Tyrosine phosphorylation and complex formation of Cbl-b upon T cell receptor stimulation

Cbl-b, a mammalian homolog of Cbl, consists of an N-terminal region (Cbl-b- N) highly homologous to oncogenic v-Cbl, a Ring finger, and a C-terminal re gion containing multiple proline-rich stretches and potential tyrosine phos phorylation sites. In the present study, we demonstrate that upon engagemen t of the T cell receptor (TCR), endogenous Cbl-b becomes rapidly tyrosine-p hosphorylated, In heterogeneous COS-1 cells, Cbl-b was phosphorylated on ty rosine residues by both Syk(Syk/Zap-70) and Src- (Fyn/Lck) family kinases, with Syk kinase inducing the most prominent effect. Syk associates and phos phorylates Cbl-b in Jurkat T cells, A Tyr-316 Chi-binding site in Syk was r equired for the association with and for the maximal tyrosine phosphorylati on of Cbl-b, Mutation at a loss-of-function site (Gly-298) in Cbl-b-N disru pts its interaction with Syk, Cbl-b constitutively binds Grb2 and becomes a ssociated with Crk-L upon TCR stimulation. The Grb2- and the Crk-L-binding regions were mapped to the C-terminus of Cbl-b, The Crk-L-binding sites wer e further determined to be (YDVP)-D-655 and (YKIP)-K-709, With th, latter b eing the primary binding site. Taken together, these results implicate that Cbl-b is involved in TCR-mediated intracellular signaling pathways.