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: 7760597
• 负责人: HONGLIN LI
• 金额: $ 17.94万
• 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 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7760597
• 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; 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; Proteins; 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; new therapeutic target; novel; novel strategies; novel therapeutic intervention; overexpression; phosphatase inhibitor; premature; promoter; public health relevance; 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一种新型蛋白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的治疗靶点的潜在利用。调控细胞周期和细胞对DNA损伤的反应对基因组稳定、正常组织稳态、肿瘤发生和癌症治疗至关重要。细胞周期调控的任何缺陷都可能导致基因组的不稳定和肿瘤的发生。细胞利用所谓的检查点机制来确保遗传物质的准确传递。G2/M检查点调节有丝分裂进入和细胞周期蛋白依赖性激酶1 （Cdk1）的激活，Cdk1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及其靶检查点激酶Chk1和Chk2在内的分子网络来唤起这样的检查点。通过磷酸化下游效应物如p53和Cdc25磷酸酶，Chk1/Chk2传播损伤信号并引发细胞周期阻滞或凋亡细胞死亡。我们最近发现Cdk5激活因子p35结合蛋白C53是细胞周期和DNA损伤反应的一种新的调节因子。一些p53缺陷肿瘤细胞用DNA损伤剂如依托泊苷（一种DNA拓扑异构酶II的有效抑制剂）处理后，会经历长时间的G2阻滞，随后出现细胞凋亡。我们的研究表明，内源性C53的消耗导致细胞周期进程缺陷和对依托泊苷和电离照射诱导的细胞凋亡的抗性。有趣的是，在没有DNA损伤的情况下，C53过表达单独诱导不均匀染色质凝聚（UCC）。此外，C53过表达通过损害DNA损伤诱导的检查点反应（包括检查点激酶的激活）使癌细胞对各种DNA损伤剂敏感，但不影响ATM/ATR活性。我们发现C53缺失增强了DNA损伤诱导的检查点激活。有趣的是，我们发现C53与Chk1和Chk2相互作用，并能够抑制它们的酶活性。根据我们的初步研究，我们假设C53通过抑制检查点激酶的激活和激酶活性而成为一种新的负调节因子。通过对抗检查点激酶，C53在正常细胞周期和DNA损伤反应中作为Cdk1激活和有丝分裂进入的启动子。为了验证我们的假设，我们的目标1：阐明c53介导的检查点激酶激活调节机制。我们将确定C53如何影响DNA损伤反应中Chk1/Chk2的激活/失活。目的2：解剖C53-Chk相互作用。通过缺失和位点特异性突变，我们将确定C53和Chk1/Chk2的哪些结构域负责它们的相互作用，以及C53抑制Chk1/Chk2激酶活性的活性。目的3：探讨caspase介导的C53裂解对有丝分裂细胞死亡的影响。这项研究将为细胞周期控制和检查点反应提供新的调控机制。这将为开发该蛋白作为新的肿瘤治疗靶点奠定基础。公共卫生相关性：细胞周期的调节对正常组织稳态、动物发育和癌症等人类疾病的发病机制至关重要。细胞周期进程是一个协调调节的过程，包括S期DNA的精确复制，有丝分裂（M期）染色体的正确分离以及细胞质分裂时子细胞的最终分离。细胞利用细胞周期检查点确保在启动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的DNA完整性至关重要，检查点的缺陷可能导致基因组不稳定并最终导致肿瘤发生。在对DNA损伤的反应中，细胞使用类似的检查点机制来停止DNA修复的细胞周期，或者在损伤无法修复时触发细胞死亡。我们最近发现了一种新的蛋白C53，它在这些基本过程中起着重要作用。对该蛋白的进一步研究将有助于揭示细胞周期和细胞死亡的一种新的调控机制。这将为进一步开发这种新型蛋白作为对抗癌症等疾病的潜在治疗靶点奠定基础。