The Wnt pathway plays critical roles in development, stem cell maintenance, and human disease. Over 90% of non-hereditary cases of colorectal cancer have been estimated to have mutations in the Wnt pathway that cause its inappropriate activation. In the prevailing model of Wnt signaling, stabilization of the transcription coactivato, b-catenin, upon Wnt pathway activation results in accumulation of nuclear b-catenin that displaces the co-repressor, Groucho(Gro)/TLE, from the TCF/Lef transcription factor. Binding of b-catenin to TCF/Lef ultimately leads to increased transcription of Wnt target genes. Recently, we performed a screen for ubiquitin E3 ligases and deubiquitinating enzymes that identified X-linked inhibitor of apoptosis (XIAP) and USP47, respectively, as essential mediators of Wnt signaling. We showed that XIAP binds TCF/Lef upon Wnt pathway activation and ubiquitinates Gro/TLE and that ubiquitinated Groucho/TLE has decreased affinity for TCF/Lef. Based on these studies, we propose a new model for transcriptional activation of the Wnt pathway: upon Wnt signaling, XIAP is recruited to TCF/Lef to promote ubiquitin-mediated removal of Gro/TLE from TCF/Lef, allowing for b-catein-TCF/Lef binding. The mechanism by which XIAP is recruited to TCF/Lef is unknown. We found that XIAP coimmunoprecipitates with TCF/Lef in the presence of lithium (inhibits GSK3) and that XIAP is phosphorylated by GSK3. We propose to test our hypothesis that, in the absence of Wnt signaling, GSK3 phosphorylates and inhibits the binding of XIAP to TCF/Lef. We will map the GSK3 phosphorylation sites on XIAP by mass spectrometry and explore the role of GSK3 in regulating binding of XIAP (and XIAP phosphomutants) to TCF/Lef in vitro and in cultured cells. Finally, we propose to perform mass spectrometry to identify proteins that regulate XIAP-TCF/Lef complex formation and Wnt pathway activation. Our proposed model suggests that Gro/TLE is the critical target for XIAP and that the ubiquitin ligase activity of XIAP is necessary for its Wnt pathway function. We will test our model by determining whether inhibition of Wnt signaling via downregulation of XIAP can be suppressed by downregulation of Gro/TLE and whether XIAP ubiquitination mutants have the capacity to mediate Wnt pathway activation. To elucidate the mechanism by which ubiquitination of Gro/TLE (by XIAP) regulates Gro/TLE activity, we also propose to identify the sites on Groucho/TLE ubiquitinated by XIAP and generate mutants that cannot be ubiquitinated by XIAP. Recently, we identified a deubiquitinating (DUB) enzyme, USP47, which is required for Wnt signaling in cultured cells and Xenopus embryos. We found that USP47 coimmunoprecipitates with XIAP. Because E3 ubiquitin ligases are often associated with DUBs that regulate their stability, we will test whether USP47 modulates Wnt signaling by binding to XIAP and regulating its stability and/or activity.
This proposal focuses on the regulation of Wnt signaling by X-linked Inhibitor of Apoptosis (XIAP), an E3 ubiquitin ligase that we identified in a screen for Wnt pathway components. Our preliminary studies have revealed that XIAP mediates Wnt signaling by ubiquitinating and promoting the dissociation of the corepressor, Groucho/TLE, from the TCF/Lef transcription factor. We propose to perform biochemistry, cell culture experiments, and Xenopus embryo studies to understand the mechanism by which XIAP binding to TCF/Lef is regulated in a Wnt-dependent manner.
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