Membrane localization of Src homology 2-containing inositol 5′-phosphatase 2 via Shc association is required for the negative regulation of insulin signaling in Rat1 fibroblasts overexpressing insulin receptors

Lipid phosphatase SHIP2 [Src homology 2 (SH2)-containing inositol 5'-phosphatase 2] has been shown to be a physiologically critical negative regulator of insulin signaling. We investigated the molecular mechanism by which SHIP2 negatively regulates insulin-induced phosphorylation of Akt, a key downstream molecule of phosphatidylinositol 3-kinase important for the biological action of insulin. Overexpression of wild-type SHIP2 (WT-SHIP2) inhibited insulin-induced phosphorylation of Akt at both Thr(308) and Ser(473) in Rat1 fibroblasts expressing insulin receptors. The degree of inhibition was less in the cells expressing either a mutant SHIP2 with R47Q change (R/Q-SHIP2) in the SH2 domain, or a mutant SHIP2 with Y987F change (Y/F-SHIP2) in the C-terminal tyrosine phosphorylation site. However, on addition of a myristoylation signal, WT-SHIP2, R/Q-SHIP2, and Y/F-SHIP2 all efficiently inhibited insulin-induced Akt phosphorylation at both residues, whereas a 5'-phosphatase-defective mutant SHIP2 (DeltaIP-SHIP2) with the myristoylation signal did not. Interestingly, the degree of inhibition of Akt phosphorylation by R/Q-SHIP2 and Y/F-SHIP2 is well correlated with the extent of their association with Shc. In addition, overexpression of WT-Shc increased the insulin-induced association of SHIP2 with Shc, whereas a decrease in the amount of Shc on expression of antisense Shc mRNA led to a reduction in the SHIP2-Shc association. Furthermore, the inhibitory effect on insulin-induced Akt phosphorylation by WT-SHIP2 was decreased in antisense-Shc cells. These results indicate that the membrane localization of SHIP2 with its 5'-phosphatase activity is required for negative regulation of insulin-induced Akt phosphorylation and that the localization is regulated, at least in part, by the association of SHIP2 with Shc in Rat1 fibroblasts.