Mass spectrometric and mutational analyses reveal lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase
Mass spectrometric and mutational analyses reveal lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase
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DOI：
10.1074/jbc.m308540200
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发表时间：
2004-02-06
影响因子：
4.8
通讯作者：
Ohta, T
中科院分区：
文献类型：
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作者：
Nishikawa, H;Ooka, S;Ohta, T
乳腺癌和卵巢癌抑制基因BRCA 1在与BARD 1结合成为RING异源二聚体时获得显著的泛素连接酶活性。虽然这种活性对于BRCA 1作为肿瘤抑制因子的作用可能很重要，但这种活性的生化后果尚不清楚。在这里，我们报告，BRCA 1-BARD 1催化赖氨酸-6-连接的多聚泛素链的形成。泛素的K6 R突变显著减少BRCA 1-BARD 1介导的多聚泛素产物。BRCA 1-BARD 1优先利用在Lys-6或Lys-29处具有单个Lys残基的泛素来介导BRCA 1的体内自泛素化。此外，质谱分析从BRCA 1-BARD 1使用野生型遍在蛋白产生的多聚遍在蛋白链中鉴定出了与Lys-6连接的分支遍在蛋白片段。BRCA 1-BARD 1介导的Lys-6连接的多聚泛素链在体外被26 S蛋白酶体去泛素化，而通过Lys-48连接的多聚泛素链自泛素化的CUL 1被降解。蛋白酶体抑制剂不会改变体内autoubiquitinated BRCA 1的稳态水平。因此，结果表明，BRCA 1-BARD 1介导的新的多聚泛素链，可以从传统的Lys-48连接的多聚泛素链不同地编辑由26 S蛋白酶体。
The breast and ovarian cancer suppressor BRCA1 acquires significant ubiquitin ligase activity when bound to BARD1 as a RING heterodimer. Although the activity may well be important for the role of BRCA1 as a tumor suppressor, the biochemical consequence of the activity is not yet known. Here we report that BRCA1-BARD1 catalyzes Lys-6-linked polyubiquitin chain formation. K6R mutation of ubiquitin dramatically reduces the polyubiquitin products mediated by BRCA1-BARD1 in vitro. BRCA1-BARD1 preferentially utilizes ubiquitin with a single Lys residue at Lys-6 or Lys-29 to mediate autoubiquitination of BRCA1 in vivo. Furthermore, mass spectrometry analysis identified the Lys-6-linked branched ubiquitin fragment from the polyubiquitin chain produced by BRCA1-BARD1 using wild type ubiquitin. The BRCA1-BARD1-mediated Lys-6-linked polyubiquitin chains are deubiquitinated by 26 S proteasome in vitro, whereas autoubiquitinated CUL1 through Lys-48-linked polyubiquitin chains is degraded. Proteasome inhibitors do not alter the steady state level of the autoubiquitinated BRCA1 in vivo. Hence, the results indicate that BRCA1-BARD1 mediates novel polyubiquitin chains that may be distinctly edited by 26 S proteasome from conventional Lys-48-linked polyubiquitin chains.