title: The DNA-PK catalytic subunit regulates Bax-mediated excitotoxic cell death by Ku70 phosphorylation.
abstract: DNA repair deficiency results in neurodegenerative disease and increased susceptibility to excitotoxic cell death, suggesting a critical but undefined role for DNA damage in neurodegeneration. We compared DNA damage, Ku70-Bax interaction, and Bax-dependent excitotoxic cell death in kainic acid-treated primary cortical neurons derived from both wild-type mice and mice deficient in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) encoded by the Prkdc gene. In both wild-type and Prkdc(-/-) neurons, kainic acid treatment resulted in rapid induction of DNA damage (53BP1 foci formation) followed by nuclear pyknosis. Bax deficiency, by either Bax shRNA-mediated knockdown or gene deletion, protected wild-type and heterozygous but not Prkdc(-/-) neurons from kainate-induced excitotoxicity. Cotransfection of DNA-PKcs with Bax shRNA restored Bax shRNA-mediated neuroprotection in Prkdc(-/-) neurons, suggesting that DNA-PKcs is required for kainate-induced activation of the pro-apoptotic Bax pathway. Immunoprecipitation studies revealed that the DNA-PKcs-nonphosphorylatable Ku70 (S6A/S51A) bound 3- to 4-fold greater Bax than wild-type Ku70, suggesting that DNA-PKcs-mediated Ku70 phosphorylation causes release of Bax from Ku70. In support of this, kainic acid induced translocation of a Bax-EGFP fusion protein to the mitochondria in the presence of a cotransfected wild-type, but not mutant Ku70 (S6A/S51A) gene when examined at 4 and 8 h following kainate addition. We conclude that DNA-PKcs links DNA damage to Bax-dependent excitotoxic cell death, by phosphorylating Ku70 on serines 6 and/or 51, to initiate Bax translocation to the mitochondria and directly activate a pro-apoptotic Bax-dependent death cascade.
authors: Program in Neuroscience and Behavior, Department of Biology, Wesleyan University, Middletown, CT 06459-0170, USA.
authors: Jia
authors: J
authors: Liu
authors: Janice R
authors: JR
authors: Naegele
authors: Stanley L
authors: SL
authors: Lin
mesh_terms: D000818:Animals; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects / Q000502:physiology; D004249:DNA Damage / Q000187:drug effects / Q000502:physiology*; D051747:DNA-Activated Protein Kinase / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006624:Hippocampus / Q000502:physiology; D007608:Kainic Acid / Q000633:toxicity; D000072200:Ku Autoantigen; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D009154:Mutation; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D009498:Neurotoxins / Q000633:toxicity; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D034961:Antigens, Nuclear; C494827:Bax protein, mouse; D004268:DNA-Binding Proteins; D009498:Neurotoxins; D009687:Nuclear Proteins; D051028:bcl-2-Associated X Protein; D051747:DNA-Activated Protein Kinase; C497264:Prkdc protein, mouse; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen; D007608:Kainic Acid