Regulators of Dishevelled Function in the Wnt Signaling Pathway

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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.