IDENT OF PROTEIN PHOSPHATASE 5 TARGETS IN THE DNA DAMAGE RESPONSE PATHWAY

DNA 损伤反应途径中蛋白磷酸酶 5 靶标的鉴定

• 批准号: 7721399
• 负责人: SANDRA ROSSIE
• 金额: $ 4.8万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-08 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721399
• 关键词: ATM gene; ATM wt Allele; Affinity Chromatography; Alleles; Amino Acids; Apoptosis; Ataxia Telangiectasia; Biochemical; Bioinformatics; Bleomycin; Cell Cycle Arrest; Cell Extracts; Cell Line; Cells; Computer Retrieval of Information on Scientific Projects Database; Condition; DNA Damage; DNA Repair; DNA Replication Damage; Data; Development; Disease; Dose; Embryo; Funding; Genes; Genotoxic Stress; Goals; Grant; Hela Cells; Human; Immune System Diseases; Institution; Ions; KRP protein; Label; Laboratories; Lead; Long-Term Effects; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Metals; Methods; Mus; Neurons; Okadaic Acid; PP5 protein-serine-threonine phosphatase; Pacific Northwest; Pathway interactions; Patients; Peptides; Phosphopeptides; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Predisposition; Premature aging syndrome; Process; Protein Overexpression; Proteins; Publications; Publishing; Research; Research Personnel; Resources; Role; Sampling; Source; Spectrometry, Mass, Electrospray Ionization; Stress; Testing; Trypsin; United States National Institutes of Health; ataxia telangiectasia mutated protein; base; carboxymethylation; cell growth; disorder risk; follow-up; liquid chromatography mass spectrometry; loss of function mutation; mutant; phosphatase inhibitor; prevent; research study; response; trizol

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A major goal of my laboratory is to identify the regulators and substrates of Ser/Thr protein phosphatase 5 (PP5), for which only a few have been identified to date. One important pathway in which PP5 has been implicated is the DNA damage response pathway. This pathway is essential for preventing the propagation of cells with damaged DNA, which can ultimately lead to cancer. Two non-redundant, functionally overlapping and closely related protein kinases, namely ATM (Ataxia Telangiectasia Mutated) and ATR (ATM and Rad3 related) are activated in response to various forms of genotoxic stress. Once activated, ATM and ATR kinases phosphorylate their substrate proteins which lead to the specific cellular response pathways mediating either cell cycle arrest and DNA repair, or apoptosis. Loss-of-function mutations in both alleles of the human ATM gene gives rise to an autosomal recessive disease called ataxia telangiectasia (A-T). Patients suffering from A-T disorder risk predisposition to cancer and also other neuronal and immune dysfunctions and premature aging. ATR is activated in response to a variety of DNA damage and replication stress pathways. Deletion of the ATR gene leads to embryonic lethality in mice indicating that this gene is essential during development or for viability. Recently, PP5 has been shown to be essential for the activation of both these kinases (ATM and ATR), but the role of PP5 in controlling these kinases is not known. The goal of this study is to identify the potential substrates and/or targets of PP5 during activation of the DNA damage pathway using whole cell phosphoproteomics, which requires the powerful high-throughput mass spectrometry and bioinformatics capabilities that are available at the Pacific Northwest National Laboratory. To do this we will compare the phosphoproteome of cells with different levels of PP5 activity and subjected to treatment with a DNA damaging agent. Two different strategies will be used to alter PP5 activity in cells. Specific Aims: 1. To compare and identify phosphoprotein targets in cells overexpressing wild type (WT) and catalytically inactive mutant PP5 following DNA damage. 2. To compare and identify phosphoprotein targets specific for PP5 from cells overexpressing WT or mutant PP5 (insensitive to okadaic acid) following treatment with a DNA damaging agent in the absence or presence of okadaic acid (at a dose that blocks WT but not mutant PP5). Methods for Specific Aim 1: Stable human HeLa cell lines overexpressing either wild type (WT) or catalytically inactive (CaIn) PP5 under inducible conditions will be used for this study. Cells induced to overexpress PP5 will be treated with or without bleomycin to activate the DNA damage response pathway. Protein extracts will be prepared using the Trizol method and digested with trypsin. Peptides will be carboxymethylated and phosphopeptides will be isolated by immobilized metal ion affinity chromatography (IMAC), and then subjected to electrospray ionization and mass spectrometry. Differential isotopic labeling, either at the level of amino acid incorporation during cell growth (SILAC) or during carboxymethylation will be used to distinguish peptides arising from the samples to be compared. Once samples have been differentially labeled, peptides can be mixed prior to IMAC and LC/MS analysis. We predict that phosphopeptides from proteins targeted by WT PP5 during activation of the DNA damage pathway will be lower in abundance than their counterparts from cells expressing CaIn PP5. In addition to PP5 substrates, we expect to identify proteins whose phosphorylation status is indirectly altered as a function of PP5 activity during activation of this pathway. Based on the mass spectrometry data, further biochemical characterization of putative targets will be performed. If successful with confirmatory results from biochemical characterizations, these data will be published. Methods for Specific Aim 2: Stable human HeLa cell lines overexpressing either WT PP5 or okadaic acid (OA) insensitive mutant PP5 [OA-insensitive] under inducible conditions will be used for this study. OA is a Ser/Thr phosphatase inhibitor. Cells induced to overexpress PP5 will be treated with or without OA. Following OA treatment, cells will be treated with or without DNA damaging agent bleomycin. OA treatment will inhibit WT PP5 and all other related Ser/Thr phosphatases in cells overexpressing WT PP5. However, in the cells overexpressing the OA-insensitive mutant PP5, the same Ser/Thr phosphatases, except for mutant PP5, will be inhibited and thus any changes in phosphoproteins in mutant PP5 overexpressing cells compared to the WT PP5 overexpressing cells can be correlated to PP5 activity. After OA treatment followed by DNA damage, whole cell extracts will be prepared and processed using mass spectrometry as mentioned in Specific Aim # 1. In contrast to Specific Aim 1 in which long term effects of altering PP5 function may be observed, this experiment can better document the rapid changes in phosphorylation mediated by PP5, because the difference in PP5 function between samples lasts only for the duration of the experiment. Follow-up experiments testing the relationship between PP5 and altered phosphoproteins (identified in this study) are expected to lead to additional publications.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 我实验室的一个主要目标是确定 Ser/Thr 蛋白磷酸酶 5 (PP5) 的调节因子和底物，迄今为止仅确定了其中的少数几个。 PP5 涉及的一条重要途径是 DNA 损伤反应途径。该途径对于防止 DNA 受损细胞的增殖至关重要，而 DNA 受损细胞最终可能导致癌症。两种非冗余、功能重叠且密切相关的蛋白激酶，即 ATM（共济失调毛细血管扩张突变）和 ATR（ATM 和 Rad3 相关）在响应各种形式的基因毒性应激时被激活。一旦激活，ATM 和 ATR 激酶就会磷酸化其底物蛋白，从而导致介导细胞周期停滞和 DNA 修复或细胞凋亡的特定细胞反应途径。人类 ATM 基因的两个等位基因的功能丧失突变会导致一种常染色体隐性遗传疾病，称为共济失调性毛细血管扩张症 (A-T)。患有 A-T 障碍的患者有患癌症、其他神经元和免疫功能障碍以及过早衰老的风险。 ATR 因多种 DNA 损伤和复制应激途径而被激活。 ATR 基因的缺失会导致小鼠胚胎死亡，表明该基因在发育或生存过程中至关重要。最近，PP5 已被证明对于这两种激酶（ATM 和 ATR）的激活至关重要，但 PP5 在控制这些激酶中的作用尚不清楚。 本研究的目标是利用全细胞磷酸蛋白质组学来识别 DNA 损伤途径激活过程中 PP5 的潜在底物和/或靶标，这需要太平洋西北国家实验室拥有强大的高通量质谱和生物信息学能力。 为此，我们将比较具有不同 PP5 活性水平并接受 DNA 损伤剂处理的细胞的磷酸蛋白质组。 将使用两种不同的策略来改变细胞中的 PP5 活性。 具体目标： 1. 比较和鉴定 DNA 损伤后过表达野生型 (WT) 和催化失活突变体 PP5 的细胞中的磷蛋白靶标。 2. 在不存在或存在大田酸的情况下（以阻断 WT 但不阻断突变型 PP5 的剂量）用 DNA 损伤剂处理后，比较和鉴定来自过表达 WT 或突变型 PP5（对大田酸不敏感）的细胞的 PP5 特异性磷蛋白靶点）。 具体目标 1 的方法： 本研究将使用在诱导条件下过表达野生型 (WT) 或催化失活 (CaIn) PP5 的稳定人类 HeLa 细胞系。 诱导过度表达 PP5 的细胞将用或不用博来霉素处理以激活 DNA 损伤反应途径。 蛋白质提取物将使用 Trizol 方法制备并用胰蛋白酶消化。肽将被羧甲基化，磷酸肽将通过固定化金属离子亲和层析（IMAC）分离，然后进行电喷雾电离和质谱分析。细胞生长期间氨基酸掺入水平 (SILAC) 或羧甲基化期间的差异同位素标记将用于区分待比较样品中产生的肽。一旦样品被差异化标记，就可以在 IMAC 和 LC/MS 分析之前混合肽。我们预测，在 DNA 损伤途径激活过程中，来自 WT PP5 靶向蛋白质的磷酸肽的丰度将低于来自表达 CaIn PP5 的细胞的对应磷酸肽。 除了 PP5 底物之外，我们还期望鉴定出在该途径激活期间其磷酸化状态作为 PP5 活性的函数而间接改变的蛋白质。根据质谱数据，将对假定目标进行进一步的生化表征。如果生化特征的验证结果成功，这些数据将被公布。 具体目标 2 的方法： 本研究将使用在诱导条件下过表达 WT PP5 或冈田酸 (OA) 不敏感突变体 PP5 [OA 不敏感] 的稳定人 HeLa 细胞系。 OA 是一种 Ser/Thr 磷酸酶抑制剂。 诱导过度表达PP5的细胞将用或不用OA处理。 OA 处理后，细胞将接受或不接受 DNA 损伤剂博莱霉素处理。 OA 治疗将抑制 WT PP5 和过表达 WT PP5 的细胞中所有其他相关的 Ser/Thr 磷酸酶。然而，在过表达 OA 不敏感突变体 PP5 的细胞中，除了突变体 PP5 之外，相同的 Ser/Thr 磷酸酶将受到抑制，因此与 WT PP5 过表达细胞相比，突变体 PP5 过表达细胞中磷蛋白的任何变化都可以与PP5活性。 OA 处理后进行 DNA 损伤，将使用质谱法制备和处理全细胞提取物，如特定目标 # 1 中所述。与特定目标 1 相比，其中可以观察到改变 PP5 功能的长期影响，该实验可以更好记录了 PP5 介导的磷酸化的快速变化，因为样品之间 PP5 功能的差异仅在实验期间持续。 测试 PP5 和改变的磷蛋白（在本研究中确定）之间关系的后续实验预计将导致更多出版物的发表。