IN NIH-3T3 FIBROBLASTS, INSULIN-RECEPTOR INTERACTION WITH SPECIFIC PROTEIN-KINASE-C ISOFORMS CONTROLS RECEPTOR INTRACELLULAR ROUTING

Insulin increased protein kinase C (PKC) activity by 2-fold in both me mbrane preparations and insulin receptor (IR) antibody precipitates fr om NIH-3T3 cells expressing human IRs (3T3(hIR)). PKC-alpha, -delta, a nd -zeta were barely detectable in IR antibody precipitates of unstimu lated cells, while increasing by 7-, 3.5-, and 3-fold, respectively, a fter insulin addition. Preexposure of 3T3(hIR) cells to staurosporine reduced insulin-induced receptor coprecipitation with PKC-alpha, -delt a, and -zeta by 3-, 4-, and 10-fold, respectively, accompanied by a 1. 5-fold decrease in insulin degradation and a similar increase in insul in retroendocytosis. Selective depletion of cellular PKC-alpha and -de lta, by 24 h of 12-O-tetradecanoylphorbol-13-acetate (TPA) exposure, r educed insulin degradation by 3-fold and similarly increased insulin r etroendocytosis, with no change in PKC-zeta. In lysates of NIH-3T3 cel ls expressing the R1152Q/K1153A IRs (3T3(Mut)), insulin-induced coprec ipitation of PKC-alpha, -delta, and -zeta with the IR was reduced by 1 0-, 7-, and 3-fold, respectively. Similar to the 3T3(Mut) cells chroni cally exposed to TPA, untreated 3T3(Mut) featured a 3-fold decrease in insulin degradation, with a 3-fold increase in intact insulin retroen docytosis. Thus, in NIH-3T3 cells, insulin elicits receptor interactio n with multiple PKC isoforms, Interaction of PKC-alpha and/or -delta w ith the IR appears to control its intracellular routing.