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
OBJECTIVE To observe the inhibitory effect on bladder tumor proliveration after transfection with the expression plasmid pcDNA3.1（＋）/Madl. METHODS Bladder tumors were induced in SD rats by intravesical instillation...OBJECTIVE To observe the inhibitory effect on bladder tumor proliveration after transfection with the expression plasmid pcDNA3.1（＋）/Madl. METHODS Bladder tumors were induced in SD rats by intravesical instillation of MNU . The tumor-bearing rats were randomly divided into 3 groups： group A, transfected with pcDNA3.1 （＋）/Mad1, group B, transfected with an empty vector and group C, transfected with saline. Rat body weight （RBW）, bladder absolute weight （BAW） and bladder relative weight （BRW） were measured and expression levels of Mad1 and TERT were assayed. Flow cytometer analysis was used to observe the effect of Mad1 on the bladder tumors. RESULTS Comparions of RBW among the 3 groups showed there were no differences （P〉0.05）. But the BAW and BRW for group A were significantly decreased （P〈0.01, P〈0.05, respectively） comparded to groups B and C. In group A, the Mad1 mRNA expression level was markedly improved, while the TERT mRNA expression level was decreased. Flow cytometry showed an increase in GJG1-phase cells and a decrease of Sphase cells after transfection with Mad1. CONCLUSION Over expression of Mad1 can inhibit the cellular proliferation of bladder tumors.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.
文摘OBJECTIVE To observe the inhibitory effect on bladder tumor proliveration after transfection with the expression plasmid pcDNA3.1（＋）/Madl. METHODS Bladder tumors were induced in SD rats by intravesical instillation of MNU . The tumor-bearing rats were randomly divided into 3 groups： group A, transfected with pcDNA3.1 （＋）/Mad1, group B, transfected with an empty vector and group C, transfected with saline. Rat body weight （RBW）, bladder absolute weight （BAW） and bladder relative weight （BRW） were measured and expression levels of Mad1 and TERT were assayed. Flow cytometer analysis was used to observe the effect of Mad1 on the bladder tumors. RESULTS Comparions of RBW among the 3 groups showed there were no differences （P〉0.05）. But the BAW and BRW for group A were significantly decreased （P〈0.01, P〈0.05, respectively） comparded to groups B and C. In group A, the Mad1 mRNA expression level was markedly improved, while the TERT mRNA expression level was decreased. Flow cytometry showed an increase in GJG1-phase cells and a decrease of Sphase cells after transfection with Mad1. CONCLUSION Over expression of Mad1 can inhibit the cellular proliferation of bladder tumors.
