EVIDENCE FOR A FUNCTIONAL-ROLE OF SHE PROTEINS IN MITOGENIC SIGNALINGINDUCED BY INSULIN, INSULIN-LIKE GROWTH-FACTOR-I, AND EPIDERMAL GROWTH-FACTOR

Shc proteins contain a single SH2 domain, lack catalytic activity, and are substrates for activated receptors for insulin, insulin-like grow th factor-1 (IGF-1), and epidermal growth factor (EGF). Treatment with these growth factors induced rapid tyrosine phosphorylation of Shc. W e investigated the potential role of Shc in mitogenic signaling. Affin ity-purified antibodies were microinjected into living Rat1 fibroblast s overexpressing human insulin receptors. Bromodeoxyuridine incorporat ion into newly synthesized DNA was subsequently studied to assess the importance of Shc. Cellular microinjection of anti-Shc antibody inhibi ted BrdU incorporation induced by insulin, IGF-1, and EGF, but did not affect cells stimulated by fetal calf serum. Microinjection of an onc ogenic p21(ras) protein (T24) into quiescent cells produced constituti vely active mitogenic signaling, and comicroinjection of T24 with the anti-Shc antibody restored insulin and EGF stimulation of DNA synthesi s. Immunoprecipitates of Shc from lysates of insulin-stimulated cells removed 70-80% of guanine nucleotide-releasing factor activity. These results indicate that Shc is an important component in a mitogenic sig nal transduction pathway that is shared by insulin, IGF-1, and EGF. Th e functional locus of Shc is either upstream of p21(ras) or lies on a distinct branch of the pathway leading to cell cycle progression.