OC 58.2 - Conformational Change in Platelet αIIbβ3 Induce PKC-dependent Serine 284 Phosphorylation of Junctional Adhesion Molecule-A and Rap1 Activation

Background: In resting platelets tyrosine phosphorylated JAM-A suppresses αIIbβ3 signaling by recruiting CSK to the αIIbβ3-Src complex. Upon platelet activation JAM-A is rapidly phosphorylated on S284 residue.

Aims: To elucidate the role of ligand-binding to αIIbβ3 in JAM-A S284 phosphorylation.

Methods: We used Mn2+/DTT/RGDS to induce conformation change in αIIbβ3 without inside-out signaling and ligand binding. Full αIIbβ3 activation was assessed by PAC-1 binding using flowcytometry. Immunoprecipitation and western blot analyses were performed to determine JAM-A Y280 or S284 phosphorylation. Jam-A null mouse platelets were used to detect Rap1-activation during outside-in signaling.

Results: We show that in resting platelets JAM-A is phosphorylated on Y280. Upon agonist-induced activation of αIIbβ3, JAM-A is rapidly dephosphorylated on Y280 and concomitantly, phosphorylated on S284 in a PKC-dependent manner. We observed that conformational change in αIIbβ3 by Mn2+ (10uM) is sufficient for JAM-A S284 phosphorylation without requiring inside-out signaling. RGDS peptide (200uM) or DTT (5mM) also induced JAM-A S284 phosphorylation similar to that induced by Mn2+. Moreover, this phosphorylation is independent of ligand binding to αIIbβ3 since the presence or absence of fibrinogen had no effect on Mn2+-induced phosphorylation. We found that αIIbβ3-dependent S284 phosphorylation of JAM-A was abolished by pharmacological inhibitors of PLC gamma, Src, and FAK suggesting that outside-in signaling is responsible for JAM-A S284 phosphorylation. Pan-PKC inhibitor bisindolylmaleimide I, but not calcium chelator BAPTA-AM inhibited S284 phosphorylation of JAM-A, highlighting the role for PKC in S284 phosphorylation of JAM-A. We found that aPKC zeta specifically associated with JAM-A upon platelet activation and this association is inhibited by PKC inhibitor. Furthermore, outside-in signaling dependent Rap1 activation did not occur in Jam-A null platelets indicating that Jam-A regulates outside-in dependent Rap1 activation.

Conclusion(s): Our results suggest that conformational change in αIIbβ3 is sufficient to induce outside-in signaling dependent S284 phosphorylation of JAM-A and Rap1 activation.