Syk Is Regulated Downstream Of FcγRIIA In Platelets By Transient Tyrosine Phosphorylation and Ubiquitylation

Platelet FcγRIIA contributes to the pathophysiology of heparin-induced thrombocytopenia (HIT) and other immune-mediated thrombocytopenia and thrombosis syndromes. Activation of FcγRIIA results in tyrosine phosphorylation of Syk in human platelets, but little is known about ubiquitylation of Syk. Protein ubiquitylation has been shown to regulate physiological and pathological cellular processes by regulating signaling networks. In the Platelet RNA and Expression-1 (PRAX1) study, we studied platelets from 154 healthy subjects and identified increased expression of mRNAs encoding proteins involved in ubiquitylation in platelets that were highly reactive to FcγRIIA stimulation. Previously, we (CD, SPK) reported that platelet stimulation via GPVI/FcRγ results in transient Syk phosphorylation and ubiquitylation, involving c-cbl and TULA-2. Briefly, c-Cbl acts as an E3 ligase transferring ubiquitin to the lysine residue of the target protein in the ubiquitylation reaction and TULA-2 is a dephosphorylating enzyme that removes phosphate group from ubiquitinated syk. In this study, we investigated Syk tyrosine phosphorylation and ubiquitylation downstream of platelet FcγRIIA engagement.

FcγRIIA on washed human platelets was activated by cross-linking with IV.3 antibody (10ug/ml) and goat anti-mouse antibody (GAM) (30ug/ml). At specific time intervals following activation, platelets were lysed and the lysates were immunoblotted for total Syk and for pY525/526 phospho-Syk. We observed phosphorylation and ubiquitylation of Syk within 15 sec, and peaking within 1 to 3 min. The MW of the Syk species was consistent with 1 to 3 ubiquitin (Ub) moieties per Syk molecule. Alternatively, platelet FcγRIIA was activated via anti-CD9; Ub-Syk was observed at 15 sec, while ubiqutinated and phosphorylated Syk was observed at 2 min. Additional ubiquitinated bands appeared by 2 min; all diminished after 5 min. To understand if the ubiquitinated Syk was degraded by the proteasomal system, human platelets were pre-treated with proteasomal inhibitors (MG132 or Epoxomicin) followed by activation of FcγRIIA. There was no accumulation of Ub-Syk observed for up to 5 minutes after FcγRIIA stimulation in the presence of proteasomal inhibitors compared to controls, indicating that Ub-Syk is not degraded by the proteasomes and inactivated. Activation of FcγRIIA by cross linking with IV.3/GAM in HEL cells, a cell line model, showed a transient increase in ubiquitylation and phosphorylation of Syk within 15 sec which decreased by 3 min. To understand if activation of Syk by upstream Src-family kinases is necessary for ubiquitylation in this model system, SFK inhibitor (PP2) (10uM) was pre-incubated with HEL cells followed by FcγRIIA activation. Western blot analysis showed elimination of phosphorylated and ubiquitinated Syk upon activation of FcγRIIA in HEL cells in presence of PP2. In contrast, ubiquitylation and phosphorylation of Syk was observed in control cells treated with PP3 or vehicle (DMSO) and as well as in untreated cells. HEL cells were also pretreated with proteasomal inhibitors (MG132 or Epoxomicin) followed by activation of FcγRIIA. There was no accumulation of phosphorylated and ubiquitinated Syk observed for up to 10 min of stimulation of FcγRIIA in HEL cells in presence of proteasomal inhibitors compared to controls indicating no degradation of Ub-syk by proteasomal complex. Finally, we directly compared the ubiquitylation and phosphorylation of Syk in GPVI/FcRγ-stimulated vs.FcγRIIA-stimulated platelets from the same donors. Of note, FcγRIIA-stimulated platelets demonstrated both different kinetics and extent of Syk ubiquitylation than did GPVI/FcRγ-stimulated platelets.

In this study, we demonstrated that FcγRIIA signaling results in transient Syk tyrosine phosphorylation and ubiquitylation in platelets and HEL cells. The ubiquitinated Syk is not destined for proteasomal degradation. Limited ubiquitylation of proteins has been noted to modulate downstream signaling events and protein-protein interactions. Further studies are needed to decipher the molecular partners of ubiquitinated Syk following FcγRIIA activation, and to elucidate the differences between GPVI/FcRγ and FcγRIIA signaling via Syk.