Expression, Purification, and Characterization of Protein Kinase C-r*

Of the recently described members of the protein kinase C (PKC) family (-6, -6, -c), no detailed properties of the purified enzymes have been presented. Here we describe the expression of PKC-c in insect cells using a baculovirus vector. The recombinant enzyme has been purified to homogeneity by sequential chromatography on DEAE-cellulose, serine-Sepharose, Mono Q, and Superose 12; the protein shows a molecular mass of 90 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PKC-t is dependent upon phospholipid and diacylglycerol (or phorbol esters) for activity and displays a pattern of specificity for these effecters similar to other PKC isotypes. Similarly, inhibition of PKC-c by staurosporine and H-7 parallels inhibition of other PKC isotypes. However, unlike PKC-(u, -8, and -7, PKC-c shows no dependence upon Ca2’. Furthermore, the substrate specificity of PKC-t is quite different from other characterized PKCs. The importance of functional diversity within the PKC family is discussed.