Role of p85 subunit of phosphatidylinositol-3-kinase as an adaptor molecule linking the insulin receptor, p62, and GTPase-activating protein.
After insulin stimulation of rat HTC hepatoma cells overexpressing normal human insulin receptors (IR), an antiserum to the p85 subunit of phosphatidylinositol-3-kinase (PIK) (alpha-p85) immunoprecipitated three major tyrosine-phosphorylated proteins: IR, insulin receptor substrate-1 (IRS-1), and a new 62-kDa protein (p62). Studies with antibodies to GTPase activating protein (alpha-GAP) and p62 GAP-associated protein suggested that p62 was the same as (or closely related to) p62 GAP-associated protein. In order to understand how p62 interacts with p85, we employed: 1) antibodies to the p110 subunit of PIK (alpha-p110); and 2) antiserum to IRS-1. To determine which subunit of PIK (p110 or p85) p62 associates with, we first immunoprecipitated insulin-treated cell lysates with alpha-p110 and subsequently immunoprecipitated with alpha-p85 followed by Western blotting analysis with anti-phosphotyrosine antibody (alpha-PY). In response to insulin, most of the tyrosine-phosphorylated p62 was complexed to p85 alone rather than with the PIK heterodimer. Moreover, p62 was absent in alpha-IRS-1 immunoprecipitates. These data suggest that: 1) p62 GAP-associated protein is tyrosine phosphorylated after insulin stimulation of cells; 2) p62 and IRS-1 form separate complexes with p85; 3) p62-GAP complex may be linked to p85 that is not bound to p110; 4) p85 may serve as an adaptor molecule in insulin receptor signaling, interacting with and regulating other intracellular proteins via SH2 domains.
Sung
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CK
Department of Medicine, Mount Zion Medical Center, University of California, San Francisco 94115.
Sánchez-Margalet
V
V
Goldfine
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ID
D000818:Animals; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D020690:GTPase-Activating Proteins; D006801:Humans; D007106:Immune Sera; D055504:Insulin Receptor Substrate Proteins; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000378:metabolism*; D011233:Precipitin Tests; D011506:Proteins / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism
D004268:DNA-Binding Proteins; C489560:DOK1 protein, human; C074561:GAP-associated protein p62; D020690:GTPase-Activating Proteins; C526292:IRS1 protein, human; D007106:Immune Sera; D055504:Insulin Receptor Substrate Proteins; C526296:Irs1 protein, rat; D010446:Peptide Fragments; D010750:Phosphoproteins; D011506:Proteins; D016601:RNA-Binding Proteins; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011972:Receptor, Insulin