Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin.
Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context of previous work, suggest that binding of GRB2/Sos to Shc may be the predominant mechanism whereby insulin as well as cytokine receptors activate Ras.
Pruett
W
W
Skirball Institute for Biomolecular Medicine, Department of Pharmacology, New York University Medical Center, New York 10016.
Yuan
Y
Y
Rose
E
E
Batzer
A G
AG
Harada
N
N
Skolnik
E Y
EY
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D016466:CHO Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D066246:ErbB Receptors / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D015847:Interleukin-4 / Q000378:metabolism / Q000494:pharmacology*; D009132:Muscles / Q000187:drug effects / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014162:Transfection; D018631:ras Proteins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495950:Grb2 protein, rat; C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526296:Irs1 protein, rat; D010750:Phosphoproteins; D011506:Proteins; D011994:Recombinant Proteins; D015847:Interleukin-4; D011494:Protein Kinases; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D018631:ras Proteins