INTERACTIONS OF CBL WITH GRB2 AND PHOSPHATIDYLINOSITOL 3'-KINASE IN ACTIVATED JURKAT CELLS

T-cell receptor (TCR) cross-linking increases tyrosine phosphorylation of multiple proteins, only a few of which have been identified. One o f the most rapidly tyrosine-phosphorylated polypeptides is the 120-kDa product of the proto-oncogene c-cbl, a cytosolic and cytoskeletal pro tein containing multiple proline-rich motifs that are potential bindin g sites for proteins containing Src homology 3 (SH3) domains. We repor t here that in cultured Jurkat T cells, Cbl is coprecipitated with ant ibody against the adapter protein Grb2. Upon activation of Jurkat T ce lls via the TCR-CD3 complex, we find that high-affinity binding of Cbl requires the N-terminal SH3 domain of GST-Grb2 fusion protein but aft er cross-linking of the TCR-CD3 and CD4 receptors, Cbl binds equally t o its SH2 domain. Grb2 antisera also precipitated p85 from serum-starv ed cells, while TCR activation increased p85 and tyrosine-phosphorylat ed Cbl but not Cbl protein in Grb2 immunocomplexes. Phosphatidylinosit ol (PI) 3-kinase activity was immunoprecipitated from serum-starved ce lls with Cbl and to a lesser extent,vith Grb2 antisera, and TCR cross- linking increased this activity severalfold. The PI 3-kinase activity associated with Cbl amounted to 5 to 10% of the total cellular activit y that could be precipitated by p85 antisera. The Pas exchange factor Son-of-sevenless 1 (Sos-1) was not found in anti-Cbl immunoprecipitate s from activated cells, and Cbl was not detectable in anti-Sos-1 preci pitates, supporting the likelihood that Sos-Grb2 and Cbl-Grb2 are pres ent as distinct complexes. Taken together, these data suggest that Cbl function in Jurkat T cells involves its constitutive association with Grb2 and its recruitment of PI 3-kinase in response to TCR activation .