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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DNA依赖性蛋白激酶催化亚基缺陷型细胞中Nocodazole诱导的Anillin和其他有丝分裂蛋白的表达和磷酸化水平降低，并通过用proTAME而不是Apcin抑制后期促进复合物/脂质体来拯救。

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公认的作用，近来还报道了非修复功能。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。产生的细胞对辐射敏感，共济失调-毛细血管扩张突变（ATM）的表达降低，并表现出多个有丝分裂缺陷。从机理上讲，在DNA-PKcs缺陷型细胞中，诺考达唑诱导的细胞周期蛋白B1，阿尼林和securin的上调减少，苏氨酸288上的Aurora A磷酸化，苏氨酸210上的Polo样激酶1（PLK1）磷酸化和磷酸化降低爪蟾Klp2（TPX2）的靶向蛋白在丝氨酸121上的表达。此外，诺考达唑诱导的阿尼林，securin，proTAME抑制后期促进复合物/环体（APC / C），从而挽救了APC / C激活蛋白Cdc20和Cdh1与APC的结合，从而挽救了细胞周期蛋白B1和PLK1，Aurora A和TPX2的磷酸化/C。总而言之，我们的研究表明，DNA-PKcs的丢失可防止诺考达唑处理的细胞中APC / C的失活。