Molecular cloning of rat SH2-containing inositol phosphatase 2 (SHIP2) andits role in the regulation of insulin signaling

SH2-containing inositol 5'-phosphatase (SHIP) plays a negative regulatory r ole in hematopoietic cells. We have now cloned the rat SHIP isozyme (SHIP2) cDNA from skeletal muscle, which is one of the most important target tissu e of insulin action. Rat SHIPS cDNA encodes a 1183-amino-acid protein that is 45% identical with rat SHIP. Rat SHIPS contains an aminoterminal SH2 dom ain, a central 5'-phosphoinositol phosphatase activity domain, and a phosph otyrosine binding (PTB) consensus sequence and a proline-rich region at the carboxyl tail. Specific antibodies to SHIPS were raised and the function o f SHIPS was studied by stably overexpressing rat SHIPS in Rat1 fibroblasts expressing human insulin receptors (HIRc). Endogenous SHIPS underwent insul in-mediated tyrosine phosphorylation and phosphorylation was markedly incre ased when SHIPS was overexpressed. Although overexpression of SHIPS did not affect insulin-induced tyrosine phosphorylation of the insulin receptor P- subunit and Shc, subsequent association of Shc with Grb2 was inhibited, pos sibly by competition between the SH2 domains of SHIPS and Grb2 for the Shc phosphotyrosine. As a result, insulin-stimulated MAP kinase activation was reduced in SHIP2-over-expressing cells. Insulin-induced tyrosine phosphoryl ation of IRS-1, IRS-1 association with the p85 subunit of PI3-kinase, and P I3-kinase activation were not affected by overexpression of SHIPS. Interest ingly, although both PtdIns-(3,4,5)P3 and PtdIns(3,4)P2 have been implicate d in the regulation of Akt activity in vitro, overexpression of SHIPS inhib ited insulin-induced Akt activation, presumably by its 5'-inositol phosphat ase activity. Furthermore, insulin-induced thymidine incorporation was decr eased by overexpression of SHIPS. These results indicate that SHIPS plays a negative regulatory role in insulin-induced mitogenesis, and regulation of the Shc Grb2 complex and of the downstream products of PI3-kinase provides possible mechanisms of SHIPS action in insulin signaling. (C) 1999 Academi c Press.