Normal and Neoplastic Regulation of Cyclin E

细胞周期蛋白 E 的正常和肿瘤调节

• 批准号: 7997178
• 负责人: Bruce E Clurman
• 金额: $ 37.1万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7997178
• 关键词: Animal Model; Area; Binding; Biochemical; Biochemistry; Biology; C-terminal; CDK2 gene; Cancer Etiology; Cell Cycle; Cell division; Complex; Cyclin A; Cyclin E; Data; Development; Engineering; Exhibits; Funding; Gene Targeting; Genes; Genetic; Genetic Screening; Goals; Health; Human; Insertional Mutagenesis; Lead; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Modeling; Molecular; Mus; Mutate; Mutation; N-terminal; Normal Cell; Oncogenic; Pathway interactions; Periodicity; Phosphorylation; Phosphotransferases; Physiological; Play; Proteins; Proteomics; Regulation; Research; Role; Signal Transduction; Sleeping Beauty; Subgroup; System; Testing; Therapeutic Studies; Tumor Suppressor Genes; Variant; Work; Yeasts; base; cancer cell; cancer therapy; cellular targeting; dimer; in vivo; insight; mouse model; neoplastic; novel; treatment strategy; tumorigenesis; ubiquitin ligase

DESCRIPTION (provided by applicant): Cyclin E, in conjunction with its catalytic partner CDK2, regulates diverse aspects of cell division. Normal cells tightly regulate cyclin E, whereas cancer cells often exhibit abnormal cyclin E activity and this directly contributes to genetic instability and tumorigenesis. The research proposed in this application uses a combined approach of biochemistry, animal models, human gene targeting, and proteomics, to understand novel aspects of cyclin E regulation and function. One critical mechanism of cyclin E regulation that is often disrupted in cancer cells is its degradation by the Fbw7 ubiquitin ligase. The interactions of cyclin E and Fbw7 are complex and regulated by two cyclin E phosphodegrons that bind to Fbw7, and the relationships between cyclin E and Fbw7 will be studied in Aim 1. The first subaim will determine the physiologic significance of the N-terminal degron through the development of a mouse knockin strain in which this degron is mutated. In the second subaim we will test the hypothesis that variations in CDK2 specific activity regulate cyclin E accessibility to Fbw7 during the cell cycle, and that this involves both CDK2 and cyclin E phosphorylation. Finally, in the third subaim we will use purified components to test the hypothesis that both cyclin E degrons can simultaneously engage an Fbw7 dimer, and determine the feasibility of a structural analysis of cyclin E bound to an Fbw7 dimer. Robust mouse models are needed for mechanistic and therapeutic studies of cyclin E- associated cancer, and these will be developed in Aim 2. In the first subaim, we will combine cyclin E degron mutations with the disruption of two tumor suppressor genes that normally restrain cyclin E: p53 and p27. The goal of the second subaim is to identify genes that cooperate with cyclin E during tumorigenesis and the pathways that suppress cyclin E-driven hyperproliferation in vivo. The approach that we will take is to use a genetic screen employing the "Sleeping Beauty" transposon system to identify cooperating genes and pathways in mice bearing cyclin E degron mutations. Approximately a dozen cyclin E-CDK2 substrates are known, and these have wide ranging cell cycle functions. Studies in yeast have revealed more than 200 CDK substrates and it is likely that many cyclin E-CDK2 substrates are unknown. We have developed a kinase engineering/mass spectrometry-based approach that efficiently identifies candidate CDK2 substrates. The goal of this aim is to utilize these methods to identify CDK2 substrates, and then to use biochemical and gene targeting methods to study the functions of a subgroup of validated substrates. These latter studies are critical, because they will determine the physiologic significance of endogenous substrates. PUBLIC HEALTH RELEVANCE The research in this proposal focuses on a protein, called cyclin E, that plays a central role in cell division and cancer. The goals of this research are to understand the functions and regulation of cyclin E in normal cells, and why loss of these normal controls causes cancer. This research may increase our understanding of why cancer develops and lead to new cancer treatment strategies.

描述（由申请人提供）：Cyclin E 与其催化伙伴 CDK2 一起调节细胞分裂的各个方面。正常细胞严格调节细胞周期蛋白 E，而癌细胞通常表现出异常的细胞周期蛋白 E 活性，这直接导致遗传不稳定和肿瘤发生。本申请中提出的研究采用生物化学、动物模型、人类基因靶向和蛋白质组学的组合方法，以了解细胞周期蛋白 E 调控和功能的新方面。癌细胞中经常被破坏的细胞周期蛋白 E 调节的一项关键机制是其被 Fbw7 泛素连接酶降解。细胞周期蛋白 E 和 Fbw7 的相互作用很复杂，并受到与 Fbw7 结合的两个细胞周期蛋白 E 磷酸化子的调节，并且细胞周期蛋白 E 和 Fbw7 之间的关系将在目标 1 中进行研究。第一个子目标将确定 N 末端降解决定子的生理意义通过开发小鼠敲入品系，其中该降解决定子发生突变。在第二个子目标中，我们将测试以下假设：CDK2 比活性的变化在细胞周期期间调节细胞周期蛋白 E 对 Fbw7 的可及性，并且这涉及 CDK2 和细胞周期蛋白 E 磷酸化。最后，在第三个子目标中，我们将使用纯化的组件来测试两个细胞周期蛋白 E 降解决定子可以同时接合 Fbw7 二聚体的假设，并确定与 Fbw7 二聚体结合的细胞周期蛋白 E 结构分析的可行性。细胞周期蛋白 E 相关癌症的机制和治疗研究需要稳健的小鼠模型，这些模型将在目标 2 中开发。在第一个子目标中，我们将结合细胞周期蛋白 E 降解决定子突变与通常抑制细胞周期蛋白的两个肿瘤抑制基因的破坏E：第 53 页和第 27 页。第二个子目标的目标是鉴定在肿瘤发生过程中与细胞周期蛋白 E 合作的基因以及抑制体内细胞周期蛋白 E 驱动的过度增殖的途径。我们将采取的方法是使用“睡美人”转座子系统的遗传筛选来识别携带细胞周期蛋白 E 降解决定子突变的小鼠中的合作基因和通路。已知大约有十几种细胞周期蛋白 E-CDK2 底物，它们具有广泛的细胞周期功能。对酵母的研究已揭示了 200 多种 CDK 底物，并且很可能许多细胞周期蛋白 E-CDK2 底物是未知的。我们开发了一种基于激酶工程/质谱的方法，可以有效识别候选 CDK2 底物。该目标的目标是利用这些方法来识别 CDK2 底物，然后使用生化和基因靶向方法来研究经过验证的底物亚组的功能。后面的这些研究至关重要，因为它们将确定内源性底物的生理意义。公共健康相关性 本提案中的研究重点是一种称为细胞周期蛋白 E 的蛋白质，它在细胞分裂和癌症中发挥着核心作用。这项研究的目标是了解正常细胞中细胞周期蛋白 E 的功能和调节，以及为什么失去这些正常控制会导致癌症。这项研究可能会增加我们对癌症发生原因的理解，并带来新的癌症治疗策略。