The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has well-established roles in DNA double-strand break repair, and recently, nonrepair functions have also been reported. To better understand its cellular functions, we deleted DNA-PKcs from HeLa and A549 cells using CRISPR/Cas9. The resulting cells were radiation sensitive, had reduced expression of ataxia-telangiectasia mutated (ATM), and exhibited multiple mitotic defects. Mechanistically, nocodazole-induced upregulation of cyclin B1, anillin, and securin was decreased in DNA-PKcs-deficient cells, as were phosphorylation of Aurora A on threonine 288, phosphorylation of Polo-like kinase 1 (PLK1) on threonine 210, and phosphorylation of targeting protein for Xenopus Klp2 (TPX2) on serine 121. Moreover, reduced nocodazole-induced expression of anillin, securin, and cyclin B1 and phosphorylation of PLK1, Aurora A, and TPX2 were rescued by inhibition of the anaphase-promoting complex/cyclosome (APC/C) by proTAME, which prevents binding of the APC/C-activating proteins Cdc20 and Cdh1 to the APC/C. Altogether, our studies suggest that loss of DNA-PKcs prevents inactivation of the APC/C in nocodazole-treated cells.

中文翻译：

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诺考达唑诱导的苯胺和其他有丝分裂蛋白的表达和磷酸化在 DNA 依赖性蛋白激酶催化亚基缺陷细胞中减少，并通过用 proTAME 而不是 Apcin 抑制后期促进复合物/环体来挽救。


DNA依赖性蛋白激酶催化亚基（DNA-PKcs）在DNA双链断裂修复中具有明确的作用，最近也报道了非修复功能。为了更好地了解其细胞功能，我们使用 CRISPR/Cas9 从 HeLa 和 A549 细胞中删除了 DNA-PKcs。所得细胞对辐射敏感，共济失调毛细血管扩张突变（ATM）表达减少，并表现出多种有丝分裂缺陷。从机制上讲，诺考达唑诱导的细胞周期蛋白 B1、苯胺和 securin 的上调在 DNA-PKcs 缺陷细胞中减少，Aurora A 在苏氨酸 288 上的磷酸化、Polo 样激酶 1 (PLK1) 在苏氨酸 210 上的磷酸化以及苏氨酸 210 上的磷酸化也减少了。爪蟾 Klp2 (TPX2) 的靶向蛋白在丝氨酸 121 上的表达。此外，通过抑制后期促进复合物/环体，可以挽救诺考达唑诱导的苯胺、Seculin 和细胞周期蛋白 B1 表达的减少以及 PLK1、Aurora A 和 TPX2 的磷酸化。 (APC/C) by proTAME，可防止 APC/C 激活蛋白 Cdc20 和 Cdh1 与 APC/C 结合。总而言之，我们的研究表明，DNA-PKcs 的丢失可以防止诺考达唑处理的细胞中 APC/C 的失活。