Toward the identification of selective modulators of protein kinase C (PKC) isozymes: Establishment of a binding assay for PKC isozymes using synthetic C1 peptide receptors and identification of the critical residues involved in the phorbol ester binding

Conventional and novel protein kinase C (PKC) isozymes contain two cysteine-rich C1 domains (C1A and C1B), both of which are candidate phorbol-12,13-dibutyrate (PDBu) binding sites. We previously synthesized Cl peptides (of approximately 50 residues) corresponding to all PKC isozymes and measured their PDBu binding affinity. While many of these peptide receptors exhibited PDBu affinities comparable to the respective complete isozyme, some of the CIA peptides could not be used because they undergo temperature dependent inactivation. This problem was however eliminated by 4 degreesC incubation or elongation of the 50-mer Cl peptides at both N- and C-termini to increase their folding efficiency and stability. These findings enabled us to determine the K-d's of PDBu for all PKC Cl peptides (except for theta -C1A) and establish the value of these peptides as readily available, stable, and easily handled surrogates of the individual isozymes. The resultant Cl peptide receptor library can be used to screen for new ligands with PKC isozyme and importantly Cl domain selectivity. Most of the CI peptide receptors showed strong PDBu binding affinities with K-d's in the nanomolar range (0.45-7.4nM). Two peptides (delta -C1A and theta -C1A) bound PDBu over 100-fold less tightly. To identify the residues that contribute to this affinity difference, several mutants of delta -C1A and theta -C1A were synthesized. Both the G9K mutant of delta -C1A and the P9K mutant of theta -C1A showed K-d's of 2-3 nM. This approach provides a useful procedure to determine the role of each C1 domain of the PKC isozymes by point mutation. (C) 2001 Elsevier Science Ltd. All rights reserved.