Covalent modification of protein kinase C isozymes by the inactivating peptide substrate analog N-biotinyl-Arg-Arg-Arg-Cys-Leu-Arg-Arg-Leu. Evidence that the biotinylated peptide is an active-site affinity label.
We recently reported that the peptide substrate analog Arg-Lys-Arg-Cys-Leu-Arg-Arg-Leu (RKRCLRRL) irreversibly inactivates the protein kinase C (PKC) isozymes alpha, beta, and gamma in a dithiothreitol-sensitive manner by an active site-directed mechanism. We hypothesized that the inactivation mechanism entailed covalent complex formation between the PKC isozyme and the inactivator peptide. In this report, N-biotinylated analogs of RKRCLRRL that inactivate Ca2+-dependent PKC activity were designed and tested for their ability to covalently label PKC isozymes. A purified PKC isozyme mixture (alpha, beta, gamma, epsilon, zeta) was incubated with the N-biotinylated peptides and then subjected to denaturing gel electrophoresis, transferred to nitrocellulose, and probed for avidin-reactive species. The Ca2+-dependent PKC subfamily members PKC-alpha, -beta, and -gamma comigrated at 82 kDa and were distinguished by isozyme-specific immunoprecipitation. N-Biotinyl-RRRCLRRL covalently labeled all of the isozymes examined. When the isozymes were denatured prior to incubation with the N-biotinylated peptides, no labeling was observed. Inactivation of the Ca2+-dependent PKC subfamily by the N-biotinylated peptides was associated with covalent labeling of the 82-kDa PKC subspecies. The concentration dependence curves observed with N-biotinyl-RRRCLRRL were similar for inactivation and covalent labeling. The rank order of potency of three N-biotinylated peptides was the same for the inactivation and covalent labeling. Both the inactivation and covalent labeling were dithiothreitol-sensitive, and they were each subject to protection by MgATP and a peptide substrate analog. The covalent label was mapped to the catalytic domain of PKC by limited proteolysis of the modified enzyme. These results provide evidence that the N-biotinylated inactivator peptides are active-site affinity labels of PKC. The inactivator peptides most likely function by S-thiolating the active-site Cys residue conserved in PKC. This is the first report to demonstrate covalent labeling of PKC by a peptide substrate analog.
Ward
N E
NE
Department of Cell Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Gravitt
K R
KR
O'Brian
C A
CA
D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry* / Q000494:pharmacology; D010718:Phosphatidylserines / Q000378:metabolism; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000737:chemistry*; D051381:Rats
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009842:Oligopeptides; D010718:Phosphatidylserines; D001710:Biotin; D011493:Protein Kinase C; D003545:Cysteine; D002118:Calcium; D004229:Dithiothreitol