The DNA-PK catalytic subunit regulates Bax-mediated excitotoxic cell death by Ku70 phosphorylation.
The DNA-PK catalytic subunit regulates Bax-mediated excitotoxic cell death by Ku70 phosphorylation.
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DOI：
10.1016/j.brainres.2009.07.101
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发表时间：
2009-11-03
期刊：
影响因子：
2.9
通讯作者：
Lin, Stanley L.
中科院分区：
文献类型：
--
作者：
Liu, Jia;Naegele, Janice R.;Lin, Stanley L.
DNA修复缺陷导致神经退行性疾病和对兴奋毒性细胞死亡的易感性增加，这表明DNA损伤在神经退行性疾病中的作用至关重要，但尚未明确。我们比较了来自野生型小鼠和缺乏Prkdc基因编码的DNA依赖性蛋白激酶催化亚基（DNA- pkcs）的小鼠的kainic酸处理的原代皮质神经元的DNA损伤、Ku70-Bax相互作用和bax依赖性兴奋毒性细胞死亡。在野生型和Prkdc−/−神经元中，kainic酸处理导致DNA损伤（53BP1病灶形成）的快速诱导，随后是核固缩。通过Bax shrna介导的敲低或基因缺失，Bax缺乏症可以保护野生型和杂合子，但不能保护Prkdc−/−神经元免受盐酸盐诱导的兴奋性毒性。DNA-PKcs与Bax shRNA共转染恢复了Bax shRNA介导的Prkdc−/−神经元的神经保护作用，这表明DNA-PKcs是盐酸盐诱导的促凋亡Bax通路激活所必需的。免疫沉淀研究显示，dna - pkcs -非磷酸化的Ku70 （S6A/S51A）结合Bax的能力是野生型Ku70的3- 4倍，这表明dna - pkcs介导的Ku70磷酸化导致Ku70释放Bax。为了支持这一观点，在添加kainate后4和8 h检测时，kainic酸诱导Bax-EGFP融合蛋白在共转染的野生型存在下向线粒体移位，而不是突变的Ku70 （S6A/S51A）基因。我们得出结论，DNA- pkcs通过磷酸化丝氨酸6和/或51上的Ku70，将DNA损伤与Bax依赖性兴奋性毒性细胞死亡联系起来，从而启动Bax向线粒体的易位，并直接激活促凋亡的Bax依赖性死亡级联。
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.
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