DELETION ANALYSIS OF PROTEIN-KINASE-C-ALPHA REVEALS A NOVEL REGULATORY SEGMENT

Using a combined pharmacological and genetic approach, we have identif ied aa 260-280 in the C2 region as a critical factor in the catalytic function of protein kinase C alpha (PKC alpha). Progressive truncation s from the N-terminus as well as selected internal deletion mutants we re expressed in Saccharomyces cerevisiae and tested for altered sensit ivity to dequalinium, a PKC inhibitor whose target site was previously mapped to the catalytic domain. PKC mutants representing truncations of up to 158 amino acid residues (aa) from the N-terminus (ND84 and ND 158) displayed 60-63% inhibition of kinase activity by 50 mu M dequali nium, somewhat more sensitive than the wild-type PKC alpha enzyme (45% inhibition). Mutant ND262, lacking N-terminal aa 1-262, was inhibited by almost 72% with 50 mu M dequalinium, but mutant ND278, which lacke d an additional 16 as, was inhibited by only 9% of total activity. Thi s result suggests that a C-terminal segment of the C2 region (aa 263-2 78) influences inhibition by dequalinium at low micromolar concentrati ons. An internal deletion mutant (D260-280) which retains the entire p rimary structure of PKC alpha except for aa 260-280, was similarly inh ibited by only 4% with 50 mu M dequalinium. In the absence of dequalin ium and despite the presence of a nearly complete regulatory domain, t his mutant exhibited constitutive activity (both in vitro and in a phe notypic assay with S. cerervisiae) that could not be further stimulate d even by the potent activator TPA, Taken together, our findings sugge st that, in the native structure of PKC alpha, the segment described b y as 260-280 regulates PKC alpha activity and influences the sensitivi ty of PKC alpha to dequalinium.