Glutathione conjugate interactions with DNA-dependent protein kinase.
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.
Shen
H
H
Department of Pharmacology, Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA.
Schultz
M P
MP
Tew
K D
KD
D000595:Amino Acid Sequence; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D002384:Catalysis; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D005978:Glutathione / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism; D009687:Nuclear Proteins; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013379:Substrate Specificity
D001386:Azides; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D009842:Oligopeptides; C024939:S-(4-azidophenacyl)glutathione; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D005978:Glutathione