Chemical genetics reveals a specific requirement for Cdk2 activity in the DNA damage response and identifies Nbs1 as a Cdk2 substrate in human cells.
Chemical genetics reveals a specific requirement for Cdk2 activity in the DNA damage response and identifies Nbs1 as a Cdk2 substrate in human cells.
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DOI：
10.1371/journal.pgen.1002935
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发表时间：
2012-08
期刊：
影响因子：
4.5
通讯作者：
Fisher RP
中科院分区：
文献类型：
--
作者：
Wohlbold L;Merrick KA;De S;Amat R;Kim JH;Larochelle S;Allen JJ;Zhang C;Shokat KM;Petrini JH;Fisher RP
促进细胞周期进程的细胞周期蛋白依赖性激酶(CDK)是DNA损伤或未复制信号负性调控的靶标，但也在DNA损伤反应中发挥积极作用。面对DNA损伤时对活性的要求意味着存在使某些CDK免受检查点抑制的机制。然而，仍然很难确定特定CDK在保护基因组完整性方面的确切功能。在哺乳动物中，CDK2在整个S和G2期都是活跃的，但由于受到其他CDK的补偿，CDK2蛋白对于生存是必不可少的。这种可塑性掩盖了人类细胞增殖中对CDK2活性的要求，我们通过用突变型CDK2替换野生型CDK2来揭示这一点，该突变版本对笨重的腺嘌呤类似物的抑制敏感。在这里，我们表明，在暴露于电离辐射(IR)后，对模拟敏感(AS)CDK2的瞬时、选择性抑制增强了对细胞的杀伤。在添加了AS激酶优先使用的ATP类似物的提取物中，CDK2AS磷酸化了奈梅亨断裂综合征基因产物Nbs1-正常DNA损伤修复和检查点信号转导所需的保守的Mre11-Rad50-Nbs1复合体的组成部分-依赖于Ser432上的共识CDK识别位点。在体内，选择性抑制CDK2延迟和减弱了S期NBS1-Ser432的磷酸化，Ser432突变为Ala或Asp增加了对IR的敏感性。因此，通过化学遗传学，我们发现了响应DNA损伤的CDK2活性的非冗余要求，以及DNA修复机制中的CDK2的特定靶点。多个细胞周期蛋白依赖性激酶(CDK)控制着人类细胞的增殖，但目前尚不清楚不同的CDK在不受干扰的细胞分裂过程中或在细胞分裂引起DNA损伤后是如何协调功能的。DNA损伤激活检查点信号通路，抑制CDK活性，阻止细胞分裂周期，从而防止遗传信息的丢失；但对损伤的有效响应还需要CDK活性来修改修复和检查点通路的组成部分。我们采用了化学遗传学的方法来研究特定的CDK，CDK2，是否在保护人类细胞基因组完整性方面发挥了特殊的、非冗余的作用。通过使CDK2对化学抑制敏感，我们能够检测到它在暴露于电离辐射(IR)后对细胞存活的催化活性的特殊要求。我们确定Nbs1是癌症易感奈梅根断裂综合征基因突变的产物，作为CDK2底物，并表明不能被CDK2修饰的Nbs1突变形式在保护细胞免受IR诱导的DNA损伤方面存在缺陷。因此，我们的工作定义了一条依赖于特定CDK在人类细胞中的催化活性的DNA损伤反应途径，并提出了一种在不引发不适当细胞分裂的情况下促进有效修复的机制。
The cyclin-dependent kinases (CDKs) that promote cell-cycle progression are targets for negative regulation by signals from damaged or unreplicated DNA, but also play active roles in response to DNA lesions. The requirement for activity in the face of DNA damage implies that there are mechanisms to insulate certain CDKs from checkpoint inhibition. It remains difficult, however, to assign precise functions to specific CDKs in protecting genomic integrity. In mammals, Cdk2 is active throughout S and G2 phases, but Cdk2 protein is dispensable for survival, owing to compensation by other CDKs. That plasticity obscured a requirement for Cdk2 activity in proliferation of human cells, which we uncovered by replacement of wild-type Cdk2 with a mutant version sensitized to inhibition by bulky adenine analogs. Here we show that transient, selective inhibition of analog-sensitive (AS) Cdk2 after exposure to ionizing radiation (IR) enhances cell-killing. In extracts supplemented with an ATP analog used preferentially by AS kinases, Cdk2as phosphorylated the Nijmegen Breakage Syndrome gene product Nbs1—a component of the conserved Mre11-Rad50-Nbs1 complex required for normal DNA damage repair and checkpoint signaling—dependent on a consensus CDK recognition site at Ser432. In vivo, selective inhibition of Cdk2 delayed and diminished Nbs1-Ser432 phosphorylation during S phase, and mutation of Ser432 to Ala or Asp increased IR–sensitivity. Therefore, by chemical genetics, we uncovered both a non-redundant requirement for Cdk2 activity in response to DNA damage and a specific target of Cdk2 within the DNA repair machinery. Multiple cyclin-dependent kinases (CDKs) control human cell proliferation, but it remains unclear how functions of different CDKs are coordinated during unperturbed cell division or after dividing cells incur DNA damage. DNA lesions activate checkpoint signaling pathways to inhibit CDK activity, arrest the cell division cycle, and thus prevent loss of genetic information; but an effective response to damage also requires CDK activity to modify components of repair and checkpoint pathways. We took a chemical-genetic approach to ask if a specific CDK, Cdk2, played a specialized, non-redundant role in protecting genomic integrity of human cells. By sensitizing Cdk2 to chemical inhibition, we were able to detect a specific requirement for its catalytic activity in survival of cells after exposure to ionizing radiation (IR). We identified Nbs1, product of the gene mutated in the cancer-predisposing Nijmegen Breakage Syndrome, as a Cdk2 substrate and showed that mutant forms of Nbs1 that cannot be modified by Cdk2 are defective in protecting cells from death due to IR–induced DNA damage. Therefore, our work defines a DNA damage response pathway that depends on catalytic activity of a specific CDK in human cells and suggests a mechanism to promote efficient repair without triggering inappropriate cell division.
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作者：
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影响因子：
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DOI：
10.1073/pnas.0809350106
发表时间：
2009-03-03
影响因子：
11.1
作者：
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