Chk2 activation dependence on Nbs1 after DNA damage
Chk2 activation dependence on Nbs1 after DNA damage
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DOI：
10.1128/mcb.21.15.5214-5222.2001
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发表时间：
2001-08-01
影响因子：
5.3
通讯作者：
Delia, D
中科院分区：
文献类型：
--
作者：
Buscemi, G;Savio, C;Delia, D
检查点激酶 Chk2 在响应 DNA 损伤而延迟细胞周期进展方面发挥着关键作用。低剂量电离辐射 (IR) 以共济失调毛细血管扩张突变 (ATM) 依赖性方式发生，Chk2 被低剂量电离辐射 (IR) 激活后，可以磷酸化抑制位点上的有丝分裂诱导磷酸酶 Cdc25C，阻止进入有丝分裂，并磷酸化调节位点上的 p53，导致 G1 停滞。在这里，我们表明，γ-辐射对 Chk2 的 ATM 依赖性激活需要 Nbs1，Nbs1 是与奈梅亨断裂综合征 (NBS) 相关的基因产物，这种疾病与 AT 共有多种表型缺陷，包括染色体脆性、辐射敏感性和抗辐射 DNA 合成。因此，在正常细胞中，Chk2 经历时间依赖性磷酸化增加并诱导针对 Cdc25C 的催化活性，而在 Nbs1 蛋白无效的 NBS 细胞中，Chk2 磷酸化和激活均存在缺陷。重要的是，NBS细胞中的这些缺陷可以通过重新引入野生型Nbs1来弥补，但既不能通过Nbs1第590位氨基酸的羧基末端缺失突变体来补充，也不能通过在ATM磷酸化位点Ser343 (S343A)处突变的Nbs1来补充，因为Nbs1不能与Mre11和Rad50形成复合物并在细胞核中转运。 NBS 细胞中 Chk2 核表达不受影响，因此排除了 Nbs1-null 细胞中 Chk2 激活失败的原因是错误定位，有趣的是，NBS 细胞中受损的 Chk2 功能与无法（与正常细胞不同）在照射后立即停止进入有丝分裂有关，这是一种检查点异常，可以通过引入野生型而非 S343A 突变形式的 Nbs1 来纠正。总之，这些发现强调了功能性 Nbs1 复合物在 Chk2 激活中的关键作用，并表明 NBS 细胞中的检查点缺陷可能是由于无法激活 Chk2 造成的。
The checkpoint kinase Chk2 has a key role in delaying cell cycle progression in response to DNA damage. Upon activation by low-dose ionizing radiation (IR), which occurs in an ataxia telangiectasia mutated (ATM)dependent manner, Chk2 can phosphorylate the mitosis-inducing phosphatase Cdc25C on an inhibitory site, blocking entry into mitosis, and p53 on a regulatory site, causing G, arrest. Here we show that the ATM-dependent activation of Chk2 by gamma- radiation requires Nbs1, the gene product involved in the Nijmegen breakage syndrome (NBS), a disorder that shares with AT a variety of phenotypic defects including chromosome fragility, radiosensitivity, and radioresistant DNA synthesis. Thus, whereas in normal cells Chk2 undergoes a time-dependent increased phosphorylation and induction of catalytic activity against Cdc25C, in NBS cells null for Nbs1 protein, Chk2 phosphorylation and activation are both defective. Importantly, these defects in NBS cells can be complemented by reintroduction of wild-type Nbs1, but neither by a carboxy-terminal deletion mutant of Nbs1 at amino acid 590, unable to form a complex with and to transport Mre11 and Rad50 in the nucleus, nor by an Nbs1 mutated at Ser343 (S343A), the ATM phosphorylation site. Chk2 nuclear expression is unaffected in NBS cells, hence excluding a mislocalization as the cause of failed Chk2 activation in Nbs1-null cells, interestingly, the impaired Chk2 function in NBS cells correlates with the inability, unlike normal cells, to stop entry into mitosis immediately after irradiation, a checkpoint abnormality that can be corrected by introduction of the wild-type but not the S343A mutant form of Nbs1, Altogether, these findings underscore the crucial role of a functional Nbs1 complex in Chk2 activation and suggest that checkpoint defects in NBS cells may result from the inability to activate Chk2.