Abstract
Many adaptor proteins involved in endocytic cargo transport exhibit additional functions in other cellular processes which may be either related to or independent from their trafficking roles. The endosomal adaptor protein Tollip is an example of such a multitasking regulator, as it participates in trafficking and endosomal sorting of receptors, but also in interleukin/Toll/NF-κB signaling, bacterial entry, autophagic clearance of protein aggregates and regulation of sumoylation. Here we describe another role of Tollip in intracellular signaling. By performing a targeted RNAi screen of soluble endocytic proteins for their additional functions in canonical Wnt signaling, we identified Tollip as a potential negative regulator of this pathway in human cells. Depletion of Tollip potentiates the activity of β-catenin/TCF-dependent transcriptional reporter, while its overproduction inhibits the reporter activity and expression of Wnt target genes. These effects are independent of dynamin-mediated endocytosis, but require the ubiquitin-binding CUE domain of Tollip. In Wnt-stimulated cells, Tollip counteracts the activation of β-catenin and its nuclear accumulation, without affecting its total levels. Additionally, under conditions of ligand-independent signaling, Tollip inhibits the pathway after the stage of β-catenin stabilization, as observed in human cancer cell lines, characterized by constitutive β-catenin activity. Finally, the regulation of Wnt signaling by Tollip occurs also during early embryonic development of zebrafish. In summary, our data identify a novel function of Tollip in regulating the canonical Wnt pathway which is evolutionarily conserved between fish and humans. Tollip-mediated inhibition of Wnt signaling may contribute not only to embryonic development, but also to carcinogenesis. Mechanistically, Tollip can potentially coordinate multiple cellular pathways of trafficking and signaling, possibly by exploiting its ability to interact with ubiquitin and the sumoylation machinery.
Highlights
Adaptor proteins act as molecular scaffolds in various intracellular processes [1]
We employed a library of endoribonuclease-prepared short interfering RNAs to downregulate expression of the selected genes in HEK293 cells stimulated with Wnt3a
Our results identify a novel inhibitory activity of Tollip towards canonical Wnt signaling in mammalian cells and in zebrafish embryonic development
Summary
Adaptor proteins act as molecular scaffolds in various intracellular processes [1]. Usually lacking enzymatic activities, adaptors mediate protein-protein and protein-lipid interactionsPLOS ONE | DOI:10.1371/journal.pone.0130818 June 25, 2015Tollip Inhibits Canonical Wnt SignalingFoundation for Polish Science within the International PhD Project 'Studies of nucleic acids and proteins from basic to applied research', co-financed from the European Union - Regional Development Fund. Adaptor proteins act as molecular scaffolds in various intracellular processes [1]. Foundation for Polish Science within the International PhD Project 'Studies of nucleic acids and proteins from basic to applied research', co-financed from the European Union - Regional Development Fund. Work in the lab of MGG was supported by the Departement d’Instruction Publique of the Canton of Geneva Ch), ERC advanced grants (Sara and Morphogen; http://erc.europa.eu), the NCCR Frontiers in Genetics and Chemical Biology programs (www.snf.ch/en/ funding/programmes/national-centres-ofcompetence-in-research-nccr/Pages/default.aspx) and a grant from Switzerland through the Swiss Contribution to the Enlarged European Union (PolishSwiss Research Programme project PSPB-094/2010; www.swiss.opi.org.pl). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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