Relative involvement of Shc tyrosine 239/240 and tyrosine 317 on insulin induced mitogenic signaling in rat1 fibroblasts expressing insulin receptors.

Shc is phosphorylated on Tyr-239/240 and/or Tyr-317, which serves as a docking site for Grb2. To clarify the relative involvement of Shc Tyr-239/240 and Tyr-317 in insulin-induced mitogenesis, we generated expression vectors for Y317F (1F)-Shc, Y239/240F (2F)-Shc, and Y239/240/317F (3F)-Shc, and stably transfected them into Rat1 fibroblasts expressing insulin receptors (HIRc). Insulin-induced Shc phosphorylation and subsequent association with Grb2 was enhanced in wild-type (WT)-Shc cell. In contrast, insulin-stimulated Shc phosphorylation and Shc.Grb2 association were significantly decreased in 1F-Shc and 3F-Shc cells, while these were only slightly affected and almost comparable in 2F cells compared with those in parental HIRc cells. The kinetics of MAP kinase activation closely paralleled the kinetics of Shc phosphorylation and Shc.Grb2 association. Thus, insulin stimulation of MAP kinase activation occurred more rapidly in WT-Shc cells, and the activation was delayed in 1F-Shc and 3F-Shc cells, while it was comparable in 2F-Shc cells compared with that in HIRc cells. Furthermore, WT-Shc cells displayed enhanced sensitivity to insulin stimulation of thymidine incorporation. Importantly, the sensitivity was significantly decreased in 1F-Shc and 3F-Shc cells, while it was almost comparable in 2F-Shc cells compared with that in HIRc cells. These results indicate that Shc Tyr-317 is more predominant insulin-induced phosphorylation site than Tyr-239/240 for coupling with Grb2 leading to MAP kinase activation and mitogenesis in Rat1 fibroblasts.