Identification of an inactivating cysteine switch in protein kinase Cε, a rational target for the design of protein kinase Cε-inhibitory cancer therapeutics

Critical roles played by some protein kinases in neoplastic transformation and progression provide a rationale for developing selective, small-molecule kinase inhibitors as antineoplastic drugs. Protein kinase C epsilon (PKC epsilon) is a rational target for cancer therapy, because it is oncogenic and prometastatic in transgenic mouse models. PKC epsilon is activated by sn-1,2-diacylglycerol (DAG). Attempts to develop selective PKC epsilon inhibitors that block activation by DAG or compete with ATP have not yet met with success, suggesting a need for new strategies. We previously reported that cystamine and a metabolic cystine precursor inactivate PKC epsilon in cells in a thiol-reversible manner. In this report, we first determined that PKC epsilon became resistant to inactivation by disulfides when Cys(452) C was replaced with alanine by site-specific mutagenesis of human PKC epsilon or a constitutively active PKC epsilon mutant. These results showed that the disulfides inactivated PKC epsilon by thioldisulfide exchange, either upon CYS452 S-thiolation or by rearrangement to an intra-protein disulfide. Mass spectrometric analysis of peptide digests of cystamine-inactivated, carbamidomethylated PKC epsilon detected a peptide S-cysteaminylated at CYS452, indicating that Cys(452) S-cysteaminylation is a stable modification. Furthermore, PKC epsilon inactivation by N-ethyl-maleimide was CYS452 dependent, providing corroborative evidence that PKC epsilon inhibitors can be designed by targeting CYS452 with small molecules that stably modify the residue. CYS452 is an active site residue that is conserved in only 11 human protein kinase genes. Therefore, the PKC epsilon-inactivating CYS452 switch is a rational target for the design of antineoplastic drugs that selectively inhibit PKC epsilon.