DELETION ANALYSIS OF PROTEIN-KINASE-C INACTIVATION BY CALPHOSTIN-C

Protein kinase C (PKC) undergoes specific inactivation by nanomolar co ncentrations of calphostin C. Both PKC-alpha (a Ca2+-dependent convent ional isoform) and PKC-epsilon (a Ca2+-independent novel isoform) are similarly inactivated by calphostin C (75-100 nM produced 50% inhibiti on), suggesting that inactivation requires a site common to both class es of PKC. We therefore performed studies to identify a critical regio n in the regulatory domain of PKC-alpha required for inactivation by c alphostin C. A series of N-terminal-truncation mutants of bovine PKC-a lpha expressed in Saccharomyces cerevisiae was tested with 500 nM calp hostin C, a concentration sufficient to inactivate wild-type PKC-alpha by 80-90%. This concentration was as effective with mutant proteins c ontaining deletions of up to 91 amino acid (aa) residues from the amin o terminus (ND91), whereas a mutant protein truncated by 140 aa (ND140 ) was inactivated by only 20%. These findings imply that the aa sequen ce 92-140 is a structural determinant of PKC-alpha inactivation by cal phostin C. This sequence contains one of the phorbol ester-binding sit es (aa 102-144), which is highly conserved among most PKC isoforms inc luding PKC-epsilon. In addition to aa 92-140, PKC-stimulating cofactor s (phosphatidylserine, phorbol ester, and Ca2+) are required for inact ivation by calphostin C even in the case of PKC muta nts that do not r equire these cofactors for enzymatic activity. These results suggest t hat cofactors provide a template that is required for productive inter action of PKC and the inhibitor. The significance of the proposed prox imity effect to calphostin C action is discussed. (C) 1995 Wiley-Liss, Inc.