Regulation of cell homeostasis by fbx4

描述（由申请人提供）：我的研究的长期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来阐明肿瘤中生长调节途径是如何被破坏的。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性 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性核细胞周期蛋白 D1 依赖性激酶的积累。多种人类癌症中细胞周期蛋白 D1 的过度表达凸显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 过度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（取决于其在 Thr286 上的磷酸化）被认为是大多数癌症的关键因素。由于缺乏有关指导细胞周期蛋白 D1 蛋白水解的 E3 泛素连接酶性质的信息，我们在表征癌症中细胞周期蛋白 D1 过度表达的机制方面取得的进展受到阻碍。我们最近鉴定了控制细胞周期蛋白 D1 泛素化和降解的 SCF(Fbx4-(B crystallin)。这一发现为评估正常组织发育过程中细胞周期蛋白 D1 积累的调节机制以及恶性转化过程中细胞周期蛋白 D1 的潜在失调的机制提供了重要的信息和工具。这种 E3 连接酶的鉴定以及我们最近的初步研究得出了一个总体假设：SCF(Fbx4 -(B crystallin)连接酶通过 Fbx4 和 (B crystallin) 协调识别磷酸周期蛋白 D1，在维持细胞生长和组织维持中发挥着关键作用。本次资助中提出的实验将确定 Fbx4 对体内组织稳态的贡献（目标 1）。我们将利用 fbx4-/- 细胞最终确定 Fbx4 在调节周期蛋白 D1 和细胞增殖中的作用（目标 2）。残基的鉴定通过对黑色素瘤中鉴定的突变 Fbx4 等位基因的表征，将鉴定 Fbx4 中底物识别所需的基因。 公共卫生相关性：人类癌症中细胞周期蛋白 D1 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而导致的。为了开发对抗这些事件的有效疗法，有必要鉴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展中的作用。我们已经确定了该机制的关键组成部分 Fbx4，它指导细胞周期蛋白 D1 蛋白的破坏，并且本提案中描述的实验将评估 Fbx4 对于人类癌症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.