Kinetochore protein MAD1 participates in the DNA damage response through ataxia-telangiectasia mutated kinase-mediated phosphorylation and enhanced interaction with KU80
Kinetochore protein MAD1 participates in the DNA damage response through ataxia-telangiectasia mutated kinase-mediated phosphorylation and enhanced interaction with KU80
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动粒蛋白 MAD1 通过共济失调毛细血管扩张突变激酶介导的磷酸化以及增强与 KU80 的相互作用参与 DNA 损伤反应
DOI：
10.20892/j.issn.2095-3941.2020.0044
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发表时间：
2020-08
影响因子：
5.5
通讯作者：
Xu Bo
中科院分区：
文献类型：
--
作者：
Xiao Mingming;Li Xuesong;Su Yang;Liu Zhuang;Han Yamei;Wang Shuai;Zeng Qinghua;Liu Hong;Hao Jianwei;Xu Bo
目的：有丝分裂阻滞缺陷蛋白1（MAD 1）是有丝分裂纺锤体检查点所必需的着丝粒蛋白。蛋白质组学研究表明，MAD 1是DNA损伤反应（DDR）途径的一个组成部分。然而，MAD 1是否以及如何直接参与DDR在很大程度上是未知的。研究方法：我们异位表达野生型，或磷酸化位点突变形式的MAD 1在MAD 1敲低细胞中寻找互补效应。我们使用彗星试验、集落形成试验、免疫荧光染色和流式细胞术来评估DDR、放射敏感性和G2/M检查点。我们采用免疫共沉淀法和质谱法来鉴定MAD 1相互作用蛋白。使用非配对Student t检验分析数据。结果如下：我们发现，MAD 1是最佳DDR所必需的，因为敲低MAD 1会导致DNA修复受损和对电离辐射（IR）的超敏反应。我们发现IR诱导的丝氨酸214磷酸化是共济失调毛细血管扩张突变（ATM）激酶依赖性的。利用质谱技术，我们鉴定了一个由MAD 1丝氨酸214磷酸化介导的蛋白复合物，其中KU 80是一个关键蛋白，在DNA损伤后，它与MAD 1的相互作用增强。最后，我们发现MAD 1与KU 80的相互作用需要丝氨酸214磷酸化，并且它是激活DNA蛋白激酶催化亚基（DNA-PKcs）所必需的。结论：ATM激酶介导的MAD 1丝氨酸214磷酸化是IR与KU 80相互作用和DNA-PKCs活化所必需的。
Objective: Mitotic arrest-deficient protein 1 (MAD1) is a kinetochore protein essential for the mitotic spindle checkpoint. Proteomic studies have indicated that MAD1 is a component of the DNA damage response (DDR) pathway. However, whether and how MAD1 might be directly involved in the DDR is largely unknown. Methods: We ectopically expressed the wild type, or a phosphorylation-site--mutated form of MAD1 in MAD1 knockdown cells to look for complementation effects. We used the comet assay, colony formation assay, immunofluorescence staining, and flow cytometry to assess the DDR, radiosensitivity, and the G2/M checkpoint. We employed co-immunoprecipitation followed by mass spectrometry to identify MAD1 interacting proteins. Data were analyzed using the unpaired Student’s t-test. Results: We showed that MAD1 was required for an optimal DDR, as knocking down MAD1 resulted in impaired DNA repair and hypersensitivity to ionizing radiation (IR). We found that IR-induced serine 214 phosphorylation was ataxia-telangiectasia mutated (ATM) kinase-dependent. Mutation of serine 214 to alanine failed to rescue the phenotypes of MAD1 knockdown cells in response to IR. Using mass spectrometry, we identified a protein complex mediated by MAD1 serine 214 phosphorylation in response to IR. Among them, we showed that KU80 was a key protein that displayed enhanced interaction with MAD1 after DNA damage. Finally, we showed that MAD1 interaction with KU80 required serine 214 phosphorylation, and it was essential for activation of DNA protein kinases catalytic subunit (DNA-PKcs). Conclusions: MAD1 serine 214 phosphorylation mediated by ATM kinase in response to IR was required for the interaction with KU80 and activation of DNA-PKCs.
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DOI：
10.32388/6xsu16
发表时间：
2020-02
期刊：
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影响因子：
--
作者：
通讯作者：
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影响因子：
11.2
作者：
Y. Iwanaga;K. Jeang
通讯作者：
Y. Iwanaga;K. Jeang
DOI：
10.32388/zy7y1f
发表时间：
2020-02
期刊：
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影响因子：
--
作者：
通讯作者：
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影响因子：
16.6
作者：
Jun Wan;Samuel Block;Christina M. Scribano;Rebecca Thiry;K. Esbona;A. Audhya;Beth A. Weaver
通讯作者：
Jun Wan;Samuel Block;Christina M. Scribano;Rebecca Thiry;K. Esbona;A. Audhya;Beth A. Weaver
DOI：
10.32388/lb62u2
发表时间：
2020-02
期刊：
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影响因子：
--
作者：
通讯作者：
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