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

描述（由申请人提供）：我们的长期目标是阐明新蛋白C53在细胞周期进程和DNA损伤反应调节中的作用，并探索这种新蛋白作为新治疗靶点的潜在用途。细胞周期和细胞对DNA损伤的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。细胞周期调控中的任何缺陷都可能导致基因组不稳定和肿瘤发生。细胞利用所谓的检查点机制来确保遗传物质的准确传输。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（Cdk 1）的激活，Cdk 1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及其靶点检查点激酶Chk 1和Chk 2的分子网络来引起这样的检查点。通过磷酸化下游效应物如p53和Cdc 25磷酸酶，Chk 1/Chk 2传播损伤信号并引起细胞周期停滞或凋亡性细胞死亡。我们最近发现Cdk 5激活剂p35结合蛋白C53作为一种新的调节细胞周期和DNA损伤反应。用DNA损伤剂如DNA拓扑异构酶II的有效抑制剂依托泊苷处理的一些p53缺陷型肿瘤细胞经历延长的G2停滞，随后是凋亡性细胞死亡。我们的研究表明，内源性C53的耗尽导致缺陷的细胞周期进程和对依托泊苷和电离辐射诱导的凋亡的抵抗。有趣的是，在没有DNA损伤的情况下，单独的C53过表达诱导不均匀染色质凝聚（UCC）。此外，C53过表达通过损害DNA损伤诱导的检查点应答（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Chk 1和Chk 2相互作用，并且能够抑制它们的酶活性。基于我们的初步研究，我们假设C53是一种新的负调节剂的检查点激酶，通过抑制其激活和激酶活性。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Cdk 1激活和有丝分裂进入的启动子。为了验证我们的假设，我们将目标1：阐明C53介导的检查点激酶激活的调节机制。我们将确定C53如何影响DNA损伤反应中Chk 1/Chk 2的激活/失活。目的2：研究C53与Chk的相互作用。通过使用缺失和位点特异性突变，我们将确定C53和Chk 1/Chk 2的哪些结构域负责它们的相互作用，以及C53抑制Chk 1/Chk 2激酶活性的活性。目的3：探讨半胱天冬酶介导的C53蛋白裂解对有丝分裂期细胞死亡的影响。这项研究将为细胞周期控制和检查点反应提供一种新的调控机制。为进一步开发该蛋白作为肿瘤治疗的新靶点奠定了基础。公共卫生相关性：细胞周期的调节对于正常组织的稳态、动物的发育和人类疾病如癌症的发病机制是至关重要的。细胞周期进程是一个协调调节的过程，包括S期DNA的准确复制、有丝分裂（M期）染色体的正确分离和胞质分裂中子细胞的最终分离。细胞利用细胞周期检查点来确保在下一个阶段开始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停止细胞周期以进行DNA修复，或者如果损伤无法修复则触发细胞死亡。我们最近发现了一种新的蛋白质C53，在这些基本过程中发挥重要作用。对该蛋白的进一步研究将为深入了解细胞周期和细胞死亡的新调控机制提供帮助。这将为进一步开发这种新蛋白作为对抗癌症等疾病的潜在治疗靶点奠定基础。

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.