Kinetochore localisation of the DNA damage response component 53BP1 during mitosis.
53BP1 is a vertebrate BRCT motif protein, originally described as a direct interactor of p53, which has recently been shown to be implicated in the early response to DNA damage. Upon DNA damage, 53BP1 re-localises to discrete nuclear foci that are thought to represent sites of DNA lesions and becomes hyperphosphorylated. Several observations suggest that 53BP1 is a direct substrate for the ataxia telangiectasia mutated (ATM) kinase. So far, 53BP1 behaviour during mitosis has not been reported in detail. We have examined 53BP1 subcellular distribution in mitotic cells using several antibodies against 53BP1, and ectopic expression of GFP-tagged 53BP1. We found that 53BP1 significantly colocalised with CENP-E to kinetochores. 53BP1 is loaded to kinetochores in prophase, before CENP-E, and is released by mid-anaphase. By expressing various GFP-tagged 53BP1 truncations, the kinetochore binding domain has been mapped to a 380 residue portion of the protein that excludes the nuclear localisation signal and the BRCT motifs. Like many kinetochore-associated proteins involved in mitotic checkpoint signalling, more 53BP1 appears to accumulate on the kinetochores of chromosomes not aligned on the metaphase plate. Finally, we show that 53BP1 is hyperphosphorylated in mitotic cells, and undergoes an even higher level of phosphorylation in response to spindle disruption with colcemid. Our data suggest that 53BP1 may have a role in checkpoint signalling during mitosis and provide the evidence that DNA damage response machinery and mitotic checkpoint may share common molecular components.
Jullien
Denis
D
The Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, The University of Edinburgh, King's Buildings, Edinburgh EH9 3JR, UK.
Vagnarelli
Paola
P
Earnshaw
William C
WC
Adachi
Yasuhisa
Y
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001324:Autoantigens; D002352:Carrier Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D002457:Cell Extracts / Q000032:analysis; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D004249:DNA Damage / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D003703:Demecolcine / Q000494:pharmacology; D005260:Female; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D018386:Kinetochores / Q000737:chemistry*; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008938:Mitosis / Q000502:physiology*; D010750:Phosphoproteins; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D008941:Spindle Apparatus / Q000187:drug effects; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection; D000071857:Tumor Suppressor p53-Binding Protein 1
D000906:Antibodies; D001324:Autoantigens; C497926:CENPB protein, human; D002352:Carrier Proteins; D002457:Cell Extracts; C497927:Cenpb protein, mouse; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D010750:Phosphoproteins; C498301:TP53BP1 protein, human; C498302:Trp53bp1 protein, mouse; D000071857:Tumor Suppressor p53-Binding Protein 1; D049452:Green Fluorescent Proteins; D003703:Demecolcine