Shp2 is required for protein kinase C-dependent phosphorylation of serine 307 in insulin receptor substrate-1.
The function of insulin receptor substrate-1 (IRS-1), a key molecule of insulin signaling, is modulated by phosphorylation at multiple serine/threonine residues. Phorbol ester stimulation of cells induces phosphorylation 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 system using a broad spectrum of PKC isoforms (alpha, beta1, beta2, delta, epsilon, eta, mu), we detected only Ser-318, but not Ser-307 phosphorylation, suggesting that phorbol ester-induced phosphorylation of this site in intact cells requires additional signaling elements and serine kinases that link PKC activation to Ser-307 phosphorylation. As we have observed recently that the tyrosine phosphatase Shp2, a negative regulator of insulin signaling, is a substrate of PKC, we studied the role of Shp2 in this context. We found that phorbol ester-induced Ser-307 phosphorylation is 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 cell model, tumor necrosis factor-alpha-induced Ser-307 phosphorylation as well depended on the presence of Shp2. 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 suggests an involvement of the phosphatidylinositol 3-kinase/mammalian target of rapamycin cascade and of JNK in this signaling pathway resulting in IRS-1 Ser-307 phosphorylation. Because the activation of these kinases does not depend on Shp2, it is concluded that the function of Shp2 is to direct these activated kinases to IRS-1.
Müssig
Karsten
K
Division of Endocrinology, Metabolism, and Pathobiochemistry, Department of Internal Medicine, University Hospital of Tübingen, Tübingen 72076, Germany.
Staiger
Harald
H
Fiedler
Hendrik
H
Moeschel
Klaus
K
Beck
Alexander
A
Kellerer
Monika
M
Häring
Hans-Ulrich
HU
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D015153:Blotting, Western; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000378:metabolism; D003720:Densitometry; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018014:Gene Transfer Techniques; D005982:Glutathione Transferase / Q000378:metabolism; D007118:Immunoassay; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000502:physiology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D051379:Mice; D010455:Peptides / Q000737:chemistry; D010703:Phorbol Esters / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism*; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000235:genetics* / Q000502:physiology*; D051381:Rats; D012190:Retroviridae / Q000235:genetics; D012694:Serine / Q000737:chemistry*; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D000077191:Wortmannin
D000730:Androstadienes; D000873:Anthracenes; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; D010455:Peptides; D010703:Phorbol Esters; D010750:Phosphoproteins; D020033:Protein Isoforms; D014409:Tumor Necrosis Factor-alpha; C432165:pyrazolanthrone; D012694:Serine; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D048031:JNK Mitogen-Activated Protein Kinases; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases; C516725:Ptpn11 protein, mouse; C516727:Ptpn11 protein, rat; D020123:Sirolimus; D000077191:Wortmannin