Normal and Neoplastic Regulation of Cyclin E

描述(申请人提供)：细胞周期蛋白E与其催化伙伴CDK2一起调节细胞分裂的不同方面。正常细胞严格调控细胞周期蛋白E，而癌细胞往往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的发生。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相结合的方法，以了解Cyclin E调节和功能的新方面。在癌细胞中，细胞周期蛋白E调节的一个关键机制是其被Fbw7泛素连接酶降解。Cyclin E和Fbw7的相互作用是复杂的，并受两个与Fbw7结合的Cyclin E磷酸降解物的调节，目的1将研究Cyclin E和Fbw7之间的关系。第一个亚目的是通过建立一个N端降解蛋白突变的小鼠敲击菌株来确定该降解蛋白的生理意义。在第二个子目标中，我们将检验一种假设，即在细胞周期中，CDK2比活性的变化调节细胞周期蛋白E对Fbw7的可及性，这涉及CDK2和细胞周期蛋白E的磷酸化。最后，在第三个子目标中，我们将使用纯化的组分来测试两个细胞周期蛋白E降解子都可以同时与Fbw7二聚体结合的假设，并确定对Fbw7二聚体结合的细胞周期蛋白E进行结构分析的可行性。细胞周期蛋白E相关癌症的机制和治疗研究需要强大的小鼠模型，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退化突变和两个通常抑制细胞周期蛋白E的肿瘤抑制基因的破坏：p53和p27。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协同的基因，以及体内抑制细胞周期蛋白E驱动的过度增殖的途径。我们将采取的方法是使用基因筛查技术，利用“睡美人”转座子系统来识别携带细胞周期蛋白E退化基因突变的小鼠的协同基因和途径。目前已知的细胞周期蛋白E-CDK2底物大约有12种，它们具有广泛的细胞周期功能。在酵母中的研究已经揭示了200多种CDK底物，很可能许多Cyclin E-CDK2底物是未知的。我们已经开发了一种基于激酶工程/质谱学的方法，可以有效地识别候选的CDK2底物。其目的是利用这些方法来鉴定CDK2底物，然后使用生化和基因打靶方法来研究一组有效底物的功能。这些后一项研究是至关重要的，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重点是一种名为细胞周期蛋白E的蛋白质，它在细胞分裂和癌症中发挥核心作用。这项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，以及为什么这些正常对照的缺失会导致癌症。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.