Regulation of cell homeostasis by fbx4

描述(申请人提供)：我研究的长期目标集中在阐明细胞外信号被细胞周期机制感知并调节正常细胞与肿瘤细胞的细胞周期进程的机制。这些信息将提供必要的框架，以阐明生长调节通路在肿瘤中是如何被颠覆的。我们目前的研究集中在生长信号通路如何调节有丝分裂反应的D型细胞周期蛋白，更具体地说，这些通路如何调节正常细胞和癌细胞中活性的、依赖于细胞周期蛋白D1的激酶的积累。细胞周期蛋白D1在多种人类肿瘤中的过度表达凸显了阐明调控细胞周期蛋白D1活性的机制的重要性。虽然细胞周期蛋白D1的过度表达是一组癌症中基因扩增和染色体易位的结果，但依赖于其对Thr286的磷酸化的细胞周期蛋白D1蛋白降解减少被认为是大多数癌症的关键因素。由于缺乏关于指导细胞周期蛋白D1蛋白分解的E3泛素连接酶的性质的信息，我们在表征细胞周期蛋白D1在癌症中过度表达的这一机制方面的进展受到了阻碍。我们最近发现了控制细胞周期蛋白D1泛素化和降解的SCF(Fbx4-(B晶状体蛋白))。这一发现为评估在正常组织发育过程中细胞周期蛋白D1的积聚调控机制以及在恶性转化过程中细胞周期蛋白D1的潜在失控机制提供了重要的信息和工具。对这种E3连接酶的鉴定以及我们最近的初步研究导致了一个重要的假设，即SCF(Fbx4-(B晶状体蛋白)连接酶通过Fbx4和(B晶状体蛋白)对磷酸化细胞周期蛋白D1的协同识别，在维持细胞生长和组织维持中发挥关键作用。这项资助中提议的实验将确定Fbx4对体内组织动态平衡的贡献(目标1)。我们将利用Fbx4-/-细胞来最终确定Fbx4在调节细胞周期蛋白D1和细胞增殖中的作用(目标2)。底物识别所需的Fbx4残基的鉴定将通过在黑色素瘤中鉴定突变的Fbx4等位基因来鉴定。 公共卫生相关性：细胞周期蛋白D1在人类癌症中的过度表达经常是由于破坏细胞周期蛋白D1蛋白的机制发生突变所致。为了开发有效的治疗方法来对抗这些事件，有必要确定指导细胞周期蛋白D1破坏的蛋白质，并确定它们在肿瘤发生发展中的作用。我们已经确定了该机制的一个关键组件，Fbx4，它指导着细胞周期蛋白D1的破坏，本提案中描述的实验将评估Fbx4在人类癌症方面的生化和生物学特性。

DESCRIPTION (provided by applicant): The long-term objective of my research centers on elucidation of the mechanisms whereby extra-cellular signals are sensed by the cell cycle machinery and regulate cell cycle progression in normal versus tumor cells. This information will provide the necessary framework to elucidate how growth regulatory pathways are subverted in neoplasia. Our current studies focus on how growth-signaling pathways regulate the mitogenically responsive D-type cyclins and more specifically, how these pathways regulate accumulation of an active, nuclear cyclin D1-dependent kinase in normal versus cancerous cells. The noted overexpression of cyclin D1 in multiple human cancers highlights the importance of elucidating the mechanisms that regulate cyclin D1 activity. While cyclin D1 overexpression is a consequence of gene amplification and chromosome translocation in a subset of cancers, decreased cyclin D1 protein degradation, which depends on its phosphorylation on Thr286, is thought to be the key factor in a majority of cancers. Our progress in the characterization of this mechanism of cyclin D1 overexpression in cancer has been hindered by lack of information regarding the nature of the E3 ubiquitin ligase that directs cyclin D1 proteolysis. We have recently identified the SCF(Fbx4-(B crystallin) that controls cyclin D1 ubiquitination and degradation. This discovery has provided essential information and tools to assess the mechanisms that contribute to regulation of cyclin D1 accumulation during normal tissue development and the potential deregulation of cyclin D1 during malignant transformation. The identification of this E3 ligase as well as our recent preliminary studies lead to the overarching hypothesis that the SCF(Fbx4 -(B crystallin) ligase, via coordinated recognition of phospho- cyclin D1 by Fbx4 and (B crystallin, plays a critical role in the maintenance of cell growth and tissue maintenance. Experiments proposed in this grant will determine the contribution of Fbx4 to tissue homeostasis in vivo (Aim 1). We will utilize fbx4-/- cells to conclusively define the role of Fbx4 in the regulation of cyclin D1 and cell proliferation (Aim 2). The identification of residues in Fbx4 necessary for substrate recognition will be identified through characterization of mutant Fbx4 alleles identified in melanoma. PUBLIC HEALTH RELEVANCE: Overexpression of cyclin D1 in human cancer occurs frequently as a consequence of mutations in the machinery that destroys the cyclin D1 protein. In order to develop effective therapies that counter these events, it is necessary to identify proteins that direct cyclin D1 destruction and determine their role in the development of neoplasia. We have identified a critical component of the machinery, Fbx4, which directs destruction of the cyclin D1 protein, and the experiments described in this proposal will evaluate the biochemical and biological properties of Fbx4 with respect to human cancer.