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....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.展开更多
为了提高2.4 m Ku频段球形充气天线的面形精度,开展了天线初始形态结构设计优化。通过建立2.4 m球形充气天线有限元模型,采用Zernike多项式及法向误差计算表面误差的方法对反射面形态进行了优化设计,使其在充气后达到预期的抛物面形状,...为了提高2.4 m Ku频段球形充气天线的面形精度,开展了天线初始形态结构设计优化。通过建立2.4 m球形充气天线有限元模型,采用Zernike多项式及法向误差计算表面误差的方法对反射面形态进行了优化设计,使其在充气后达到预期的抛物面形状,确保了表面误差在0.5 mm以内的要求。仿真结果表明,在400 Pa压差和下半球气压7000 Pa的不同角度工况下,面形精度满足Ku频段表面误差的精度要求。经上述研究,优化设计后的反射面在充气后能够实现较高的面形精度,满足工程应用需求。展开更多
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))+4 种基金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 Statesthe Tianjin Medical University Cancer Institute and Hospital Innovation Fund(Grant No.1803)。
文摘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.
