Vol. 290, Issue 3, 1101-1106, September 1999

Glutathione Conjugate Interactions with DNA-Dependent Protein Kinase¹

Hongxie Shen, Mary P. Schultz² and Kenneth D. Tew

Department of Pharmacology, Fox Chase Cancer Center, Philadelphia, Pennsylvania

A photoactivatable glutathione-drug conjugate ³⁵S-labeled-azidophenacyl-glutathione (APA-SG) was synthesized and used to identify protein(s) involved in recognition and/or transport of glutathione conjugates of electrophilic drug species. A ~460-kDa protein was found to be highly labeled by ³⁵S-labeled APA-SG in an Adriamycin-resistant HL-60 (HL-60/ADR) cell line and identified as the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) by amino acid sequence analysis, Western blot, and immunoprecipitation with specific antibodies. Binding specificity was confirmed by competition isotope dilution assays with purified proteins. A 15- to 20-fold increase 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 other glutathione conjugates and analogs inhibited the DNA-PK-mediated phosphorylation of an in vitro peptide substrate in a concentration-dependent manner. Using different antibodies to immunoprecipitate the individual components 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 dissociating the catalytic subunit from the Ku heterodimer. These data suggest that the kinase-mediated signaling is inhibited when glutathione conjugates bind to DNA-PKcs and may also indicate a possible strategy for design of novel DNA-PK inhibitors.