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Mechanism of SHIP-Mediated Inhibition of Insulin- and Platelet-Derived Growth Factor-Stimulated Mitogen-Activated Protein Kinase Activity in 3T3-L1 Adipocytes

Department of Medicine (P.M.S., H.-S.S., S.U., J.M.O.), University of California, San Diego, La Jolla, California 92093; and ICN Pharmaceuticals, Inc. (W.R.), Costa Mesa, California 92626

Address all correspondence and requests for reprints to: Prem M. Sharma, Department of Medicine (0673), University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0673. E-mail: psharma{at}ucsd.edu.

The Src homology 2-containing 5' inositolphosphatases (SHIP and SHIP2) dephosphorylate 3'-phosphorylated PtdIns on the 5' position, decreasing intracellular levels of PtdIns 3,4,5-P3. In the current study, we investigated the role of SHIP in insulin and platelet-derived growth factor (PDGF) signaling by expressing wild-type (WT) and catalytically inactive SHIPIP in 3T3-L1 adipocytes, utilizing adenoviral infection. Insulin and PDGF both stimulated tyrosine phosphorylation of SHIP-WT and of SHIPIP, and tyrosine phosphorylation of SHIP-associated proteins increased after ligand stimulation. Tyrosine-phosphorylated PDGFR, IR, and insulin receptor substrate-1 all immunoprecipitated with SHIP. Expression of WT and IP mutant SHIP did not affect tyrosine phosphorylation of either the insulin or the PDGF receptor, or the expression of insulin receptor substrate-1 and Shc proteins. Both SHIP-WT and SHIPIP blocked insulin and PDGF-induced MAPK and MAPK kinase phosphorylation as well as, GTP-bound Ras activity, suggesting that the catalytic activity of SHIP is not necessary for these effects. SHIP associated with Shc upon ligand stimulation, indicating that the SHIP-Shc association is phosphorylation dependent. This association was primarily between the SHIP-SH2 domain and the phosphorylated tyrosine residues of Shc because no association was observed when the 3YF-Shc mutant was coexpressed with SHIP. The ShcGrb2 association was not compromised by SHIP expression, despite complete inhibition of the Ras/MAPK pathway. Interestingly, son-of-sevenless (SOS) protein normally found in Grb2 complexes was markedly reduced in SHIP expressing cells, whereas the displaced SOS was recovered when the post-Grb2-IP supernatants were blotted with anti-SOS antibody. Thus, SHIP competes son-of-sevenless (SOS) away from Shc-Grb2. In summary, 1) SHIP-WT and SHIPIP expression inhibit insulin and PDGF stimulated Ras, MAPK kinase, and MAPK activities; 2) SHIP associates with tyrosine phosphorylated Shc, and the proline-rich sequences in SHIP associate with Grb2 and titrate out SOS to form ShcGrb2SHIP complexes; and 3) dissociation of SOS from the ShcGrb2 complex inhibits Ras GTP loading, leading to decreased signaling through the MAPK pathway.