Site-specific phosphorylation of the DNA damage response mediator rad9 by cyclin-dependent kinases regulates activation of checkpoint kinase 1.
Site-specific phosphorylation of the DNA damage response mediator rad9 by cyclin-dependent kinases regulates activation of checkpoint kinase 1.
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DOI：
10.1371/journal.pgen.1003310
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发表时间：
2013-04
期刊：
影响因子：
4.5
通讯作者：
Lowndes NF
中科院分区：
文献类型：
--
作者：
Abreu CM;Kumar R;Hamilton D;Dawdy AW;Creavin K;Eivers S;Finn K;Balsbaugh JL;O'Connor R;Kiely PA;Shabanowitz J;Hunt DF;Grenon M;Lowndes NF
DNA损伤反应(DDR)的介体被控制细胞增殖的激酶高度磷酸化，但人们对这一调控的作用知之甚少。在这里，我们证明了典型的DDR介导物酿酒酵母Rad9的细胞周期磷酸化依赖于细胞周期蛋白依赖性激酶(CDK)复合体。我们发现，一种特定的G2/M形式的CDC28在体外可以在9个共同的CDK磷酸化位点上磷酸化Rad9的N-末端区域。我们发现，CDK共识位点的完整性和CDC28的活性对于Chk1检查点激酶的激活及其与Rad9的相互作用都是必需的。我们已经确定T125和T143是Rad9中这种Rad9/Chk1相互作用的重要残基。T143的磷酸化是促进Rad9/Chk1相互作用的最重要特征，而邻近T125残基的更丰富的磷酸化阻碍了Rad9/Chk1的相互作用。我们提出了一种新的Chk1激活模型，其中CDC28调节Rad9和Chk1的本构相互作用。然后，在DNA损伤部位招募Rad9/Chk1复合体，其中Chk1的激活需要额外的DDR特异性蛋白激酶。人类细胞激活DNA损伤反应(DDR)来修复DNA损伤，并防止DNA损伤的细胞增殖。DDR的改变与癌症的发生密切相关。利用发芽酵母模型系统，我们研究了关键的DDR组分Rad9的调控如何整合到细胞周期控制中。调控酵母细胞周期的细胞周期蛋白依赖性激酶CDC28在细胞周期进程中也广泛地磷酸化Rad9。我们发现，在重要的下游DNA损伤效应蛋白激酶Chk1的激活过程中，CDC28控制着Rad9的功能。在CDc28调控的Rad9和Chk1之间的物理相互作用中，Rad9 N端的两个磷酸化位点是至关重要的。我们提出了一个新的Chk1激活模型，在该模型中，Rad9和Chk1的子集在没有DNA损伤的情况下进行结构性相互作用。Rad9/Chk1复合体被招募到DNA损伤部位，其中Chk1的激活涉及额外的DDR特异性蛋白激酶。人类细胞含有多个Rad9样蛋白，在没有外源DNA损伤的情况下，这些蛋白也被认为是细胞周期磷酸化的，这表明我们的观察可能对人类细胞的DDR调控具有重要意义。
The mediators of the DNA damage response (DDR) are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK) complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR–specific protein kinases. Human cells activate the DNA damage response (DDR) to repair DNA damage and to prevent cells with DNA damage from proliferating. Alterations to the DDR are strongly implicated in the development of cancer. Using the budding yeast model system, we have studied how the regulation of the key DDR component Rad9 is integrated into cell cycle control. The cyclin-dependent kinase Cdc28 that regulates the yeast cell cycle also extensively phosphorylates Rad9 during cell cycle progression. We show here that Cdc28 controls Rad9 function in the activation of the important downstream DNA damage effector kinase Chk1. Two sites of phosphorylation in the N-terminus of Rad9 are crucial for the physical interaction between Rad9 and Chk1 regulated by Cdc28. We propose a novel model for Chk1 activation whereby a subset of Rad9 and Chk1 interacts constitutively in the absence of DNA damage. The Rad9/Chk1 complex is recruited to sites of DNA damage where activation of Chk1 involves additional DDR–specific protein kinases. Human cells contain multiple Rad9-like proteins that are also known to be cell cycle phosphorylated in the absence of exogenous DNA damage, suggesting that our observations may have important implications for DDR regulation in human cells.
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影响因子：
2.3
作者：
Enserink JM;Kolodner RD
通讯作者：
Kolodner RD
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16
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Lowndes, NF
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