Serine 559 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

Conformational changes in von Willebrand factor (VWF) exposed at the injured vessel wall initiate glycoprotein (GP) Ib-IX-dependent platelet adhesion, and simultaneously trigger signaling cascades leading to integrin α_IIbβ_III activation, platelet spreading, and thrombus formation. The intracellular signaling protein 14-3-3ζ and the membrane skeleton protein filamin A have been confirmed to interact with the cytoplasmic domain of GPIbα and play key roles in regulating the VWF binding function of GPIb-IX. Several phosphoserine containing motifs in GPIb-IX complex, such as Ser⁶⁰⁹ of GPIbα and Ser¹⁶⁶ of GPIbβ, have been confirmed to bind to 14-3-3ζ and involved in the regulation of VWF binding function. We have recently identified a novel 14-3-3ζ binding site located in the filamin A binding domain of GPIbα at Ser⁵⁵⁹. Phosphorylation of GPIbα at Ser⁵⁵⁹ not only is important for 14-3-3ζ binding, but also plays a key role in the regulation of VWF binding function of GPIb-IX. However, the protein kinase responsible for Ser⁵⁵⁹ phosphorylation remains unknown. Here we show that a peptide corresponding to the Arg⁵⁵⁷-Pro⁵⁶¹ region of GPIbα and purified GPIbα 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 VWF binding to the CHO cells expressing GPIb-IX mutant replacing Ser⁵⁵⁹ of GPIbα (S559A) with alanine, while reduced in the CHO cells of Ser¹⁶⁶ of GPIbβ (S166A) with alanine. 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 GPIbα inhibited platelet spreading on VWF matrix and GPIb-IX-VWF interaction-induced association of Src with GPIb-IX. These data indicate that Ser⁵⁵⁹ of GPIbα is phosphorylated by PKA which plays key roles in regulating VWF binding, surface expression, and signaling of GPIb-IX.