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

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Abstract
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.