EVIDENCE THAT THE MODULATION OF MEMBRANE-ASSOCIATED PROTEIN-KINASE-C ACTIVITY BY AN ENDOGENOUS INHIBITOR PLAYS A ROLE IN N1E-115 MURINE NEUROBLASTOMA CELL-DIFFERENTIATION

Murine neuroblastoma cells, N1E-115, were induced to differentiate int o neuron-like cells by serum deprivation for 18 h, As previous studies have shown that the suppression of protein kinase C (PKC) activity by selective inhibitors or neutralizing antibodies induces neuroblastoma cells to differentiate, we tested the hypothesis that serum deprivati on may cause a rapid loss in membrane PKC activity that occurs well be fore the morphological changes that are characteristic of cell differe ntiation, A significant reduction in particulate (membrane) PKC activi ty was indeed observed within 3 h of serum withdrawal when enzyme acti vity was measured in intact native membranes by the recently described in vitro ''direct'' assay, This rapid reduction in enzyme activity wa s confirmed by the decreased phosphorylation of the MARCKS protein, an endogenous PKC-selective substrate, in intact cells. The decrease in membrane PKC activity occurred without any loss in the amount of membr ane-associated enzyme, suggesting that some factor(s) resident in neur oblastoma membranes was suppressing PKC activity, Indeed, results indi cate the presence of an endogenous inhibitor of PKC tightly associated with neuroblastoma membranes, This inhibitory activity increased in t he membranes of cells subjected to serum deprivation, raising the poss ibility that it was likely responsible for the decline in membrane PKC activity in differentiating N1E-115 cells, Preliminary characterizati on indicated that the inhibitory activity is a protein and is localize d mainly in the membrane fraction, Thus, these results demonstrate dir ectly that endogenous inhibitor can regulate membrane-associated PKC a ctivity in cells and thereby modulate PKC-related neuronal functions.