Characterization of Platelet Lipid Rafts Leads to the Identification of RGS-19, Which Forms a Phosphorylation-Dependent Complex with 14-3-3ζ.

Abstract

Lipid rafts are specialised membrane regions, rich in dynamic multi-protein complexes that are implicated in the regulation of platelet activation processes, particularly those mediated by GPVI and GPIb/IX/V. To elucidate the role of rafts in platelet function, we carried out a comprehensive characterization of the detergent-insoluble proteins associated with rafts from control and VWF-activated platelets using liquid chromatography coupled to tandem mass spectrometry. Over 160 proteins were identified including 72 proteins unique to platelet rafts and several novel platelet signaling and vesicle transport proteins e.g., rab 5, rab 8 and syntaxin 11. Indeed, the high level of signaling and trafficking proteins identified implicates rafts as concentrating platforms for the critical platelet functions of activation and secretion. Moreover, activation through GPIb-IX-V resulted in the translocation of many proteins into or out of rafts. Proteins recruited into rafts upon VWF-activation included GPIbαand regulator of G-protein signalling-19 (RGS-19). We confirmed that the novel platelet protein, RGS-19, upon VWF activation resides exclusively within the raft fraction and using confocal microscopy, RGS-19 co-localised with GPIbα and the raft-marker flottilin, to both the plasma and vesicle membranes of VWF-activated platelets. The phosphorylation-dependent interaction of RGS proteins with 14-3-3 leads to the inhibition of their GTPase-activating protein (GAP) activity. Aligning RGS-19 to other members of the RGS family, we identified that it contained a novel 14-3-3 binding motif. Immunoprecipitation with an antibody to 14-3-3ζ demonstrated that RGS-19 and GPIbα co-immunoprecipitated with 14-3-3ζ in VWF-activated rafts. Furthermore, we demonstrated that acid phosphatase treatment could significantly reduce the co-immunoprecipitation of RGS-19 with 14-3-3ζ from activated rafts. Thus, within 15 sec of VWF activation we are suggesting maximal suppression of the GAP activity of RGS-19 in rafts through a phosphorylation-dependent association with 14-3-3ζ. Our results provide the basis for a new hypothesis concerning how RGS proteins might govern signaling within platelet rafts.