Normal and Neoplastic Regulation of Cyclin E

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。正常细胞严格调节周期蛋白E，而癌细胞通常表现出异常的周期蛋白E活性，这直接导致遗传不稳定和肿瘤发生。本研究采用生物化学、动物模型、人类基因靶向和蛋白质组学相结合的方法来了解周期蛋白E调控和功能的新方面。癌细胞中周期蛋白E调控的一个关键机制是它被Fbw7泛素连接酶降解。cyclin E和Fbw7的相互作用是复杂的，并受到两个结合Fbw7的cyclin E磷酸化子的调控，我们将在Aim 1中研究cyclin E和Fbw7之间的关系。第一个子目标将通过开发一种小鼠敲除蛋白菌株来确定n端degron的生理意义，其中该degron发生突变。在第二个子目标中，我们将测试CDK2特异性活性的变化调节细胞周期中周期蛋白E对Fbw7的可及性的假设，并且这涉及CDK2和周期蛋白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验证两个cyclin E度可以同时与Fbw7二聚体结合的假设，并确定结合Fbw7二聚体的cyclin E结构分析的可行性。周期蛋白E相关癌症的机制和治疗研究需要健壮的小鼠模型，这些将在Aim 2中开发。在第一个子目标中，我们将结合cyclin E degron突变和通常抑制cyclin E的两个肿瘤抑制基因p53和p27的破坏。第二个子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。我们将采用的方法是使用使用“睡美人”转座子系统的基因筛选来识别携带周期蛋白E退化突变的小鼠中的合作基因和途径。已知大约有12种周期蛋白E-CDK2底物，它们具有广泛的细胞周期功能。在酵母中的研究已经发现了200多种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，可以有效地识别候选CDK2底物。本目的目的是利用这些方法鉴定CDK2底物，然后使用生化和基因靶向方法研究验证底物亚群的功能。后一项研究至关重要，因为它们将确定内源性底物的生理意义。

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.