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: 8553033
• 负责人: ETTORE APPELLA
• 金额: $ 65.51万
• 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/8553033
• 关键词: Affect; Alanine; Antineoplastic Agents; Binding; Biochemical; Biological Assay; CDKN2A gene; Calorimetry; Cell Aging; Cell Death; Cells; Chemicals; DNA Damage; Dependence; Development; Disease; Dissociation; Embryo; Environment; Enzymes; Exhibits; Exposure to; Feedback; Fibroblasts; Genes; Homeostasis; Human; Immune system; In Vitro; Insulin; Ions; Kinetics; Knockout Mice; Laboratories; MAP Kinase Gene; MAPK14 gene; Malignant Neoplasms; Masks; Measures; Messenger RNA; Metabolism; Metal Binding Site; Metal Ion Binding; Metals; Methods; Modeling; Molecular Models; Molecular Weight; Mus; Mutation; Normal tissue morphology; Organ; Oxygen; PPM1D gene; Pathway interactions; Peptides; Phosphopeptides; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Play; Process; Prodrugs; Protein Dephosphorylation; Protein Serine/Threonine Phosphatase; Protein p53; Proteins; Publishing; Pyrroles; Radio; Regulation; Role; S Phase; Scheme; Screening procedure; Serine; Signal Transduction; Site-Directed Mutagenesis; Solutions; Specific qualifier value; Structure; Substrate Specificity; Testing; Threonine; Time; Tissues; Translation Initiation; Tumor Cell Line; Tumor Suppressor Proteins; Work; analog; base; biological adaptation to stress; cancer therapy; cell type; design; divalent metal; improved; inhibitor/antagonist; inorganic phosphate; interest; molecular modeling; neoplastic cell; novel; overexpression; p19ARF; premature; pressure; prevent; promoter; protein phosphatase 2C; recombinase; response; senescence; small molecule; transcription factor; tumor; tumorigenesis; upstream kinase; uptake

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 activityThe transcriptional induction of Wip1 following exposure to DNA damaging agents requires functional p53 protein. p53 and Wip1 form a negative feedback loop, with Wip1 phosphatase activity promoting the return to homeostasis by removing activating phosphorylations of p53 itself and those of its upstream kinases, ATM, Chk1 and Chk2. Following exposure to IR, the level of Wip1 protein changes more dramatically than its mRNA levels. 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. These studies suggest that Wip1 is regulated on many levels.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 p21, p16Ink4a and p19Arf. The absence of p38 MAPK activation implies that Wip1 may regulate the Ink4a/Arf locus through a novel pathway. These findings suggest that Wip1 prevents cellular senescence by regulating both the DNA Damage Response and p16Ink4a/p19Arf signaling.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 generating these conditional knock-out mice, which will be used to study the effects of Wip1 in several mouse tumor models. Wip1 phosphatase activity and substrate identificationWip1 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. In addition to its role in the response to DNA damage, ATM regulates the cellular response to insulin through phosphorylation of Ser112 of the inhibitor 4EBP1, resulting in its dissociation from the translation initiation protein, eIF-4E. We are investigating Ser112 of 4EBP1 as a potential Wip1 substrate. Currently, the majority of anticancer therapies use the p53 pathway to induce tumor cells death. We have explored an alternative approach, based on activation of Wip1 phosphatase, toward sensitizing tumors that lack wildtype p53 to anticancer drugs, while at the same time protecting normal tissues. In response to anticancer drugs in tumors with inactive p53, Wip1 overexpression led to the induction of Bax through dephosphorylation-dependent activation of the transcription factor RUNX2. We found that Wip1 interacted with RUNX2 in Saos-2-Wip1-on cells. Mutation of RUNX2 Ser432 to alanine resulted in significantly increased transcriptional activity of the Bax promoter, compared with WT RUNX2. To confirm that phospho-Ser432 of RUNX2 was a target for Wip1, we synthesized a human RUNX2 peptide containing phosphorylated Ser432 and measured the peptide concentration dependence of Wip1 phosphatase activity. The resulting kinetic constants were similar to those obtained with the Chk1 345pS phosphopeptide, a well known substrate for Wip1.Wip1 Inhibitors and structure of the Wip1 catalytic domainMany human tumors in which the PPM1D gene is amplified and/or overexpressed contain wild type p53. Our published results indicate that Wip1 phosphatase promotes tumorigenesis through inactivation of wild-type p53. Phosphatases in general play critical physiological roles as antagonists of kinase activity and, as such, represent important targets in a number of diseases, including cancer. We have continued to develop pyrrole-based small molecule inhibitors of Wip1. The synthesis of these inhibitors has been improved by decreasing the number of synthetic steps and optimization of the synthetic scheme for solution phase synthesis. The new synthesis is scalable, allowing us to prepare 100 mg amounts. We have already synthesized 30 different analogs. The new synthesis has been adapted to the synthesis of a pro-drug, in which the phosphate groups are masked to improve cellular uptake. We have already synthesized a pro-drug form of the pyrrole-based inhibitors and have successfully tested its activity in a cell-based assay. 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 PPM1D phosphatases. We have used site-directed mutagenesis, molecular modeling, calorimetry, and phosphatase activity assays to characterize additional metal binding site(s). These results suggest that the binding of additional metal ions to human PP2C phosphatases is essential for catalytic activity and open prospects for the development of specific inhibitors.

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鉴定为 DNA 损伤后的诱导需要野生型 p53 的基因。 PPM1D 基因在多种类型的人类癌症中被扩增和/或过度表达。我们的结果表明 Wip1 磷酸酶通过 p53 失活促进肿瘤发生。作为一种酶，Wip1 有可能被低分子量化合物失活。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 表达和活性的调节，确定 Wip1 磷酸酶活性的靶标，并开发 Wip1 磷酸酶活性的抑制剂。Wip1 表达和活性的调节暴露于 DNA 损伤剂后 Wip1 的转录诱导需要功能性 p53 蛋白。 p53 和 Wip1 形成负反馈环，Wip1 磷酸酶活性通过消除 p53 本身及其上游激酶 ATM、Chk1 和 Chk2 的激活磷酸化来促进体内平衡的恢复。 暴露于红外线后，Wip1 蛋白水平的变化比其 mRNA 水平的变化更为显着。 在我们目前的工作中，我们已经确定，在 Jurkat 和 U2OS 细胞（两种不同的人类肿瘤细胞系）中，Wip1 蛋白水平在 S 期增加约 4 倍，而 Wip1 mRNA 水平没有明显变化。这些研究表明，Wip1 在多个层面上受到调控。在常规培养条件下，Wip1-/- 小鼠胚胎成纤维细胞 (MEF) 会过早衰老。 我们研究了 Wip1 减少 MEF 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，早期传代的 Wip1-/- MEF 在 20% 和 3% 氧气条件下均表现出 p53 活化增加以及 p21、p16Ink4a 和 p19Arf 水平增加。 p38 MAPK 激活的缺失意味着 Wip1 可能通过一种新途径调节 Ink4a/Arf 位点。这些发现表明，Wip1 通过调节 DNA 损伤反应和 p16Ink4a/p19Arf 信号传导来防止细胞衰老。当前表征 Wip1 缺失对小鼠影响的结果基于非条件敲除小鼠。尽管这种小鼠很实用，但它不允许我们确定 Wip1 缺失对单个组织的具体影响。小鼠体内普遍存在的 Wip1 缺失会影响免疫系统、机体代谢和肿瘤微环境，其中任何一个都可能影响感兴趣器官的肿瘤发生。为了克服这些限制，我们开发了一种条件敲除小鼠，其中可以通过 Cre 重组酶的组织特异性表达或通过 Cre 重组酶的诱导表达在特定时间诱导缺失，将 Wip1 缺失定向到单个组织。 我们正在培育这些条件性基因敲除小鼠，它们将用于研究 Wip1 在几种小鼠肿瘤模型中的作用。 Wip1 磷酸酶活性和底物鉴定Wip1 使 pTXpY 和 pTQ/pSQ 基序内的丝氨酸和苏氨酸残基去磷酸化，我们使用生化方法来表征其底物特异性。许多已知的 Wip1 pTQ/pSQ 底物均被 ATM 磷酸化。除了在 DNA 损伤反应中发挥作用外，ATM 还通过抑制剂 4EBP1 的 Ser112 磷酸化来调节细胞对胰岛素的反应，导致其与翻译起始蛋白 eIF-4E 解离。我们正在研究 4EBP1 的 Ser112 作为潜在的 Wip1 底物。 目前，大多数抗癌疗法使用p53途径诱导肿瘤细胞死亡。我们探索了一种基于 Wip1 磷酸酶激活的替代方法，使缺乏野生型 p53 的肿瘤对抗癌药物敏感，同时保护正常组织。为了应对 p53 失活肿瘤中的抗癌药物，Wip1 过度表达通过转录因子 RUNX2 的去磷酸化依赖性激活诱导 Bax。我们发现 Wip1 在 Saos-2-Wip1-on 细胞中与 RUNX2 相互作用。 与 WT RUNX2 相比，RUNX2 Ser432 突变为丙氨酸导致 Bax 启动子的转录活性显着增加。为了确认 RUNX2 的磷酸化 Ser432 是 Wip1 的靶标，我们合成了含有磷酸化 Ser432 的人 RUNX2 肽，并测量了 Wip1 磷酸酶活性的肽浓度依赖性。 所得动力学常数与使用 Chk1 345pS 磷酸肽（Wip1 的众所周知的底物）获得的动力学常数相似。Wip1 抑制剂和 Wip1 催化结构域的结构许多 PPM1D 基因被扩增和/或过表达的人类肿瘤含有野生型 p53。我们发表的结果表明，Wip1 磷酸酶通过野生型 p53 的失活促进肿瘤发生。一般来说，磷酸酶作为激酶活性的拮抗剂发挥着重要的生理作用，因此是包括癌症在内的许多疾病的重要靶标。我们继续开发基于吡咯的 Wip1 小分子抑制剂。通过减少合成步骤数和优化溶液相合成的合成方案，改进了这些抑制剂的合成。新的合成方法具有可扩展性，使我们能够制备 100 毫克的量。我们已经合成了 30 种不同的类似物。新的合成方法已适应前药的合成，其中磷酸基团被掩蔽以改善细胞摄取。我们已经合成了基于吡咯的抑制剂的前药形式，并在基于细胞的测定中成功测试了其活性。 PP2C 丝氨酸/苏氨酸蛋白磷酸酶是应激反应的关键调节因子，其特点是体外磷酸酶活性的二价金属离子依赖性刺激。 在人类中，PP2C-α (PPM1A) 作为肿瘤抑制因子发挥作用，而 Wip1 (PPM1D) 对多种肿瘤抑制因子具有负调节作用。 尽管人 PP2C-α 的晶体结构显示含有两个结合的 Mn2+ 离子，但催化机制的细节和底物特异性的决定因素仍未完全了解。 最近，对几种原核 PP2C 磷酸酶的结构研究表明存在三个或四个结合的金属离子。 由于其他金属离子的大多数配位残基高度保守，因此这些结果预计人 PP2C-α 和 PPM1D 磷酸酶中存在其他金属结合位点。 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其他金属结合位点。 这些结果表明，额外的金属离子与人 PP2C 磷酸酶的结合对于催化活性至关重要，并为开发特异性抑制剂开辟了前景。