Deletion analysis of protein kinase C inactivation by calphostin C.
Protein kinase C (PKC) undergoes specific inactivation by nanomolar concentrations of calphostin C. Both PKC-alpha (a Ca(2+)-dependent conventional isoform) and PKC-epsilon (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-alpha required for inactivation by calphostin C. A series of N-terminal-truncation mutants of bovine PKC-alpha expressed in Saccharomyces cerevisiae was tested with 500 nM calphostin C, a concentration sufficient to inactivate wild-type PKC-alpha 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-alpha 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-epsilon. In addition to aa 92-140, PKC-stimulating cofactors (phosphatidylserine, phorbol ester, and Ca2+) 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.
Rotenberg
S A
SA
Department of Chemistry and Biochemistry, Queens College, City University of New York, Flushing 11367.
Huang
M H
MH
Zhu
J
J
Su
L
L
Riedel
H
H
D000818:Animals; D001483:Base Sequence; D029742:Caenorhabditis elegans Proteins; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins; D002417:Cattle; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D008969:Molecular Sequence Data; D009281:Naphthalenes; D010718:Phosphatidylserines / Q000494:pharmacology; D011083:Polycyclic Compounds / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000737:chemistry; D011955:Receptors, Drug / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017384:Sequence Deletion; D018411:Spodoptera; D013329:Structure-Activity Relationship; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D007527:Isoenzymes; D009281:Naphthalenes; D010718:Phosphatidylserines; D011083:Polycyclic Compounds; D011955:Receptors, Drug; D011994:Recombinant Proteins; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D011493:Protein Kinase C; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium