Intracellular redistribution and modification of proteins of the Mre11/Rad50/Nbs1 DNA repair complex following irradiation and heat-shock
Intracellular redistribution and modification of proteins of the Mre11/Rad50/Nbs1 DNA repair complex following irradiation and heat-shock
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DOI：
10.1002/jcp.10475
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发表时间：
2004-05-01
影响因子：
5.6
通讯作者：
Dynlacht, JR
中科院分区：
文献类型：
--
作者：
Seno, JD;Dynlacht, JR
Mre11、Rad50和Nbs1形成一个紧密的复合体，均匀分布在哺乳动物细胞的细胞核中。然而，照射后，Mre11/Rad50/Nbs1(M/R/N)复合体迅速迁移到双链断裂(DSB)的位置，形成病灶，直到DSB修复完成。Mre11和Rad50在DSB修复中起直接作用，而Nbs1似乎参与损伤信号转导。高温使哺乳动物细胞对电离辐射敏感。热休克引起的放射增敏被认为是通过抑制DSB修复而起作用的。虽然热休克抑制修复的机制尚不清楚，但最近的报道表明，M/R/N复合体可能是热抑制DSB修复和辐射增敏的靶点。我们现在证明，当人类U-1黑色素瘤细胞在42.5或45.5℃的温度下加热时，Mre11、Rad50和Nbs1迅速从细胞核转移到细胞质。有趣的是，当细胞在热处理前暴露于电离辐射(12GyX射线)时，当处理后立即分析细胞核和细胞质部分的蛋白质时，易位的程度和动力学都会增加。当细胞在37℃下孵育30min至7h时，每种蛋白质的移位和随后的重定位回到细胞核的动力学是不同的，这表明这些蛋白质是独立分布的。然而，很大一部分转位蛋白以三重复合体的形式存在于细胞质中。用软霉素B(LMB)处理可抑制Mre11、Rad50和Nbs1向细胞质的移位，这使我们推测这些蛋白向细胞质的重新定位是通过CRM1介导的核输出发生的。此外，虽然Nbs1在受照射细胞的细胞核中迅速磷酸化，并且对正常的DNA损伤反应至关重要，但我们发现Nbs1在受热照射细胞的细胞质中迅速磷酸化，而不是在细胞核中。细胞质Nbs1的磷酸化不能被Wortmannin抑制，在加热、照射的细胞中似乎是一种独特的翻译后修饰，再加上我们新的观察到Mre11、Rad50和Nbs1移位到细胞质，进一步支持了M/R/N复合体在热辐射增敏和抑制DSB修复中的作用。J.Cell.物理。199：157-170,2004。(C)2004年Wiley-Liss公司
Mre11, Rad50, and Nbs1 form a tight complex which is homogeneously distributed throughout the nuclei of mammalian cells. However, after irradiation, the Mre11/ Rad50/Nbs1 (M/R/N) complex rapidly migrates to sites of double strand breaks (DSBs), forming foci which remain until DSB repair is complete. Mre11 and Rad50 play direct roles in DSB repair, while Nbs1 appears to be involved in damage signaling. Hyperthermia sensitizes mammalian cells to ionizing radiation. Radio-sensitization by heat shock is believed to be mediated by an inhibition of DSB repair. While the mechanism of inhibition of repair by heat shock remains to be elucidated, recent reports suggest that the M/R/N complex may be a target for inhibition of DSB repair and radiosensitization by heat. We now demonstrate that when human U-1 melanoma cells are heated at 42.5 or 45.5degreesC, Mre11, Rad50, and Nbs1 are rapidly translocated from the nucleus to the cytoplasm. interestingly, when cells were exposed to ionizing radiation (12 Gy of X-rays) prior to heat treatment, the extent and kinetics of translocation were increased when nuclear and cytoplasmic fractions of protein were analyzed immediately after treatment. The kinetics of the translocation and subsequent relocalization back into the nucleus when cells were incubated at 37degreesC from 30 min to 7 h following treatment were different for each protein, which suggests that the proteins redistribute independently. However, a significant fraction of the translocated proteins exist as a triple complex in the cytoplasm. Treatment with leptomycin B (LMB) inhibits the translocation of Mre11, Rad50, and Nbs1 to the cytoplasm, leading us to speculate that the relocalization of the proteins to the cytoplasm occurs via CRM1-mediated nuclear export. In addition, while Nbs1 is rapidly phosphorylated in the nuclei of irradiated cells and is critical for a normal DNA damage response, we have found that Nbs1 is rapidly phosphorylated in the cytoplasm, but not in the nucleus, of heated irradiated cells. The phosphorylation of cytoplasmic Nbs1, which cannot be inhibited by wortmannin, appears to be a unique post-translational modification in heated, irradiated cells, and coupled with our novel observations that Mre11, Rad50, and Nbs1 translocate to the cytoplasm, lend further support for a role of the M/R/N complex in thermal radiosensitization and inhibition of DSB repair. J.Cell. Physiol. 199: 157-170, 2004. (C) 2004 Wiley-Liss, Inc.