In NIH-3T3 fibroblasts, insulin receptor interaction with specific protein kinase C isoforms controls receptor...

Insulin increased protein kinase C (PKC) activity by 2-fold in both membrane preparations and insulin receptor (IR) antibody precipitates from NIH-3T3 cells expressing human IRs (3T3hIR). PKC-alpha, -delta, and -zeta were barely detectable in IR antibody precipitates of unstimulated cells, while increasing by 7-, 3.5-, and 3-fold, respectively, after insulin addition. Preexposure of 3T3hIR cells to staurosporine reduced insulin-induced receptor coprecipitation with PKC-alpha, -delta, and -zeta by 3-, 4-, and 10-fold, respectively, accompanied by a 1.5-fold decrease in insulin degradation and a similar increase in insulin retroendocytosis. Selective depletion of cellular PKC-alpha and -delta, by 24 h of 12-O-tetradecanoylphorbol-13-acetate (TPA) exposure, reduced insulin degradation by 3-fold and similarly increased insulin retroendocytosis, with no change in PKC-zeta. In lysates of NIH-3T3 cells expressing the R1152Q/K1153A IRs (3T3Mut), insulin-induced coprecipitation of PKC-alpha, -delta, and -zeta with the IR was reduced by 10-, 7-, and 3-fold, respectively. Similar to the 3T3hIR cells chronically exposed to TPA, untreated 3T3Mut featured a 3-fold decrease in insulin degradation, with a 3-fold increase in intact insulin retroendocytosis. Thus, in NIH-3T3 cells, insulin elicits receptor interaction with multiple PKC isoforms. Interaction of PKC-alpha and/or -delta with the IR appears to control its intracellular routing.