Interactive roles of Ras, insulin receptor substrate-1, and proteins with Src homology-2 domains in insulin signaling in Xenopus oocytes.
Insulin receptor substrate-1 (IRS-1) serves as the major immediate substrate of insulin/insulin-like growth factor (IGF)-1 receptors and following tyrosine phosphorylation binds to specific Src homology-2 (SH2) domain-containing proteins including the p85 subunit of phosphatidylinositol (PI) 3-kinase and GRB2, a molecule believed to link IRS-1 to the Ras pathway. To investigate how these SH2-containing signaling molecules interact to regulate insulin/IGF-1 action, IRS-1, glutathione S-transferase (GST)-SH2 domain fusion proteins and Ras proteins were microinjected into Xenopus oocytes. We found that pleiotropic insulin actions are mediated by IRS-1 through two independent, but convergent, pathways involving PI 3-kinase and GRB2. Thus, microinjection of GST-fusion proteins of either p85 or GRB2 inhibited IRS-1-dependent activation of mitogen-activated protein (MAP) and S6 kinases and oocyte maturation, although only the GST-SH2 of p85 reduced insulin-stimulated PI 3-kinase activation. Co-injection of a dominant negative Ras (S17N) with IRS-1 inhibited insulin-stimulated MAP and S6 kinase activation. Micro-injection of activated [Arg12,Thr59]Ras increased basal MAP and S6 kinase activities and sensitized the oocytes to insulin-stimulated maturation without altering insulin-stimulated PI 3-kinase. The Ras-enhanced oocyte maturation response, but not the elevated basal level of MAP and S6 kinase, was partially blocked by the SH2-p85, but not SH2-GRB2. These data strongly suggest that IRS-1 can mediate many of insulin's actions on cellular enzyme activation and cell cycle progression requires binding and activation of multiple different SH2-domain proteins.
Research Division, Joslin Diabetes Center, Boston, Massachusetts 02215.
L M
LM
Chuang
S F
SF
Hausdorff
M G
MG
Myers
M F
MF
White
M J
MJ
Birnbaum
C R
CR
Kahn
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D051380:GRB2 Adaptor Protein; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D019893:Ribosomal Protein S6 Kinases; D015398:Signal Transduction; D029867:Xenopus Proteins; D014982:Xenopus laevis
D048868:Adaptor Proteins, Signal Transducing; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D051380:GRB2 Adaptor Protein; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D017346:Protein Serine-Threonine Kinases; D011506:Proteins; D016283:Proto-Oncogene Proteins p21(ras); D019893:Ribosomal Protein S6 Kinases; D029867:Xenopus Proteins; C526294:irs1 protein, Xenopus