Wu Ching-Shyi, Ouyang Jian, Mori Eiichiro, Nguyen Hai Dang, Maréchal Alexandre, Hallet Alexander, Chen David J, Zou Lee
Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, Massachusetts 02129, USA;
Division of Molecular Radiation Biology, Department of Radiation Oncology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA;
Genes Dev. 2014 Jul 1;28(13):1472-84. doi: 10.1101/gad.238535.114.
The ATR (ATM [ataxia telangiectasia-mutated]- and Rad3-related) checkpoint is a crucial DNA damage signaling pathway. While the ATR pathway is known to transmit DNA damage signals through the ATR-Chk1 kinase cascade, whether post-translational modifications other than phosphorylation are important for this pathway remains largely unknown. Here, we show that protein SUMOylation plays a key role in the ATR pathway. ATRIP, the regulatory partner of ATR, is modified by SUMO2/3 at K234 and K289. An ATRIP mutant lacking the SUMOylation sites fails to localize to DNA damage and support ATR activation efficiently. Surprisingly, the ATRIP SUMOylation mutant is compromised in the interaction with a protein group, rather than a single protein, in the ATR pathway. Multiple ATRIP-interacting proteins, including ATR, RPA70, TopBP1, and the MRE11-RAD50-NBS1 complex, exhibit reduced binding to the ATRIP SUMOylation mutant in cells and display affinity for SUMO2 chains in vitro, suggesting that they bind not only ATRIP but also SUMO. Fusion of a SUMO2 chain to the ATRIP SUMOylation mutant enhances its interaction with the protein group and partially suppresses its localization and functional defects, revealing that ATRIP SUMOylation promotes ATR activation by providing a unique type of protein glue that boosts multiple protein interactions along the ATR pathway.
ATR（与共济失调毛细血管扩张症突变基因ATM及Rad3相关的蛋白）检查点是一条关键的DNA损伤信号通路。虽然已知ATR通路通过ATR-Chk1激酶级联传递DNA损伤信号，但除磷酸化之外的翻译后修饰对此通路是否重要仍 largely unknown。在此，我们表明蛋白质SUMO化在ATR通路中起关键作用。ATR的调节伙伴ATRIP在K234和K289位点被SUMO2/3修饰。一个缺乏SUMO化位点的ATRIP突变体无法定位于DNA损伤处，也无法有效地支持ATR激活。令人惊讶的是，ATRIP SUMO化突变体在与ATR通路中的一组蛋白质而非单个蛋白质的相互作用中受损。多种与ATRIP相互作用的蛋白质，包括ATR、RPA70、TopBP1以及MRE11-RAD50-NBS1复合物，在细胞中与ATRIP SUMO化突变体的结合减少，并且在体外对SUMO2链表现出亲和力，这表明它们不仅与ATRIP结合，还与SUMO结合。将SUMO2链融合到ATRIP SUMO化突变体上可增强其与这组蛋白质的相互作用，并部分抑制其定位和功能缺陷，这表明ATRIP SUMO化通过提供一种独特类型的蛋白质胶水来促进ATR激活，这种胶水可增强ATR通路中多种蛋白质的相互作用。
