FUNCTIONAL IMPORTANCE OF SHC TYROSINE-317 ON INSULIN SIGNALING IN RAT1 FIBROBLASTS EXPRESSING INSULIN-RECEPTORS

Shc is phosphorylated on Tyr-317, which serves as a docking site for G rb2. To investigate the specific role of Shc phosphorylation and Shc.G rb2 coupling on insulin signaling, we generated expression vectors for wild-type (WT-Shc) and a mutant Shc with a Tyr-317 --> Phe substituti on (317Y/F-Shc) and stably transfected them into Rat1 fibroblasts expr essing insulin receptors (HIRc). From different clonal cell lines, cel ls expressing 10 times greater amounts of WT-Shc or 317Y/F-Shc compare d with endogenous Shc were chosen for analysis of insulin signaling. I nsulin-induced Shc phosphorylation and subsequent association with Grb 2 was enhanced in WT-Shc cells, Because of competition between Shc and IRS-1 for interaction with the insulin receptor, insulin-stimulated t yrosine phosphorylation of IRS-1 was decreased in WT-Shc cells compare d with that in HIRc cells, Likewise, reduction of endogenous Shc expre ssion by antisense Shc mRNA resulted in increased insulin stimulation of IRS-1 phosphorylation, Although 317Y/F-Shc was also able to interac t with insulin receptor, decreased amounts of Shc phosphorylation and Shc association with Grb2 were observed in 317Y/F-Shc cells, indicatin g that 317Y/F-Shc functions as a dominant-negative mutant. The kinetic s of mitogen-activated protein (MAP) kinase activation closely paralle led the kinetics of Shc phosphorylation. Thus, insulin stimulation of MAP kinase activation occurred more rapidly and was prolonged in WT-Sh c cells, while the activation was delayed and transient in 317Y/F-Shc cells compared with that in HIRc cells, Importantly, WT-Shc cells disp layed enhanced sensitivity to insulin stimulation of thymidine and bro modeoxyuridine incorporation, whereas the sensitivity was decreased in 317Y/F-Shc cells. These results indicate that Shc Tyr-317 phosphoryla tion plays an important role, via coupling with Grb2 and competition w ith IRS-1, in signal transduction to MAP kinase by insulin, ultimately leading to mitogenesis in Rat1 fibroblasts.