Shp2 Is Required for Protein Kinase C-dependent Phosphorylation of Serine 307 in Insulin

The function of insulin receptor substrate-1 (IRS-1), a keymolecule of insulin signaling, is modulated by phosphorylation at multiple serine/ threonineresidues.Phorbolesterstimulationofcells inducesphosphorylation of two inhibitory serine residues in IRS-1, i.e. Ser-307 and Ser-318, suggesting that both sites may be targets of protein kinase C (PKC) isoforms.However, in an in vitro systemusingabroad spectrumofPKC isoforms ( , 1, 2, , , , ), we detected only Ser-318, but not Ser-307 phosphorylation, suggesting that phorbol ester-inducedphosphorylation of this site in intact cells requires additional signalingelements andserine kinases that link PKC activation to Ser-307 phosphorylation. As we have observedrecentlythatthetyrosinephosphataseShp2,anegativeregulator ofinsulinsignaling,isasubstrateofPKC,westudiedtheroleofShp2inthis context.Wefoundthatphorbolester-inducedSer-307phosphorylationis reduced markedly in Shp2-deficient mouse embryonic fibroblasts (Shp2 / ) whereas Ser-318 phosphorylation is unaltered. The Ser-307 phosphorylation was rescued by transfection of mouse embryonic fibroblasts with wild-type Shp2 or with a phosphatase-inactive Shp2 mutant, respectively. In this cellmodel, tumor necrosis factor-induced Ser-307 phosphorylationaswelldependedonthepresenceofShp2.Furthermore, Shp2-dependent phorbol ester effects on Ser-307 were blocked by wortmannin, rapamycin, and the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125.This suggestsan involvementof thephosphatidylinositol3-kinase/mammaliantargetofrapamycincascadeandof JNKinthissignaling pathway resulting in IRS-1 Ser-307 phosphorylation. Because the activation of these kinases does not depend on Shp2, it is concluded that the functionof Shp2 is todirect these activatedkinases to IRS-1.