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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我的实验室的一个主要目标是确定丝氨酸/苏氨酸蛋白磷酸酶5（PP 5）的调节剂和底物，迄今为止只有少数几个已经确定。PP 5参与的一个重要途径是DNA损伤反应途径。这种途径对于防止DNA受损的细胞繁殖至关重要，而DNA受损的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Rad 3相关）在响应各种形式的遗传毒性应激时被激活。一旦被激活，ATM和ATR激酶磷酸化其底物蛋白，这导致介导细胞周期停滞和DNA修复或凋亡的特异性细胞反应途径。人类ATM基因的两个等位基因的功能丧失突变引起称为共济失调毛细血管扩张症（A-T）的常染色体隐性遗传疾病。患有A-T障碍的患者有患癌症以及其他神经元和免疫功能障碍和过早衰老的风险。ATR响应于多种DNA损伤和复制应激途径而被激活。ATR基因的缺失导致小鼠胚胎死亡，表明该基因在发育过程中或对于生存力是必需的。最近，PP 5已被证明是这两种激酶（ATM和ATR）的激活所必需的，但PP 5在控制这些激酶中的作用尚不清楚。 本研究的目的是使用全细胞磷酸蛋白质组学在DNA损伤途径激活过程中识别PP 5的潜在底物和/或靶点，这需要太平洋西北国家实验室提供的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水平的PP 5活性的细胞的磷酸化蛋白质组，并进行DNA损伤剂的处理。 将使用两种不同的策略来改变细胞中的PP 5活性。 具体目标： 1. 比较和鉴定DNA损伤后过表达野生型（WT）和无催化活性突变型PP 5的细胞中的磷蛋白靶点。 2. 比较和鉴定在不存在或存在冈田酸（以阻断WT但不阻断突变型PP 5的剂量）的情况下用DNA损伤剂处理后，过表达WT或突变型PP 5（对冈田酸不敏感）的细胞中PP 5特异性磷蛋白靶标。 具体目标1的方法： 在诱导条件下过表达野生型（WT）或无催化活性（CaIn）PP 5的稳定人HeLa细胞系将用于本研究。 诱导过表达PP 5的细胞将用或不用博来霉素处理以激活DNA损伤反应途径。 将使用Trizol方法制备蛋白质提取物，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色谱法（IMAC）分离，然后进行电喷雾电离和质谱分析。在细胞生长期间（SILAC）或羧甲基化期间的氨基酸掺入水平上的差异同位素标记将用于区分来自待比较样品的肽。一旦样品被差异标记，可以在IMAC和LC/MS分析之前混合肽。我们预测，在DNA损伤途径的激活过程中，WT PP 5靶向的蛋白质的磷酸肽的丰度将低于表达CaIn PP 5的细胞的对应物。 除了PP 5底物，我们希望确定蛋白质的磷酸化状态是间接改变的PP 5活性的功能，在激活这一途径。根据质谱数据，将对推定靶点进行进一步生化表征。如果成功获得生化表征的确证性结果，则将发表这些数据。 具体目标2的方法： 本研究将使用在诱导条件下过表达WT PP 5或冈田酸（OA）不敏感突变体PP 5 [OA不敏感]的稳定人HeLa细胞系。OA是一种Ser/Thr磷酸酶抑制剂。 诱导过表达PP 5的细胞将用或不用OA处理。在OA处理后，将用或不用DNA损伤剂博来霉素处理细胞。OA处理将抑制WT PP 5和过表达WT PP 5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PP 5的细胞中，除了突变体PP 5之外，相同的Ser/Thr磷酸酶将被抑制，因此与WT PP 5过表达细胞相比，突变体PP 5过表达细胞中磷蛋白的任何变化都可以与PP 5活性相关。 在OA处理后进行DNA损伤，将制备全细胞提取物，并使用质谱法进行处理，如具体目标#1所述。 与可以观察到改变PP 5功能的长期作用的特异性目标1相比，该实验可以更好地记录由PP 5介导的磷酸化的快速变化，因为样品之间PP 5功能的差异仅持续实验的持续时间。 后续实验测试PP 5和改变磷蛋白（在本研究中确定）之间的关系，预计将导致更多的出版物。