INSULIN AND INSULIN-LIKE GROWTH FACTOR-I SIGNAL-TRANSDUCTION REQUIRES P21(RAS)

We have investigated the role of cellular p21(ras) protein in insulin and insulin-like growth factor-I (IGF-I) signaling pathways. Insulin stimulation increased Ras-GTP formation in Rat-1 fibroblasts overexpressing normal human insulin receptors (HIRc-B), far greater than in parental Rat-1 fibroblasts, indicating that competent insulin receptors mediate this response, Cellular microinjection of a dominant-negative mutant p21(ras) protein (N17 ras) or anti-pa21(ras) monoclonal antibody (Y13-259) into HIRc-B cells reduced insulin- and IGF-I-stimulated DNA synthesis by 75-90%. Insulin-induced c-fos protein expression was also inhibited by 74%. Microinjection of oncogenic p2l(ras) (T-24 ras) into HIRc-B cells activated the mitogenic pathway, and coinjection of N17 ras and T-24 ras showed that oncogenic p21(ras) rescued the cells from the N17 ras blockade. This later finding indicates that T-24 ras acts downstream of N17 ras. In conclusion, 1) microinjection of a dominant interferring ras mutant into quiescent cells abrogated subsequent insulin and IGF-I mitogenic signaling; 2) oncogenic ras protein rescued cells from the N17 ras blockade, indicating that T24 ras action is downstream of the site of N17 inhibition; and 3) p21(ras) is an intermediate signaling molecule in the insulin/IGF-I signal transduction pathway and is required for gene expression and DNA synthesis.