Abstract
Protein-tyrosine phosphatase TULA-2 has been shown to regulate receptor signaling in several cell types, including platelets. Platelets are critical for maintaining vascular integrity; this function is mediated by platelet aggregation in response to recognition of the exposed basement membrane collagen by the GPVI receptor, which is non-covalently associated with the signal-transducing FcRγ polypeptide chain. Our previous studies suggested that TULA-2 plays an important role in negatively regulating signaling through GPVI-FcRγ and indicated that the tyrosine-protein kinase Syk is a key target of the regulatory action of TULA-2 in platelets. However, the molecular basis of the down-regulatory effect of TULA-2 on Syk activation via FcRγ remained unclear. In this study, we demonstrate that suppression of Syk activation by TULA-2 is mediated, to a substantial degree, by dephosphorylation of Tyr(P)346, a regulatory site of Syk, which becomes phosphorylated soon after receptor ligation and plays a critical role in initiating the process that yields fully activated Syk. TULA-2 is capable of dephosphorylating Tyr(P)346 with high efficiency, thus controlling the overall activation of Syk, but is less efficient in dephosphorylating other regulatory sites of this kinase. Therefore, dephosphorylation of Tyr(P)346 may be considered an important “checkpoint” in the regulation of Syk activation process. Putative biological functions of TULA-2-mediated dephosphorylation of Tyr(P)346 may include deactivation of receptor-activated Syk or suppression of Syk activation by suboptimal stimulation.
Highlights
Protein-tyrosine phosphatase TULA-2 has been shown to regulate receptor signaling in several cell types, including platelets
To rule out the possibility that the effects observed in our experiments were at least partially due to the lack of TULA, which can be present in platelets at a low level, we analyzed the effects of the single TULA and TULA-2 KOs on platelet responses to two strong agonists of the GPVI collagen receptor, convulxin (CVX), a snake venom protein (Fig. 1A), and the synthetic collagen-related peptide (CRP) (Fig. 1B), both mimicking collagen, which is a natural ligand of GPVI
Effects of TULA-2 KO and TULA KO on GPVI signaling in platelets, which was assessed by phosphorylation of Syk in experiments examining its time course (Fig. 1C) or ligand concentration dependence (Fig. 1D), were in agreement with their effects on physiological responses; activation-induced Sykphosphorylation in TULA KO platelets was not significantly different from that in WT platelets, whereas activation-induced Syk phosphorylation in TULA-2 KO platelets was significantly different from that in WT and TULA KO platelets
Summary
Protein-tyrosine phosphatase TULA-2 has been shown to regulate receptor signaling in several cell types, including platelets. Our previous studies suggested that TULA-2 plays an important role in negatively regulating signaling through GPVI-FcR␥ and indicated that the tyrosine-protein kinase Syk is a key target of the regulatory action of TULA-2 in platelets.
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