Photoinduced inactivation of protein kinase C by dequalinium identifies the RACK-1-binding domain as a recognition site.

Abstract 1,1′-Decamethylenebis-4-aminoquinaldinium diiodide (DECA; dequalinium) is an anti-tumor agent and protein kinase C (PKC) inhibitor whose mechanism of action with PKC is unknown. This study reports that with human PKCα, DECA exhibited competitive inhibition (K i = 11.5 ± 5 μm) with respect to RACK-1 (receptor for activatedC kinase-1), an adaptor protein that has been proposed to bind activated PKC following translocation (Ron, D., Luo, J., and Mochly-Rosen, D. (1995) J. Biol. Chem. 270, 24180–24187). When exposed to UV light, DECA covalently modified and irreversibly inhibited PKC (α or β), with IC50 = 7–18 μm. UV/DECA treatment of synthetic peptides modeled after the RACK-1-binding site in the C2 region of PKCβ induced modification of Ser218-Leu-Asn-Pro-Glu-Trp-Asn-Glu-Thr226, but not of a control peptide. This modification occurred at a tryptophan residue (Trp223) that is conserved in all conventional PKC isoforms. In overlay assays with native RACK-1 that had been immobilized on nitrocellulose, UV-treated control PKCα bound well to RACK-1, whereas UV/DECA-inactivated PKCα had reduced binding activity. The significance of these findings is shown with adenocarcinoma cells, which, when pretreated with 10 μmDECA and UV light, exhibited diminished 12-O-tetradecanoylphorbol-13-acetate-induced PKCα translocation. Overall, this work identifies DECA as a tool that prevents PKC translocation by inhibiting formation of the PKC·RACK-1 complex.