Ray Hind, Moreau Karen, Dizin Eva, Callebaut Isabelle, Venezia Nicole Dalla
Laboratoire de Génétique Moléculaire, Signalisation et Cancer, CNRS UMR 5201, Faculté de Médecine Rockefeller, 8 Avenue Rockefeller, 69373 Lyon cedex 08, France.
J Mol Biol. 2006 Jun 16;359(4):973-82. doi: 10.1016/j.jmb.2006.04.010. Epub 2006 Apr 25.
The tumour suppressor gene BRCA1 encodes a 220 kDa protein that participates in multiple cellular processes. The BRCA1 protein contains a tandem of two BRCT repeats at its carboxy-terminal region. The majority of disease-associated BRCA1 mutations affect this region and provide to the BRCT repeats a central role in the BRCA1 tumour suppressor function. The BRCT repeats have been shown to mediate phospho-dependant protein-protein interactions. They recognize phosphorylated peptides using a recognition groove that spans both BRCT repeats. We previously identified an interaction between the tandem of BRCA1 BRCT repeats and ACCA, which was disrupted by germ line BRCA1 mutations that affect the BRCT repeats. We recently showed that BRCA1 modulates ACCA activity through its phospho-dependent binding to ACCA. To delineate the region of ACCA that is crucial for the regulation of its activity by BRCA1, we searched for potential phosphorylation sites in the ACCA sequence that might be recognized by the BRCA1 BRCT repeats. Using sequence analysis and structure modelling, we proposed the Ser1263 residue as the most favourable candidate among six residues, for recognition by the BRCA1 BRCT repeats. Using experimental approaches, such as GST pull-down assay with Bosc cells, we clearly showed that phosphorylation of only Ser1263 was essential for the interaction of ACCA with the BRCT repeats. We finally demonstrated by immunoprecipitation of ACCA in cells, that the whole BRCA1 protein interacts with ACCA when phosphorylated on Ser1263.
肿瘤抑制基因BRCA1编码一种220 kDa的蛋白质，该蛋白质参与多种细胞过程。BRCA1蛋白在其羧基末端区域包含两个BRCT重复序列串联。大多数与疾病相关的BRCA1突变影响该区域，并使BRCT重复序列在BRCA1肿瘤抑制功能中发挥核心作用。已表明BRCT重复序列介导磷酸依赖性蛋白质-蛋白质相互作用。它们使用跨越两个BRCT重复序列的识别凹槽识别磷酸化肽段。我们之前鉴定出BRCA1 BRCT重复序列串联与ACCA之间存在相互作用，这种相互作用被影响BRCT重复序列的种系BRCA1突变所破坏。我们最近表明，BRCA1通过其与ACCA的磷酸依赖性结合来调节ACCA活性。为了确定ACCA中对其活性受BRCA1调节至关重要的区域，我们在ACCA序列中寻找可能被BRCA1 BRCT重复序列识别的潜在磷酸化位点。通过序列分析和结构建模，我们提出Ser1263残基是六个残基中最有可能被BRCA1 BRCT重复序列识别的候选者。使用实验方法，如用Bosc细胞进行的GST下拉实验，我们清楚地表明只有Ser1263的磷酸化对于ACCA与BRCT重复序列的相互作用至关重要。我们最终通过在细胞中对ACCA进行免疫沉淀证明，当Ser1263磷酸化时，整个BRCA1蛋白与ACCA相互作用。
