Normal and Neoplastic Regulation of Cyclin E

描述(申请人提供)：细胞周期蛋白E控制细胞周期从G1期到S期的转变。细胞周期蛋白E活性异常会导致细胞分裂异常，而导致细胞周期蛋白E-CDK2调控异常的突变在大多数人类癌症中都被发现。这项建议中描述的研究试图了解在正常细胞和肿瘤中控制细胞周期蛋白E的机制。我们对细胞周期调控和肿瘤发生中细胞周期蛋白E磷酸化的调节和作用特别感兴趣。多个位点特异性的磷酸化控制细胞周期蛋白E，但特定的有丝分裂和信号转导通路在调节这些磷酸化中的作用尚不清楚。第一个目标是研究细胞周期蛋白E在正常细胞和肿瘤细胞中的磷酸化调控。我们将开发识别Cyclin E磷酸化形式的抗体，这些试剂将用于检测正常细胞中Cyclin E的磷酸化，并确定其在肿瘤细胞中是否异常。我们对磷酸化的细胞周期蛋白E和Fbw7泛素连接酶之间的关系特别感兴趣。由于大多数关于细胞周期蛋白E磷酸化的研究都是利用过表达的细胞周期蛋白E，所以很难区分磷酸化的作用和过度表达的后果。因此，我们将使用“敲入”模型，其中磷酸化位点在内源性细胞周期蛋白E基因座的背景下发生突变，以研究特定磷酸化的功能。这些研究将在小鼠身上使用同源重组技术，并在人类细胞中开发基于腺相关病毒载体的新方法。后一种方法可能广泛适用于在不需要或不需要小鼠模型的系统中进行蛋白质磷酸化的研究。细胞周期蛋白E相关的肿瘤发生机制目前尚不清楚。这一目标的总体目标是使用Cyclin E转基因和敲入菌株来建立Cyclin E相关癌症的模型。由于我们已经发现了一种稳定的p53依赖的反应，可以抑制细胞周期蛋白E的过度活性，所以我们将专门检验这样一种假设，即p53功能的丧失是细胞周期蛋白E表达异常的肿瘤发生发展中不可或缺的一步。这些模型将被用来研究周期蛋白E相关的肿瘤发生机制，并可能促进新的治疗策略的开发。

DESCRIPTION (provided by applicant): The cyclin E protein controls the transition from the G1 to the S-phase of the cell cycle. Deregulated cyclin E activity causes abnormal cell division, and mutations leading to aberrant cyclin E-cdk2 regulation are found in most human cancers. The research described in this proposal seeks to understand the mechanisms that govern cyclin E in normal cells and tumors. We are particularly interested in the regulation and function of cyclin E phosphorylation in cell cycle control and tumorigenesis. Multiple site-specific phosphorylations control cyclin E, but the role of specific mitogenic and signal transduction pathways in regulating these phosphorylations is unknown. The goal of the first aim is to characterize the regulation of cyclin E phosphorylation in normal cells and in tumor cells. We will develop antibodies that recognize phosphorylated forms of cyclin E, and these reagents will be used to examine cyclin E phosphorylation in normal cells, and to determine if it is abnormal in tumor cells. We are particularly interested in the relationships between phosphorylated cyclin E and the Fbw7 ubiquitin ligase. Because most studies on cyclin E phosphorylation have utilized overexpressed cyclin E, it has been difficult to distinguish the role of phosphorylation from the consequences of overexpression. We will thus use "knock-in" models in which phosphorylation sites are mutated in the context of the endogenous cyclin E locus to study the function of specific phosphorylations. These studies will use homologous recombination techniques in mice, as well as develop new methods based on adeno-associated virus vectors in human cells. The latter method may be broadly applicable to studies of protein phosphorylation in systems where mouse models are unnecessary or undesirable. The mechanisms of cyclin E-associated tumorigenesis are largely unknown. The overall goal of this aim is to use cyclin E transgenic and knock-in strains to develop models of cyclin E-associated cancer. Because we have discovered a homeostatic p53-dependent response that restrains excess cyclin E activity, we will specifically test the hypothesis that loss of p53 function is an integral step in the development of tumors with deregulated cyclin E expression. These models will be used to study the mechanisms of cyclin E-associated tumorigenesis and may facilitate the development of new therapeutic strategies.