Insulin-dependent tyrosine phosphorylation of the vav proto-oncogene product in cells of hematopoietic origin

Insulin activates the ras signaling pathway and promotes hematopoietic cell proliferation. One possible mediator in such signaling is the vav proto- oncogene product (p95(vav)), which is specifically expressed in cells of hematopoietic origin and contains domains typical of guanine nucleotide exchange factors as well as Src homology 2 and Src homology 3 domains. We studied the tyrosine phosphorylation of p95(vav) in hematopoietic cells expressing insulin receptors. Immunoblotting experiments with an antiphosphotyrosine monoclonal antibody disclosed that insulin induces rapid and transient tyrosine phosphorylation of p95(vav) in the human U-266 myeloma cell line. These findings were confirmed by immunoprecipitation experiments performed with ³²P-labeled cells and phosphoamino acid analysis of the bands corresponding to p95(vav). Similarly, insulin-dependent tyrosine phosphorylation of p95(vav) was observed in the human IM-9 and mouse J558L hematopoietic cell lines. Furthermore, insulin treatment of cells led to the association of the Src homology 2 domain of p95(vav) with the activated β- subunit of the insulin receptor in vitro. Altogether, these data suggest that p95(vav) is a substrate for the insulin receptor tyrosine kinase and may be involved in an insulin signaling pathway linking receptor-generated signals to Ras or other GTP-binding proteins in cells of hematopoietic origin.