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Identification of an Inactivating Cysteine Switch in Protein Kinase C, a Rational Target for the Design of Protein Kinase C–Inhibitory Cancer Therapeutics

Departments of ¹ Cancer Biology and ² Molecular Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas

Requests for reprints: Catherine A. O'Brian, Department of Cancer Biology, M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 173, Houston, TX 77030. Phone: 713-792-7969; E-mail: cobrian{at}mdanderson.org.

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 (PKC) is a rational target for cancer therapy, because it is oncogenic and prometastatic in transgenic mouse models. PKC is activated by sn-1,2-diacylglycerol (DAG). Attempts to develop selective PKC 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 in cells in a thiol-reversible manner. In this report, we first determined that PKC became resistant to inactivation by disulfides when Cys⁴⁵² was replaced with alanine by site-specific mutagenesis of human PKC or a constitutively active PKC mutant. These results showed that the disulfides inactivated PKC by thiol-disulfide 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 detected a peptide S-cysteaminylated at Cys⁴⁵², indicating that Cys⁴⁵² S-cysteaminylation is a stable modification. Furthermore, PKC inactivation by N-ethylmaleimide was Cys⁴⁵² dependent, providing corroborative evidence that PKC 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-inactivating Cys⁴⁵² switch is a rational target for the design of antineoplastic drugs that selectively inhibit PKC.