SYK-DEPENDENT AND LYN-DEPENDENT PHOSPHORYLATION OF SYK ON MULTIPLE TYROSINES FOLLOWING B-CELL ACTIVATION INCLUDES A SITE THAT NEGATIVELY REGULATES SIGNALING

The Syk protein tyrosine kinase is an essential component of the B cel l Ag receptor signaling pathway, Syk is phosphorylated on tyrosine fol lowing B cell activation. However, the sites that are modified and the kinases responsible for these modifications have Set to be determined . To approach this problem, we used a mapping strategy based on the el ectrophoretic separation of peptides on alkaline polyacrylamide gels t o identify the tryptic phosphopeptides derived from metabolically labe led Syk. In this work, we report that Syk from activated B cells is ph osphorylated principally on six tyrosines: one located between the tan dem SH2 domains (Tyr(130)); three in the linker region (Tyr(317), Tyr( 342), and Tyr(346)); and two in the catalytic domain (Tyr(519) and Tyr (520)). The linker region sites are the primary targets of the Src fam ily protein tyrosine kinase, Lyn, and include a site that negatively ( Tyr(317)) regulates receptor signaling. Efficient phosphorylation of t he catalytic domain and inter-SH2 domain tyrosines is catalyzed primar ily by SSk itself, but only occurs to an appreciable extent in cells t hat express Lyn. We propose that these sites are phosphorylated follow ing the binding of SSk to immunoreceptor tyrosine-based activation mot if.