Glutathione conjugate interactions with DNA-dependent protein kinase

A photoactivatable glutathione-drug conjugate S-35-labeled-azidophenacyl-gl utathione (APA-SG) was synthesized and used to identify protein(s) involved in recognition and/or transport of glutathione conjugates of electrophilic drug species. A similar to 460-kDa protein was found to be highly labeled by S-35-labeled APA-SG in an Adriamycin-resistant HL-60 (HL-60/ADR) cell li ne and identified as the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) by amino acid sequence analysis, Western blot, and immunoprecipi tation with specific antibodies. Binding specificity was confirmed by compe tition isotope dilution assays with purified proteins. A 15- to 20-fold inc rease in DNA-PKcs expression in the HL-60/ADR cell line was accompanied by an equivalent increase in 35S-labeled APA-SG binding. APA-SG, along with ot her glutathione conjugates and analogs inhibited the DNA-PK-mediated phosph orylation of an in vitro peptide substrate in a concentration-dependent man ner. Using different antibodies to immunoprecipitate the individual compone nts of the DNA-PK complex (DNA-PKcs, Ku70, and Ku80), it was shown that APA -SG caused a destabilization of the trimeric holoenzyme complex by dissocia ting the catalytic subunit from the Ku heterodimer. These data suggest that the kinase-mediated signaling is inhibited when glutathione conjugates bin d to DNA-PKcs and may also indicate a possible strategy for design of novel DNA-PK inhibitors.