Functional Regulation of the GPIb-IX-V Complex by 14-3-3ζ: Role of Protein Kinase A Phosphorylation of Multiple GPIbα Cytoplasmic Residues.

Platelet adhesion to the site of vascular injury is essential to prevent blood loss. The initial step of adhesion is mediated by the glycoprotein (GP) Ib-IX-V complex on the platelet surface, binding von Willebrand factor (VWF) on the exposed subendothelium. This interaction is transitory, resulting in platelet rolling, and elicits “inside-out” activation of the integrin αIIβ3, thus instigating stable arrest of platelets on fibrinogen and/or VWF and their subsequent spreading and aggregation. The GPIb-IX-V complex consists of 4 polypeptides: GPIbα disulfide linked to GPIbβ, GPIX, and GPV. While recent effort has focused on elucidation of GPIb-IX-V-generated signals, much remains to be learned. Each component of the complex is a type I transmembrane protein, possessing a C-terminal cytoplasmic tail. Of these, GPIbα’s is the longest at 96 amino acids, and associates with both signaling molecules (PI 3-kinase and Src kinase) and structural proteins (filamin A and 14-3-3ζ). Yet, the GPIbα cytoplasmic sequence lacks domains used by other receptors to recruit signaling molecules. Its only tyrosine residue, at amino acid 605, is not within a known consensus sequence for phosphorylation. However, 10 serine and 8 threonine residues are contained within the cytoplasmic domain. Of these, S587, S590, and S609 are known to be stably phosphorylated in resting platelets and to facilitate 14-3-3ζ binding. S609 does not reside in a consensus motif for phosphorylation, whereas S590 and S587 are within consensus motifs for Casein kinase I and the cAMP-dependent protein kinase A (PKA), respectively. Two other residues, T547 and S566, also reside within consensus sites for PKA. PKA has previously been demonstrated to phosphorylate S166 of GPIbβ and thereby inhibit platelet function. We hypothesized that phosphorylation of GPIbα by PKA also regulates platelet function and 14-3-3ζ binding. To test this, we produced a recombinant protein comprising the cytoplasmic domain of GPIbα (amino acids 515–610) fused to glutathione S-transferase at its N-terminus and evaluated the ability of PKA to phosphorylate the protein in vitro. Once we established that PKA could indeed phosphorylate the protein, we produced the recombinant in bacteria also expressing the PKA catalytic domain in an effort to phosphorylate the recombinant GPIbα cytoplasmic domain de novo to avoid cumbersome in vitro phosphorylations and increase the yield. Analysis using a phosphoS609 antibody demonstrated that GPIbα was phosphorylated on S609. We also examined 14-3-3 binding to wild type and mutant GPIbα expressed as part of the GPIb-IX complex in CHO cells (CHOαβIX) by evaluating which proteins were pulled down with GST-14-3-3ζ from lysates. 14-3-3ζ was able to pull down wild-type GPIbα, but only 5–10% as much of GPIbα S609A. Combined mutation of T547 and S609, each to A, completely abrogated 14-3-3ζ binding, as did combined mutation to A of T547, S566, S587, S590 and S609. Interestingly, approximately 20% residual binding was observed for GPIbα S587A/S590A and GPIbα T547A/S587A/S590A/S609A. These results indicate that PKA phosphorylates T547, S566, S587, S590 and S609 in vitro and at least S609 de novo in bacteria. They also demonstrate that 14-3-3ζ can associate with the cytoplasmic tail of GPIbα via residues T547, S566, S587, S590 and S609. This suggests a complex pattern of functional regulation of the GPIb-IX-V complex by PKA mediated through differential binding of 14-3-3ζ, involving both GPIbα and GPIbβ.