Regulators of Dishevelled Function in the Wnt Signaling Pathway

描述（由申请人提供）：在动物的发育和体内平衡过程中，Wnt信号通路调节了一系列惊人的过程，其失调导致人类许多综合征和退行性疾病。Wnt配体如何激活Wnt受体复合物下游的上下文特异性信号尚不清楚。这项工作的长期目标是了解在发育过程中介导环境依赖性Wnt信号传导的分子机制。disheveled （Dvl）蛋白是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。这种神秘蛋白在Wnt通路中的调控仍然是该领域长期未解决的问题之一。最近的研究强调了细胞质Dvl点对通过Wnt/？-catenin信号通路，但这些点是如何影响的呢？-连环蛋白的核化鲜为人知。在海胆中，斑Dvl聚集在植物极卵皮质的一个特殊区域，这些结构与高度受限的Wnt/？植物卵裂球中的-catenin信号。此外，功能研究表明，Dvl仅能激活Wnt/?？植物极卵裂球中的-连环蛋白信号。这些观察结果表明，植物皮质点上与Dvl共定位的分子可能在Wnt/?? ？连环蛋白信号。申请人的实验室已发展出一套程序，以分离海胆卵皮质的生化量，并附上内源性的小点藻。因此，本应用程序的总体目标是鉴定斑点中的Dvl伴侣蛋白

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.