Phosphorylation of Xenopus Rad1 and Hus1 defines a readout for ATR activation that is independent of claspin and the Rad9 carboxy terminus
Phosphorylation of Xenopus Rad1 and Hus1 defines a readout for ATR activation that is independent of claspin and the Rad9 carboxy terminus
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DOI：
10.1091/mbc.e05-09-0865
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发表时间：
2006-04-01
影响因子：
3.3
通讯作者：
Cimprich, KA
中科院分区：
文献类型：
--
作者：
Lupardus, PJ;Cimprich, KA
DNA损伤检查点通路感知并响应DNA损伤以确保基因组的稳定性。ATR激酶是其中一个通路的中心调节因子，并磷酸化许多在细胞周期进程和DNA修复中起作用的蛋白质。利用爪蟾卵提取液系统，研究了Rad1/Hus1/ Rad9复合物的调控作用。我们发现Rad1和Hus1的磷酸化以ATR-和topbp1依赖的方式发生在Rad1的T5上。Hus1的S219和T223。这些位点的突变对ATR对Chk1的磷酸化没有影响。有趣的是，Rad1的磷酸化独立于Claspin和Rad9羧基端，两者都是Chk1磷酸化所必需的。这些数据表明，活跃的ATR信号复合物存在于缺乏Rad9的羧基末端的情况下，并且该羧基末端结构域可能是Chk1磷酸化的特定要求，而不是所有ATR介导的信号事件所必需的。因此，Rad1磷酸化为ATR激活的研究提供了一个替代的早期读数。
The DNA damage checkpoint pathways sense and respond to DNA damage to ensure genomic stability. The ATR kinase is a central regulator of one such pathway and phosphorylates a number of proteins that have roles in cell cycle progression and DNA repair. Using the Xenopus egg extract system, we have investigated regulation of the Rad1/Hus1/ Rad9 complex. We show here that phosphorylation of Rad1 and Hus1 occurs in an ATR- and TopBP1-dependent manner on T5 of Rad1. and S219 and T223 of Hus1. Mutation of these sites has no effect on the phosphorylation of Chk1 by ATR. Interestingly, phosphorylation of Rad1 is independent of Claspin and the Rad9 carboxy terminus, both of which are required for Chk1 phosphorylation. These data suggest that an active ATR signaling complex exists in the absence of the carboxy terminus of Rad9 and that this carboxy-terminal domain may be a specific requirement for Chk1 phosphorylation and not necessary for all ATR-mediated signaling events. Thus, Rad1 phosphorylation provides an alternate and early readout for the study of ATR activation.