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 wildtype mice and mice deficient in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) encoded by the Prkdc gene. In both wildtype 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 wildtype and heterozygous, but not Prkdc−/− neurons from kainate-induced excitotoxicity. Co-transfection 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-non-phosphorylatable Ku70 (S6A/S51A) bound 3- to 4-fold greater Bax than wildtype 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 co-transfected wildtype, 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.

The DNA-dependent protein kinase (DNA-PK): More than just a case of making ends meet?

“…138 The link between Ku70 and cell death is also noted in a neurodegenerative disease model where DNA-PK CS links DNA damage to Bax-dependent cell death, by phosphorylating Ku70 on serines 6 and/or 51, initiating Bax translocation to the mitochondria and directly activating a pro-apoptotic Bax-dependent death cascade. 139 These reports complement the described role of DNA-PK particularly in regard to the maintenance of chromosomes. As previously considered, telomerase-deficient (Terc -/-) mice show widespread germ cell line apoptosis, however a Terc -/-DNA-PK CS -/-double knockout mouse strain does not show increased apoptosis indicating a clear role of DNA-PK CS in mediating apoptosis (that is independent of Ku) in cell lines with critically shortened telomeres.…”

New Insights into the Link Between DNA Damage and Apoptosis

“…In this scenario, the specific post-translational modification of Ku70 could account for both its dual functions, in DNA repair and in suppressing Bax-mediated apoptosis, and decide the cell fate in a condition of severe DNA damage. Moreover, it is worth mentioning that Liu et al have demonstrated that, on DNA damage induced by kainic acid, Ku70 is phosphorylated by DNA-PKcs, resulting in Bax release and translocation into the mitochondria to initiate apoptosis (46). There is further evidence highlighting Ku's involvement in the crosstalk between the DNA repair machinery and the DNA damage signaling regulators controlling apoptosis.…”

DNA-PKcs, ATM, and ATR Interplay Maintains Genome Integrity during Neurogenesis

“…staurosporine and kainic acid (Chechlacz et al, 2001;Vemuri et al, 2001;Liu et al, 2009). We therefore sought to determine how DNA-PKcs functions during the neural DDR, and additionally in the context of the related kinases ATM and ATR.…”