Mutant of insulin receptor substrate-1 incapable of activating phosphatidylinositol 3-kinase did not mediate insulin-stimulated maturation of Xenopus laevis oocytes.
Insulin receptor substrate-1 (IRS-1) is rapidly phosphorylated on multiple tyrosine residues in response to insulin and binds several Src homology 2 domain-containing proteins, thereby initiating downstream signaling. To assess the tyrosine phosphorylation sites that mediate relevant downstream signaling and biological effects, we created site-directed mutants of IRS-1 and overexpressed them in the Xenopus laevis oocyte. In oocytes overexpressing IRS-1 or IRS-1-895F (Tyr-895 replaced with phenylalanine), insulin activated phosphatidylinositol (PI) 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and induced oocyte maturation. In contrast, in oocytes overexpressing IRS-1-4F (Tyr-460, Tyr-608, Tyr-939, and Tyr-987 of IRS-1 replaced with phenylalanine), insulin did not activate PI 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and failed to induce oocyte maturation. These observations indicate that in X. laevis oocytes overexpressing IRS-1, the association of PI 3-kinase rather than Grb2 (growth factor-bound protein 2) with IRS-1 plays a major role in insulin-induced oocyte maturation. Activation of PI 3-kinase may lie upstream of mitogen-activated protein kinase activation and p70 S6 kinase activation in response to insulin.
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
R
R
Yamamoto-Honda
Z
Z
Honda
K
K
Ueki
K
K
Tobe
Y
Y
Kaburagi
Y
Y
Takahashi
H
H
Tamemoto
T
T
Suzuki
K
K
Itoh
Y
Y
Akanuma
Y
Y
Yazaki
T
T
Kadowaki
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation; D051380:GRB2 Adaptor Protein; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009865:Oocytes / Q000187:drug effects* / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000235:genetics*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases; D029867:Xenopus Proteins; D014982:Xenopus laevis
D048868:Adaptor Proteins, Signal Transducing; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004268:DNA-Binding Proteins; D051380:GRB2 Adaptor Protein; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009419:Nerve Tissue Proteins; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D017346:Protein Serine-Threonine Kinases; D011506:Proteins; D019893:Ribosomal Protein S6 Kinases; D029867:Xenopus Proteins; C526294:irs1 protein, Xenopus; C100394:neuron-specific p150; C100393:neuron-specific p65