Quantitative phosphoproteomics of the ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia-mutated and rad3-related (ATR) dependent DNA damage response in Arabidopsis thaliana.
Quantitative phosphoproteomics of the ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia-mutated and rad3-related (ATR) dependent DNA damage response in Arabidopsis thaliana.
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DOI：
10.1074/mcp.m114.040352
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发表时间：
2015-03
期刊：
影响因子：
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通讯作者：
Mechtler K
中科院分区：
文献类型：
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作者：
Roitinger E;Hofer M;Köcher T;Pichler P;Novatchkova M;Yang J;Schlögelhofer P;Mechtler K
蛋白质丝氨酸、苏氨酸和酪氨酸残基上的可逆磷酸化是一种重要的生物调节机制。在基因组完整性的背景下，磷酸化驱动的信号级联对于 DNA 修复的协调和调节至关重要。两种丝氨酸/苏氨酸蛋白激酶共济失调毛细血管扩张突变 (ATM) 和共济失调毛细血管扩张突变和 Rad3 相关 (ATR) 是这一过程中的关键因素，每种激酶都针对不同类型的 DNA 损伤。它们在真核生物中是保守的，介导细胞周期检查点的激活、染色质修饰和 DNA 修复蛋白的调节。我们设计了一种基于质谱的新型磷酸蛋白质组学方法来研究拟南芥 DNA 损伤修复。该方案结合了过滤辅助样品制备、固定金属亲和色谱、金属氧化物亲和色谱和强阳离子交换色谱，用于磷酸肽的生成、富集和分离。使用 iTRAQ（用于相对和绝对定量的同量异位标签）进行同量异位标记，用于分析 atm atr 双突变体和野生型植物在常规生长条件或受到辐射挑战下的磷酸蛋白质组。总共鉴定了 10,831 种蛋白质，并对 15,445 种独特的磷酸肽进行了定量，其中包含 134 种上调和 38 种下调的 ATM/ATR 依赖性磷酸肽。我们鉴定了已知和新的 ATM/ATR 靶标，例如 LIG4 和 MRE11（抵抗电离辐射所需）、PIE1 和 SDG26（与染色质重塑有关）、PCNA1、WAPL 和 PDS5（与 DNA 复制有关）以及 ASK1 和 HTA10（与减数分裂有关）。
The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is an important biological regulatory mechanism. In the context of genome integrity, signaling cascades driven by phosphorylation are crucial for the coordination and regulation of DNA repair. The two serine/threonine protein kinases ataxia telangiectasia-mutated (ATM) and Ataxia telangiectasia-mutated and Rad3-related (ATR) are key factors in this process, each specific for different kinds of DNA lesions. They are conserved across eukaryotes, mediating the activation of cell-cycle checkpoints, chromatin modifications, and regulation of DNA repair proteins. We designed a novel mass spectrometry-based phosphoproteomics approach to study DNA damage repair in Arabidopsis thaliana. The protocol combines filter aided sample preparation, immobilized metal affinity chromatography, metal oxide affinity chromatography, and strong cation exchange chromatography for phosphopeptide generation, enrichment, and separation. Isobaric labeling employing iTRAQ (isobaric tags for relative and absolute quantitation) was used for profiling the phosphoproteome of atm atr double mutants and wild type plants under either regular growth conditions or challenged by irradiation. A total of 10,831 proteins were identified and 15,445 unique phosphopeptides were quantified, containing 134 up- and 38 down-regulated ATM/ATR dependent phosphopeptides. We identified known and novel ATM/ATR targets such as LIG4 and MRE11 (needed for resistance against ionizing radiation), PIE1 and SDG26 (implicated in chromatin remodeling), PCNA1, WAPL, and PDS5 (implicated in DNA replication), and ASK1 and HTA10 (involved in meiosis).