title: Huwe1-mediated ubiquitylation of dishevelled defines a negative feedback loop in the Wnt signaling pathway.
abstract: Wnt signaling plays a central role in development, adult tissue homeostasis, and cancer. Several steps in the canonical Wnt/β-catenin signaling cascade are regulated by ubiquitylation, a protein modification that influences the stability, subcellular localization, or interactions of target proteins. To identify regulators of the Wnt/β-catenin pathway, we performed an RNA interference screen in Caenorhabditis elegans and identified the HECT domain-containing ubiquitin ligase EEL-1 as an inhibitor of Wnt signaling. In human embryonic kidney 293T cells, knockdown of the EEL-1 homolog Huwe1 enhanced the activity of a Wnt reporter in cells stimulated with Wnt3a or in cells that overexpressed casein kinase 1 (CK1) or a constitutively active mutant of the Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6). However, knockdown of Huwe1 had no effect on reporter gene expression in cells expressing constitutively active β-catenin, suggesting that Huwe1 inhibited Wnt signaling upstream of β-catenin and downstream of CK1 and LRP6. Huwe1 bound to and ubiquitylated the cytoplasmic Wnt pathway component Dishevelled (Dvl) in a Wnt3a- and CK1ε-dependent manner. Mass spectrometric analysis showed that Huwe1 promoted K63-linked, but not K48-linked, polyubiquitination of Dvl. Instead of targeting Dvl for degradation, ubiquitylation of the DIX domain of Dvl by Huwe1 inhibited Dvl multimerization, which is necessary for its function. Our findings indicate that Huwe1 is part of an evolutionarily conserved negative feedback loop in the Wnt/β-catenin pathway.
authors.affiliation: 1Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center Utrecht, Uppsalalaan 8, 3584CT Utrecht, the Netherlands.
authors.forename: Reinoud E A
authors.initials: RE
authors.lastname: de Groot
authors.forename: Ranjani S
authors.initials: RS
authors.lastname: Ganji
authors.forename: Ondrej
authors.initials: O
authors.lastname: Bernatik
authors.forename: Bethan
authors.initials: B
authors.lastname: Lloyd-Lewis
authors.forename: Katja
authors.initials: K
authors.lastname: Seipel
authors.forename: Kateřina
authors.initials: K
authors.lastname: Šedová
authors.forename: Zbyněk
authors.initials: Z
authors.lastname: Zdráhal
authors.forename: Vishnu M
authors.initials: VM
authors.lastname: Dhople
authors.forename: Trevor C
authors.initials: TC
authors.lastname: Dale
authors.forename: Hendrik C
authors.initials: HC
authors.lastname: Korswagen
authors.forename: Vitezslav
authors.initials: V
authors.lastname: Bryja
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism; D000072261:Dishevelled Proteins; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D010750:Phosphoproteins / Q000378:metabolism*; D034622:RNA Interference; D015398:Signal Transduction; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D060449:Wnt Signaling Pathway; D051176:beta Catenin / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D029742:Caenorhabditis elegans Proteins; D000072261:Dishevelled Proteins; D010750:Phosphoproteins; D025521:Tumor Suppressor Proteins; D051176:beta Catenin; C521393:EEL-1 protein, C elegans; C502830:HUWE1 protein, human; D044767:Ubiquitin-Protein Ligases