Abstract
We have studied the phosphatidylinositol 3-kinase (PtdIns 3-kinase) in insulin-stimulated Chinese hamster ovary (CHO) cells expressing normal (CHO/IR) and mutant human insulin receptors. Insulin stimulation of CHO/IR cells results in an increase in PtdIns 3-kinase activity associated with anti-phosphotyrosine (alpha PY) immunoprecipitates, which has been previously shown to correlate with the in vivo production of PtdIns(3,4)P2, and PtdIns(3,4,5)P3 (Ruderman, N., Kapeller, R., White, M.F., and Cantley, L.C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1411-1415). Stimulation was maximal within 1 min and showed a dose response identical to that of insulin receptor autophosphorylation. The PtdIns 3-kinase also associated with the insulin receptor in an insulin-stimulated manner, as approximately 50% of the total alpha PY-precipitable activity could be specifically immunoprecipitated with anti-insulin receptor antibody. Mutant insulin receptors displayed variable ability to stimulate the PtdIns 3-kinase, but in all cases the presence of PtdIns 3-kinase in alpha PY immunoprecipitates correlated closely with the tyrosyl phosphorylation of the endogenous substrate pp185. In CHO cells expressing a kinase-deficient mutant (IRA1018), there was no observable insulin stimulation of PtdIns 3-kinase activity in alpha PY immunoprecipitates and no tyrosyl phosphorylation of pp185. Substitution of Tyr1146 in the insulin receptor regulatory region with phenylalanine partially impaired receptor autophosphorylation, pp185 phosphorylation, and insulin-stimulated increases in alpha PY-precipitable PtdIns 3-kinase activity. In contrast, a deletion mutant lacking 12 amino acids from the juxtamembrane region (IR delta 960) displayed normal in vivo autophosphorylation but failed to stimulate the PtdIns 3-kinase or phosphorylate pp185. Finally, a mutant receptor from which the C-terminal 43 amino acids had been deleted (IR delta CT) exhibited normal insulin-stimulated autophosphorylation, pp185 phosphorylation, and stimulation of the PtdIns 3-kinase activity in alpha PY immunoprecipitates. These data suggest that the PtdIns 3-kinase is itself a substrate of the insulin receptor kinase or associates preferentially with a substrate. A comparison of the biological activities of the mutant receptors with their activation of the PtdIns 3-kinase furthermore suggests that the PtdIns 3-kinase may be linked to insulin's ability to regulate DNA synthesis and cell growth.
Highlights
From the Research Division, Joslin Diabetes Center, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston,Massachusetts 02215
Recent work 3-kinase, but in all cases the presence of PtdIns 3- in this laboratory and others (Wilden et al, 1990; Maegawa kinase in aPY immunoprecipitates correlated closely et al, 1988) suggests thatthe insulin signal is mediated with the tyrosyl phosphorylation of the endogenous through multiple signal transduction pathways
The presence of PtdIns 3-kinase activity in anti-phosphotyrosine and anti-insulin receptor immunoprecipitates from insulin-stimulated cells suggests that the PtdIn3s-kinase is a direct substrate of the insulin receptor kinase or associates with the receptor or another protein that undergoes tyrosine phosphorylation during insulin stimulationI.n thisregard, we have recently shown thatthePtdIns 3-kinase associates strongly with IRS-1, presumably through phosphorylated YMXM residues (Sun et al, 1991);the role of this association inthe activation of thePtdIns 3-kinase is not yet clear
Summary
Transfection of CHO Cells-The normal human insulin receptor (IR) expression plasmid pCVSVHIRc and the expression plasmid encoding mutant human insulin receptors IRAlOls, IRa960, IR~1146a, nd IRA~Thave been described previously (Chou et al, 1987;Backer et al, 1990; Wildenet al., 1990;McClain et al, 1987).CHO cells were grown in 10-cm dishes in F-12 medium containing 10% fetal bovine serum (GIBCO)and transfected as described previously (White etal., 1988). [32PJPhosphateLabelingof CHO Cells Expressing Wild-typeInsulin Receptors-Confluent monolayers of transfected CHO cells in 10- or 15-cm dishes (Nunc) at37 "C were labeled for 2 h with 0.5 mCi/ml with ["Plphosphate (Du Pont-New England Nuclear) as described previously (Backer et al, 1990). The cells were incubated for additional periods of time in the presence of 100nM insulin, rapidly frozen with liquid nitrogen, and solubilized in 100 mM Tris, pH8.0, containing 2 mM sodium vanadate, 0.34 mg/ml phenylmethylsulfonyl fluoride, 100 pg/ml aprotinin, 1pg/ml leupeptin, and 1%Triton X-100. Tyr(P)-containingproteinswere immunoprecipitated with anti-phosphotyrosine antibody (aPY), andprecipitated proteins were reduced with dithiothreitol and analyzed by SDS-PAGE (White and Backer, 1991). The autophosphorylation reactions were terminated after10min at 20 "Cby boiling in Laemmli sample buffer containing 100 mM dithiothreitol (Laemmli, 1970); alternatively, samples were immunoprecipitated with aPY or antiinsulin receptor antibodies (aIR) andeluted by boiling as above.
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