Ubiquitylation in Canonical Wnt Signaling

Abstract

The Wnt/b-catenin (canonical) signaling pathway is an evolutionarily conserved pathway that is critical for normal development, and misregulation of Wnt signaling can lead to diseases in humans. Many components of the Wnt pathway undergo post-translation modification to control key signaling events. Herein, I describe how phosphorylation of the ubiquitin ligase XIAP by GSK3 and how deubiquitylation of the Wnt co-receptor LRP6 by the USP46 complex promote canonical Wnt signaling. Our lab had previously identified XIAP as a positive nuclear regulator of Wnt signaling and demonstrated that upon Wnt activation, XIAP monoubiquitylates the transcriptional co-repressor, Gro/TLE. The mechanism by which XIAP is recruited to monoubiquitylate Gro/TLE was unknown. It was previously shown that GSK3 interacts with XIAP. Thus, I explored whether GSK3 phosphorylates XIAP and whether XIAP phosphorylation was important for its activity in Wnt signaling. Using an in vitro kinase assay followed by mass spectrometry analysis, multiple phosphorylation sites were identified. Mutational analysis indicated that threonine 180 on XIAP is critical for its activity in the Wnt pathway in vitro and in vivo. XIAPT180A showed a decreased interaction and ubiquitylation of Gro/TLE in mammalian cells. In apoptosis assays, XIAPT180A exhibited identical activity to wildtype XIAP, indicating that phosphorylation of threonine 180 on XIAP is not required for its role in apoptosis. In a separate set of studies, I determined the mechanism by which the Wnt co-receptor, LRP6, is deubiquitylated. Ubiquitylation of the Wnt receptors, Frizzled and LRP6, had previously been shown to be important for their stability and activity. Although the E3 ligases have been identified, the deubiquitylases were not known. Our lab identified members of the USP46 complex (USP46, WDR20, and UAF1) as positive regulators in a screen for novel Wnt components. I found that the USP46 complex interacts with and deubiquitylates LRP6 to promote Wnt signaling. Cell-based and biochemical studies show that the USP46 complex plays a critical role in stabilizing the Wnt co-receptor, LRP6 as well as promoting the formation of “LRP6 signalosomes” during Wnt signaling. Together, regulation of XIAP and the USP46 complex highlight the role of post-translational mechanisms in controlling canonical Wnt signaling.