Role of the Src homology 2 (SH2) domain and C-terminus tyrosine phosphorylation sites of SH2-containing inositol phosphatase (SHIP) in the regulationof insulin-induced mitogenesis

To examine the role of SHIP in insulin-induced mitogenic signaling, we used a truncated SHIP lacking the SH2 domain (Delta SH2-SHIP) and a Y917/1020F- SHIP (2F-SHIP) in which two tyrosines contributing to Shc binding were muta ted to phenylalanine. Wild-type (WT)-, ;Delta SH2-, and 2F-SHIP were transi ently transfected into Rat1 fibroblasts overexpressing insulin receptors (H IRc). Insulin-stimulated tyrosine phosphorylation of WT-SHIP and Delta SH2- SHIP, whereas ty tyrosine phosphorylation of BF-SHIP was not detectable, in dicating that 917/1020-Tyr are key phosphorylation sites on SHIP. Although SHIP can bind via its 917/1020-Tyr residues and SH2 domain to Shc PTB domai n and 317-Tyr residue, respectively, insulin-induced SHIP association with Shc was more greatly decreased in BF-SHIP cells than that in Delta SH2-SHIP cells. Insulin stimulation of Shc association with Grb2, which is importan t for p21ras-MAP kinase activation, was decreased by overexpression of WT- and 2F-SHIP. Importantly, insulin-induced Shc Grb2 association was not dete ctably reduced in Delta SH2-SHIP cells. In accordance with the extent of Sh c association with Grb2, insulin-induced MAP kinase activation was relative ly decreased in both WT-SHIP and 2F-SHIP cells, but not in Delta SH2-SHIP c ells. To examine the functional role of SHIP in insulin's biological action , insulin-induced mitogenesis was compared among these transfected cells. I nsulin stimulation of thymidine incorporation and bromodeoxyuridine incorpo ration was decreased in WT-SHIP cells compared with that of control HIRc ce lls. Expression of 2F-SHIP also significantly reduced insulin-induced mitog enesis, whereas it was only slightly affected by overexpression of Delta SH 2-SHIP. Furthermore, the reduction of insulin-induced mitogenesis in WT-SHI P cells was partly compensated by coexpression of Shc. These results indica te that SHIP plays a negative regulatory role in insulin-induced mitogenesi s and that the SH2 domain of SHIP is important for its negative regulatory function.