Phosphatidylinositol 3-kinase functions upstream of Ras and Raf in mediating insulin stimulation of c-fos transcription.

Insulin treatment of Chinese hamster ovary (CHO) cells expressing high levels of the insulin receptor (CHO-IR) was found to markedly activate (10-20-fold) transcription of the luciferase reporter gene (Luc) driven by the serum response element (SRE) of the c-fos promoter. SRE-Luc expression was also strongly activated by co-transfection with expression plasmids encoding for either v-Ras or v-Raf. In contrast, insulin-stimulated SRE-Luc activity was inhibited by expression of a negative-dominant Ras (RasAsn-17)- or a negative-dominant Raf (p301-1)-encoding plasmid. Furthermore, the negative-dominant Raf mutant blocked v-Ras activation whereas the negative-dominant Ras mutant had no significant effect on v-Raf activation. Together, these data demonstrate that insulin utilizes the Ras and Raf signaling pathways and that Ras functions upstream of Raf in terms of transcriptional activation of the serum response element. To assess the role of the phosphatidylinositol (PI) 3-kinase in this insulin signaling pathway, CHO-IR cells were co-transfected with the p85 regulatory subunit of the PI 3-kinase. Expression of the p85 subunit inhibited the insulin stimulation of SRE-Luc activity without affecting v-Ras or v-Raf activation. Thus, these data demonstrate that the PI 3-kinase is necessary for insulin signaling of transcriptional events and that, in a linear model of intracellular signaling, the PI 3-kinase functions upstream of both Ras and Raf.