Translocation of protein kinase C from the soluble to the membrane-bound state is synergistically regulated by Ca⁺⁺ and phorbol esters

Stimulation of protein kinase C in whole cells has been associated with translation of the enzyme from the cytoplasm to the plasma membrane. We developed a reconstitution system as a model to study the mechanism of this translocation. By using purified protein kinase C and inside-out vesicles from human erythrocytes, the enzyme was found to bind to the membranes after raising the free calcium concentration from 100 nM. to 500 nM. The association with the membrane was rapid (requiring less than 30 sec) and reversible in the same time range after removing Ca⁺⁺. Band 4.1 of erythrocyte membranes was the major substrate for the bound enzyme. Active phorbol esters enhance the sensitivity of the binding to the membrane at lower Ca⁺⁺ concentrations and increase total membrane binding at higher Ca⁺⁺ concentrations. The calcium concentrations required for activation of protein kinase C are about 50 times higher than those required for membrane binding, suggesting the existence of two calcium-binding sites on the enzyme.