Tyr612 and Tyr632 in Human Insulin Receptor Substrate-1 Are Important for Full Activation of Insulin-Stimulated Phosphatidylinositol 3-Kinase Activity and Translocation of GLUT4 in Adipose Cells
To examine contributions of specific YXXM motifs in human in- sulin receptor substrate-1 (IRS-1) to mediating the metabolic actions of insulin, we studied IRS-1 mutants containing various substitutions of Phe for Tyr. In transfected NIH-3T3IR cells, insulin stimulation caused a 5-fold increase in phosphatidylinositol 3-kinase (PI3K) ac- tivity coimmunoprecipitated with wild-type IRS-1. No PI3K activity was associated with IRS1-F6 (Phe substituted for Tyr at positions 465, 612, 632, 662, 941, and 989). Adding back both Tyr612 and Tyr632 fully restored IRS-1-associated PI3K activity, whereas adding back either Tyr612 or Tyr632 alone was associated with intermediate PI3K activ- ity. In rat adipose cells transfected with epitope-tagged GLUT4, in- sulin stimulation caused a 2-fold increase in cell surface GLUT4-HA. Cotransfection of cells with GLUT4-HA and either wild-type IRS-1 or IRS1-Y612/Y632 increased basal cell surface GLUT4-HA (in the ab- sence of insulin) to approximately 80% of the levels seen in insulin- stimulated control cells, whereas overexpression of IRS1-F6 had no effect on the insulin dose-response curve. Overexpression of IRS1- Y612 or IRS1-Y632 caused intermediate effects. Thus, both Tyr612 and Tyr632 are important for IRS-1 to fully activate PI3K and mediate