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Medientyp: E-Artikel Titel: Deletion analysis of protein kinase c inactivation by calphostin C Beteiligte: Rotenberg, Susan A.; Huang, Michael H.; Zhu, Jianwei; Su, Lihe; Riedel, Heimo Erschienen: Wiley, 1995 Erschienen in: Molecular Carcinogenesis Sprache: Englisch DOI: 10.1002/mc.2940120107 ISSN: 0899-1987; 1098-2744 Schlagwörter: Cancer Research ; Molecular Biology Entstehung: Anmerkungen: Beschreibung: <jats:title>Abstract</jats:title><jats:p>Protein kinase C (PKC) undergoes specific inactivation by nanomolar concentrations of calphostin C. Both PKC‐α (a Ca<jats:sup>2+</jats:sup>‐dependent conventional isoform) and PKC‐α (a Ca<jats:sup>2+</jats:sup>‐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 <jats:italic>Saccharomyces cerevisiae</jats:italic> 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 (ND 140) 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‐α. In addition to aa 92–140, PKC‐stimulating cofactors (phosphatidylserine, phorbol ester, and Ca<jats:sup>2+</jats:sup>) 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 of calphostin C action is discussed. © 1995 Wiley‐Liss Inc.</jats:p>