A peptide substrate-based affinity label blocks protein kinase C-catalyzedATP 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 protei n kinase catalysis and have ruled out a role for Cys residues in the cataly tic mechanism. Protein kinase C (PKC) is a Ser/Thr protein kinase isozyme f amily. We recently reported that the peptide-substrate analog N-biotinyl-Ar g-Arg-Arg-Cys-Leu-Arg-Arg-Leu (N-biotinyl-RRRCLRRL) spontaneously forms int ermolecular disulfide bridges with the active-site region of PKC isozymes c oncomitant 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 b ulky PKC-catalyzed histone phosphorylation reaction, the comparatively less bulky PKC-catalyzed phosphorylation of a series of octapeptide, hexapeptid e, and pentapeptide substrates, the intramolecular autophosphorylation reac tion of PKC, and the least bulky PKC-catalyzed reaction, ATP hydrolysis, in a dithiothreitol-sensitive manner with comparable efficacy. Our results pr ovide evidence that the covalent linkage of N-biotinyl-RRRCLRRL to the acti ve-site region of PHC sterically hinders PKC catalysis, even in the absence of peptide and protein substrates. (C) 1999 Academic Press.