Protein kinase C-zeta and protein kinase B regulate distinct steps of insulin endocytosis and intracellular sorting.

We have investigated the molecular mechanisms regulating insulin internalization and intracellular sorting. Insulin internalization was decreased by 50% upon incubation of the cells with the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. PI3K inhibition also reduced insulin degradation and intact insulin release by 50 and 75%, respectively. Insulin internalization was reduced by antisense inhibition of protein kinase C- (PKC) expression and by overexpression of a dominant negative PKC mutant (DN-PKC). Conversely, overexpression of PKC increased insulin internalization as a function of the PKC levels achieved in the cells. Expression of wild-type protein kinase B (PKB)- or of a constitutively active form (myr-PKB) did not significantly alter insulin internalization and degradation but produced a 100% increase of intact insulin release. Inhibition of PKB by a dominant negative mutant (DN-PKB) or by the pharmacological inhibitor ML-9 reduced intact insulin release by 75% with no effect on internalization and degradation. In addition, overexpression of Rab5 completely rescued the effect of PKC inhibition on insulin internalization but not that of PKB inhibition on intact insulin recycling. Indeed, PKC bound to and activated Rab5. Thus, PI3K controls different steps within the insulin endocytic itinerary. PKC appears to mediate the PI3K effect on insulin internalization in a Rab5-dependent manner, whereas PKB directs intracellular sorting toward intact insulin release.