Protease associated domain of RNF43 is not necessary for the suppression of Wnt/\u03b2-catenin signaling in human cells

Tomasz Radaszkiewicz,Vítězslav Bryja

doi:10.1186/s12964-020-00559-0

Abstract

BackgroundRNF43 and its homolog ZNRF3 are transmembrane E3 ubiquitin ligases frequently mutated in many human cancer types. Their main role relays on the inhibition of canonical Wnt signaling by the negative regulation of frizzled receptors and LRP5/6 co-receptors levels at the plasma membrane. Intracellular RING domains of RNF43/ZNRF3 mediate the key enzymatic activity of these proteins, but the function of the extracellular Protease Associated (PA) fold in the inhibition of Wnt/β-catenin pathway is controversial up-to date, apart from the interaction with secreted antagonists R-spondin family proteins shown by the crystallographic studies.MethodsIn our research we utilised cell-based approaches to study the role of RNF43 lacking PA domain in the canonical Wnt signalling pathway transduction. We developed controlled overexpression (TetON) and CRISPR/Cas9 mediated knock-out models in human cells.ResultsRNF43ΔPA mutant activity impedes canonical Wnt pathway, as manifested by the reduced phosphorylation of LRP6, DVL2 and DVL3 and by the decreased β-catenin-dependent gene expression. Finally, rescue experiments in the CRISPR/Cas9 derived RNF43/ZNRF3 double knock-out cell lines showed that RNFΔPA overexpression is enough to inhibit activation of LRP6 and β-catenin activity as shown by the Western blot and Top flash dual luciferase assays. Moreover, RNF43 variant without PA domain was not sensitive to the R-spondin1 treatment.ConclusionTaken together, our results help to understand better the mode of RNF43 tumor suppressor action and solve some discrepancies present in the field.1QAtRBXJHQCFGvAUYCKAWHVideo Graphical

Highlights

Really Interesting New Gene (RING) finger protein 43 (RNF43) and its homolog Zinc/ Ring finger protein 3 (ZNRF3) are transmembrane E3 ubiquitin ligases frequently mutated in many human cancer types
Inducible RNF43 and RNF43ΔPA expression in T-REx 293 cell line In order to address the role of Protease Associated (PA) domain of RNF43 we generated T-Rex 293 stable cell lines overexpressing human wild type RNF43 and RNF43 variant lacking its PA domain (Δ87–187), RNF43ΔPA, in a tetracycline (Tet)-controlled manner (TetON) (Fig. 1a)
This was confirmed in the Top flash dual luciferase assay, by which we showed that RNF43ΔPA was not sensitive to the recombinant RSPO1 treatment

Summary

Introduction

RNF43 and its homolog ZNRF3 are transmembrane E3 ubiquitin ligases frequently mutated in many human cancer types. Their main role relays on the inhibition of canonical Wnt signaling by the negative regulation of frizzled receptors and LRP5/6 co-receptors levels at the plasma membrane. RING finger protein 43 (RNF43) and its homolog Zinc/ Ring finger protein 3 (ZNRF3) are E3 ubiquitin ligases belonging to the PA-TM-RING family [1]. These enzymes bear a resemblance to the typical type I transmembrane receptors. It was proposed that RNF43 can act downstream from the plasma membrane receptor complexes by tethering the T-cell factor 4 (TCF4) to the nuclear membrane, preventing gene transcription [12]

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