Mordes Daniel A, Glick Gloria G, Zhao Runxiang, Cortez David
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Genes Dev. 2008 Jun 1;22(11):1478-89. doi: 10.1101/gad.1666208.
The ATR (ATM and Rad3-related) kinase and its regulatory partner ATRIP (ATR-interacting protein) coordinate checkpoint responses to DNA damage and replication stress. TopBP1 functions as a general activator of ATR. However, the mechanism by which TopBP1 activates ATR is unknown. Here, we show that ATRIP contains a TopBP1-interacting region that is necessary for the association of TopBP1 and ATR, for TopBP1-mediated activation of ATR, and for cells to survive and recover DNA synthesis following replication stress. We demonstrate that this region is functionally conserved in the Saccharomyces cerevisiae ATRIP ortholog Ddc2, suggesting a conserved mechanism of regulation. In addition, we identify a domain of ATR that is critical for its activation by TopBP1. Mutations of the ATR PRD (PIKK [phosphoinositide 3-kinase related kinase] Regulatory Domain) do not affect the basal kinase activity of ATR but prevent its activation. Cellular complementation experiments demonstrate that TopBP1-mediated ATR activation is required for checkpoint signaling and cellular viability. The PRDs of ATM and mTOR (mammalian target of rapamycin) were shown previously to regulate the activities of these kinases, and our data indicate that the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) PRD is important for DNA-PKcs regulation. Therefore, divergent amino acid sequences within the PRD and a unique protein partner allow each of these PIK kinases to respond to distinct cellular events.
ATR（ATM与Rad3相关）激酶及其调节伴侣ATRIP（ATR相互作用蛋白）协调对DNA损伤和复制应激的检查点反应。TopBP1作为ATR的一般激活剂发挥作用。然而，TopBP1激活ATR的机制尚不清楚。在此，我们表明ATRIP包含一个TopBP1相互作用区域，该区域对于TopBP1与ATR的结合、TopBP1介导的ATR激活以及细胞在复制应激后存活和恢复DNA合成是必需的。我们证明该区域在酿酒酵母ATRIP直系同源物Ddc2中功能保守，提示存在保守的调节机制。此外，我们鉴定出ATR的一个对其被TopBP1激活至关重要的结构域。ATR PRD（PIKK[磷酸肌醇3激酶相关激酶]调节结构域）的突变不影响ATR的基础激酶活性，但会阻止其激活。细胞互补实验表明，检查点信号传导和细胞活力需要TopBP1介导的ATR激活。先前已表明ATM和mTOR（雷帕霉素的哺乳动物靶标）的PRD调节这些激酶的活性，我们的数据表明DNA-PKcs（DNA依赖性蛋白激酶催化亚基）的PRD对DNA-PKcs的调节很重要。因此，PRD内不同的氨基酸序列和独特的蛋白质伴侣使这些PIK激酶中的每一种都能对不同的细胞事件做出反应。
