Dissecting the Molecular Mechanisms of Canonical Wnt Signaling

描述(由申请人提供)：癌细胞的一个中心特征是细胞在正常功能中失去调控，随后经历快速分裂(肿瘤生长)和迁移行为(肿瘤转移)的改变。一些信号分子及其信号通路的组成部分被认为是导致这些疾病的原因之一，其中之一就是Wnt蛋白。有重要证据表明Wnt信号在癌症中的作用，因为Wnt蛋白可以转化乳腺细胞，并且Wnt信号通路成分的突变，如2-catenin，已被证明在人类癌症中起到致病作用。事实上，第一个Wnt蛋白，Wnt-1，被确定为在乳腺中异常表达时导致乳腺癌的一个因素。因此，我们认为，确定Wnt信号的分子组成并了解其机制对于我们理解人类癌症的形成仍然至关重要。研究表明，Wnt信号通过细胞质蛋白紊乱(DVL)诱导2-连环蛋白(2-cat)的稳定和核移位，进而调节Wnt靶基因的转录诱导。然而，对2-CAT核转位的调控仍不明确，对2-CAT转录活性的调控也不完全清楚。该建议针对WNT信令的新调制器的特征。我们通过酵母双杂交筛选，以DVL为诱饵，鉴定了这种被称为DBP的分子，即蓬乱的结合分子。在哺乳动物细胞中，DBP与Wnt信号通路的关键成分相互作用，包括DVL和酪蛋白激酶1(CK1)，并拮抗Wnt刺激的转录诱导。DBP的细胞定位研究表明，该蛋白通常存在于细胞的胞浆和胞核中。然而，作为对Wnt刺激的反应，DBP主要定位于细胞核。此外，DBP影响2-CAT的亚细胞定位，这是Wnt-靶基因转录诱导所需的关键因素。在非洲爪哇发育过程中对DBP的体内分析扰乱了胚胎模式的形成，DBP的耗尽阻止了胚胎头部的形成。根据我们的初步研究，我们假设DBP是Wnt信号转导所必需的成分。我们在这项拨款提案中提出了两个特定的目标，我们的研究针对DBP的生化、细胞生物学和体内特性，以描绘其作用模式以及最近发现的DBP的效应器。这些研究将利用哺乳动物培养细胞和非洲爪哇胚胎作为模型系统。为此，我们的研究旨在揭示和理解Wnt信号通路的这两个新组成部分的作用。我们认为，分析这些新的成分将有助于定义Wnt信号从质膜到细胞核的分子性质，并最终导致更好地理解解除调控的Wnt信号如何导致癌症的形成。 公共卫生相关性：理解人类癌症的发展仍然依赖于识别导致这种病理的关键信号分子及其信号转导途径。已被证明在人类癌症中起致病作用的一个关键信号分子是Wnt蛋白。我们的研究集中在了解两个我们已经确定的在Wnt信号通路中发挥作用的新分子，我们认为这些研究可以为癌症的形成提供重要的新见解。

DESCRIPTION (provided by applicant): A central characteristic of cancer cells is the ability of the cells to become deregulated in their normal function and subsequently undergo rapid division (tumor growth) and changes in their migratory behavior (tumor metastasis). Several signaling molecules along with components of their signaling pathways have been implicated as causative factors for, one of these being Wnt proteins. There is important evidence for a role of Wnt signaling in cancers as Wnt proteins can transform mammary cells and mutations in components of the Wnt signaling pathway such as 2-Catenin have been shown to play causative roles in cancers in humans. In fact, the first Wnt protein, Wnt-1, was identified as a factor when aberrantly expressed in the mammary gland resulted in breast cancer. We therefore propose that identifying the molecular components and understanding the mechanisms of Wnt signaling remains crucial for our understanding of human cancer formation. Studies have shown that Wnt signaling through the cytoplasmic protein Dishevelled (Dvl) induces the stabilization and nuclear translocation of 2-catenin (2-cat), which then regulates transcriptional induction of Wnt-target genes. However, the regulation of 2-cat nuclear translocation remains poorly defined and also the regulation of 2-cat transcriptional activity is not fully understood. This proposal is directed towards the characterization of a new modulator of Wnt signaling. We have identified this molecule termed DBP, for Dishevelled Binding Molecule, via a yeast-two hybrid screen using Dvl as a bait. In mammalian cells, DBP interacts with key components of the Wnt signaling pathway including Dvl and Casein kinase 1 (CK1), and antagonizes Wnt-stimulated transcriptional induction. Cellular localization studies of DBP demonstrate that this protein is normally present in both the cytoplasmic and nuclear compartments of cells. However, in response to Wnt stimulation, DBP becomes predominantly localized to the nucleus. Furthermore, DBP influences the subcellular localization of 2- cat, a crucial factor required for the transcriptional induction of Wnt-target genes. In vivo analysis of DBP during Xenopus development perturbs embryonic pattern formation and depletion of DBP prevents embryonic head formation. Based on our preliminary studies, we hypothesize that DBP is an essential component required for Wnt signaling. We propose two specific aims in this grant proposal and our studies are directed towards a biochemical, cell biological and in-vivo characterization of DBP to delineate its mode of action along with a recently identified effector for DBP. These studies will utilize mammalian culture cells and the Xenopus embryo as model systems. Towards this end, our studies are directed towards uncovering and understanding the role of these two new components of the Wnt signaling pathway. We propose that analyzing these new components will help to define the molecular nature of Wnt signaling from the plasma membrane to the cell nucleus and ultimately lead to a better understanding of how deregulated Wnt signaling results in cancer formation. PUBLIC HEALTH RELEVANCE: Understanding the development of human cancers remains dependent on identifying key signaling molecules and their signal transduction pathways that contribute to this pathology. One key-signaling molecule that has been demonstrated to play causative roles in human cancer is the Wnt protein. Our studies are focused on understanding two new molecules that we have identified that function in the Wnt signaling pathway, and we propose that these studies can provide important new insights into cancer formation.