Association of the insulin receptor and phosphatidylinositol 3-kinase requires a third component.
We have studied the interactions between the insulin receptor and PtdIns 3-kinase by a reconstitution system in vitro composed of highly purified PtdIns 3-kinase from rat liver and highly purified insulin receptors bound to insulin-agarose or to antibodies against insulin receptors. As a positive control, receptors for platelet-derived growth factor, which bind and phosphorylate PtdIns 3-kinase, were studied in parallel with insulin receptors. Our results indicate that the insulin receptor, regardless of its phosphorylation state, does not directly associate with purified PtdIns 3-kinase, whereas the autophosphorylated receptor does associate with PtdIns 3-kinase present in the crude CHO-cell lysate. Also, we could not detect phosphorylation of PtdIns 3-kinase by the insulin receptor, even through the receptor readily underwent autophosphorylation and phosphorylated an insulin-receptor substrate, poly(Glu-Tyr) (4:1). These findings argue that one or more cytosolic components link the receptor and the enzyme. Insulin-receptor substrate-1 (IRS-1) was evaluated as a potential linking protein. In the absence of ATP, IRS-1 did not facilitate the coupling of the phosphorylated insulin receptor to PtdIns 3-kinase. Thus IRS-1 is unlikely to be the component in crude CHO-cell lysate that couples PtdIns 3-kinase to the phosphorylated insulin receptor. However, the addition of ATP, which allows phosphorylation of IRS-1 by the insulin receptor, also enhances the coupling of PtdIns 3-kinase to the insulin receptor. In support of this idea, immunoprecipitates of IRS-1 from insulin-treated CHO cells were found to contain both the insulin receptor and PtdIns 3-kinase. In conclusion, the insulin receptor does not appear to phosphorylate or bind directly to PtdIns 3-kinase, regardless of the receptor's state of phosphorylation. Association of PtdIns 3-kinase with the insulin receptor is mediated by one or more components, one of which may involve an unidentified factor in cell lysate and another that apparently involves phosphorylated IRS-1.
Liu
R
R
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642.
Livingston
J N
JN
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D066298:In Vitro Techniques; D055504:Insulin Receptor Substrate Proteins; D046911:Macromolecular Substances; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D055504:Insulin Receptor Substrate Proteins; C526296:Irs1 protein, rat; D046911:Macromolecular Substances; D010750:Phosphoproteins; D011994:Recombinant Proteins; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011972:Receptor, Insulin; D017479:Receptors, Platelet-Derived Growth Factor