Regulation of cell homeostasis by fbx4

描述（由申请人提供）：我的长期研究目标集中在阐明细胞周期机制感知细胞外信号并调节正常细胞和肿瘤细胞周期进程的机制。这一信息将为阐明肿瘤生长调控通路是如何被破坏提供必要的框架。我们目前的研究重点是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与癌细胞中活跃的核细胞周期蛋白d1依赖性激酶的积累。细胞周期蛋白D1在多种人类癌症中的过度表达突出了阐明调节细胞周期蛋白D1活性的机制的重要性。虽然细胞周期蛋白D1过表达是一部分癌症中基因扩增和染色体易位的结果，但细胞周期蛋白D1蛋白降解的减少（依赖于其在Thr286上的磷酸化）被认为是大多数癌症的关键因素。由于缺乏关于E3泛素连接酶的性质的信息，我们在癌症中细胞周期蛋白D1过表达机制的表征方面的进展受到阻碍，该泛素连接酶指导细胞周期蛋白D1蛋白水解。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结晶蛋白））。这一发现为评估正常组织发育过程中cyclin D1积累的调节机制以及恶性转化过程中cyclin D1的潜在失调提供了必要的信息和工具。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总体假设，即SCF(Fbx4 -（B结晶蛋白）连接酶通过Fbx4和（B结晶蛋白）协调识别磷酸化细胞周期蛋白D1，在维持细胞生长和组织维持中起着关键作用。本基金提出的实验将确定Fbx4对体内组织稳态的贡献（目的1）。我们将利用fbx4-/-细胞最终确定fbx4在调节cyclin D1和细胞增殖中的作用（目的2）。Fbx4中识别底物所必需的残基的鉴定将通过对黑色素瘤中发现的突变Fbx4等位基因的表征来确定。

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.