Pertussis toxin-sensitive and insensitive intracellular signalling pathways in undifferentiated 3T3-L1 cells stimulated by insulin converge with phosphatidylinositol 3-kinase upstream of the Ras mitogen-activated protein kinase cascade.
We have previously reported that pertussis toxin (PTX)-sensitive GTP binding protein (G-protein) and phosphatidylinositol 3-kinase (PI 3-K) are involved in adipocyte differentiation of 3T3-L1 cells induced by insulin/dexamethasone/methylisobutyl xanthine. The aim of this study was to examine the effect of PTX on the tyrosine kinase cascade stimulated by insulin acting through insulin-like growth factor-I (IGF-I) receptors in undifferentiated 3T3-L1 cells. A high level of mitogen-activated protein kinase (MAPK) activation was sustained for up to 4 h after insulin treatment, and mobility shifted and tyrosine phosphorylated MAPK was also detected. MAPK kinase activity measured by the incorporation of 32P into kinase-negative recombinant MAPK was enhanced by insulin treatment. We previously discovered that insulin activates Ras and that this is mediated by wortmannin-sensitive PI 3-K. Tyrosine-phosphorylation of IRS-1 and Shc also occurred in response to insulin. Subsequently, we investigated the effects of PTX on the activation of these proteins by insulin. Interestingly, treating 3T3-L1 cells with PTX attenuates the activation by insulin of both the Ras-MAPK cascade and PI 3-K. In contrast, neither tyrosine-phosphorylation of IRS-1 and Shc nor the interaction between IRS-1 and PI 3-K is sensitive to PTX. However, activation of the Ras-MAPK cascade and tyrosine-phosphorylation of Shc by epidermal growth factor are insensitive to PTX. These results indicate that there is another pathway which regulates PI 3-K and Ras-MAPK, independent of the pathway mediated by IGF-I receptor kinase. These findings suggest that in 3T3-L1 fibroblasts, PTX-sensitive G-proteins cross-talk with the Ras-MAPK pathway via PI 3-K by insulin acting via IGF-I receptors.
Uehara
T
T
Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Japan.
Tokumitsu
Y
Y
Nomura
Y
Y
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D051380:GRB2 Adaptor Protein; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*; D018631:ras Proteins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; D011506:Proteins; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D010566:Virulence Factors, Bordetella; C060974:phosphatidylinositol 3,4,5-triphosphate; D014443:Tyrosine; D037342:Pertussis Toxin; D011494:Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D019204:GTP-Binding Proteins; D018631:ras Proteins