PROTEIN-KINASE-C CONTROLS THE PRIMING STEP OF REGULATED EXOCYTOSIS INADRENAL CHROMAFFIN CELLS

1. To investigate the mechanism whereby protein kinase C enhances secr etory function in adrenal chromaffin, cells, we examined the effects o f 12-O-tetradecanoylphorbor13-acetate, (TPA) on Ca2+-induced catechola mine release from digitonin-permeabilized cells, resolving the release into a MgATP-dependent priming step and a MgATP-independent Ca2+-trig gered step. Treatment with TPA selectively potentiated the priming act ivity of MgATP, with little increase in the MgATP-independent release; The potentiation by TPA of the MgATP-dependent priming was blocked by [Ser(25)]protein kinase C(19-31), a specific; substrate of protein ki nase C. Go, 6976, an inhibitor selective for protein kinase C alpha an d beta isoforms, also blocked the potentiation by TPA. These results. suggest that activation of protein kinase C, probably the alpha isofor m, potentiates the MgATP-dependent priming step. 2. The antibody raise d against GAP-43, a known substrate of protein kinase C, also potentia ted the MgATP-dependent priming. The effect of TPA and that of the. an ti-GAP-43 antibody were not additive. Calmodulin, which binds to GAP-4 3 and inhibits its phosphorylation by protein kinase C, abolished:the effect of TPA. Thus, the present results suggest that protein kinase C potentiates MgATP-dependent priming, at least in part, through phosph orylation of GAP-43.