The DNA damage repair protein Ku70 interacts with FOXO4 to coordinate a conserved cellular stress response
The DNA damage repair protein Ku70 interacts with FOXO4 to coordinate a conserved cellular stress response
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DOI：
10.1096/fj.10-158717
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发表时间：
2010-11-01
期刊：
影响因子：
4.8
通讯作者：
Burgering, Boudewijn M. T.
中科院分区：
文献类型：
--
作者：
Brenkman, Arjan B.;van den Broek, Niels J. F.;Burgering, Boudewijn M. T.
在这项研究中，我们寻找调节肿瘤抑制因子和寿命调节因子FOXO 4的蛋白质。通过无偏串联亲和纯化策略结合质谱，我们确定了异二聚体Ku 70/Ku 80（Ku），DNA双链断裂修复组件。使用生化相互作用的研究，我们发现Ku 70是必要的和充分的相互作用。FOXO 4通过细胞周期阻滞p27（kip 1）基因的转录调节介导其肿瘤抑制功能。免疫印迹、荧光素酶报告基因分析和流式细胞术显示，Ku 70抑制FOXO 4介导的p27（kip 1）转录和细胞周期阻滞诱导> 40%。相反，Ku 70 RNAi而非对照RNAi显著增加p27（kip 1）转录。此外，与野生型小鼠胚胎干（ES）细胞相比，Ku 70（-/-）ES细胞表现出显著增加的FOXO活性，这是由Ku 70再表达拯救的。免疫荧光研究表明，Ku 70螯合FOXO 4在细胞核中。有趣的是，Ku 70-FOXO 4相互作用的化学计量学遵循一个非线性的剂量-反应曲线过氧化氢产生的氧化应激。低水平的氧化应激增加相互作用的化学计量高达75%，峰值在50 μ M，之后发生解离。由于线虫中的Ku 70直系同源物被证明可以调节线虫的寿命，elegans FOXO，我们的研究结果表明，FOXO功能的保守关键Ku 70作用，对生存程序的协调，由氧化损伤的大小调节。Brenkman，A. B.，货车登布鲁克，N. J. F.，de Keizer，P. L. J.，货车Gent，D. C.的方法，汉堡店，B。M. T. DNA损伤修复蛋白Ku 70与FOXO 4相互作用以协调保守的细胞应激反应。FASEB J.24，4271-4280（2010）。www.fasebj.org
In this study, we searched for proteins regulating the tumor suppressor and life-span regulator FOXO4. Through an unbiased tandem-affinity purification strategy combined with mass spectrometry, we identified the heterodimer Ku70/Ku80 (Ku), a DNA double-strand break repair component. Using biochemical interaction studies, we found Ku70 to be necessary and sufficient for the interaction. FOXO4 mediates its tumor-suppressive function in part through transcriptional regulation of the cell cycle arrest p27(kip1) gene. Immunoblotting, luciferase reporter assays, and flow cytometry showed that Ku70 inhibited FOXO4-mediated p27(kip1) transcription and cell cycle arrest induction by >40%. In contrast, Ku70 RNAi but not control RNAi significantly increased p27(kip1) transcription. In addition, in contrast to wild-type mouse embryonic stem (ES) cells, Ku70(-/-) ES cells showed significantly increased FOXO activity, which was rescued by Ku70 reexpression. Immunofluorescence studies demonstrated that Ku70 sequestered FOXO4 in the nucleus. Interestingly, the Ku70-FOXO4 interaction stoichiometry followed a nonlinear dose-response curve by hydrogen peroxide-generated oxidative stress. Low levels of oxidative stress increased interaction stoichiometry up to 75%, peaking at 50 mu M, after which dissociation occurred. Because the Ku70 ortholog in the roundworm Caenorhabditis elegans was shown to regulate life span involving C. elegans FOXO, our findings suggest a conserved critical Ku70 role for FOXO function toward coordination of a survival program, regulated by the magnitude of oxidative damage.-Brenkman, A. B., van den Broek, N. J. F., de Keizer, P. L. J., van Gent, D. C., Burgering, B. M. T. The DNA damage repair protein Ku70 interacts with FOXO4 to coordinate a conserved cellular stress response. FASEB J. 24, 4271-4280 (2010). www.fasebj.org