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

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.

The Short Isoform of Nuclear Mitotic Apparatus Protein 1 Functions as a Putative Tumor Suppressor

Background：Nuclear mitotic apparatus protein 1 （NuMA 1） had been reported to produce three groups of isoforms categorized as long,middle,and short groups,of which short NuMA displayed distinct localization patterns compared to long and middle isoforms.However,the function of short NuMA was not clear in the progress of cancer formation.This study aimed to unveil the role of short NuMA in cancer pathogenesis.Methods：The expression levels of short isoforms were explored in paired gastric carcinoma （GC） samples and different cell lines.Furthermore,the short isoform behaved as a putative tumor suppressor based on cell proliferation and cell colony formation assays.Pull-down assay and whole-genome gene expression analysis were carried out to search candidate interaction partners of short NuMA.Results：The expression of short NuMA was highly expressed in S and G2 phases of the cell cycle;compared with nontumor tissues,short NuMA downregulated in nine GCs （GC 1 [0.131,P =5 × 10^-4];GC2 [0.316,P =3 × 10^-5];GC3 [0.111,P =6 × 10^-4];GC4 [0.456,P =0.011];GC5 [0.474,P=0.001];GC6 [0.311,P=0.004];GC7 [0.28,P=3 × 10^-5];GC8 [0.298,P=0.007];and GC9 [0.344,P=0.002]）.Besides,high expression of short NuMA significantly inhibits cell growth （2.43 × 10^-5 vs.2.97 × 10^-5,P =0.0029） and cell clone information in vitro （70 vs.2,P =1.67 × 10^-45）.Short NuMA could bind with alpha-actinin-4 （ACTN4）,a putative tumor promoting gene.Overexpression of short NuMA could tremendously decrease the expression of MYB proto-oncogene like 2 （MYBL2） of about 92-fold,which played an important role in the cell cycles.Conclusions：Short isoform of NuMA might be functioned as a putative role of tumor suppressor.Further studies should be made to illuminate the relationship between ACTN4,MYBL2,and tumor progression.

A Long Type of TBCK Is a Novel Cytoplasmic and Mitotic Apparatus-Associated Protein Likely Suppressing Cell Proliferation

Autoantibodies from patients with various connective tissue diseases have been shown to be specific probes that can detect cellular structures, including centrosome, centromere/kineto- chore, spliceosome, Golgi complex and the rough endoplasmic reticulum （Louvard et al., 1982; Rattner et al., 1998;