The protein kinase C (PKC) family of enzymes plays a crucial role in cellular signal transduction
and tumor promotion. Conventional and novel PKC isozymes consist of a catalytic domain for protein
phosphorylation and a regulatory domain which binds the endogenous messenger diacyl glycerol or exogenous
agents such as phorbol esters. The N-terminal regulatory region of these isozymes contains two cysteine-rich
domains (C1A and C1B, also known as CRD1 and CRD2), both of which are candidates for the phorbol
ester-binding site. To determine the phorbol ester-binding sites of these isozymes and to elucidate the structural
requirements for isozyme selective PKC modulation, the C1 peptides,
consisting of ca. 50 amino acids of all
conventional and novel PKCs,
along with those of atypical PKCs have been synthesized by a
solid
-
phase
Fmoc strategy. Exceptionally high overall yields (10−20%) were achieved in the syntheses of most of the C1
peptides on a Pioneer Peptide Synthesizer (PerSeptive Biosystems) through the use of HATU as a coupling
reagent. These peptides were successfully folded by zinc treatment, as monitored by CD spectroscopy.
Importantly, only the C1Bs of all conventional and
novel PKCs, except for PKCγ, bound [
3
H]phorbol-12,13-dibutyrate
(PDBu) with high affinities, comparable to those of the native
isozymes. Of special significance,
both C1 peptides of PKCγ (i.e., γ-C1A and γ-C1B) exhibited high-affinity binding, providing the structural
basis for a novel approach to PKCγ-selective modulators, compounds of potential significance for the treatment
of neuropathic pain. The effects of metal cations other than zinc on the binding of these isozymes were also
investigated. Only the PKCγ surrogates (γ-C1A and γ-C1B), when treated with cadmium, exhibited no binding,
while other similarly treated conventional and novel PKC surrogates strongly bound PDBu, as did the zinc-folded peptides. These results suggest that cadmium ion could serve as a new and effective tool for controlling
the activation of PKCγ.
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