Synthesis and characterization of the regulatory domains of protein kinase C

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Protein kinase C (PKC) isozymes are major receptors of tumor-promoting phorbol esters. Conventional and novel PKC isozymes (α, β, γ, δ, ε, η, θ) contain two cysteine-rich C1 domains (C1A and C1B), both of which are candidate phorbol-12,13-dibutyrate (PDBu) binding sites. To determine the phorbol ester binding sites of these isozymes, the C1 domains consisting of about 50 amino acids of all PKC isozymes have been synthesized by a solid phase Fmoc strategy. All C1B peptides except for α-C1B were successfully folded by zinc treatment as monitored by CD and ESI-MS spectroscopy, and showed potent PDBu binding affinities with the dissociation constants (K_d) of nanomolar range (0.45-1.5 nM), comparable to those of the native PKC isozymes. However, the K_d values of PDBu for many of the C1A peptides could not be determined. We found that some of the C1A peptides experience the temperature dependent inactivation and that elongation of the 50-mer C1 peptides at both N-and C-termini increases their folding efficiency. These findings enabled us to determine the K_d's of PDBu for all PKC C1 peptides except for θ-C1A.The major PDBu binding sites of novel PKC isozymes (δ, ε, η, θ) were C1B domains with K_d values of 0.45-0.81 nM.In contrast, all C1A peptides of conventional PKC isozymes (α, β, γ) exhibited nanomolar K_d values (0.97-1.3 nM). It is noteworthy that both C1 peptides of PKCβ and PKCγ showed strong PDBu binding affinity of nanomolar range. The above results provide a structural blueprint for the rational design of PKCγ-selective modulators for the treatment of neuropathic pain. To extend this approach, the 116-mer peptide containing the double cysteine-rich motifs of PKCγ (γ-C1A-C1B) has been synthesized for the first time.