THE PHORBOL ESTER TPA MARKEDLY ENHANCES THE BINDING OF CALCIUM TO THEREGULATORY DOMAIN OF PROTEIN-KINASE-C BETA-1 IN THE PRESENCE OF PHOSPHATIDYLSERINE

Activation of protein kinase enzyme activity by Ca2+ and diacylglycero l or phorbol esters is a feature of certain isoforms of protein kinase C (PKC). Although the binding sites of phorbol ester on the regulator y domain of PKC have been extensively studied, little is known about t he actual mechanism of Ca2+ binding and how this leads to enzyme activ ation. We previously reported that high affinity binding of Ca-45(2+) to the regulatory domain of PKC beta 1, expressed as a GST fusion prot ein in Escherichia coli, is dependent on the presence of phosphatidyls erine (PS) or 12-O-tetradecanoylphorbol-13-acetate (TPA). In the prese nt study we have used this system to further analyze Ca2+ binding. Usi ng various deletions, we found that different domains in the regulator y domain of PKC beta 1 are involved in TPA-induced Ca2+ binding, depen ding on whether or not PS was also present in the binding assay. In ad dition, Ca2+ binding in the presence of TPA alone displayed very diffe rent kinetics than Ca2+ binding in the presence of TPA and PS. Scatcha rd analysis indicated that in the presence of TPA, the K-d value for C a2+ binding was 51.9 mu M. However, in the presence of both TPA and PS , the K-d value dropped to 0.23 mu M. These results provide direct evi dence that TPA activates certain isoforms of PKC by enhancing PS-depen dent Ca2+ binding, thus decreasing the K-d value for Ca2+ binding to a physiological level.