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 bindin g protein (G-protein) and phosphatidylinositol 3-kinase (PI 3-K) are involv ed in adipocyte differentiation of 3T3-L1 cells induced by insulin/dexameth asone/methylisobutyl xanthine. The aim of this study was to examine the eff ect of PTX on the tyrosine kinase cascade stimulated by insulin acting thro ugh insulin-like growth factor-I (IGF-I) receptors in undifferentiated 3T3- L1 cells. A high level of mitogen-activated protein kinase (MAPK) activatio n was sustained for up to 4 h after insulin treatment, and mobility shifted and tyrosine phosphorylated MAPK was also detected. MAPK kinase activity m easured by the incorporation of P-32 into kinase-negative recombinant MAPK was enhanced by insulin treatment. We previously discovered that insulin ac tivates Ras and that this is mediated by wortmannin-sensitive PI 3-K. Tyros ine-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 attenua tes the activation by insulin of both the Ras-MAPK cascade and PI 3-K. In c ontrast, neither tyrosine-phosphorylation of IRS-1 and Shc nor the interact ion between IRS-1 and PI 3-K is sensitive to PTX. However, activation of th e 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 me diated by IGF-I receptor kinase. These findings suggest that in 3T3-L1 fibr oblasts, PTX-sensitive G-proteins cross-talk with the Ras-MAPK pathway via PI 3-K by insulin acting via IGF-I receptors.