Claspin operates downstream of TopBP1 to direct ATR signaling towards Chk1 activation
Claspin operates downstream of TopBP1 to direct ATR signaling towards Chk1 activation
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DOI：
10.1128/mcb.00492-06
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发表时间：
2006-08-01
影响因子：
5.3
通讯作者：
Lukas, Jiri
中科院分区：
文献类型：
--
作者：
Liu, Shizhou;Bekker-Jensen, Simon;Lukas, Jiri
TopBP1和Claspin是ATR激酶在基因毒性胁迫下促进Chk1磷酸化的衔接蛋白。尽管已确定它们需要Chk1激活，但TopBP1和Claspin控制Chk1磷酸化的确切方式尚不清楚。我们发现，在DNA损伤产生的不同核亚室中，TopBP1与ATR紧密共定位。尽管通过RNA干扰（RNAi）耗尽TopBP1会严重损害多个ATR靶点的磷酸化，包括Chk1、Nbs1、Smc1和H2AX，但它不会干扰DNA损伤位点的ATR组装。这些发现挑战了目前ATR通过招募受损DNA激活的概念。相比之下，Claspin和Chk1一样，在DNA损伤前后仍然分布在整个细胞核中。与此一致的是，rnai介导的Claspin消融选择性地破坏了ATR磷酸化Chk1的能力，但没有破坏其他ATR靶点。此外，Claspin的下调通过诱导自发性DNA损伤模拟Chk1失活。最后，我们发现TopBP1是DNA损伤诱导的Claspin和Chk1相互作用所必需的。总之，这些结果表明，虽然TopBP1是ATR的一般调节剂，但Claspin在TopBP1的下游有选择性地调节chk1控制的基因毒性应激反应分支。
TopBP1 and Claspin are adaptor proteins that facilitate phosphorylation of Chk1 by the ATR kinase in response to genotoxic stress. Despite their established requirement for Chk1 activation, the exact way in which TopBP1 and Claspin control Chk1 phosphorylation remains unclear. We show that TopBP1 tightly colocalizes with ATR in distinct nuclear subcompartments generated by DNA damage. Although depletion of TopBP1 by RNA interference (RNAi) strongly impaired phosphorylation of multiple ATR targets, including Chk1, Nbs1, Smc1, and H2AX, it did not interfere with ATR assembly at the sites of DNA damage. These findings challenge the current concept of ATR activation by recruitment to damaged DNA. In contrast, Claspin, like Chk1, remained distributed throughout the nucleus both before and after DNA damage. Consistently, the RNAi-mediated ablation of Claspin selectively abrogated ATR's ability to phosphorylate Chk1 but not other ATR targets. In addition, downregulation of Claspin mimicked Chk1 inactivation by inducing spontaneous DNA damage. Finally, we show that TopBP1 is required for the DNA damage-induced interaction between Claspin and Chk1. Together, these results suggest that while TopBP1 is a general regulator of ATR, Claspin operates downstream of TopBP1 to selectively regulate the Chk1-controlled branch of the genotoxic stress response.