Feng Zhihui, Kachnic Lisa, Zhang Junran, Powell Simon N, Xia Fen
Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA.
J Biol Chem. 2004 Jul 2;279(27):28574-84. doi: 10.1074/jbc.M404137200. Epub 2004 Apr 15.
The tumor suppressor gene BRCA1 plays an important role in the response to DNA damage. BRCA1 function is regulated by a variety of mechanisms including transcriptional control, phosphorylation, and protein-protein interactions. Recent studies have shown that BRCA1 is a nuclear-cytoplasmic shuttle protein. Its subcellular localization is controlled by a nuclear localization signal-mediated nuclear import via the importin receptor pathway and a nuclear export signal-facilitated nuclear export through a CRM1-dependent pathway. Using the human breast cancer cell line, MCF7, the subcellular distribution of BRCA1 was assessed by immunohistochemical staining and Western blotting analyses of fractionated subcellullar extracts. Ionizing radiation stimulated BRCA1 nuclear export in a dose-dependent manner. This DNA damage-induced BRCA1 nuclear export utilized a CRM1-dependent mechanism and also required wild-type p53, whose function was abrogated by the E6 protein in MCF7 cells. In addition, the dependence on p53 was confirmed using a second cell type operating a tetracycline-inducible system. The effect of ionizing radiation on BRCA1 export was observed in every phase of the cell cycle, although BRCA1 localization did vary between the G(1), S, and G(2)/M phases. These results imply that, in addition to ATM-, ATR-, and Chk2-dependent phosphorylations, cytoplasmic relocalization of BRCA1 protein is a mechanism whereby BRCA1 function is regulated in response to DNA damage.
肿瘤抑制基因BRCA1在对DNA损伤的反应中起重要作用。BRCA1的功能受多种机制调控，包括转录控制、磷酸化以及蛋白质-蛋白质相互作用。最近的研究表明，BRCA1是一种穿梭于细胞核与细胞质之间的蛋白质。其亚细胞定位由核定位信号介导的通过输入蛋白受体途径的核输入以及核输出信号促进的通过依赖CRM1的途径的核输出所控制。利用人乳腺癌细胞系MCF7，通过免疫组织化学染色和对分级分离的亚细胞提取物进行蛋白质印迹分析来评估BRCA1的亚细胞分布。电离辐射以剂量依赖的方式刺激BRCA1的核输出。这种DNA损伤诱导的BRCA1核输出利用了一种依赖CRM1的机制，并且还需要野生型p53，其功能在MCF7细胞中被E6蛋白所废除。此外，使用第二种操作四环素诱导系统的细胞类型证实了对p53的依赖性。尽管BRCA1在G(1)、S和G(2)/M期的定位确实有所不同，但在细胞周期的每个阶段都观察到了电离辐射对BRCA1输出的影响。这些结果表明，除了依赖ATM、ATR和Chk2的磷酸化之外，BRCA1蛋白的细胞质重新定位是一种在DNA损伤反应中调控BRCA1功能的机制。
