Functional study of C53 protein as a novel regulator of checkpoint kinases

C53 蛋白作为检查点激酶新型调节剂的功能研究

• 批准号: 7463681
• 负责人: HONGLIN LI
• 金额: $ 23.99万
• 依托单位: LURIE CHILDREN'S HOSPITAL OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7463681
• 关键词: Affect; Animals; Apoptosis; Apoptotic; Ataxia-Telangiectasia-Mutated protein kinase; Behavior; Binding Proteins; CDC2 Protein Kinase; Caspase; Cell Cycle; Cell Cycle Arrest; Cell Cycle Checkpoint; Cell Cycle Progression; Cell Cycle Regulation; Cell Death; Cell division; Cells; Cessation of life; Checkpoint kinase 1; Chromatin; Chromosome Segregation; Cytokinesis; DNA; DNA Damage; DNA Repair; DNA biosynthesis; DNA topoisomerase II alpha; Defect; Development; Disease; Ensure; Etoposide; Event; Foundations; Genetic Materials; Genome Stability; Genomic Instability; Goals; Hela Cells; Homeostasis; Hour; Lead; Ligands; Light; Malignant Neoplasms; Mediating; Mitosis; Mitotic; Mutagens; Mutation; Normal Cell; Normal tissue morphology; Pathogenesis; Pathway interactions; Phase; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physical condensation; Play; Process; Protein Dephosphorylation; Protein Overexpression; Proteins; Public Health; Regulation; Reporting; Resistance; Role; Signal Transduction; Site; System; TNF-related apoptosis-inducing ligand; TP53 gene; Testing; Topoisomerase II; Transducers; Work; ataxia telangiectasia mutated protein; base; cancer cell; cancer therapy; cdc25 Phosphatase; daughter cell; driving force; fighting; human disease; in vitro Assay; inhibitor/antagonist; insight; irradiation; neoplastic cell; novel; novel strategies; novel therapeutics; phosphatase inhibitor; promoter; receptor; repaired; response; sensor; therapeutic target; tissue culture; transmission process; tumorigenesis

DESCRIPTION (provided by applicant): Our long-term goal is to elucidate the roles of a novel protein C53 in regulation of cell cycle progression and DNA damage response, and to explore potential utilization of this novel protein as a novel therapeutic target. Regulation of cell cycle and cellular response to DNA damage is critical for genome stability, normal tissue homeostasis, tumorigenesis and cancer treatment. Any defects in cell cycle regulation may lead to genome instability and oncogenesis. Cells utilize the so-called checkpoint mechanisms to ensure accurate transmission of genetic material. The G2/M checkpoint regulates mitotic entry and activation of cyclin- dependent kinase 1 (Cdk1), a driving force for cell division. DNA damage usually evokes such checkpoints through activating a network of molecules including protein kinases ATM/ATR and their targets checkpoint kinases Chk1 and Chk2. By phosphorylating downstream effectors such as p53 and Cdc25 phosphatases, Chk1/Chk2 spread damage signal and elicit either cell cycle arrest or apoptotic cell death. We have recently identified Cdk5 activator p35 binding protein C53 as a novel regulator of cell cycle and DNA damage response. Some p53 deficient tumor cells treated with DNA damaging agents such as etoposide, a potent inhibitor of DNA topoisomerase II, undergo prolonged G2 arrest, and followed by apoptotic cell death. Our study demonstrated that depletion of endogenous C53 leads to defective cell cycle progression and resistance to apoptosis induced by etoposide and ionizing irradiation. Interestingly, C53 overexpression alone induces uneven chromatin condensation (UCC) in the absence of DNA damage. Moreover, C53 overexpression sensitizes cancer cells to various DNA damage agents via impairing DNA damage-induced checkpoint response, including activation of checkpoint kinases, but does not affect ATM/ATR activity. We found that C53 depletion enhances DNA damage-induced checkpoint activation. Intriguingly, we found that C53 interacts with both Chk1 and Chk2, and is able to inhibit their enzymatic activity. Based upon our preliminary study, we hypothesize that C53 is a novel negative regulator of checkpoint kinases via inhibiting their activation and kinase activity. By counteracting checkpoint kinases, C53 acts as a promoter of Cdk1 activation and mitotic entry during normal cell cycle and DNA damage response. To test our hypothesis, we will Aim 1: To elucidate the mechanism of C53-mediated regulation of checkpoint kinase activation. We will determine how C53 affects activation/inactivation of Chk1/Chk2 in DNA damage response. Aim 2: To dissect the C53-Chk interaction. By using deletion and site-specific mutations, we will determine which domains of C53 and Chk1/Chk2 are responsible for their interaction, and the C53 activity to inhibit Chk1/Chk2 kinase activity. Aim 3: To investigate the effect of caspase-mediated cleavage of C53 on mitotic cell death. This study will provide insight on a novel regulatory mechanism for cell cycle control and checkpoint response. It will lay a foundation for exploitation of this protein as a novel therapeutic target in cancer treatment. PUBLIC HEALTH RELEVANCE: Regulation of cell cycle is critical for normal tissue homeostasis, animal development, and the pathogenesis of human diseases such as cancer. Cell cycle progression is a coordinately regulated process that includes accurate replication of DNA during the S phase, correct segregation of chromosomes during mitosis (M phase) and final separation of daughter cells in cytokinesis. Cells utilize the cell cycle checkpoints to ensure the completion of critical event of a particular phase before the next phase is initiated. The checkpoints are crucial for maintaining DNA integrity in normal cells, and defects in the checkpoints may cause genomic instability and ultimately tumorigenesis. In response to DNA damage, cells use similar checkpoint mechanisms to halt cell cycle for DNA repair, or trigger cell death if the damage is beyond repair. We have recently identified a novel protein C53 that plays important roles in these fundamental processes. Further study of this protein will provide insight into a novel regulatory mechanism for cell cycle and cell death. It will lay a foundation for further exploitation of this novel protein as a potential therapeutic target in fighting diseases such as cancer.

描述（由申请人提供）：我们的长期目标是阐明新型蛋白C53在调节细胞周期进程和DNA损伤反应中的作用，并探索这种新型蛋白作为一种新型治疗靶标的潜在利用。细胞周期和对DNA损伤的细胞反应的调节对于基因组稳定性，正常组织稳态，肿瘤发生和癌症治疗至关重要。细胞周期调节中的任何缺陷都可能导致基因组不稳定性和肿瘤发生。细胞利用所谓的检查点机制来确保遗传物质的准确传播。 G2/M检查点调节细胞周期依赖性激酶1（CDK1）的有丝分裂进入和激活，这是细胞分裂的驱动力。 DNA损伤通常通过激活包括蛋白激酶ATM/ATR的分子网络及其目标检查点激酶CHK1和CHK2引起此类检查点。通过磷酸化下游效应子，例如p53和Cdc25磷酸酶，CHK1/CHK2传播损伤信号并引起细胞周期停滞或凋亡细胞死亡。我们最近将CDK5激活剂p35结合蛋白C53鉴定为细胞周期和DNA损伤反应的新调节剂。一些p53缺乏DNA损伤剂（例如依托泊苷）处理的p53缺陷肿瘤细胞，一种有效的DNA拓扑异构酶II的抑制剂，经历了延长的G2停滞，随后是凋亡细胞死亡。我们的研究表明，内源性C53的耗竭会导致细胞周期的进展和对依托泊苷和电离照射引起的细胞凋亡的抗性。有趣的是，在没有DNA损伤的情况下，单独的C53过表达会诱导不均匀的染色质凝结（UCC）。此外，C53的过表达通过损害DNA损伤引起的检查点响应（包括激活检查点激酶）将癌细胞敏感到各种DNA损伤剂，但不会影响ATM/ATR活性。我们发现C53耗竭增强了DNA损伤诱导的检查点激活。有趣的是，我们发现C53与CHK1和CHK2都相互作用，并且能够抑制其酶促活性。根据我们的初步研究，我们假设C53是通过抑制其激活和激酶活性的检查点激酶的新型负调节剂。通过抵消检查点激酶，C53在正常细胞周期和DNA损伤响应中充当CDK1激活和有丝分裂进入的启动子。为了检验我们的假设，我们将目标1：阐明C53介导的检查点激酶激活调节的机理。我们将确定C53如何影响CHK1/CHK2在DNA损伤反应中的激活/灭活。目标2：剖析C53-CHK相互作用。通过使用缺失和位点特异性突变，我们将确定哪些C53和CHK1/CHK2的域负责它们的相互作用，以及C53活性抑制CHK1/CHK2激酶活性。目标3：研究caspase介导的C53裂解对有丝分裂细胞死亡的影响。这项研究将提供有关细胞周期控制和检查点响应的新型调节机制的见解。它将为剥削该蛋白作为癌症治疗中的新治疗靶点奠定基础。公共卫生相关性：细胞周期的调节对于正常组织稳态，动物发育以及人类疾病（例如癌症）的发病机理至关重要。细胞周期进程是一个协同调节的过程，包括在S期间准确复制DNA，在有丝分裂（M期）（M期）正确分离染色体（M期）和细胞因子中子细胞的最终分离。细胞利用细胞周期检查点来确保在开始下一阶段之前完成特定阶段的关键事件。检查点对于维持正常细胞中的DNA完整性至关重要，并且检查点中的缺陷可能导致基因组不稳定性并最终导致肿瘤发生。为了应对DNA损伤，细胞使用类似的检查点机制来阻止细胞周期进行DNA修复，或者如果损伤无法修复，则触发细胞死亡。我们最近确定了一种新型的蛋白C53，该蛋白在这些基本过程中起着重要作用。对该蛋白质的进一步研究将为细胞周期和细胞死亡的新调节机制提供深入的了解。它将为进一步剥削这种新型蛋白质作为对抗癌症等疾病的潜在治疗靶点的基础。