Regulation and Function of WIP1 Phosphatase and its Role in Tumor Cells

WIP1磷酸酶的调控、功能及其在肿瘤细胞中的作用

• 批准号: 8763392
• 负责人: ETTORE APPELLA
• 金额: $ 59.18万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8763392
• 关键词: 5' Untranslated Regions; Active Sites; Affect; Affinity; Alleles; Amino Acids; Binding; Biochemical; Biological Assay; C-terminal; Calorimetry; Cancer Patient; Catalytic Domain; Cell Aging; Cells; Chemicals; Cyclin-Dependent Kinase Inhibitor; Cytoplasm; DNA Damage; Databases; Development; Embryo; Environment; Enzymes; Exhibits; Exons; Exposure to; Fibroblasts; Genes; Genetic Transcription; Homeostasis; Human; Immune system; In Vitro; Ions; Knockout Mice; Laboratories; Location; Maintenance; Malignant Neoplasms; Measures; Messenger RNA; Metal Binding Site; Metal Ion Binding; Metals; Methods; Missense Mutation; Molecular Models; Molecular Weight; Mus; Mutation; Nature; Organ; Oxygen; PPM1D gene; Phosphoproteins; Phosphoric Monoester Hydrolases; Population; Premature aging syndrome; Process; Production; Protein Serine/Threonine Phosphatase; Protein p53; Proteins; Proteomics; Publishing; Radio; Regulation; Response Elements; Role; S Phase; Serine; Signal Transduction; Single Nucleotide Polymorphism; Site; Site-Directed Mutagenesis; Somatic Mutation; Source; Specific qualifier value; Stress; Structure; Substrate Specificity; TP53 gene; Threonine; Time; Tissues; Transcriptional Activation; Translating; Tumor Cell Line; Tumor Suppressor Proteins; Work; base; biological adaptation to stress; cancer therapy; cell type; design; divalent metal; gain of function; in vivo; inhibitor/antagonist; interest; loss of function mutation; magnesium ion; molecular modeling; mutant; neoplastic cell; novel; overexpression; premature; pressure; prevent; promoter; protein phosphatase 2C; recombinase; response; screening; senescence; tumor; tumor metabolism; tumorigenesis

Wip1, the product of the PPM1D gene, is a PP2C serine/threonine protein phosphatase that was first identified in my laboratory as a gene whose induction following DNA damage required wild-type p53. The gene PPM1D is amplified and/or overexpressed in several types of human cancers. Our results suggest that Wip1 phosphatase promotes tumorigenesis through inactivation of p53. As an enzyme, Wip1 has the potential to be inactivated by low molecular weight chemical compounds. We have identified specific Wip1 inhibitors through combined use of rational design and screening assays and are pursuing various optimization strategies. To better understand the connection between Wip1 activity and tumorigenesis, we are investigating the regulation of Wip1 expression and activity, identifying targets of Wip1 phosphatase activity, and developing inhibitors of Wip1 phosphatase activity. Regulation of Wip1 expression and activity. The transcriptional induction of Wip1 following exposure to DNA damaging agents requires functional p53 protein. Previously, we characterized a functional p53 response element in the Wip1 promoter that is located downstream of the usual start of transcription. Following the activation of DNA damage response (DDR) signaling, p53-dependent transcriptional activation of the Wip1 promoter results in the production of mRNAs with shorter 5' untranslated regions that may be more efficiently translated. The increased levels of Wip1 negatively regulate p53 activity through suppression of DDR signaling, enabling the return to cellular homeostasis. Following exposure to IR, the Wip1 protein levels increase more dramatically than its mRNA levels, resulting from both increased translational efficiency and reduced degradation. In our current work, we have established that in Jurkat and U2OS cells, two disparate human tumor cell lines, the level of Wip1 protein increased about 4-fold during S phase without appreciable change in the level of Wip1 mRNA. The S phase-specific increase in Wip1 protein levels resulted, at least partially, from increased protein stability. Recent published work has established that in some cancer patients, somatic mutations result in prematurely terminated Wip1 proteins that are enzymatically active, but lack a portion of the terminal protein-encoding exon. Interestingly, these truncated forms of Wip1 show increased stability; cells expressing the truncated forms of Wip1 accumulate high levels of the phosphatase and exhibit severely compromised activation of p53 (Ruark et al. 1013, Nature 493:06-410). To investigate the relationship between Wip1 mutations and activity, we interrogated public databases to identify missense mutations of Wip1. We identified four single nucleotide polymorphisms present in the human population that resulted in amino acid changes and characterized the effects of these changes on the activity of Wip1. For two of the mutations, L120F and P322Q, the altered amino acids are located within the structured portion of the catalytic domain and the mutant proteins exhibited drastically reduced catalytic activity. The location of a third mutation, S82A, is in an unstructured loop far from the active site and did not affect phosphatase activity. The fourth mutation, I496V, is located in the poorly characterized C-terminal domain of Wip1 and increased the amount of the protein localized to the cytoplasm. To investigate the relationship between Wip1 mutations and primary human cancers, we identified 41 mutation sites in Wip1 among cancer cases. Two thirds of the cases were located in the C-terminal domain and about one fourth were located in the phosphatase domain. In general, cases with loss of function mutations of Wip1 were likely to harbor mutations of p53, whereas mutations resulting in gain of function for Wip1, often through increased protein stability, were likely to contain no mutations in the p53 gene. These results indicate that the stability of Wip1 is important in its contribution to tumorigenesis. Under conventional culture conditions, Wip1-/- mouse embryonic fibroblasts (MEFs) undergo premature senescence. We have investigated the mechanism by which Wip1 reduces premature senescence in MEFs. We found that reduced oxygen pressure only partially suppressed premature senescence. Compared with wild type cells, early passage Wip1-/- MEFs under both 20% and 3% oxygen conditions exhibited increased activation of p53 and increased levels of cyclin-dependent kinase inhibitors. These findings suggest that Wip1 prevents cellular senescence by regulating DDR signaling resulting from endogenous sources of DNA damage. The premature aging exhibited by Wip1 knockout mice suggests that the ability of Wip1 to forestall senescence is also important in maintaining tissue maintenance capabilities in vivo. Current results characterizing the effects of Wip1 deletion in mice are based on a non-conditional knockout mouse. Despite the utility of this mouse, it does not allow us to determine the specific effects of Wip1 deletion in a single tissue. Ubiquitous Wip1 deletion in mice affects the immune system, organismal metabolism and the tumor micro-environment, any of which may affect tumorigenesis in the organ of interest. To overcome these limitations, we have developed a conditional knock-out mouse in which Wip1 deletion can be directed to a single tissue through tissue-specific expression of Cre recombinase or through inducible expression of Cre recombinase to induce deletion at a specified time. We are in the process of crossing these conditional knock-out mice with mice bearing an allele for the tissue specific expression of the Cre recombinase. Wip1 phosphatase activity and substrate identification Wip1 dephosphorylates serine and threonine residues within pTXpY and pTQ/pSQ motifs, and we have used biochemical methods to characterize its substrate specificity. Many of the known pTQ/pSQ substrates of Wip1 are phosphorylated by ATM. To provide an unbiased characterization of the substrate specificity of the Wip1 phosphatase, we have undertaken a quantitative phospho-proteomic analysis of the phosphoproteins present in cells following a stress under conditions of high or low Wip1 activity. Structure of the Wip1 catalytic domain PP2C serine/threonine protein phosphatases are critical regulators of stress responses and are distinguished by divalent metal ion-dependent stimulation of in vitro phosphatase activity. In humans, PP2C-alpha (PPM1A) functions as a tumor suppressor whereas Wip1 (PPM1D) negatively regulates several tumor suppressors. Although a crystal structure of human PP2C-alpha was shown to contain two bound Mn2+ ions, details of the catalytic mechanism and determinants of substrate specificity remain incompletely understood. Recently, structural studies of several prokaryotic PP2C phosphatases demonstrated the presence of three or four bound metal ions. As most of the coordinating residues for the additional metal ions are highly conserved, these results anticipate additional metal binding sites in human PP2C-alpha and Wip1 phosphatases. We have used site-directed mutagenesis, molecular modeling, calorimetry, and phosphatase activity assays to characterize the millimolar-affinity binding of magnesium ions to PP2C-alpha. Interestingly, mutation of the corresponding active site residues in PP2C-alpha and Wip1 has similar effects on the catalytic activity of each enzyme, as measured in vitro using purified proteins. These results suggest that the binding of a third metal ion to these phosphatases is essential for catalytic activity, involves amino acids conserved in both enzymes and identifies a critical process that could be abrogated by the binding of a specific inhibitor.

Wip1是PPM1D基因的产物，是一种PP2C丝氨酸/苏氨酸蛋白磷酸酶，在我的实验室中首次被确定为一种基因，其DNA损伤后的诱导需要野生型p53。基因PPM1D在几种类型的人类癌症中被扩增和/或过度表达。我们的研究结果表明Wip1磷酸酶通过p53的失活促进肿瘤的发生。作为一种酶，Wip1具有被低分子量化合物灭活的潜力。我们通过合理设计和筛选试验的结合，确定了特定的Wip1抑制剂，并正在寻求各种优化策略。为了更好地了解Wip1活性与肿瘤发生之间的联系，我们正在研究Wip1表达和活性的调控，确定Wip1磷酸酶活性的靶点，并开发Wip1磷酸酶活性的抑制剂。Wip1表达和活性的调控。暴露于DNA损伤剂后Wip1的转录诱导需要功能性p53蛋白。之前，我们在Wip1启动子中发现了一个功能性p53应答元件，它位于通常转录开始的下游。在DNA损伤反应（DDR）信号激活后，Wip1启动子的p53依赖性转录激活导致产生具有更短的5'非翻译区域的mrna，这些mrna可能更有效地翻译。Wip1水平的增加通过抑制DDR信号负调控p53活性，使细胞恢复稳态。暴露于IR后，Wip1蛋白水平比mRNA水平显著增加，这是由于翻译效率的提高和降解的减少。在我们目前的工作中，我们已经确定在Jurkat和U2OS细胞这两种不同的人类肿瘤细胞系中，Wip1蛋白的水平在S期增加了约4倍，而Wip1 mRNA的水平没有明显变化。S期特异性Wip1蛋白水平的增加，至少部分是由于蛋白稳定性的提高。最近发表的研究已经证实，在一些癌症患者中，体细胞突变导致Wip1蛋白过早终止，这些蛋白具有酶活性，但缺乏末端蛋白编码外显子的一部分。有趣的是，这些截断形式的Wip1表现出更高的稳定性；表达截断形式Wip1的细胞积累了高水平的磷酸酶，并表现出p53的严重受损激活（Ruark等，1013,Nature 493:06-410）。为了研究Wip1突变与活性之间的关系，我们查询了公共数据库以识别Wip1的错义突变。我们确定了人类群体中存在的四个单核苷酸多态性，这些多态性导致氨基酸变化，并表征了这些变化对Wip1活性的影响。对于两个突变，L120F和P322Q，改变的氨基酸位于催化结构域的结构部分，突变蛋白表现出显著降低的催化活性。第三个突变S82A位于远离活性位点的非结构化环中，不影响磷酸酶活性。第四个突变I496V位于Wip1的c端结构域，增加了定位于细胞质的蛋白数量。为了研究Wip1突变与原发性人类癌症之间的关系，我们在癌症病例中鉴定了41个Wip1突变位点。三分之二的病例位于c端结构域，约四分之一位于磷酸酶结构域。一般来说，Wip1功能突变丧失的病例可能包含p53突变，而导致Wip1功能获得的突变（通常是通过增加蛋白质稳定性）可能不包含p53基因突变。这些结果表明，Wip1的稳定性在其对肿瘤发生的贡献中是重要的。在常规培养条件下，Wip1-/-小鼠胚胎成纤维细胞（mef）会发生过早衰老。我们已经研究了Wip1减少mef中过早衰老的机制。我们发现，降低氧压只能部分抑制过早衰老。与野生型细胞相比，在20%和3%氧气条件下，早期传代的Wip1-/- MEFs表现出p53激活增加和细胞周期蛋白依赖性激酶抑制剂水平增加。这些发现表明，Wip1通过调节内源性DNA损伤引起的DDR信号来防止细胞衰老。Wip1敲除小鼠表现出的早衰表明，Wip1预防衰老的能力在维持体内组织维持能力方面也很重要。目前表征Wip1缺失对小鼠影响的结果是基于非条件敲除小鼠。尽管这种小鼠具有实用性，但它不能让我们确定Wip1缺失在单个组织中的具体影响。小鼠中普遍存在的Wip1缺失影响免疫系统、机体代谢和肿瘤微环境，其中任何一个都可能影响目标器官的肿瘤发生。为了克服这些限制，我们开发了一种条件敲除小鼠，在这种小鼠中，通过组织特异性表达Cre重组酶或通过诱导Cre重组酶在特定时间诱导Wip1缺失，可以将Wip1缺失定向到单个组织中。我们正在将这些条件敲除小鼠与携带Cre重组酶组织特异性表达等位基因的小鼠杂交。Wip1磷酸化pTXpY和pTQ/pSQ基序中的丝氨酸和苏氨酸残基，我们使用生化方法来表征其底物特异性。许多已知的Wip1的pTQ/pSQ底物被ATM磷酸化。为了对Wip1磷酸酶的底物特异性进行公正的描述，我们对在高或低Wip1活性条件下应激后细胞中存在的磷酸化蛋白进行了定量的磷酸化蛋白质组学分析。Wip1催化结构域PP2C丝氨酸/苏氨酸蛋白磷酸酶的结构是应激反应的关键调节因子，并且通过二价金属离子依赖性刺激体外磷酸酶活性来区分。在人类中，pp2c - α (PPM1A)作为肿瘤抑制因子发挥作用，而Wip1 （PPM1D）负调控几种肿瘤抑制因子。虽然人类pp2c - α的晶体结构被证明包含两个结合的Mn2+离子，但催化机制的细节和底物特异性的决定因素仍然不完全清楚。最近，对几种原核生物PP2C磷酸酶的结构研究表明存在三到四个结合的金属离子。由于附加金属离子的大多数配位残基是高度保守的，这些结果预测了人类pp2c - α和Wip1磷酸酶中额外的金属结合位点。我们使用定点诱变、分子建模、量热法和磷酸酶活性测定来表征镁离子与pp2c - α的毫摩尔亲和力结合。有趣的是，pp2c - α和Wip1中相应活性位点残基的突变对每种酶的催化活性有相似的影响，这是用纯化蛋白在体外测量的。这些结果表明，第三种金属离子与这些磷酸酶的结合对催化活性至关重要，涉及两种酶中保守的氨基酸，并确定了一个可以被特定抑制剂结合所废除的关键过程。