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与其催化伴侣CDK2结合使用，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，而癌细胞通常表现出异常的细胞周期蛋白E活性，这直接有助于遗传不稳定性和肿瘤发生。该应用中提出的研究采用了生物化学，动物模型，人类基因靶向和蛋白质组学的综合方法，以了解细胞周期蛋白E调节和功能的新方面。 Cyclin E调节的一种关键机制通常会在癌细胞中破坏，这是FBW7泛素连接酶的降解。细胞周期蛋白E和FBW7的相互作用是复杂的，并由与FBW7结合的两个环蛋白E磷降氧化物的调节，细胞周期蛋白E和FBW7之间的关系将在AIM 1中研究。第一个Subaim将通过这种degron在该degron中的开发来确定N末端Degron的生理意义。在第二个SubiaM中，我们将测试以下假设：CDK2特异性活性的变化调节细胞周期中FBW7的可及性，这涉及CDK2和Cyclin e磷酸化。最后，在第三个Subaim中，我们将使用纯化的组件来检验以下假设：两个细胞周期蛋白DEGRON都可以同时参与FBW7二聚体，并确定与FBW7二聚体结合的结构性分析的可行性。需要强大的小鼠模型来对细胞周期蛋白相关的癌症进行机械和治疗研究，这些模型将在AIM 2中开发。在第一个SubiaM中，我们将结合细胞周期蛋白E DEGRON突变与两个通常抑制细胞周期蛋白E：p53和p27的肿瘤抑制基因的破坏。第二个Subaim的目的是鉴定肿瘤发生过程中与细胞周期蛋白E合作的基因以及抑制细胞周期蛋白E驱动的体内过度增殖的途径。我们将采用的方法是使用使用“睡美人”转座系统系统的遗传筛选来识别带有细胞周期蛋白E DEGRON突变的小鼠中的合作基因和途径。已知大约十二个细胞周期蛋白E-CDK2底物，并且具有广泛的细胞周期功能。在酵母中的研究表明，有200多个CDK底物，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，该方法有效地识别了候选CDK2底物。该目的的目的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。这些后一种研究至关重要，因为它们将确定内源性底物的生理意义。公共卫生相关性该提案中的研究重点是一种称为细胞周期蛋白E的蛋白质，该蛋白在细胞分裂和癌症中起着核心作用。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，以及为什么这些正常对照的丧失会导致癌症。这项研究可能会增加我们对癌症为什么发展和导致新的癌症治疗策略的理解。