The USP46 complex deubiquitylates LRP6 to promote Wnt/ß-catenin signaling.

The relative abundance of Wnt receptors plays a crucial role in controlling Wnt signaling in tissue homeostasis and human disease. While the ubiquitin ligases that ubiquitylate Wnt receptors are well-characterized, the deubiquitylase that reverses these reactions remains unclear. Herein, we identify USP46, UAF1, and WDR20 (USP46 complex) as positive regulators of Wnt signaling in cultured human cells. We find that the USP46 complex is similarly required for Wnt signaling in Xenopus and zebrafish embryos. We demonstrate that Wnt signaling promotes the association between the USP46 complex and cell surface Wnt coreceptor, LRP6. Knockdown of USP46 decreases steady-state levels of LRP6 and increases the level of ubiquitylated LRP6. In contrast, overexpression of the USP46 complex blocks ubiquitylation of LRP6 by the ubiquitin ligases RNF43 and ZNFR3. Size exclusion chromatography studies suggest that the size of the USP46 cytoplasmic complex increases upon Wnt stimulation. Finally, we show that USP46 is essential for Wnt-dependent intestinal organoid viability, likely via its role in LRP6 receptor homeostasis. We propose a model in which the USP46 complex increases the steady-state level of cell surface LRP6 and facilitates the assembly of LRP6 into signalosomes via a pruning mechanism that removes sterically hindering ubiquitin chains.

USP47 deubiquitylates Groucho/TLE to promote Wnt-ß-catenin signaling.

The Wnt-ß-catenin signal transduction pathway is essential for embryonic development and adult tissue homeostasis. Wnt signaling converts TCF from a transcriptional repressor to an activator in a process facilitated by the E3 ligase XIAP. XIAP-mediated monoubiquitylation of the transcriptional corepressor Groucho (also known as TLE) decreases its affinity for TCF, thereby allowing the transcriptional coactivator ß-catenin to displace it on TCF. Through a genome-scale screen in cultured Drosophila melanogaster cells, we identified the deubiquitylase USP47 as a positive regulator of Wnt signaling. We found that USP47 was required for Wnt signaling during Drosophila and Xenopus laevis development, as well as in human cells, indicating evolutionary conservation. In human cells, knockdown of USP47 inhibited Wnt reporter activity, and USP47 acted downstream of the ß-catenin destruction complex. USP47 interacted with TLE3 and XIAP but did not alter their amounts; however, knockdown of USP47 enhanced XIAP-mediated ubiquitylation of TLE3. USP47 inhibited ubiquitylation of TLE3 by XIAP in vitro in a dose-dependent manner, suggesting that USP47 is the deubiquitylase that counteracts the E3 ligase activity of XIAP on TLE. Our data suggest a mechanism by which regulated ubiquitylation and deubiquitylation of TLE enhance the ability of ß-catenin to cycle on and off TCF, thereby helping to ensure that the expression of Wnt target genes continues only as long as the upstream signal is present.