LETTERS RNF146 is a poly(ADP-ribose)-directed E3 ligase that regulates axin degradation and Wnt signalling Yue Zhang1, Shanming Liu1, Craig Mickanin1, Yan Feng1, Olga Charlat1, Gregory A. Michaud1, Markus Schirle1, Xiaoying Shi1, Marc Hild1, Andreas Bauer1, Vic E. Myer1, Peter M. Finan1, Jeffery A. Porter1, Shih-Min A. Huang1,2 and Feng Cong1,3 The Wnt=β-catenin signalling pathway plays essential roles thus representing the concentration-limiting factor for complex in embryonic development and adult tissue homeostasis, and assembly3. As a key node of the Wnt pathway, the concentration of deregulation of this pathway has been linked to cancer. Axin is a axin needs to be tightly regulated. Indeed, axin2 is a major target concentration-limiting component of the β-catenin destruction gene of β-catenin4 and activation of the Wnt pathway itself leads to complex, and its stability is regulated by tankyrase. However, degradation of axin5. Using a chemical genetics approach, we have the molecular mechanism by which tankyrase-dependent recently discovered that tankyrases (TNKS1 and TNKS2) regulate axin poly(ADP-ribosyl)ation (PARsylation) is coupled abundance, and that tankyrase inhibitor XAV939 potently inhibits to ubiquitylation and degradation of axin remains undefined. Wnt signalling through stabilization of axin6. Tankyrases belong to Here, we identify RNF146, a RING-domain E3 ubiquitin the poly(ADP-ribose) polymerase (PARP) family of proteins, which ligase, as a positive regulator of Wnt signalling. RNF146 function by synthesizing ADP-ribose polymers onto protein acceptors7. promotes Wnt signalling by mediating tankyrase-dependent This modification, called poly(ADP-ribosyl)ation or PARsylation, degradation of axin. Mechanistically, RNF146 directly interacts is emerging as an important regulatory mechanism and is gaining with poly(ADP-ribose) through its WWE domain, and promotes increasing attention8. Tankyrase is implicated in many important degradation of PARsylated proteins. Using proteomics cellular functions, such as telomere homeostasis, mitosis and vesicle approaches, we have identified BLZF1 and CASC3 as further trafficking7. Tankyrase promotes ubiquitylation and degradation of substrates targeted by tankyrase and RNF146 for degradation. its substrates, such as axin, TRF1 and tankyrase itself through an Thus, identification of RNF146 as a PARsylation-directed unknown mechanism, as no PARsylation-directed E3 ligase has ever E3 ligase establishes a molecular paradigm been identified. A better understanding of PARsylation-dependent that links tankyrase-dependent PARsylation to ubiquitylation. ubiquitylation would provide further insights into axin homeostasis RNF146-dependent protein degradation may emerge and may yield further targets for modulating Wnt signalling. as a major mechanism by which tankyrase exerts its function. To identify the E3 ligase that mediates tankyrase-dependent axin degradation, we carried out a short interfering RNA (siRNA) screen The evolutionarily conserved Wnt=β-catenin signalling pathway plays against 258 genes of the ubiquitin conjugation system using a Wnt3a- essential roles during embryonic development and adult tissue induced Super TOPFlash (STF) luciferase reporter assay in HEK293 homeostasis, and it is often aberrantly activated in cancers1,2. The main cells. In this screen, two independent siRNAs against RNF146, which function of this pathway is to regulate proteolysis of β-catenin. In the encodes a putative RING-domain E3 ligase, significantly inhibited the absence of Wnt ligands, β-catenin is associated with the multiprotein STF reporter. Both RNF146 siRNAs strongly inhibited the Wnt3a- β-catenin destruction complex that contains axin, glycogen synthase induced STF reporter without inhibiting (the TNF-α tumour-necrosis kinase 3 (GSK3) and adenomatous polyposis coli (APC). In this factor-α)-induced (NF-κB nuclear factor-κB) reporter (Fig. 1a), complex, β-catenin is constitutively phosphorylated and degraded by whereas siRNAs against NEDD4, which encodes a control E3 ligase, had the ubiquitin–proteasome pathway. Wnt ligands induce dissociation no effect on the STF reporter (Supplementary Fig. S1a). Depletion of of the β-catenin degradation complex, which leads to stabilization and RNF146 also blocked Wnt3a-induced β-catenin accumulation (Fig. 1b) subsequent nuclear translocation of β-catenin. Within this complex, and axin2 expression (Fig. 1c). We next tested whether RNF146 siRNA the concentration of axin is much lower than that of other components, inhibits Wnt signalling through stabilizing axin. Indeed, depletion of 1Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts 02139, USA. 2Present address: Sanofi-Aventis Oncology, Cambridge, Massachusetts 02139, USA. 3Correspondence should be addressed to F.C. (e-mail: [email protected]) Received 10 May 2010; accepted 4 February 2011; published online 10 April 2011; DOI: 10.1038/ncb2222 NATURE CELL BIOLOGY VOLUME 13 j NUMBER 5 j MAY 2011 623 © 2011 Macmillan Publishers Limited. All rights reserved. LETTERS a bc300 3.0 NF-κB (TNF-α) 250 2.5 STF (Wnt3a conditioned medium) 200 axin2 2.0 -catenin siRNA: pGL2 pGL2 RNF146-A RNF146-B TNKS1+2 β 150 1.5 Wnt3a –+++++ Mr (K) 100 mRNA level activity 1.0 115 Relative β-catenin 50 Relative luciferase 0.5 0 Wnt3a –++ + + 0 Tubulin 64 siRNA: siRNA: pGL2 pGL2 pGL2 pGL2 RNF146-ARNF146-B RNF146-ARNF146-B -catenin β RNF146-A deRNF146-B siRNA: -catenin pGL2 RNF146-A RNF146-B TNKS1+2 β M (K) r dsRNA: Axin1 115 White White DRNF146-A DRNF146-A DRNF146-B DRNF146-B Mr (K) HA Daxin–3×HA 182 TNKS1 82 TNKS ∗ TNKS2 115 64 Tubulin Tubulin 64 Figure 1 RNF146 positively regulates Wnt signalling by affecting the upregulation. Error bars denote the s.d. between triplicates. (d) Depletion of protein level of axin. (a) Depletion of RNF146 specifically inhibits the RNF146 increases the protein level of axin and TNKS1/2 in HEK293 cells. Wnt3a-induced STF reporter, but not the TNF-α-induced NF-κB reporter The asterisk indicates a background band. (e) Knockdown of Drosophila in HEK293 cells. Error bars denote the s.d. between four replicates. RNF146 (CG8786) using independent dsRNAs increases the protein level of (b) Depletion of RNF146 blocks Wnt3a-induced accumulation of cytosolic HA-tagged Drosophila axin in Drosophila S2 cells. dsRNA against white was β-catenin in HEK293 cells. Co-depletion of TNKS1 and TNKS2 was used used as a control. Uncropped images of blots are shown in Supplementary as a control. (c) Depletion of RNF146 abolishes Wnt3a-induced axin2 Fig. S7. RNF146 markedly increased the protein level, but not the messenger that it may interact with poly(ADP-ribose) (PAR) and function as a RNA level, of axin1 in HEK293 cells (Fig. 1d and Supplementary Fig. substrate recognition module. Indeed, overexpressed RNF146, but not S1b). Similar results were also obtained in PA-1 cells (Supplementary RNF1461WWE, was immunoprecipitated by anti-PAR antibodies, Fig. S1c,d). In addition, knockdown of the Drosophila orthologue indicating that RNF146 may interact with PARsylated proteins through of RNF146 (CG8786) using double-stranded RNAs (dsRNAs) in the WWE domain (Fig. 2c). Sequence alignment revealed several S2 cells increased the protein level, but not the mRNA level, of positively charged amino-acid residues at the carboxy terminus of the exogenously expressed Drosophila axin (Fig. 1e and Supplementary WWE domain (Supplementary Fig. S2). As PAR is negatively charged, Fig. S1e), supporting an evolutionarily conserved role for RNF146 these residues may be critical for PAR interaction. We mutated these in the regulation of axin. It has been shown that autoPARsylation of residues and found that RNF146R163A was no longer precipitated by tankyrase leads to its degradation9. Interestingly, depletion of RNF146 anti-PAR antibodies, whereas RNF146R161A behaved similarly to the increased the protein levels, but not mRNA levels, of TNKS1 and wild-type protein (Fig. 2c).We next carried out a dot-blot experiment TNKS2 (Fig. 1d and Supplementary Fig. S1b–d). These results indicate using recombinant glutathione S-transferase (GST)–WWE proteins that RNF146 may target both tankyrase and axin for degradation and purified PAR, and showed that GST–WWE, but not BSA, bound through the same mechanism. to PAR in vitro (Fig. 2d). Importantly, the R163A mutation, but not RNF146 has two distinct domains, a RING domain, which is the R161A mutation, completely abolished the interaction between predicted to interact with an E2, and a Trp–Trp–Glu (WWE) domain the WWE domain and PAR (Fig. 2d). Surface plasmon resonance with unknown function (Fig. 2a). A complementary DNA rescue analysis further demonstrated that GST–WWE and GST–RNF146, but experiment indicated that both domains are critical for the function of not GST–WWER163A or GST–RNF146R163A, bound to PAR efficiently RNF146. Expression of siRNA-resistant full-length RNF146 completely (Fig. 2e and Supplementary Fig. S3), indicating that RNF146 binds to rescued the effect of RNF146 siRNA on axin1 and tankyrase, whereas PAR through its WWE domain. expression of either RNF1461RING or RNF1461WWE failed to do We next examined the interaction between axin and RNF146 using so (Fig. 2b). Note that the protein level of RNF1461RING was much a co-immunoprecipitation assay. As shown in Fig. 2f, axin1 interacted higher than that of full-length RNF146, consistent with a critical role of with RNF146, but not RNF146R163A, and this interaction was abolished the RING