Glutathione conjugate interactions with DNA-dependent protein kinase

• Published in: The Journal of pharmacology and experimental therapeutics Vol. 290; no. 3; p. 1101
• Main Authors: Shen, H, Schultz, M P, Tew, K D
• Format: Journal Article
• Language: English
• Published: United States 01-09-1999
• Subjects: Amino Acid Sequence; Azides - chemical synthesis; Azides - metabolism; Azides - pharmacology; Binding Sites; Catalysis; DNA-Activated Protein Kinase; DNA-Binding Proteins; Glutathione - analogs & derivatives; Glutathione - chemical synthesis; Glutathione - metabolism; Glutathione - pharmacology; HL-60 Cells; Humans; Molecular Sequence Data; Neoplasm Proteins - metabolism; Nuclear Proteins; Oligopeptides - metabolism; Phosphorylation; Protein Binding; Protein-Serine-Threonine Kinases - antagonists & inhibitors; Protein-Serine-Threonine Kinases - metabolism; Substrate Specificity
• ISSN: 0022-3565

A photoactivatable glutathione-drug conjugate (35)S-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 approximately 460-kDa protein was found to be highly labeled by (35)S-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 (35)S-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.