Glutathione Conjugate Interactions with DNA-Dependent Protein Kinase1
Abstract
A photoactivatable glutathione-drug conjugate35S-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 by35S-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.
Footnotes
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Send reprint requests to: Dr. Kenneth D. Tew, Department of Pharmacology, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111. E-mail: kd_tew{at}fccc.edu
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↵1 This work was supported in part by National Institutes of Health Grants CA06927 and RR05539, National Institutes of Health Grant CA53893 (to K.D.T.), and by an appropriation from the Commonwealth of Pennsylvania.
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↵2 Present address: Merck & Co., Westpoint, PA 19486.
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Received for publication February 26, 1999.
- Abbreviations:
- GSH
- glutathione
- GS (or SG)
- GSH in conjugated form
- GSSG
- oxidized glutathione
- MRP
- multidrug resistance-associated protein
- EA
- ethacryic acid
- γ-GCS
- γ-glutamylcysteine synthetase
- GST
- glutathioneS-transferase
- LT
- leukotriene
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- APA-SG
- azidophenacyl-glutathione
- DNA-PK
- DNA-dependent protein kinase
- DNA-PKcs
- catalytic subunit of DNA-PK
- PI3-kinase
- phosphatidylinositol-3-kinase
- TER117
- γ-glutamyl-S-(benzyl)cysteinyl-RS-(benzyl)cysteinyl-R(−)phenylglycine diethyl ester
- PAGE
- polyacrylamide gel electrophoresis
- WCL
- whole-cell lysate
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- Accepted May 11, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
