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首页> 外文期刊>Molecular & cellular proteomics: MCP >Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen
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Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen

机译：活性氧（ROS）激活的ATM依赖的大规模磷酸化蛋白质组学筛选的细胞质基质的磷酸化。

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摘要

Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM-dependence for translocation from the cytoplasm to the nucleus. These data provide new insights into the activation of ATM by oxidative stress through identification of novel substrates for ATM in the cytoplasm.
机译：共济失调-毛细血管扩张，突变（ATM）蛋白在磷酸化蛋白网络中起着核心作用，以响应DNA损伤。这些蛋白质通过控制细胞周期检查点，启动DNA修复和调节基因表达，在旨在维持基因组稳定性并最小化疾病风险的信号传导途径中起作用。 ATM激酶可以被多种刺激激活，包括氧化应激。在这里，我们证实了通过多个部位的自磷酸化激活了细胞质ATM。然后，我们采用了一种全球定量的磷酸化蛋白质组学方法，来确定在质控和共济失调毛细血管扩张（A-T）细胞中，由于氧化损伤而磷酸化状态改变的胞质蛋白。我们证明了ATM被细胞质和细胞核中的氧化损伤所激活，并鉴定出总共9,833个磷酸化位点，其中包括6,686个高可信位点，它们映射到2,536个独特蛋白质。总共鉴定出62种差异磷酸化的肽。其中，有43个在对照中被磷酸化，但在A-T细胞中没有，而19个在磷酸化水平上有所不同。磷酸肽的基序富集分析表明，共有的ATM丝氨酸谷氨酰胺位点被过度代表。当考虑磷酸化事件时，仅在对照细胞中观察到（在A-T细胞中未观察到），且预测的ATM位点为磷酸丝氨酸/磷酸苏氨酸谷氨酰胺，我们将该列表缩小为11种候选ATM依赖性细胞质蛋白。在这11种物质中，有两种以前被描述为ATM底物（HMGA1和UIMCI / RAP80），在全细胞提取物磷酸蛋白质组学筛选中鉴定出另外五种，而之前在DNA损伤反应筛选中尚未鉴定出其余四种蛋白质。我们验证了H2O2暴露后，这三种蛋白质（氧化应激反应1（OSR1），HDGF和ccdc82）的磷酸化为ATM依赖性，而另一种蛋白质（S100A11）则证明了ATM从细胞质向细胞核的依赖性。这些数据通过鉴定细胞质中ATM的新底物，为氧化应激激活ATM提供了新的见解。

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