LETTERS RNF146 is a poly(ADP-ribose)-directed E3 ligase that regulates axin degradation and Wnt signalling Yue Zhang 1 , Shanming Liu 1 , Craig Mickanin 1 , Yan Feng 1 , Olga Charlat 1 , Gregory A. Michaud 1 , Markus Schirle 1 , Xiaoying Shi 1 , Marc Hild 1 , Andreas Bauer 1 , Vic E. Myer 1 , Peter M. Finan 1 , Jeffery A. Porter 1 , Shih-Min A. Huang 1,2 and Feng Cong 1,3 The Wnt/β-catenin signalling pathway plays essential roles in embryonic development and adult tissue homeostasis, and deregulation of this pathway has been linked to cancer. Axin is a concentration-limiting component of the β-catenin destruction complex, and its stability is regulated by tankyrase. However, the molecular mechanism by which tankyrase-dependent poly(ADP-ribosyl)ation (PARsylation) is coupled to ubiquitylation and degradation of axin remains undefined. Here, we identify RNF146, a RING-domain E3 ubiquitin ligase, as a positive regulator of Wnt signalling. RNF146 promotes Wnt signalling by mediating tankyrase-dependent degradation of axin. Mechanistically, RNF146 directly interacts with poly(ADP-ribose) through its WWE domain, and promotes degradation of PARsylated proteins. Using proteomics approaches, we have identified BLZF1 and CASC3 as further substrates targeted by tankyrase and RNF146 for degradation. Thus, identification of RNF146 as a PARsylation-directed E3 ligase establishes a molecular paradigm that links tankyrase-dependent PARsylation to ubiquitylation. RNF146-dependent protein degradation may emerge as a major mechanism by which tankyrase exerts its function. The evolutionarily conserved Wnt/β-catenin signalling pathway plays essential roles during embryonic development and adult tissue homeostasis, and it is often aberrantly activated in cancers 1,2 . The main function of this pathway is to regulate proteolysis of β-catenin. In the absence of Wnt ligands, β-catenin is associated with the multiprotein β-catenin destruction complex that contains axin, glycogen synthase kinase 3 (GSK3) and adenomatous polyposis coli (APC). In this complex, β-catenin is constitutively phosphorylated and degraded by the ubiquitin–proteasome pathway. Wnt ligands induce dissociation of the β-catenin degradation complex, which leads to stabilization and subsequent nuclear translocation of β-catenin. Within this complex, the concentration of axin is much lower than that of other components, 1 Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts 02139, USA. 2 Present address: Sanofi-Aventis Oncology, Cambridge, Massachusetts 02139, USA. 3 Correspondence should be addressed to F.C. (e-mail: feng.cong@novartis.com) Received 10 May 2010; accepted 4 February 2011; published online 10 April 2011; DOI: 10.1038/ncb2222 thus representing the concentration-limiting factor for complex assembly 3 . As a key node of the Wnt pathway, the concentration of axin needs to be tightly regulated. Indeed, axin2 is a major target gene of β-catenin 4 and activation of the Wnt pathway itself leads to degradation of axin 5 . Using a chemical genetics approach, we have recently discovered that tankyrases (TNKS1 and TNKS2) regulate axin abundance, and that tankyrase inhibitor XAV939 potently inhibits Wnt signalling through stabilization of axin 6 . Tankyrases belong to the poly(ADP-ribose) polymerase (PARP) family of proteins, which function by synthesizing ADP-ribose polymers onto protein acceptors 7 . This modification, called poly(ADP-ribosyl)ation or PARsylation, is emerging as an important regulatory mechanism and is gaining increasing attention 8 . Tankyrase is implicated in many important cellular functions, such as telomere homeostasis, mitosis and vesicle trafficking 7 . Tankyrase promotes ubiquitylation and degradation of its substrates, such as axin, TRF1 and tankyrase itself through an unknown mechanism, as no PARsylation-directed E3 ligase has ever been identified. A better understanding of PARsylation-dependent ubiquitylation would provide further insights into axin homeostasis and may yield further targets for modulating Wnt signalling. To identify the E3 ligase that mediates tankyrase-dependent axin degradation, we carried out a short interfering RNA (siRNA) screen against 258 genes of the ubiquitin conjugation system using a Wnt3a- induced Super TOPFlash (STF) luciferase reporter assay in HEK293 cells. In this screen, two independent siRNAs against RNF146, which encodes a putative RING-domain E3 ligase, significantly inhibited the STF reporter. Both RNF146 siRNAs strongly inhibited the Wnt3a- induced STF reporter without inhibiting (the TNF-α tumour-necrosis factor-α)-induced (NF-κB nuclear factor-κB) reporter (Fig. 1a), whereas siRNAs against NEDD4, which encodes a control E3 ligase, had no effect on the STF reporter (Supplementary Fig. S1a). Depletion of RNF146 also blocked Wnt3a-induced β-catenin accumulation (Fig. 1b) and axin2 expression (Fig. 1c). We next tested whether RNF146 siRNA inhibits Wnt signalling through stabilizing axin. Indeed, depletion of NATURE CELL BIOLOGY VOLUME 13 | NUMBER 5 | MAY 2011 623 © 20 11 M acmillan Publishers Limited. All rights reserved.