Physical and functional association of the cbl protooncogene product with an Src-family protein tyrosine kinase, p53/56(lyn), in the B cell antigen receptor-mediated signaling

To identify novel signal transducers involved in signaling mediated by the Src-family protein tyrosine kinases (PTKs), we used a yeast two-hybrid system with a probe corresponding to the regulatory region of p56(lyn), a member of Src-family PTKs. One of the isolated clones contained the COOH-terminal 470 amino acid residues of p120(c-cbl), the product of the cellular homologue of the v-cbl retroviral oncogene. p120(c-cbl) is a cytoplasmic protein with nuclear protein-like motifs. Here we show in vivo association of p120(c-cbl) with p53/56(lyn). After stimulation of the B cell antigen receptor (BCR), p120(c-cbl) was rapidly tyrosine phosphorylated. Studies with lyn- or syk-negative chicken B cells demonstrated that p53/56(lyn), but not p72(syk), was crucial for tyrosine phosphorylation of p120(c-cbl) upon stimulation of the BCR. We also show the importance of p59(fyn) in tyrosine phosphorylation of p120(c-cbl) in the T-cell receptor-mediated signaling, using fyn-overexpressing T cell hybridomas and splenic T cells from fyn-deficient mice. These results suggest that p120(c-cbl) is an important substrate of Src-family PTKs in the intracellular signaling mediated by the antigen receptors.