Kinetochore protein MAD1 participates in the DNA damage response through ataxia-telangiectasia mutated kinase-mediated phosphorylation and enhanced interaction with KU80  

作　　者：Mingming Xiao Xuesong Li Yang Su Zhuang Liu Yamei Han Shuai Wang Qinghua Zeng Hong Liu Jianwei Hao Bo Xu 

机构地区：[1]Department of Biochemistry and Molecular Biology,Key Laboratory of Breast Cancer Prevention and Therapy,Ministry of Education,Tianjin Medical University Cancer Institute and Hospital,National Clinical Research Center for Cancer,Key Laboratory of Cancer Prevention and Therapy,Tianjin,Tianjin's Clinical Research Center for Cancer,Tianjin 300060,China [2]Department of Oncology,Southern Research Institute,Birmingham,AL 35205,USA [3]Department of Neurosurgery,Tianjin Huanhu Hospital,Tianjin 300350,China [4]Tianjin Key Laboratory of Cerebral Vascular and Neurodegenerative Diseases,Tianjin 300350,China [5]Center for Intelligent Oncology,Chongqing University Cancer Hospital,Chongqing University School of Medicine,Chongqing 400030,China

出　　处：《Cancer Biology & Medicine》2020年第3期640-651,共12页癌症生物学与医学（英文版）

基　　金：This work was supported by the National Natural Science Foundation of China(Grant Nos.81672743 and 81974464);Beijing Tianjin Hebei Basic Research Cooperation Project(Grant No.19JCZDJC64500(Z));Shenzhen Basic Research Project(Grant No.JCYJ20160331114230843);Tianjin Municipal Health Commission(Grant Nos.2015KR11 and 2013KG134);Tianjin Municipal Science and Technology Bureau(Grant No.18JCYBJC27800);US NIH grant RO 1 CAI33093,the Alabama Innovation Fund of the United States;the Tianjin Medical University Cancer Institute and Hospital Innovation Fund(Grant No.1803)。

摘　　要：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'st-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.

关 键 词：DNA damage response ataxia-telangiectasia mutated kinase(ATM) mitotic arrest-deficient protein 1(MAD1) KU80 protein DNA-PKCS 

分 类 号：R363[医药卫生—病理学]