Abstract
Protein kinase C (PKC) undergoes specific inactivation by nanomolar concentrations of calphostin C. Both PKC-α (a Ca 2+ -dependent conventional isoform) and PKC-ε (a Ca 2+ -independent novel isoform) are similarly inactivated by calphostin C (75-100 nM produced 50% inhibition), suggesting that inactivation requires a site common to both classes of PKC. We therefore performed studies to identify a critical region in the regulatory domain of PKC-α required for inactivation by calphostin C. A series of N-terminal-truncation mutants of bovine PKC-α expressed in Saccharomyces cerevisiae was tested with 500 nM calphostin C, a concentration sufficient to inactivate wild-type PKC-α by 80-90%. This concentration was as effective with mutant proteins containing deletions of up to 91 amino acid (aa) residues from the amino terminus (ND91), whereas a mutant protein truncated by 140 aa (ND140) was inactivated by only 20%. These findings imply that the aa sequence 92-140 is a structural determinant of PKC-α inactivation by calphostin C. This sequence contains one of the phorbol ester-binding sites (aa 102-144), which is highly conserved among most PKC isoforms including PKC-ε In addition to aa 92-140, PKC-stimulating cofactors (phosphatidylserine, phorbol ester, and Ca 2+ ) are required for inactivation by calphostin C even in the case of PKC mutants that do not require these cofactors for enzymatic activity. These results suggest that cofactors provide a template that is required for productive interaction of PKC and the inhibitor. The significance of the proposed proximity effect to calphostin C action is discussed.