Tyr(612) and Tyr(632) 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 insulin 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-3T3(IR) cells, insulin stimulation caused a 5-fold increase in phosphatidylinositol 3-kinase (PI3K) activity 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 Tyr(612) and Tyr(632) fully restored IRS-1-associated PI3K activity, whereas adding back either Tyr(612) or Tyr(632) alone was associated with intermediate PI3K activity. In rat adipose cells transfected with epitope-tagged GLUT4, insulin 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 absence 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 Tyr(612) and Tyr(632) are important for IRS-1 to fully activate PI3K and mediate translocation of GLUT4 in response to insulin.
Esposito
D L
DL
Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Li
Y
Y
Cama
A
A
Quon
M J
MJ
D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D004789:Enzyme Activation; D051275:Glucose Transporter Type 4; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D008297:Male; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics* / Q000502:physiology*; D051381:Rats
D051275:Glucose Transporter Type 4; C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D010750:Phosphoproteins; C495692:SLC2A4 protein, human; C495693:Slc2a4 protein, mouse; C495694:Slc2a4 protein, rat; D019869:Phosphatidylinositol 3-Kinases