Regulators of Dishevelled Function in the Wnt Signaling Pathway

描述(由申请人提供)：Wnt信号通路在动物的发育和动态平衡过程中调节着一系列令人震惊的过程，它的错误调节导致了人类的许多症状和退行性疾病。Wnt配体如何激活Wnt受体复合体下游的上下文特定信号尚不清楚。这项工作的长期目标是了解在发育过程中调节上下文依赖的Wnt信号的分子机制。杂乱蛋白(DVL)是Wnt信号转导的中心枢纽，通过不同的细胞质级联整合和转导上游信号。这一谜团蛋白在Wnt途径中的调控仍然是该领域中一个长期悬而未决的问题。最近的工作强调了细胞质DVL斑点对Wnt信号通路的重要作用，但这些斑点如何影响连环蛋白核化却知之甚少。在海胆中，DVL斑点堆积在位于植物极点的卵皮质中的一个特殊区域，这些结构与植物卵裂球中Wnt/β-catenin信号的高度受限激活有关。此外，功能研究表明，DVL仅能激活植物极胚球中的Wnt/？？-catenin信号。这些观察结果表明，在Wnt/？-catenin信号转导过程中，与DVL共定位于植物皮质斑点的分子可能调节DVL的活动。申请人的实验室已开发出一种程序，以分离生化数量的海胆卵皮质与内源DVL点状附着物。因此，这项应用的总体目标是鉴定斑点蛋白中的DVL伙伴蛋白。 在胚胎发生过程中介导DVL局部激活的分离的卵皮层。中心假说是DVL斑点蛋白是局部激活Wnt/β-连环蛋白信号的支架。 早期胚胎，斑点中的DVL相关蛋白在这一途径中调节DVL的功能。这项研究的基本原理是斑点中的DVL相关蛋白很有吸引力。 DVL活性的特定背景调节的候选分子。利用实验室生产的DVL免疫试剂，利用DVL免疫共沉淀和质谱法，从鸡蛋和皮质裂解物中鉴定了几种可能的DVL相关蛋白。在这项应用中，将对其中两种蛋白质进行功能分析，以确定 它们在调节DVL在早期胚胎发育中的定位、稳定性和激活中的作用。这项拟议的研究的贡献在于确定了关键的DVL伙伴蛋白，这些蛋白可能调节其在Wnt信号中的活性。这一贡献的意义在于，它将为胚胎发育和组织内稳过程中上下文相关的DVL调节和Wnt信号的机制提供新的见解。由于Wnt信号是高度保守和广泛部署的，我们的结果可能会被外推，以更好地理解其他物种的发育方面以及许多人类疾病的分子基础。这些进展将为这些疾病的药物干预确定新的靶点。

DESCRIPTION (provided by applicant): The Wnt signaling pathway regulates an astounding array of processes during development and homeostasis in animals, and its misregulation leads to numerous syndromes and degenerative diseases in humans. How Wnt ligands can activate context-specific signals downstream of the Wnt receptor complex is not well understood. The long-term goal of this work is to understand the molecular mechanisms that mediate context- dependent Wnt signaling during development. The Dishevelled (Dvl) protein is a central hub of Wnt signal transduction that integrates and transduces upstream signals through distinct cytoplasmic cascades. The regulation of this enigmatic protein in the Wnt pathway remains one of the enduring unsolved problems in this field. Recent work has emphasized the critical importance of cytoplasmic Dvl puncta for Wnt signaling via the Wnt/?-catenin signaling pathway, but how these puncta influence ?-catenin nuclearization is poorly understood. In sea urchins, Dvl puncta accumulate in a specialized domain in the egg cortex at the vegetal pole, and these structures correlate with highly restricted activation of Wnt/??-catenin signaling in vegetal blastomeres. In addition, functional studies have shown that Dvl is only able to activate Wnt/??-catenin signaling in vegetal pole blastomeres. These observations suggest that molecules that co-localize with Dvl in puncta at the vegetal cortex may regulate Dvl activity during Wnt/??-catenin signaling. A procedure has been developed in the applicant's laboratory to isolate biochemical quantities of sea urchin egg cortices with attached endogenous Dvl puncta. Hence, the overall objective of this application is to identify Dvl partner proteins in puncta from isolated egg cortices that mediate localized activation of Dvl during embryogenesis. The central hypothesis is that Dvl puncta are scaffolds for localized activation of Wnt/??-catenin signaling in early embryos, and that Dvl-associated proteins in puncta regulate Dvl function in this pathway. The rationale that underlies this research is that Dvl-associated proteins in puncta are attractive candidate molecules for context-specific regulation of Dvl activity. Using excellent immuno-reagents to Dvl produced in the laboratory, several putative Dvl associated proteins from egg and cortex lysates have been identified using Dvl Co-Immunoprecipitation followed by mass spectrometry. In this application, two of these proteins will be functionally analyzed to determine their roles in regulating Dvl localization, stability, and activation during early embryogenesis. Te contribution of the proposed study is identification of critical Dvl partner proteins that may regulate its activity in Wnt signaling. The significance of this contribution is that it will provie novel insight into mechanisms of context-dependent Dvl regulation and Wnt signaling during embryonic development and tissue homeostasis. As Wnt signaling is highly conserved and broadly deployed, our results may be extrapolated to better understand aspects of development in other species as well as the molecular basis for many human diseases. These advances will identify novel targets for pharmacological interventions of these diseases.