Synergistic role of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase cascade in the regulation of insulin receptor trafficking

To examine the molecular mechanism of insulin receptor trafficking, we inve stigated the intracellular signaling molecules that regulate this process i n Rat1 fibroblasts overexpressing insulin receptors. Cellular localization of insulin receptors was assessed by confocal laser microscopy with indirec t immunofluorescence staining. Insulin receptors were visualized diffusely in the basal state. Insulin treatment induced the change of insulin recepto r localization to perinuclear compartment. This insulin-induced insulin rec eptor trafficking was not affected by treatment of the cells with PI3-kinas e inhibitor (wortmannin), whereas treatment with MEK [mitogen-activated pro tein (MAP) kinase-Erk. kinase] inhibitor (PD98059) partly inhibited the pro cess in a dose-dependent manner. Interestingly, treatment with both wortman nin and PD98059 almost completely inhibited insulin receptor trafficking. T he functional importance of PI3-kinase and MAP kinase in the trafficking pr ocess was directly assessed by using single cell microinjection analysis. M icroinjection of p85-SH2 and/or catalytically inactive MAP kinase ([K71A]Er k1) GST fusion protein gave the same results as treatment with wortmannin a nd PD98059. Furthermore, to determine the crucial step for the requirement of PI3-kinase and MAP kinase pathways, the effect of wortmannin and PD98059 on insulin receptor endocytosis was studied. Insulin internalization from the plasma membrane and subsequent insulin degradation were not affected by treatment with wortmannin and PD98059. In contrast, insulin receptor down- regulation from the cell surface and insulin receptor degradation, after pr olonged incubation with insulin, were markedly impaired by the treatment. T hese results suggest that PI3-kinase and MAP kinase pathways synergisticall y regulate insulin receptor trafficking at a step subsequent to the recepto r internalization.