Regulation of Canonical Wnt Signaling by Ubiquitylation

Abstract

Canonical Wnt signaling regulates many fundamental developmental processes and is misregulated in a variety of disease states in humans. Ubiquitylation has been shown to play critical roles in the regulation of Wnt signal transduction, and many components of the Wnt pathway are known to be ubiquitylated. At the time I began my thesis work, however, little was known about how the ubiquitin system regulates Wnt signaling. Thus, I designed a RNA interference (RNAi) screen in Drosophila S2 cells to identify novel E3 ligases and deubiquitylases involved in Wg/Wnt signaling and identified XIAP (an E3 ligase) and USP47 (a deubiquitylase) as novel Wnt pathway components.

<P>A key event in Wnt signaling is conversion of TCF/Lef from a transcriptional repressor to an activator, yet how this switch occurs is not well understood. Here, I describe an unanticipated role for X-linked Inhibitor of Apoptosis (XIAP) in regulating this critical Wnt signaling event that is independent of its anti-apoptotic function. I identified DIAP1 as a positive regulator of Wingless signaling in a Drosophila S2 cell-based RNAi screen and show that XIAP, its vertebrate homolog, is similarly required for Wnt signaling in cultured mammalian cells and in Xenopus embryos, indicating evolutionary conservation of function. I also demonstrate that upon Wnt pathway activation, XIAP is recruited to TCF/Lef where it mono-ubiquitylates Groucho/TLE: this modification decreases the affinity of Groucho/TLE for TCF/Lef. These data reveal a transcriptional switch involving XIAP-mediated ubiquitylation of Groucho/TLE that facilitates its removal from TCF/Lef, thus allowing assembly of β-catenin-TCF/Lef complexes and initiation of a Wnt-specific transcriptional program.</P>

<P>In addition to the discovery of XIAP as a novel Wnt pathway component, I also identified the de-ubiquitylase Ubp64E as a positive regulator of Wingless signaling in the Drosophila S2 cell-based RNAi screen. USP47, its vertebrate homolog, is similarly required for Wnt signaling in cultured mammalian cells and in Xenopus embryos, indicating evolutionary conservation of function. My data indicate that USP47 likely functions at the level of transcription in the nucleus potentially through its interaction with the E3 ligases β-TRCP or XIAP or an as yet unidentified target.</P>