FYN, YES, AND SYK PHOSPHORYLATION SITES IN C-CBL MAP TO THE SAME TYROSINE RESIDUES THAT BECOME PHOSPHORYLATED IN ACTIVATED T-CELLS

Protooncogenic protein c-Cbl undergoes tyrosine phosphorylation in res ponse to stimulation through the receptors for antigens, immunoglobuli ns, cytokines, and growth factors as well as through the integrins. Ty rosine phosphorylation of c-Cbl may play a functional role in signal t ransduction, since c-Cbl interacts with many crucial signaling molecul es including protein tyrosine kinases, adaptor proteins, and phosphati dylinositol 3'-kinase. Therefore, it is essential for our understandin g of the functions of c-Cbl in signal transduction to identify its tyr osine phosphorylation sites, to determine the protein-tyrosine kinases that phosphorylate these sites, and to elucidate the role of these si tes in the interactions of c-Cbl with other signaling proteins. In thi s report, we demonstrate that tyrosines 700, 731, and 774 are the majo r tyrosine phosphorylation sites of c-Cbl in T cells in response to pe rvanadate treatment, as well as in response to TcR/CD3 ligation. Coexp ression experiments in COS cells demonstrate that among T cell-express ed Src-and Syk-related protein-tyrosine kinases, Fyn, Yes, and Syk app ear to play a major role in phosphorylation of c-Cbl, whereas Lck and Zap phosphorylate c-Cbl ineffectively. Fyn, Yes, and Syk phosphorylate the same sites of c-Cbl that become phosphorylated in stimulated T ce lls. Among these kinases, Fyn and Yes demonstrate strong binding to c- Cbl, which involves both phosphotyrosine-dependent and phosphotyrosine -independent mechanisms.