Regulation of Syk Function by Ubiquitination in Human Platelets.

Abstract Platelets have an integral function in maintaining hemostasis. Signaling pathways are strictly regulated to ensure the cessation of bleeding without precipitating a thrombotic episode. An important initial physiological step in this process is the interaction of platelets with freshly exposed subendothelial collagen after vascular injury. A major platelet receptor involved is the GPVI/FcRγ-chain complex. Upon activation of this receptor, the immunoreceptor tyrosine-based activation motif (ITAM) present in the FcRγ-chain is phosphorylated by Src family kinases. This causes the tyrosine kinase Syk to bind to the ITAM where it is autophosphorylated, initializing a signaling cascade that activates a number of proteins including PLCγ2, PI-3 kinase and small G proteins. Syk appears to play an early pivotal role in GPVI/FcRγ-chain signal transduction and its regulation is crucial. Recent studies have shown that platelet aggregation, in response to collagen-related peptide (CRP), is potentiated in c-Cbl knockout mice. c-Cbl, a protein containing an E3 ligase responsible for substrate recognition for ubiquitin, appears to regulate numerous cytoplasmic kinases in other cell systems. This protein modification has emerged as one of the most common regulatory processes in all eukaryotes, second only to possibly phosphorylation. It appears to target proteins for proteosomal degradation although recently other mechanisms have been described, ranging from protein kinase activation to translation control. In the work presented here, we demonstrate that Syk associates with c-Cbl after GPVI/FcRγ-chain activation and therefore explored the possibility that Syk is ubiquitinated during GPVI/FcRγ-chain stimulation in human platelets. We have found that Syk is rapidly ubiquitinated upon activation by collagen, CRP and the snake venom protein convulxin, but not thrombin. PP2 and SU6656, two Src kinase inhibitors, prevented Syk phosphorylation and its ubiquitination, indicating that the process is downstream of Src kinases and probably requires Syk phosphorylation. The ubiquitination of Syk did not cause any apparent degradation of the protein as evidenced by the lack of effect of proteosomal and lysosomal inhibitors. We have been able to separate ubiquitinated Syk from its non-ubiquitinated counterpart. We have used an in vitro kinase assay to compare the activity of ubiquitinated Syk to non-ubiquitinated Syk. Surprisingly, when we compared the specific activity of the two Syk fractions, we found that the ubiquitinated Syk appeared to be about five-fold more active. Using a phosphospecific antibody to Syk (Tyr525/Tyr526) that measures activated Syk, we found that the majority (75%) of the active Syk is in the ubiquitinated fraction. In addition we found that Syk is not ubiquitinated in c-Cbl deficient mice. We therefore propose that c-Cbl plays a regulatory role in GPVI/FcRγ-chain stimulation through ubiquitination of Syk. The fact that 75% of active Syk is ubiquitinated suggests that ubiquitination plays an essential role in the regulation of its function. A possible function of ubiquitination may be targeting Syk to appropriate signaling molecules.