To examine the functional role of Shc tyrosine phosphorylation in IGF-1 signaling, wild-type (WT)-Shc and Y239,240,317F (3F)-Shc were transiently transfected into L6 myoblasts. IGF-1 signaling was compared among the transfected cells. IGF-1-induced tyrosine phosphorylation of Shc and its subsequent association with Grb2 were increased in WT-Shc cells, whereas they were decreased in 3F-Shc cells compared with those in parental L6 cells. Consistent with their changes, IGF-1-induced MAPK activation and thymidine incorporation were enhanced in WT-Shc cells, whereas they were again decreased in 3F-Shc cells. It is possible that Shc and insulin receptor substrate (IRS)-1 can interact competitively, via their phosphotyrosine binding (PTB) domains, with the activated IGF-1 receptor. In this regard, IGF-1-induced tyrosine phosphorylation of IRS-1 was decreased by overexpressing both WT-Shc and 3F-Shc cells. Consistent with the decrease, IGF-1-induced IRS-1 association with the p85 subunit of PI3K and activation of PI3K and Akt were reduced in both WT-Shc and 3F-Shc cells. As a result, IGF-1-induced glycogen synthesis was also decreased in both cells. Furthermore, expression of Shc PTB domain alone inhibited IGF-1 stimulation of Akt and glycogen synthesis. These results indicate that tyrosine phosphorylation of Shc is important for IGF-1 stimulation of MAPK leading to mitogenesis and that Shc, via its PTB domain, negatively regulates IGF-1-induced glycogen synthesis by competing with IRS-1, which is not relevant to Shc tyrosine phosphorylation.