Regulators of Dishevelled Function in the Wnt Signaling Pathway

描述（由申请人提供）：Wnt信号通路在动物发育和体内平衡期间调节一系列惊人的过程，其失调导致人类多种综合征和退行性疾病。Wnt配体如何激活Wnt受体复合物下游的环境特异性信号还不清楚。这项工作的长期目标是了解在发育过程中介导背景依赖性Wnt信号传导的分子机制。Dishevelled（Dvl）蛋白是Wnt信号转导的中心枢纽，其通过不同的细胞质级联整合和转导上游信号。Wnt通路中这种神秘蛋白的调节仍然是该领域中持久未解决的问题之一。最近的工作强调了细胞质Dvl点对于Wnt信号转导的重要性。连环蛋白信号通路，但这些斑点如何影响？对连环蛋白核化了解甚少。在海胆中，Dvl puncta在植物极的卵皮质中的专门结构域中积累，并且这些结构与高度限制的Wnt/puncta激活相关。植物性卵裂球中的连环蛋白信号。此外，功能研究表明，Dvl仅能够激活Wnt/β？植物极卵裂球中的连环蛋白信号。这些观察结果表明，与Dvl共定位在植物皮层斑点中的分子可能在Wnt/β-Dvl过程中调节Dvl活性。连环蛋白信号传导。在申请人的实验室中已经开发了一种方法来分离具有附着的内源性Dvl斑点的海胆卵皮质的生化量。因此，本申请的总体目标是鉴定来自大肠杆菌的斑点中的Dvl配偶体蛋白。 在胚胎发生期间介导Dvl的局部激活的分离的卵皮质。中心假设是Dvl点是Wnt/p53局部激活的支架。catenin信号通路 早期胚胎，Dvl相关蛋白在斑点调节Dvl在这一途径中的功能。这项研究的基本原理是，Dvl相关蛋白在斑点是有吸引力的 Dvl活性的背景特异性调节的候选分子。使用实验室中产生的针对Dvl的优良免疫试剂，已经使用Dvl共免疫沉淀随后质谱鉴定了来自卵和皮质裂解物的几种推定的Dvl相关蛋白。在本申请中，将对这些蛋白质中的两种进行功能分析，以确定 它们在早期胚胎发生过程中调节Dvl定位、稳定性和激活的作用。所提出的研究的贡献是鉴定可能调节其在Wnt信号传导中的活性的关键Dvl伴侣蛋白。这一贡献的意义在于，它将提供新的见解的背景依赖性Dvl调控和Wnt信号在胚胎发育和组织稳态的机制。由于Wnt信号是高度保守和广泛部署的，我们的研究结果可以外推到更好地了解其他物种的发展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。 公共卫生相关性：拟议的研究与公共卫生有关，因为发现Wnt信号传导的背景依赖性激活的新机制将增加对当Wnt信号传导出错时人类如何发展特定退行性疾病和癌症的理解。因此，拟议中的研究与NIH的使命的一部分有关，该使命涉及开发有助于治疗人类疾病的基础知识。Dvl蛋白的背景依赖性调节物的鉴定将在Wnt信号传导在人类疾病进展期间被错误调节时提供用于治疗干预的额外靶标。

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. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because the discovery of novel mechanisms of context- dependent activation of Wnt signaling will increase understanding of how specific degenerative diseases and cancers develop in humans when Wnt signaling goes awry. Thus, the proposed research is relevant to the part of NIH's mission that pertains to developing fundamental knowledge that will help with treatment of human disease. The identification of context-dependent regulators of the Dvl protein will provide additional targets for therapeutic intervention when Wnt signaling is misregulated during human disease progression.