Synthesis and characterization of the regulatory domains of protein kinase C

蛋白激酶C （PKC）同工酶是促肿瘤磷酸酯的主要受体。传统的和新型的PKC同工酶（α, β, γ, δ, ε, η, θ）含有两个富含半胱氨酸的C1结构域（C1A和C1B），这两个结构域都是候选的phorl -12,13-二丁酸酯（PDBu）结合位点。为了确定这些同工酶的磷酯结合位点，采用固相Fmoc策略合成了所有PKC同工酶的C1结构域，该结构域由大约50个氨基酸组成。除α-C1B外，其余的C1B多肽经锌处理后均成功折叠，其解离常数（K_d）在纳摩尔范围（0.45 ~ 1.5 nM），与天然PKC同工酶相当。然而，许多C1A肽的PDBu的K_d值无法确定。我们发现一些C1A肽经历了温度依赖的失活，并且50-mer C1肽在n和c端的延伸增加了它们的折叠效率。这些发现使我们能够确定除θ-C1A外所有PKC C1肽的PDBu的K_d。新型PKC同工酶的主要PDBu结合位点（δ、ε、η、θ）为C1B结构域，K_d值为0.45 ~ 0.81 nM。相比之下，常规PKC同工酶（α, β, γ）的所有C1A肽的K_d值均为纳摩尔（0.97-1.3 nM）。值得注意的是，PKCβ和PKCγ的C1肽均表现出很强的纳米摩尔范围内的PDBu结合亲和力。上述结果为合理设计pkc - γ选择性调节剂治疗神经性疼痛提供了结构蓝图。为了扩展这一方法，首次合成了含有PKCγ双半胱氨酸基序的116聚肽（γ-C1A-C1B）。

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.