Comparison of chemical characteristics of the first and the second cysteine-rich domains of protein kinase Cγ Original Research Article

Protein kinase C (PKC) is a key enzyme family involved in cellular signal transduction. The binding of endogenous diacyl glycerol (DAG) to the cysteine-rich domain (CRD) of PKC is associated with normal cell signaling and function. In contrast, the binding of exogenous phorbol esters to the CRD of PKC is considered to be a key initiating event in tumor promotion. Conventional PKC isozymes (PKCα, βI, βII, and γ) contain two CRDs, both of which are candidates for the phorbol ester binding site. In order to elucidate the binding requirements of phorbol esters and to obtain information on the phorbol ester binding site in native PKCγ, several key chemical characteristics of the first and the second CRDs consisting of ca. 50 amino acids of rat PKCγ (γ-CRD1 and γ-CRD2) were examined. In the presence of Zn2+ and phosphatidylserine (PS), both CRDs gave similar Kd values (65.3 nM for γ-CRD1, 44.1 nM for γ-CRD2) in phorbo1 12,13-dibutyrate (PDBu) binding assays. In comparison, the binding affinity of PDBu for native rat PKCγ was found to be 6.8 nM. Zn2+ was shown to play an important role in the folding and PDBu binding of both CRDs. A Zn2+-induced conformational change was observed for the first time by CD spectroscopic analysis of the complexed and uncomplexed CRDs. Relative to the pronounced Zn2+ effect, most divalent first row transition metal ions along with Ca2+, Mg2+, and Al3+ were ineffective in folding either CRD. Notably, however, Co2+ exhibited a γ-CRD1-selective effect, suggesting that metal ions, not unlike extensively used organic probes, might also become effective tools for controlling isozyme selective activation of PKC. Moreover, group Ib (Cu2+ and Ag+) and group IIb element ions other than Zn2+ (Cd2+ and Hg2+) were found to abolish PDBu binding of both CRDs. Importantly, these inhibitory effects of Cu2+, Ag+, Cd2+, and Hg2+ were also observed with native PKCγ. These results indicate that recent reports on the modulation of conventional PKC by heavy metal ions could be explained by their coordination to the CRDs. While the similar affinities of γ-CRD1 and γ-CRD2 for PDBu suggest that either site qualifies as the PDBu binding site, new molecular probes of these CRDs have now been identified that provide information on the preferred site. These novel ligands (5a and 5b) were synthesized by aza-Claisen rearrangement of (−)-N13-desmethyl-N13-allylindolactam-G (4). These compounds did not significantly affect the specific PDBu binding of γ-CRD1 but did inhibit that of γ-CRD2 with similar potency to (−)-indolactam-V. Moreover, these new probes did not significantly inhibit the PDBu binding of native PKCγ. (−)-Indolactam-V itself bound almost equally to γ-CRD1, γ-CRD2, and native PKCγ. These results suggest that the major PDBu binding site in native PKCγ is the first CRD, not the second CRD, unlike the novel PKCs.