Hyperphosphorylation of the BARD1 tumor suppressor in mitotic cells
Hyperphosphorylation of the BARD1 tumor suppressor in mitotic cells
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DOI：
10.1074/jbc.m502446200
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发表时间：
2005-07-01
影响因子：
4.8
通讯作者：
Baer, R
中科院分区：
文献类型：
--
作者：
Choudhury, AD;Xu, H;Baer, R
尽管BRCA 1肿瘤抑制因子与许多细胞过程有关，但它作为细胞周期检查点和DNA修复途径的调节剂在DNA损伤反应中起着特别重要的作用。在体内，BRCA 1与BARD 1蛋白以异源二聚体形式存在，其许多生物学功能都是由BRCA 1-BARD 1复合物介导的。在这里，我们表明，BARD 1是磷酸化的细胞周期依赖性的方式和BARD 1的过度磷酸化的形式占主导地位，在M期。通过迁移率变动分析和质谱分析，我们已经确定了BARD 1内的7个有丝分裂磷酸化位点。所有位点都存在于SP或TP序列中，其中两个位点类似于细胞周期蛋白依赖性激酶识别的共有基序。为了检查BARD 1磷酸化的功能后果，我们使用基因靶向敲入方法来产生表达野生型或突变形式的BARD 1多肽的等基因细胞系。在克隆形成存活测定中对这些细胞系的分析显示，具有磷酸化位点突变的细胞对丝裂霉素C（一种诱导链间DNA交联的遗传毒性剂）超敏。这些结果暗示BARD 1磷酸化在细胞对DNA损伤的反应中。
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.