Regulation of the Wnt/Wingless receptor LRP6/Arrow by the deubiquitylating complex Usp46

The evolutionarily conserved Wnt/Wingless signal transduction pathway is critical for the proper development of all animals and implicated in numerous diseases in adulthood. Upon binding of the Wnt/Wingless ligand, a cascade of events culminates in inactivation of the destruction complex, a negative regulator of the pathway, and the subsequent formation of singalosomes which mediate pathway activation. A critical component of signalosome formation is the Wnt/Wingless receptor LRP6/Arrow. Upon canonical pathway activation, LRP6/Arrow undergoes activation via phosphorylation by several kinases and complexes with another Wnt/Wingless receptor Frizzled, along with several cytoplasmic components. While many studies have investigated the regulatory mechanisms of ligand synthesis and secretion, Wnt/Wingless receptor regulation remains incompletely understood. Here we describe a role for the evolutionarily conserved Usp46-Uaf1-Wdr20 deubiquitylating complex in Wingless signaling. Analysis of RNAi-mediated knockdown of the complex in the Drosophila third instar larval wing disc revealed the requirement of its individual components for proper Wingless pathway activation. Next, CRISPR-generated Drosophila lines harboring null mutant alleles of Usp46, Uaf1, and Wdr20 facilitated in vivo analysis of each component’s conditional loss in the adult Drosophila intestine. We identified the requirement of the Usp46 complex for tightly controlled Wingless concentration-dependent responses necessary for proper signaling. In cells where the Usp46 complex was inactivated, we observed decreased levels of both Arrow andβ-catenin/Armadillo, the Wnt/Wingless signaling pathway’s primary transcription factor. Thus, the proper control of precise Wnt/Wingless signal transduction within a Wnt/Wingless morphogen gradient requires the Usp46 complex for stabilization of Arrow and consequent accumulation of Arm/β-catenin. We also investigated the mechanism through which the Usp46 complex acts on Arrow using several cell culture methods. We found the enzymatic deubiquitylation of Arrow to be crucial in its regulation and that the Usp46 complex deubiquitylates mature Arrow involved in Wingless signal transduction. Together, these findings suggest a model where the deubiquitylation of plasma membrane-associated Arrow by Usp46 acts to stabilize the receptor and potentiate Wingless signaling