Hyperphosphorylation of the BARD1 tumor suppressor in mitotic cells.
Although the BRCA1 tumor suppressor has been implicated in a number of cellular processes, it plays an especially important role in the DNA damage response as a regulator of cell cycle checkpoints and DNA repair pathways. In vivo, BRCA1 exists as a heterodimer with the BARD1 protein, and many of its biological functions are mediated by the BRCA1-BARD1 complex. Here, we show that BARD1 is phosphorylated in a cell cycle-dependent manner and that the hyperphosphorylated forms of BARD1 predominate during M phase. By mobility shift analysis and mass spectrometry, we have identified seven sites of mitotic phosphorylation within BARD1. All sites exist within either an SP or TP sequence, and two sites resemble the consensus motif recognized by cyclin-dependent kinases. To examine the functional consequences of BARD1 phosphorylation, we used a gene targeting knock-in approach to generate isogenic cell lines that express either wild-type or mutant forms of the BARD1 polypeptide. Analysis of these lines in clonogenic survival assays revealed that cells bearing phosphorylation site mutations are hypersensitive to mitomycin C, a genotoxic agent that induces interstrand DNA cross-links. These results implicate BARD1 phosphorylation in the cellular response to DNA damage.
Choudhury
Atish D
AD
Institute for Cancer Genetics and the Departments of Pathology and Anatomy and Cell Biology, Columbia University Medical Center, New York, New York 10032, USA.
Xu
Hong
H
Modi
Ami P
AP
Zhang
Wenzhu
W
Ludwig
Thomas
T
Baer
Richard
R
D002453:Cell Cycle; D002455:Cell Division; D002460:Cell Line; D002469:Cell Separation; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D004252:DNA Mutational Analysis; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005822:Genetic Vectors; D005838:Genotype; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D016685:Mitomycin / Q000737:chemistry / Q000494:pharmacology; D008938:Mitosis; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry*
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D010455:Peptides; D025521:Tumor Suppressor Proteins; D013912:Threonine; D012694:Serine; D016685:Mitomycin; D004247:DNA; C102449:BARD1 protein, human; D044767:Ubiquitin-Protein Ligases