A peptide substrate-based affinity label blocks protein kinase C-catalyzed ATP hydrolysis and peptide-substrate phosphorylation.
Studies focused on the cAMP-dependent protein kinase (PKA) have led to the identification of conserved active-site residues involved in Ser/Thr protein kinase catalysis and have ruled out a role for Cys residues in the catalytic mechanism. Protein kinase C (PKC) is a Ser/Thr protein kinase isozyme family. We recently reported that the peptide-substrate analog N-biotinyl-Arg-Arg-Arg-Cys-Leu-Arg-Arg-Leu (N-biotinyl-RRRCLRRL) spontaneously forms intermolecular disulfide bridges with the active-site region of PKC isozymes concomitant with inactivation of histone kinase catalysis. Because Cys does not participate in PKC catalysis, one can analyze the active-site topology of PKC by examining which catalytic reactions are sterically hindered when the inactivator peptide is tethered to Cys in the active-site region of the enzyme. In this report, we show that N-biotinyl-RRRCLRRL inactivates the bulky PKC-catalyzed histone phosphorylation reaction, the comparatively less bulky PKC-catalyzed phosphorylation of a series of octapeptide, hexapeptide, and pentapeptide substrates, the intramolecular autophosphorylation reaction of PKC, and the least bulky PKC-catalyzed reaction, ATP hydrolysis, in a dithiothreitol-sensitive manner with comparable efficacy. Our results provide evidence that the covalent linkage of N-biotinyl-RRRCLRRL to the active-site region of PKC sterically hinders PKC catalysis, even in the absence of peptide and protein substrates.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D001921:Brain / Q000201:enzymology*; D004220:Disulfides; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D006656:Protamine Kinase / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity