Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.
Cell Cycle. 2013 May 15;12(10):1560-8. doi: 10.4161/cc.24675. Epub 2013 Apr 17.
Claspin is a critical mediator protein in the DNA replication checkpoint, responsible for ATR-dependent activation of the effector kinase Chk1. Cdc7, an essential kinase required for the initiation of DNA replication, can also interact with and phosphorylate Claspin. In this study we use small-molecule inhibitors of Cdc7 kinase to further understand the relationship between Cdc7, Claspin and Chk1 activation. We demonstrate that inhibition of Cdc7 kinase delays HU-induced phosphorylation of Chk1 but does not affect the maintenance of the replication checkpoint once it is established. We find that while chromatin association of Claspin is not affected by Cdc7 inhibition, Claspin phosphorylation is attenuated following HU treatment, which may be responsible for the altered kinetics of HU-induced Chk1 phosphorylation. We demonstrate that Claspin is an in vitro substrate of Cdc7 kinase, and using mass-spectrometry, we identify multiple phosphorylation sites that help to define a Cdc7 phosphorylation motif. Finally, we show that the interaction between Claspin and Cdc7 is not dependent on Cdc7 kinase activity, but Claspin interaction with the DNA helicase subunit Mcm2 is lost upon Cdc7 inhibition. We propose Cdc7-dependent phosphorylation regulates critical protein-protein interactions and modulates Claspin's function in the DNA replication checkpoint.
Claspin 是 DNA 复制检查点的关键调节蛋白，负责 ATR 依赖性激活效应激酶 Chk1。CDC7 是起始 DNA 复制所必需的激酶，也可以与 Claspin 相互作用并磷酸化它。在这项研究中，我们使用 CDC7 激酶的小分子抑制剂来进一步了解 CDC7、Claspin 和 Chk1 激活之间的关系。我们证明，CDC7 激酶的抑制会延迟 HU 诱导的 Chk1 磷酸化，但不会影响复制检查点的维持，一旦它建立起来。我们发现，虽然 Claspin 与染色质的关联不受 CDC7 抑制的影响，但在 HU 处理后，Claspin 的磷酸化被减弱，这可能是 HU 诱导的 Chk1 磷酸化动力学改变的原因。我们证明 Claspin 是 CDC7 激酶的体外底物，并通过质谱分析，确定了多个有助于定义 CDC7 磷酸化模体的磷酸化位点。最后，我们表明 Claspin 与 CDC7 的相互作用不依赖于 CDC7 激酶活性，但 CDC7 抑制后，Claspin 与 DNA 解旋酶亚基 Mcm2 的相互作用丧失。我们提出 CDC7 依赖性磷酸化调节关键的蛋白质-蛋白质相互作用，并调节 Claspin 在 DNA 复制检查点中的功能。
