PROJECT SUMMARY: The evolutionarily conserved Wnt pathway plays a critical role during metazoan development and stem cell maintenance in the adult. Mutations in the Wnt pathway leading to its misregulation in humans have been shown to contributes to both developmental defects and cancers. In the latter case, over 90% of all non-hereditary forms of colorectal cancers are initiated by mutations in the Wnt pathway leading to its inappropriate activation. There are no FDA-approved drugs that inhibit the Wnt pathway, which is likely due to our incomplete understanding of the mechanism of Wnt signaling and the lack of suitable drug targets. The primary focus of my lab is to understand the biological function of the Wnt signaling pathway by deciphering its molecular mechanism and identifying new Wnt components. Our ultimate goal is to use this information to understand how deregulation of the Wnt pathway can lead to Wnt- driven diseases in humans. Over more than a decade, my lab has made significant contributions to our fundamental understanding of Wnt signaling. These breakthroughs were accomplished via the development of the first biochemical system (using Xenopus egg extract), the reconstitution of active purified proteins, the development of the first mathematical model (Lee-Heinrich model), and the identification of small molecule inhibitors (CK1 agonists), one of which has been designated by the FDA as an orphan drug for a familial precancerous disease (familial adenomatous polyposis). Our current work focused on determining the role of the USP46/UAF1/WDR20 deubiquitinase complex in regulating LRP6 receptor turnover, the mechanism by which APC regulates Wnt receptor activity, the role of the -catenin degradation complex in mediating bistable pathway behavior, and the roles of USP47 and STK38, in regulating Wnt signaling. In our renewal application, we now propose to 1) reconstitute LRP6 ubiquitylation and identify its evolutionarily conserved E3 ligase, 2) understand the basis for signal compensation upon Wnt receptor loss in APC mutant cells, 3) provide evidence for the role of bistability in generating morphogenetic signaling, and 4) uncover the roles of the STK38 and TRIP12 in nuclear Wnt signaling.