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

Abstract

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 C1 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 C1A peptides could not be used because they undergo temperature dependent inactivation. This problem was however eliminated by 4 °C incubation or elongation of the 50-mer C1 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 C1 peptides (except for θ-C1A) and establish the value of these peptides as readily available, stable, and easily handled surrogates of the individual isozymes. The resultant C1 peptide receptor library can be used to screen for new ligands with PKC isozyme and importantly C1 domain selectivity. Most of the C1 peptide receptors showed strong PDBu binding affinities with K d's in the nanomolar range (0.45–7.4 nM). Two peptides (δ-C1A and θ-C1A) bound PDBu over 100-fold less tightly. To identify the residues that contribute to this affinity difference, several mutants of δ-C1A and θ-C1A were synthesized. Both the G9K mutant of δ-C1A and the P9K mutant of θ-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.