Normal and Neoplastic Regulation of Cyclin E

描述（由申请人提供）：细胞周期蛋白E与其催化伴侣CDK 2一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综合方法，以了解细胞周期蛋白E调控和功能的新方面。在癌细胞中经常被破坏的细胞周期蛋白E调节的一个关键机制是其被Fbw 7泛素连接酶降解。细胞周期蛋白E和Fbw 7的相互作用是复杂的，并通过两个细胞周期蛋白E磷酸降解决定子结合Fbw 7进行调节，细胞周期蛋白E和Fbw 7之间的关系将在目标1中研究。第一个子目标将通过开发其中该降解决定子突变的小鼠敲入品系来确定N-末端降解决定子的生理学意义。在第二个子目标，我们将测试的假设，在细胞周期中，CDK 2的特异性活性的变化调节细胞周期蛋白E的Fbw 7的可及性，这涉及CDK 2和细胞周期蛋白E磷酸化。最后，在第三subaim，我们将使用纯化的组件来测试的假设，这两个细胞周期蛋白E降解决定子可以同时从事Fbw 7二聚体，并确定结合Fbw 7二聚体的细胞周期蛋白E的结构分析的可行性。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，我们将联合收割机将细胞周期蛋白E降解决定子突变与通常抑制细胞周期蛋白E的两个肿瘤抑制基因p53和p27的破坏相结合。第二个子目标的目标是确定在肿瘤发生过程中与细胞周期蛋白E合作的基因和抑制细胞周期蛋白E驱动的体内过度增殖的途径。我们将采取的方法是使用遗传筛选，采用“睡美人”转座子系统，以确定在小鼠轴承细胞周期蛋白E降解决定子突变的合作基因和途径。大约有十几种细胞周期蛋白E-CDK 2底物是已知的，并且这些具有广泛的细胞周期功能。在酵母中的研究已经揭示了超过200种CDK底物，并且很可能许多细胞周期蛋白E-CDK 2底物是未知的。我们已经开发了一种基于激酶工程/质谱的方法，可以有效地识别候选CDK 2底物。本研究的目的是利用这些方法鉴定CDK 2底物，然后使用生物化学和基因靶向方法研究经验证底物亚组的功能。这些后面的研究是至关重要的，因为它们将确定内源性底物的生理意义。公共卫生相关性这项提案中的研究集中在一种名为细胞周期蛋白E的蛋白质上，这种蛋白质在细胞分裂和癌症中起着核心作用。本研究的目的是了解细胞周期蛋白E在正常细胞中的功能和调节，以及为什么失去这些正常对照会导致癌症。这项研究可能会增加我们对癌症发展原因的理解，并导致新的癌症治疗策略。

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.