by the associations of 14-3-3ζ and filamin A with the cytoplasmic domain of GPIba. We reported recently that phosphorylation at Ser⁵⁵⁹ of GPIba is important for 14-3-3ζ binding, however, the protein kinase responsible for Ser⁵⁵⁹ phosphorylation remains unknown. Here we show that a peptide corresponding to the Arg557-Pro561 region of GPIba and purified GPIba could be phosphorylated at Ser⁵⁵⁹ by the catalytic subunit of protein kinase A (PKA). Ser⁵⁵⁹ phosphorylation was enhanced by PKA activators and reduced by PKA inhibitors in platelets and Chinese hamster ovary (CHO) cells expressing GPIb-IX. Furthermore, PKA inhibitor enhanced, however, reduced VWF binding to two kinds of CHO cells expressing GPIb-IX mutant replacing Ser⁵⁵⁹ of GPIba (S559A) or Ser¹⁶⁶ of GPIbβ (S166A) with alanine, respectively. GPIb-IX association with filamin A and membrane expression were enhanced in S559A cells, whereas reduced in S166A cells. The peptide disrupting the interaction of 14-3-3ζ with Ser⁵⁵⁹ of GPIba inhibited platelet spreading on VWF matrix and GPIb-IX-VWF interaction-induced association of Src with GPIb-IX. These data indicate that Ser⁵⁵⁹ of GPIba is phosphorylated by PKA which plays key roles in regulating VWF binding, surface expression, and signaling of GPIb-IX.