RNF146 is a poly(ADP-ribose)-directed E3 ligase that regulates...

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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 ubiquitinproteasome 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: [email protected]) 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 © 2011 Macmillan Publishers Limited. All rights reserved.

Transcript of RNF146 is a poly(ADP-ribose)-directed E3 ligase that regulates...

Page 1: RNF146 is a poly(ADP-ribose)-directed E3 ligase that regulates …basicmed.med.ncku.edu.tw/admin/up_img/1129-1.pdf · 2011-10-20 · Here, we identify RNF146, a RING-domain E3 ubiquitin

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RNF146 is a poly(ADP-ribose)-directed E3 ligase thatregulates axin degradation and Wnt signallingYue 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 rolesin embryonic development and adult tissue homeostasis, andderegulation of this pathway has been linked to cancer. Axin is aconcentration-limiting component of the β-catenin destructioncomplex, and its stability is regulated by tankyrase. However,the molecular mechanism by which tankyrase-dependentpoly(ADP-ribosyl)ation (PARsylation) is coupledto ubiquitylation and degradation of axin remains undefined.Here, we identify RNF146, a RING-domain E3 ubiquitinligase, as a positive regulator of Wnt signalling. RNF146promotes Wnt signalling by mediating tankyrase-dependentdegradation of axin. Mechanistically, RNF146 directly interactswith poly(ADP-ribose) through its WWE domain, and promotesdegradation of PARsylated proteins. Using proteomicsapproaches, we have identified BLZF1 and CASC3 as furthersubstrates targeted by tankyrase and RNF146 for degradation.Thus, identification of RNF146 as a PARsylation-directedE3 ligase establishes a molecular paradigmthat links tankyrase-dependent PARsylation to ubiquitylation.RNF146-dependent protein degradation may emergeas a major mechanism by which tankyrase exerts its function.

The evolutionarily conserved Wnt/β-catenin signalling pathway playsessential roles during embryonic development and adult tissuehomeostasis, and it is often aberrantly activated in cancers1,2. The mainfunction of this pathway is to regulate proteolysis of β-catenin. In theabsence of Wnt ligands, β-catenin is associated with the multiproteinβ-catenin destruction complex that contains axin, glycogen synthasekinase 3 (GSK3) and adenomatous polyposis coli (APC). In thiscomplex, β-catenin is constitutively phosphorylated and degraded bythe ubiquitin–proteasome pathway. Wnt ligands induce dissociationof the β-catenin degradation complex, which leads to stabilization andsubsequent nuclear translocation of β-catenin. Within this complex,the concentration of axin is much lower than that of other components,

1Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts 02139, USA. 2Present address: Sanofi-AventisOncology, 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

thus representing the concentration-limiting factor for complexassembly3. As a key node of the Wnt pathway, the concentration ofaxin needs to be tightly regulated. Indeed, axin2 is a major targetgene of β-catenin4 and activation of the Wnt pathway itself leads todegradation of axin5. Using a chemical genetics approach, we haverecently discovered that tankyrases (TNKS1 and TNKS2) regulate axinabundance, and that tankyrase inhibitor XAV939 potently inhibitsWnt signalling through stabilization of axin6. Tankyrases belong tothe poly(ADP-ribose) polymerase (PARP) family of proteins, whichfunction by synthesizing ADP-ribose polymers onto protein acceptors7.This modification, called poly(ADP-ribosyl)ation or PARsylation,is emerging as an important regulatory mechanism and is gainingincreasing attention8. Tankyrase is implicated in many importantcellular functions, such as telomere homeostasis, mitosis and vesicletrafficking7. Tankyrase promotes ubiquitylation and degradation ofits substrates, such as axin, TRF1 and tankyrase itself through anunknown mechanism, as no PARsylation-directed E3 ligase has everbeen identified. A better understanding of PARsylation-dependentubiquitylation would provide further insights into axin homeostasisandmay yield further targets formodulatingWnt signalling.To identify the E3 ligase that mediates tankyrase-dependent axin

degradation, we carried out a short interfering RNA (siRNA) screenagainst 258 genes of the ubiquitin conjugation system using a Wnt3a-induced Super TOPFlash (STF) luciferase reporter assay in HEK293cells. In this screen, two independent siRNAs against RNF146, whichencodes a putative RING-domain E3 ligase, significantly inhibited theSTF reporter. Both RNF146 siRNAs strongly inhibited the Wnt3a-induced STF reporter without inhibiting (the TNF-α tumour-necrosisfactor-α)-induced (NF-κB nuclear factor-κB) reporter (Fig. 1a),whereas siRNAs againstNEDD4, which encodes a control E3 ligase, hadno effect on the STF reporter (Supplementary Fig. S1a). Depletion ofRNF146 also blockedWnt3a-induced β-catenin accumulation (Fig. 1b)and axin2 expression (Fig. 1c). We next tested whether RNF146 siRNAinhibits Wnt signalling through stabilizing axin. Indeed, depletion of

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Figure 1 RNF146 positively regulates Wnt signalling by affecting theprotein level of axin. (a) Depletion of RNF146 specifically inhibits theWnt3a-induced STF reporter, but not the TNF-α-induced NF-κB reporterin HEK293 cells. Error bars denote the s.d. between four replicates.(b) Depletion of RNF146 blocks Wnt3a-induced accumulation of cytosolicβ-catenin in HEK293 cells. Co-depletion of TNKS1 and TNKS2 was usedas a control. (c) Depletion of RNF146 abolishes Wnt3a-induced axin2

upregulation. Error bars denote the s.d. between triplicates. (d) Depletion ofRNF146 increases the protein level of axin and TNKS1/2 in HEK293 cells.The asterisk indicates a background band. (e) Knockdown of DrosophilaRNF146 (CG8786) using independent dsRNAs increases the protein level ofHA-tagged Drosophila axin in Drosophila S2 cells. dsRNA against white wasused as a control. Uncropped images of blots are shown in SupplementaryFig. S7.

RNF146 markedly increased the protein level, but not the messengerRNA level, of axin1 in HEK293 cells (Fig. 1d and Supplementary Fig.S1b). Similar results were also obtained in PA-1 cells (SupplementaryFig. S1c,d). In addition, knockdown of the Drosophila orthologueof RNF146 (CG8786) using double-stranded RNAs (dsRNAs) inS2 cells increased the protein level, but not the mRNA level, ofexogenously expressed Drosophila axin (Fig. 1e and SupplementaryFig. S1e), supporting an evolutionarily conserved role for RNF146in the regulation of axin. It has been shown that autoPARsylation oftankyrase leads to its degradation9. Interestingly, depletion of RNF146increased the protein levels, but not mRNA levels, of TNKS1 andTNKS2 (Fig. 1d and Supplementary Fig. S1b–d). These results indicatethat RNF146 may target both tankyrase and axin for degradationthrough the same mechanism.RNF146 has two distinct domains, a RING domain, which is

predicted to interact with an E2, and a Trp–Trp–Glu (WWE) domainwith unknown function (Fig. 2a). A complementary DNA rescueexperiment indicated that both domains are critical for the function ofRNF146. Expression of siRNA-resistant full-length RNF146 completelyrescued the effect of RNF146 siRNA on axin1 and tankyrase, whereasexpression of either RNF1461RING or RNF1461WWE failed to doso (Fig. 2b). Note that the protein level of RNF1461RING was muchhigher than that of full-length RNF146, consistent with a critical role ofthe RING domain in auto-ubiquitylation and degradation of RNF146.The protein level of axin1 was modestly increased on overexpressionof RNF1461RING, and it was further enhanced by RNF146 siRNA(Fig. 2b), indicating that RNF1461RING has a dominant negativeeffect. The WWE domain is a conserved globular domain foundin multiple PARPs and E3 ligases10. As it is the only recognizabledomain within RNF146 apart from the RING domain, we speculated

that it may interact with poly(ADP-ribose) (PAR) and function as asubstrate recognition module. Indeed, overexpressed RNF146, but notRNF1461WWE, was immunoprecipitated by anti-PAR antibodies,indicating that RNF146may interact with PARsylated proteins throughthe WWE domain (Fig. 2c). Sequence alignment revealed severalpositively charged amino-acid residues at the carboxy terminus of theWWE domain (Supplementary Fig. S2). As PAR is negatively charged,these residues may be critical for PAR interaction. We mutated theseresidues and found that RNF146R163A was no longer precipitated byanti-PAR antibodies, whereas RNF146R161A behaved similarly to thewild-type protein (Fig. 2c).We next carried out a dot-blot experimentusing recombinant glutathione S-transferase (GST)–WWE proteinsand purified PAR, and showed that GST–WWE, but not BSA, boundto PAR in vitro (Fig. 2d). Importantly, the R163A mutation, but notthe R161A mutation, completely abolished the interaction betweenthe WWE domain and PAR (Fig. 2d). Surface plasmon resonanceanalysis further demonstrated that GST–WWE and GST–RNF146, butnot GST–WWER163A or GST–RNF146R163A, bound to PAR efficiently(Fig. 2e and Supplementary Fig. S3), indicating that RNF146 binds toPAR through its WWE domain.We next examined the interaction between axin and RNF146 using

a co-immunoprecipitation assay. As shown in Fig. 2f, axin1 interactedwith RNF146, but not RNF146R163A, and this interaction was abolishedby tankyrase inhibitor XAV939. Furthermore, axin1 was PARsylatedin vivo and its PARsylation was abolished by XAV939 (Fig. 2f).These results indicate that the interaction between RNF146 and axinis mediated by the WWE domain and PAR moiety. Importantly,siRNA-resistant RNF146R163A failed to rescue the effect of RNF146siRNA in a cDNA rescue experiment, whereas RNF146R161A behavedsimilarly to the wild-type protein (Fig. 2g). Together, these results

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Figure 2 Interaction between the WWE domain and PAR is essentialfor RNF146-dependent regulation of axin in vivo. (a) Schematicrepresentation of the domain structure of RNF146. (b) Expressionof siRNA-resistant wild-type RNF146, but not RNF146 mutantswith either the RING domain or the WWE domain deleted (1RINGand 1WWE), prevents RNF146 -siRNA-induced stabilization of axin1and TNKS1/2 in HEK293 cells. (c) Immunoprecipitation (IP) ofRNF146 and RNF146R161A, but not RNF1461WWE or RNF146R163A,by anti-PAR antibodies in HEK293 cells. WB, western blot; TCL,

total cell lysates. (d) Dot-blot analysis of PAR-binding activities ofGST–WWE proteins with BSA used as a control. (e) Binding of PARwith wild-type WWE, but not WWER163A, as shown by surface plasmonresonance. (f) Axin1 and RNF146 interact with each other in aPARsylation-dependent and WWE-domain-dependent manner. Theasterisk indicates a nonspecific band. (g) Expression of RNF146R161A,but not RNF146R163A, prevents RNF146 -siRNA-induced stabilizationof axin1 and TNKS1/2 in HEK293 cells. Uncropped images of blotsare shown in Supplementary Fig. S7.

indicate that RNF146 recognizes PARsylated proteins for degradationthrough a direct binding between theWWEdomain and PARmoiety.Our previous study demonstrated that tankyrase promotes ubiq-

uitylation and degradation of axin6. However, whether endogenousaxin is PARsylated and subsequently degraded in vivo is less clear.To address this question, we took advantage of the strong bindingbetween the WWE domain and PAR, and used GST–WWE as a toolto isolate PARsylated proteins from cells. Lysates of cells expressingFlag–axin1 were pulled down by GST–WWE, eluted and recapturedby anti-Flag antibody. As a control, fractions of cell lysates weredirectly immunoprecipitated with anti-Flag antibody. Compared withstraight immunoprecipitation, sequential pulldown brought downmuch more PARsylated axin1 (Fig. 3a), indicating that GST–WWEbinds and enriches PARsylated axin1. We then used GST–WWEto purify PARsylated proteins from cells treated with tankyraseinhibitor or RNF146 RNA interference (RNAi). As reported earlier6,treatment of XAV939 led to stabilization of endogenous axin1 andtankyrase (Fig. 3b,c). However, the amount of axin1 or tankyrasepulled down with GST–WWE was significantly decreased on XAV939

treatment (Fig. 3b,c), indicating that endogenous axin is PARsylatedin a tankyrase-dependent manner in vivo. We next reasoned that ifPARsylated proteins were targeted for degradation they should greatlyaccumulate when the E3 ligase responsible for their degradation isinhibited. Indeed, depletion of RNF146 using a doxycycline (DOX)-induced RNF146 short hairpin RNA (shRNA) markedly increased thelevel of axin1 or tankyrase pulled down by GST–WWE, although thetotal level was only moderately increased (Fig. 3b,c). By comparingthe amounts of proteins in the input and GST–WWE precipitates, itis clear that only a very small fraction of axin1 was pulled down byGST–WWE(Supplementary Fig. S4a), whereas the fraction of tankyrasepulled down by GST–WWE was significantly higher (SupplementaryFig. S4b). Presumably, autoPARsylation of tankyrase is more efficientthan tankyrase-dependent PARsylation of axin. Taken together, thesedata strongly indicate that RNF146 is responsible for degradation ofPARsylated axin and tankyrase in cells.We then investigated whether RNF146 is required for ubiquitylation

of axin in vivo. To enhance the detection of ubiquitylation ofendogenous axin, we pretreated SW480 cells with XAV939 to increase

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Figure 3 RNF146 is required for PARsylation-dependent degradationof axin and tankyrase in vivo. (a) Enrichment of PARsylatedaxin1 using GST–WWE in a sequential pulldown analysis. Straightimmunoprecipitation with anti-Flag antibody was used as a control.(b,c) Inhibition of tankyrase by XAV939 or depletion of RNF146 usingDOX-inducible RNF146 shRNA increases the protein levels of axin(b) and tankyrase (c) in HEK293 cells. XAV939 reduces, whereas

depletion of RNF146 markedly increases, the amount of axin (b) ortankyrase (c) pulled down by GST–WWE. GST–WWER163A was usedas a control. DMSO, dimethylsulphoxide. (d) RNF146 is requiredfor tankyrase-dependent ubiquitylation and degradation of axin in acompound wash-off experiment. (e) RNF146 siRNA stabilizes axin2 inSW480 cells in a pulse-chase analysis. Uncropped images of blots areshown in Supplementary Fig. S7.

the protein level of axin2. Axin2 was quickly degraded after XAV939was washed off, and the proteasome inhibitor MG132 blocked axin2degradation and induced accumulation of polyubiquitylated axin2(Fig. 3d). Significantly, depletion of RNF146 suppressed compoundwash-off-induced degradation of axin2 and completely blockedMG132-dependent accumulation of polyubiquitylated axin2 (Fig. 3d).These results indicate that RNF146 is required for PARsylation-dependent ubiquitylation of axin. A pulse-chase experiment furtherdemonstrated that depletion of RNF146 significantly increased thehalf-life of axin2 protein (Fig. 3e). Note that the half-life of newlysynthesized axin2 (Fig. 3e) is longer than that of total axin2 (Fig. 3d).It is possible that pre-existing total axin2 forms a tight complexwith stabilized tankyrase and therefore is quickly PARsylated oncompound wash-off, whereas newly synthesized axin2 would needmore time to bind to tankyrase tightly, leading to a lower efficiencyof PARsylation and degradation. Together, these results indicate thatRNF146 promotes ubiquitylation and degradation of axin in vivo.To gain further insights into the mechanism of PARsylation-

dependent ubiquitylation, we established an in vitro ubiquitylationassay using GST–RNF146, E1, the E2 ubiquitin-conjugating enzymeUbcH5a and haemagglutinin (HA)–ubiquitin (Supplementary Fig.S5a). Using this ubiquitylation assay, we showed that RNF1461RINGno longer possessed E3 ligase activity, whereas RNF146R163A, which isdeficient in PAR interaction, had similar E3 ligase activity to the wild-type protein (Fig. 4a). We then examined the link between PARsylationand ubiquitylation. We found that purified PAR markedly increasedauto-ubiquitylation of RNF146 (Fig. 4b,c) and this enhancement

was not observed with RNF146R163A (Fig. 4b). As a control, PARhad no effect on auto-ubiquitylation of the ubiquitin ligases MDM2(Mdm2 p53 binding protein homologue) and SMURF1 (SMADspecific E3 ubiquitin protein ligase 1; Supplementary Fig. S5b),demonstrating the specificity of this effect. We next tested the effectof PARsylated TNKS2 on auto-ubiquitylation of RNF146. His-taggedTNKS2 was subjected to autoPARsylation followed by pulldown withNi–nitrilotriacetic acid beads and extensive washes. TNKS2-coatedbeads were subsequently added to the in vitro ubiquitylation assay.Compared with unmodified TNKS2, PARsylated TNKS2 significantlyincreased auto-ubiquitylation of RNF146, but not RNF146R163A

(Supplementary Fig. S5c,d). Consistent with these in vitro results,XAV939 decreased auto-ubiquitylation of exogenously expressedRNF146 in HEK293 cells (Supplementary Fig. S5e). Together, theseresults indicate that interaction between RNF146 and PAR enhancesauto-ubiquitylation of RNF146. This observation can be explained bythe ‘glue’ effect of PAR. PAR contains multiple ADP-ribose units, and itcan bind to multiple RNF146 molecules at the same time. Presumably,PAR holds multiple RNF146 molecules together, increases their localconcentration and promotes cross-ubiquitylation of RNF146.Direct examination of PARsylation-dependent ubiquitylation of

TNKS2 is not practical, as both PARsylation and polyubiquitylationof TNKS2 led to the formation of indistinct, high-molecular-weightspecies as assessed by SDS gel electrophoresis. To overcome thisproblem, we decreased the concentration of NAD+ in the PARsylationreaction to prevent the build-up of poly(ADP-ribose) chains on TNKS2.A significant increase of high-molecular-weight species of TNKS2

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Figure 4 PARsylation-dependent ubiquitylation in vitro.(a) Auto-ubiquitylation of RNF146 in an in vitro ubiquitylationassay. (b) PAR increases auto-ubiquitylation of RNF146,but not RNF146R163A. (c) Time course of PAR-enhanced

auto-ubiquitylation of RNF146. (d) Ubiquitylation ofTNKS2 by wild-type RNF146, but not RNF146R163A, in aPARsylation-dependent manner. Uncropped images of blots areshown in Supplementary Fig. S7.

was observed when partially PARsylated TNKS2 was subjected to theubiquitylation assay with wild-type RNF146 (Fig. 4d), which probablyrepresents the formation of polyubiquitylatedTNKS2. Importantly, thisincrease was not observed with RNF146R163A (Fig. 4d). These resultsprovided direct evidence that RNF146 recognizes PARsylated TNKS2and mediates its subsequent ubiquitylation.To further understand the physiological roles of tankyrase and

RNF146, we sought to identify their other cellular substrates usinga quantitative affinity proteomics approach. We reasoned thatdepletion of RNF146 should stabilize PARsylated proteins targetedfor degradation, and these PARsylated proteins can be pulled downby GST–WWE and analysed by quantitative mass spectrometry. Wecarried out the experiment as described in Fig. 5a, and identifiedmultiple proteins that were significantly enriched in cells depletedof RNF146 (Fig. 5b, highlighted). Identification of TNKS1, a knownsubstrate of RNF146, validated the experimental design. After fusingcandidate proteins with GFP and examining their expression ontreatment with XAV939, we identified BLZF1 (basic leucine zippernuclear factor 1) as the only validated hit (data not shown). As seenin Fig. 5c, tankyrase inhibitors such as XAV939 and IWR-1 (ref. 11)significantly increased the protein level of GFP–BLZF1, whereasABT-888, a PARP inhibitor with minimal activity on tankyrase6, hadno effect. Furthermore, depletion of TNKS1/2 or RNF146 increasedthe protein level of GFP–BLZF1 (Fig. 5d). Significantly, the amountof PARsylated BLZF1 pulled down by GST–WWE was decreased onXAV939 treatment, and greatly increased on depletion of RNF146(Fig. 5e). On the basis of similarity to known tankyrase-bindingmotifs12, we identified an evolutionarily conserved tankyrase-bindingdomain (TBD) at the C terminus of BLZF1 (Fig. 5f). Deletion of theTBD abolished the interaction between BLZF1 and tankyrase (Fig. 5g),and led to stabilization of BLZF1 (Fig. 5h). These results stronglyindicate that BLZF1 is targeted for degradation by tankyrase andRNF146 in a PARsylation-dependent manner.As an alternative way to identify substrates of tankyrase and RNF146,

we mined a yeast two-hybrid database for tankyrase interactors, andtested the effect of XAV939 on their expression (data not shown).Using this approach, we identified CASC3 (cancer susceptibilitycandidate 3) as another protein targeted by tankyrase and RNF146

for degradation. The protein level of GFP–CASC3 was increased byXAV939 (Supplementary Fig. S6a) or siRNA against TNKS1/2 orRNF146 (Supplementary Fig. S6b). Deletion of the TBD of CASC3(Supplementary Fig. S6c) blocked the interaction between CASC3and tankyrase (Supplementary Fig. S6d), and abolished tankyrase andRNF146-dependent regulation of CASC3 (Supplementary Fig. S6e,f).Together, our study shows that BLZF1 and CASC3 are also degraded

by tankyrase and RNF146. BLZF1, also called Golgin-45, is locatedon the surface of the medial cisternae of the Golgi complex, and isimportant for the maintenance of Golgi complex structure13. Notably,TNKS1 has been shown to be a Golgi-associated protein14. CASC3 isa component of the exon junction complex, which is assembled onsplicedmRNAs and plays important roles in post-splicing events15. Ourfindings therefore indicate that tankyrase regulates many biologicalprocesses that have not previously been associatedwith tankyrase.Various post-translational modifications can serve as a signal for

the ubiquitin conjugation system, thus coupling protein turnoverwith cell signalling16–19. Our previous study revealed an unexpectedrole of tankyrase in regulating axin stability, indicating thatPARsylation can also earmark proteins for degradation. Here, we havediscovered RNF146 as the E3 ligase that mediates tankyrase-dependentdegradation of axin and two further tankyrase substrates. Our study hasidentified the first PAR-directed E3 ligase and provided mechanisticinsight into how PARsylation leads to ubiquitylation.The biological functions of PARsylation are diverse and

growing8,20–22. Various PAR-binding domains may exist to read thePAR modification. So far, three PAR-binding domains have beendescribed: the macro domain23; eight-amino-acid motifs that havebeen identified within histones, the DNA repair protein XRCC1 (X-rayrepair cross-complementing protein 1) and p53 (refs 22,24); and thePAR-binding zinc-finger (PBZ) domain25. Each of these domains hasimportant roles in the DNA-damage response. Here we demonstratethat the WWE domain of RNF146 is another PAR-binding domain.Interestingly, the WWE domain is identified in multiple PARPs(ref. 20). These PARPs may interact with the PAR moiety on theirsubstrates through this domain, which may regulate their activities,such as chain elongation. In addition, the WWE domain is found inseveral other E3 ligases, such as DTX1–4 (deltex homologues 1–4),

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Figure 5 BLZF1 is identified as a substrate of tankyrase and RNF146 usingquantitative mass spectrometry. (a) A quantitative proteomics approach toidentify substrates of RNF146 through combining RNF146 knockdown andGST–WWE pulldown. (b) A scatter plot depicting proteins identified andquantified in a quantitative proteomics experiment. Proteins significantlyenriched in the DOX-treated condition are highlighted as potential substratesof RNF146. (c) Inhibition of tankyrase by either XAV939 or IWR-1 increasesthe protein level of GFP–BLZF1 in SW480 cells. (d) Depletion of RNF146

or co-depletion of TNKS1 and TNKS2 increases the protein level ofGFP–BLZF1. (e) XAV939 decreases, whereas RNF146 siRNA markedlyincreases, the amount of GFP–BLZF1 pulled down by GST–WWE, which ispresumably PARsylated. (f) The TBD of BLZF1 is evolutionarily conserved.(g) Deletion of the TBD abolishes the interaction between TNKS1 andBLZF1 in a co-immunoprecipitation assay. (h) Deletion of the TBD stabilizesGFP–BLZF1 in SW480 cells. Uncropped images of blots are shown inSupplementary Fig. S7.

HUWE1 (HECT, UBA and WWE domain containing 1, also knownas MULE) and TRIP12 (thyroid hormone receptor interactor 12;ref. 10). These E3 ligases may also regulate protein turnover in aPARsylation-dependent manner.Axin is the concentration-limiting factor in the β-catenin

degradation complex. The discovery of RNF146 as the E3 ligaseresponsible for axin degradation hints at a potential role for RNF146in Wnt-related diseases. An association between Wnt signalling andbreast cancer is known. Wnt1 itself was first identified as a mammaryoncogene26, and forced expression of Wnt1 in transgenic mice causedthe development of mammary tumours27. Accumulating literaturehas also indicated a role of aberrant Wnt signalling in humanbreast cancer28,29. Intriguingly, a genome-wide gene-association studyidentified a breast-cancer locus at 6q22.33, which contains only twogenes, RNF146 and ECHD1 (enoyl CoA hydratase domain containing1; ref. 30). The major haplotype of the locus confers protection fromdisease, whereas the minor haplotype confers risks. It is possiblethat these two haplotypes confers cancer protection or risk throughaffecting RNF146 and Wnt signalling. A potential role of RNF146in mammary-gland development and mammary-tumour formationshould be examined in future studies. �

METHODSMethods and any associated references are available in the onlineversion of the paper at http://www.nature.com/naturecellbiology/

Note: Supplementary Information is available on the Nature Cell Biology website

ACKNOWLEDGEMENTSWe thank D. Patel, C. Xin, E. McWhinnie, S. Zhao, J. Murphy, Y. Mishinaand J. Klekota for technical assistance and W. Shao, F. Stegmeier, J. Tallarico,T. Bouwmeester and M. Kirschner for comments and advice.

AUTHOR CONTRIBUTIONSY.Z., C.M., Y.F., G.A.M., M.S., M.H., A.B., V.E.M, P.M.F., J.A.P., S-M.A.H and F.C.conceived and designed the study. Y.Z., S.L., C.M., Y.F., O.C., G.A.M.,M.S., X.S. andF.C. designed and implemented experiments. Y.Z. and F.C. wrote the paper.

COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.

Published online at http://www.nature.com/naturecellbiologyReprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/

1. Logan, C. Y. & Nusse, R. The Wnt signalling pathway in development and disease.Annu. Rev. Cell Dev. Biol. 20, 781–810 (2004).

2. Clevers, H. Wnt/β-catenin signalling in development and disease. Cell 127,469–480 (2006).

3. Lee, E., Salic, A., Kruger, R., Heinrich, R. & Kirschner, M. W. The roles of APC andAxin derived from experimental and theoretical analysis of the Wnt pathway. PLoS.Biol. 1, E10 (2003).

4. Leung, J. Y. et al. Activation of AXIN2 expression by β-catenin-T cell factor.A feedback repressor pathway regulating Wnt signalling. J. Biol. Chem. 277,21657–21665 (2002).

5. Willert, K., Shibamoto, S. & Nusse, R. Wnt-induced dephosphorylation of axinreleases β-catenin from the axin complex. Genes Dev. 13, 1768–1773 (1999).

6. Huang, S. M. et al. Tankyrase inhibition stabilizes axin and antagonizes Wntsignalling. Nature 461, 614–620 (2009).

7. Hsiao, S. J. & Smith, S. Tankyrase function at telomeres, spindle poles, and beyond.Biochimie 90, 83–92 (2008).

8. Gagne, J. P., Hendzel, M. J., Droit, A. & Poirier, G. G. The expanding role ofpoly(ADP-ribose) metabolism: current challenges and new perspectives. Curr. Opin.Cell Biol. 18, 145–151 (2006).

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10. Aravind, L. The WWE domain: a common interaction module in protein ubiquitinationand ADP ribosylation. Trends Biochem. Sci. 26, 273–275 (2001).

11. Chen, B. et al. Small molecule-mediated disruption of Wnt-dependent signalling intissue regeneration and cancer. Nat. Chem. Biol. 5, 100–107 (2009).

12. Sbodio, J. I. & Chi, N. W. Identification of a tankyrase-binding motif sharedby IRAP, TAB182, and human TRF1 but not mouse TRF1. NuMA containsthis RXXPDG motif and is a novel tankyrase partner. J. Biol. Chem. 277,31887–31892 (2002).

13. Short, B. et al. A GRASP55-rab2 effector complex linking Golgi structure tomembrane traffic. J. Cell Biol. 155, 877–883 (2001).

14. Chi, N. W. & Lodish, H. F. Tankyrase is a golgi-associated mitogen-activated proteinkinase substrate that interacts with IRAP in GLUT4 vesicles. J. Biol. Chem. 275,38437–38444 (2000).

15. Palacios, I. M., Gatfield, D., St, J. D. & Izaurralde, E. An eIF4AIII-containing complexrequired for mRNA localization and nonsense-mediated mRNA decay. Nature 427,753–757 (2004).

16. Hunter, T. The age of crosstalk: phosphorylation, ubiquitination, and beyond.Mol. Cell 28, 730–738 (2007).

17. Min, J. H. et al. Structure of an HIF-1 α-pVHL complex: hydroxyproline recognitionin signalling. Science 296, 1886–1889 (2002).

18. Ikura, T. et al. DNA damage-dependent acetylation and ubiquitination of H2AXenhances chromatin dynamics. Mol. Cell Biol. 27, 7028–7040 (2007).

19. Yoshida, Y. et al. E3 ubiquitin ligase that recognizes sugar chains. Nature 418,438–442 (2002).

20. Schreiber, V., Dantzer, F., Ame, J. C. & de, M. G. Poly(ADP-ribose): novel functionsfor an old molecule. Nat. Rev. Mol. Cell Biol. 7, 517–528 (2006).

21. Hassa, P. O. & Hottiger, M. O. The diverse biological roles of mammalian PARPS,a small but powerful family of poly-ADP-ribose polymerases. Front. Biosci. 13,3046–3082 (2008).

22. Scovassi, A. I. The poly(ADP-ribosylation) story: a long route from Cinderella toPrincess. Riv. Biol. 100, 351–360 (2007).

23. Karras, G. I. et al. The macro domain is an ADP-ribose binding module. EMBO J. 24,1911–1920 (2005).

24. Pleschke, J. M., Kleczkowska, H. E., Strohm, M. & Althaus, F. R. Poly(ADP-ribose)binds to specific domains in DNA damage checkpoint proteins. J. Biol. Chem. 275,40974–40980 (2000).

25. Ahel, I. et al. Poly(ADP-ribose)-binding zinc finger motifs in DNA repair/checkpointproteins. Nature 451, 81–85 (2008).

26. Nusse, R., van, O. A., Cox, D., Fung, Y. K. & Varmus, H. Mode of proviral activationof a putative mammary oncogene (int-1) on mouse chromosome 15. Nature 307,131–136 (1984).

27. Tsukamoto, A. S., Grosschedl, R., Guzman, R. C., Parslow, T. & Varmus, H. E.Expression of the int-1 gene in transgenic mice is associated with mammarygland hyperplasia and adenocarcinomas in male and female mice. Cell 55,619–625 (1988).

28. Mohinta, S., Wu, H., Chaurasia, P. & Watabe, K. Wnt pathway and breast cancer.Front. Biosci. 12, 4020–4033 (2007).

29. Howe, L. R. & Brown, A. M. Wnt signalling and breast cancer. Cancer Biol. Ther. 3,36–41 (2004).

30. Gold, B. et al. Genome-wide association study provides evidence for a breast cancerrisk locus at 6q22.33. Proc. Natl Acad. Sci. USA 105, 4340–4345 (2008).

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METHODS DOI: 10.1038/ncb2222

METHODSPlasmids. STF reporter, 3×HA-taggedDrosophila axin, Flag-taggedTNKS11PARPand His-tagged human TNKS2 were generated as previously described6. siRNA-resistant full-length RNF146, RNF1461WWE (missing amino acids 100–175),RNF1461RING (missing amino acids 36–73), RNF146R161A and RNF146R163A weretagged with anHA epitope at the C termini and cloned into pcDNA4-TO or pCMV6.BLZF1, BLZF11TBD (missing amino acids 18–23), CASC3 and CASC31TBD(missing amino acids 146–151) were fused with GFP epitope at the amino terminiand cloned into a lentiviral vector under the control of themetallothionein promoter.N-terminal GST-tagged RNF146, RNF1461RING, RNF146R163A, WWE (aminoacids 100–175), WWER161A and WWER163A bacterium expression constructs weregenerated by cloning into pGEX-6p.

RNA interference, transfection, luciferase assay and inducible shRNA cell-line generation. HEK293 and SW480 cells were grown in DMEM supplementedwith 10% FCS. Plasmid or siRNA transfection was done using Fugene 6 (Roche) orDharmafect 1 (Dharmacon).

Sequences of siRNAs used are listed as follows: TNKS1, sense, 5′-GCAUGGAGCUUGUGUUAAUUU-3′, antisense 5′-AUUAACACAAGCUCCAUGCUU-3′ (Dharmacon); TNKS2, sense, 5′-GGAAAGACGUAGUUGAAUAUU-3′,antisense, 5′-UAUUCAACUACGUCUUUCCUU-3′ (Dharmacon); CTNNB1,sense, 5′-UGUGGUCACCUGUGCAGCUdTdT-3′, antisense, 5′-AGCUGCACAG-GUGACCACAdTdT-3′ (Qiagen); RNF146-A, sense, 5′-GGCUAGACUGUGAUGC-UAAdTdT-3′, antisense, 5′-UUAGCAUCACAGUCUAGCCdTdA-3′ (Qiagen);RNF146-B, sense, 5′-GCACGUUUUCUGCUAUCUAdTdT-3′, antisense, 5′-UAGAUAGCAGAAAACGUGCdTdT-3′ (Qiagen); pGL2, sense, 5′-CGUACGCGG-AAUACUUCGAdTdT-3′, antisense, 5′-UCGAAGUAUUCCGCGUACGdTdT-3′

(Dharmacon); NEDD4-A, sense, 5-GGAGGGAACAUACAAAGUAUU-3′, anti-sense, 5′-TACTTTGTATGTTCCCTCCUU-3′ (Dharmacon); NEDD4-B, sense,5′-GAACUAGAGCUUCUUAUGUUU-3′, antisense, 5′-ACATAAGAAGCTCTAG-TTCUU-3′ (Dharmacon).

To test the effect of RNF146 depletion in Drosophila cells, S2R cells stably trans-fectedwithDaxin-3×HAwere seeded in 24-well plates and treatedwith the indicateddsRNA for 5 d. DsRNAs were produced from the polymerase chain reaction prod-ucts using T7-linked primers (for White, forward, 5′-ACCTGTGGACGCCAAGG-3′, reverse, 5′-AAAAGAAGTCGACGGCTTC-3′; for DRNF146-A, forward, 5′-TGTCGCTGGTCACCTGGTT-3′, reverse, 5′-CTAGCCACCATACCCAAAAAG-G-3′; for DRNF146-B, forward, 5′-TGTCGCTGGTCACCTGGTT-3′, reverse,5′-CTAGCCACCATACCCAAAAAGG-3′) using a MEGAscript high-yield tran-scription kit (Ambion).

STF luciferase assays were carried out using aDual Luciferase Assay kit (Promega)according to the manufacturer’s instructions.

RNF146 shRNA cell lines were generated by infecting HEK293 or PA-1 cellswith lentivirus containing DOX-inducible shRNA targeting RNF146 followed byselectionwith puromycin. The targeting sequence ofRNF146 shRNA is the following(amino acids 2046–2066): 5′-GCTTTGCTGTCTAGTCTTATA-3′.

Surface plasmon resonance. GST-tagged proteins were coupled to a Biacore CM5sensor chip coated with anti-GST antibody. 625 nM PAR (Trevigen) was thenprofiled at a flow rate of 30mlmin−1 for 300 s, followed by 600 s flow of washbuffer. After analysis in BiaEvalution (Biacore), the normalized resonance units wereplotted over time with the assumption of one-to-one binding.

Poly(ADP-ribose) dot-blot analysis. Purified GST–WWE proteins or BSA wereblotted onto nitrocellulose membrane (Invitrogen). The nitrocellulose membranewas rinsed with TBST buffer (10mM Tris-HCl at pH 7.4, 150mM NaCl and 0.05%Tween 20) three times. The membrane was incubated with purified PAR (Trevigen)for 1 h at room temperature. After extensive washes with TBST and TBST containing1M NaCl, the membrane was blocked with 5% milk followed by immunoblottingwith mouse anti-PAR antibody (Trevigen).

Quantitative polymerase chain reaction with reverse transcription. TotalRNA from siRNA-treated cells was extracted using the RNeasy Plus Mini Kit(Qiagen) and reverse transcribed with Taqman reverse-transcription reagents(Applied Biosystems) according to the manufacturer’s instructions. Transcriptlevels were assessed using the ABI PRISM 7900HT sequence detection system.Real-time polymerase chain reaction was carried out in 12 µl reactions consistingof 0.6 µl of 20× Assay-on-Demand mix (premixed concentration of 18 µM foreach primer and 5 µM probe), 6 µl Taqman Universal PCR Master Mix and5.4 µl cDNA template. The thermocycling conditions used were 2min at 50 ◦Cand 10min at 95 ◦C, followed by 40 cycles of 15 s at 95 ◦C and 1min at 60 ◦C.All experiments were carried out in triplicate. Gene expression analysis wascarried out using the comparative cycle threshold method with the housekeepinggene GUSB (glucuronidase, β) for normalization. The Assay-on-Demand reagentsused were as follows: AXIN1 (Hs00394718_m1), AXIN2 (Hs00610344_m1),

TNKS (Hs00186671_m1), TNKS2 (Hs00228829_m1), CTNNB1 (Hs00170025_m1),RNF146 (Hs00258475_s1).

Immunoblotting, immunoprecipitation and GST pulldown assay. Total celllysates were prepared in RIPA buffer (50mM Tris-HCl at pH 7.4, 150mM NaCl,1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 1mM EDTA). Lysateswere normalized for protein concentration, resolved by SDS–polyacrylamide gelelectrophoresis (PAGE), transferred onto nitrocellulose membranes and probedwith the indicated antibodies.

For co-immunoprecipitation experiments, cells were lysed in either RIPA bufferor EBC buffer (50mM Tris-HCl at pH 7.4, 150mM NaCl, 0.5% NP-40 and1mM EDTA). Cleared cell lysates were incubated with the indicated antibodiesand protein G-Sepharose beads overnight at 4 ◦C. Beads were washed six timeswith lysis buffer. The bound proteins were dissolved in SDS sample buffer,resolved by SDS–PAGE and immunoblotted with the indicated antibodies. For theRNF146–axin1 co-immunoprecipitation experiment, cells were treatedwithMG132to block protein degradation.

The compound wash-off assay was carried out as previously described6. Briefly,SW480 cells transfected with the indicated siRNA were pretreated overnightwith 3 µM XAV939. The compound was then washed and incubated withmedium containing indicated reagents for 1 h. Cells were lysed with RIPAbuffer supplemented with 5mM N -ethylmaleimide and 5 µM ADP-HPD (ADP-(hydroxymethyl)pyrrolidinediol; Alexis) to block the activities of deubiquitylasesand PARG (poly(ADP-ribose) glycohydrolase) and immunoprecipitated with theindicated antibodies.

For the GST pulldown experiments, GST–WWE and GST–WWER163A re-combinant proteins were produced in Escherichia coli and purified using glu-tathione–agarose beads (GE Healthcare). HEK293 cells or SW480 cells expressingGFP–BLZF1 were subjected to the indicated treatments, lysed in RIPA buffersupplemented with 5 µMADP-HPD and incubated with glutathione–agarose beadscharged with GST fusion proteins for 4 h at 4 ◦C. The beads were then washed sixtimes with lysis buffer. Bound materials were resolved by SDS–PAGE and blottedwith the indicated antibodies.

In all experiments, 1× protease inhibitor cocktail (Sigma) and 1× phosphataseinhibitor cocktail (Upstate) were added to the lysis buffers. Commercial antibodiesused in this study include goat anti-axin1 (1:1,000; R&D Systems), mouse anti-TNKS (1:1,000; Abcam), rat anti-HA (1:1,000; Roche), mouse anti-GST (1:1,000;Upstate), rabbit anti-axin2 (76G6; 1:1,000 for WB; 1:100 for IP), rabbit anti-GFP(1:1,000), mouse anti-ubiquitin (1:1,000; Cell Signaling Technology), mouse anti-β-catenin and rabbit anti-poly(ADP-ribose) (1:1,000; BD Pharmingen), mouseanti-tubulin (1:1,000) and anti-Flag (M2; 1:1,000; Sigma), EZview Red anti-FlagM2 Affinity Gel (1:100 for IP, Sigma), mouse anti-poly(ADP-ribose) (1:100 for IP,Trevigen), mouse anti-mdm2 (D-12; 1:1,000; Santa Cruz) and agarose-conjugatedanti-multi-ubiquitin antibodies (1:100 for IP, MBL).

In vitro auto-PARsylation, ubiquitylation assay. Recombinant GST–RNF146proteins were produced in E. coli and purified using glutathione–agarose beads. Thein vitro auto-ubiquitylation assay of RNF146 was carried out in 1× ubiquitylationbuffer (50mM Tris-HCl at pH 8.0, 100mM NaCl, 5mM MgCl2-ATP and 1mMdithiothreitol). Various forms of GST–RNF146 at 0.6 µM were incubated with125 nM E1, 2 µM E2 (UbcH5a) and HA–ubiquitin (Boston Biochem) at 37 ◦C for6 h. Reduced amounts of enzymes were used for PAR-enhanced auto-ubiquitylationassay (E1, 62.5 nM; E2, 250 nM; E3, 256 nM).The reaction was stopped byaddition of 2× SDS sample loading buffer and subject to SDS–PAGE followed byimmunoblotting with indicated antibodies.

To examine PARsylation-dependent ubiquitylation, recombinant His-taggedTNKS2 was first subjected to an in vitro auto-PARsylation assay in the presenceor absence of 1mM NAD+, as described previously6. PARsylated or unmodifiedTNKS2 was then pulled down by Ni–nitrilotriacetic acid beads (Qiagen), extensivelywashed and subjected to an in vitro ubiquitylation assay as described above. To blockthe build-up of poly(ADP-ribose) chains, the concentration of NAD+ was reducedto 100 µM and the reaction was shortened to 30min.

Pulse-chase analysis. SW480 cells were pretreated with XAV939. The com-pound was then washed off and pulse-chase analysis was carried out as de-scribed previously6.

Statistical analysis. Results are expressed as the means± s.d. from an appropriatenumber of experiments as indicated in the figure legends. The statistical analysis wasdone using an unpaired Student t -test and P < 0.05 was considered significant.

GST pulldown and mass spectrometry. HEK293 cells containing an inducibleshRNA targeting RNF146 were grown in medium with or without DOX for threedays followed by overnight treatment with 1 µM ABT-888, a PARP1/2 inhibitor, tosuppress PARsylation mediated by PARP1/2. Cells were then washed with ice-cold

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DOI: 10.1038/ncb2222 METHODS

PBS twice and lysed in RIPA buffer supplemented with 5 µM ADP-HPD (Alexis).For each sample, cell lysates from 30×15 cm dishes of cells were used for pulldownwith glutathione–agarose beads charged with 30 µg of GST–WWE. The mixturewas incubated for 6 h at 4 ◦C and washed six times with 1× PBS containing 0.05%Tween 20. The bound materials were resolved by SDS–PAGE. Complete gel laneswere excised and samples were subjected to in-gel tryptic digestion. Peptide extractsof controls (no RNF146 knockdown) were labelled with TMT Reagents 131 andcombined with extracts from corresponding samples of RNF146-knockdown laneslabelled with TMT Reagents 129. Peptide sequencing was carried out by liquidchromatography–tandem mass spectrometry on an Eksigent 1D+ high-pressureliquid chromatography system coupled to an LTQ-Orbitrap XL mass spectrometer(Thermo Scientific). Peptide mass and fragmentation data acquired by pulsed-Q

dissociation were searched against the IPI database using Mascot (Matrix Science).For each peptide sequence and modification state, reporter-ion signal intensitiesfrom all spectral matches were summed for each reporter-ion type and correctedaccording to the isotope correction factors given by themanufacturer. Only peptidesunique to a given protein within the total data set of identified proteins wereused for relative protein quantification. Peptide fold changes over the control(TMT 131) were calculated and subsequently renormalized using the median foldchange of all quantified peptides to compensate for differences in total proteinyield for each affinity purification. Protein fold changes were derived as medianpeptide fold change, P values were calculated using a one-way t -test and data werevisualized for further analysis using Spotfire DXP. All identified proteins are shownin Supplementary Table S1.

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DOI: 10.1038/ncb2222

Figure S1 Depletion of RNF146 increased the protein level, but not mRNA level, of axin1 and TNKS1/2. (a) Knockdown of NEDD4 does not affect Wnt3a-induced STF in 293 cells. Error bars denote the standard deviation (s.d.) between four replicates. (b) Depletion of RNF146 does not have a significant effect on the mRNA level of axin1 or TNKS1/2 in HEK293 cells. Error bars denote the standard deviation (s.d.) between triplicates.

(c) Depletion of RNF146 by inducible shRNA increases the protein level of axin1 and TNKS1/2 in PA1cells. Inducible b-catenin shRNA was used as a control. (d) Depletion of RNF146 does not affect the mRNA level of axin1 or TNKS1/2 in PA1 cells. (e) Knockdown of Drosophila RNF146 (CG8786) does not affect the mRNA level of exogenously expressed Drosophila axin. Error bars represent the standard deviation (s.d.) between four replicates.

a

d e

cb

0

0.2

0.4

siRNA:

0.6

0.8

1

1.2

pGL2

NEDD4-B

NEDD4-A

STF(Wnt3a CM)

Rel

ativ

e lu

cife

rase

act

ivity

RNF146-A

RNF146-B β-c

at

020406080

100120140

axin1

Rel

ativ

e m

RN

A le

vel

shRNA

Probe

β-ca

tsh

+DO

Xβ-

cats

h -D

OX

RN

F146

sh +

DO

XR

NF1

46sh

-DO

Xβ-

cats

h +D

OX

β-ca

tsh

-DO

XR

NF1

46sh

+D

OX

RN

F146

sh -D

OX

β-ca

tsh

+DO

Xβ-

cats

h -D

OX

RN

F146

sh +

DO

XR

NF1

46sh

-DO

Xβ-

cats

h +D

OX

β-ca

tsh

-DO

XR

NF1

46sh

+D

OX

RN

F146

sh -D

OX

β-ca

tsh

+DO

Xβ-

cats

h -D

OX

RN

F146

sh +

DO

XR

NF1

46sh

-DO

X

β-cat RNF146 TNKS TNKS2

120140

020406080

100

axin1

Rel

ativ

e m

RN

A le

vel

siRNA

Probe

TNK

S1+

2

pGL2

RN

F146

-AR

NF1

46-B

TNK

S1+

2

pGL2

RN

F146

-AR

NF1

46-B

TNK

S1+

2

pGL2

RN

F146

-AR

NF1

46-B

TNK

S1+

2

pGL2

RN

F146

-AR

NF1

46-B

RNF146 TNKS1 TNKS2

11582

182

115

axin1

64

49PA1

TNKS

Tubulin

TNKS1TNKS2

β-ca

teni

n sh

+D

OX

β-ca

teni

n sh

-DO

X

RN

F146

sh +

DO

X

RN

F146

sh -D

OX

020406080

100120140

DR

NF1

46-A

DR

NF1

46-B

DR

NF1

46-A

DR

NF1

46-B

DRNF146 Daxin-3XHA

Rel

ativ

e m

RN

A le

vel

dsRNA

Probe

Whi

te

Whi

te

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Figure S2 Sequence alignment of WWE domains. Three highly conserved amino acid residues, which WWE domain is named after, are highlighted in red. Note

positively charged amino acid residues at the carboxyl terminal of the WWE domain. Arg161 and Arg163 of the WWE domain of RNF146 are labeled.

hRNF146 EELKAASRGNGEYAW YYEGRNG--WWQYDERTSR---------ELEDAFSKGKKN--------TEMLIAGFLYVADLENMVQYRRNEH--GRRRKIKRdRNF146 EDICTTRATEDGFQ W YYEGRNG--WWQYDDRT---------SQDIEDAFKKGDK--------SCTILVAGYVYVVDLEQLVQQRQNEP--TRCRRVKRhDTX4 NYYDPSSAPGKGVV W EWENDNGS-WTPYDMEV---------GITIQHAYEKQHP--------WIDLTSIGFSYVIDFNTMGQINRQTQ---RQRRVRRhDTX1 NFYDPSSAPGKGIV W EWENDGGA-WTAYDMDI---------CITIQNAYEKQHP--------WLDLSSLGFCYLIYFNSMSQMNRQTR---RRRRLRRhDTX2 HLFPQHSAPGRGVV W EWLSDDGS-WTAYEASV---------CDYLEQQVARGNQ--------LVDLAPLGYNYTVNYTTHTQTNKTSS---FCRSVRR

hPARP11 NEVDDMDTSDTQWG W FYLAECGK-WHMFQPDTNSQC--SVSSEDIEKSFKTNPCGS-------ISFTTSKFSYKIDFAEMKQMNLTTG---KQRLIKRhPARP12 SVTKPPHFILTTDWI W YWSDEFGS-WQEYGRQGTVHPVTTVSSSDV EKAYLAYCTPGSDGQAATLK FQAGKHNYELDFKAFVQKNLVYGT--TKKVCRR

hPARP13 SVTKPANSVFTTKWI W YWKNESGT-WIQYGEEKDKRKNS NVDSSYLESLYQSCPRG-------VVPFQAGSRNYELSFQGMIQTNIASKT--QKDVIRR

hPARP14 EQESRADCISEFIE W QYNDNNTS--HCFNKMT---------NLKLEDARREKKK--------TVDVKINHRHYTVNLNTYTATDTKGHSLS VQRLTKS

hMULE1 RAQMTKYLQSNSNN W RWFDDRSGRWCSYSASN---------NSTIDSAWKSGET--------SVRFTAGRRRYTVQFTTMVQVNEETG---NRRPVMLhTRIP12 MLKKGNAQNTDGAI W QWRDDRGL-WHPYNRIDS---------RIIEQINEDTGTAR------AIQRKPNPLANSNTSGYSESKKDDARAQLMKEDPEL

hPARP7 STPPSSNVNSIYHTV W KFFCRDHFGWREYPESVI---------RLIEEANSRGLK--------EVRFMMWNNHYILHNSFFRREIKRRP---LFRSCFI

161 163W W E

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Figure S3 Full-length RNF146 interacts with PAR. Binding of PAR with GST-RNF146, but not GST-RNF146R163A, as shown by surface plasmon resonance. The experiment was performed as in Fig. 2e.

16

1012

8

4

0

-4

Res

onan

ce U

nits

0 100 200 300 400 500 600 700 800 900Time (s)

RNF146

RNF146R163A

18

14

6

2

-2

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Figure S4 Depletion of RNF146 increases the amount of axin1 and TNKS pulled down by GST-WWE. The experiment was performed as in Fig. 3b and 3c. Note that only a very small fraction of axin1 was pulled down by GST-WWE.

a b0.1%

116

-DO

X

+DO

X

GST-WWE

GST-WWER163A

WB:axin1

-DO

X

+DO

X

-DO

X+D

OX

cell lysate

RNF146 shRNA: Mar

ker

Mar

ker

182115

182

115

short exposure

long exposure

0.1%

-DO

X

+DO

X

GST-WWE

GST-WWER163A

-DO

X

+DO

X

-DO

X+D

OX

cell lysate

RNF146 shRNA: Mar

ker

Mar

ker

WB:TNKS

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Figure S5 PARsylated proteins increase autoubiquitination of RNF146 in vitro and in vivo. (a) A screen for compatible E2s in an in vitro RNF146 autoubiquitination assay reveals UbcH5a as a functional partner for RNF146. (b) PAR does not affect autoubiquitination of MDM2 and SMURF1. (c) PARsylated or unmodified His-tagged TNKS2 (TNKS2-PAR or TNKS2) was generated in an in vitro PARsylation reaction by addition of ATP and NAD+, recovered by Ni-NTA beads, extensively washed to remove residual NAD+, and

subjected to in vitro ubiquitination assay. Western blot analysis with anti-HA antibody revealed that PARsylated TNKS2 enhances total ubiquitination induced by RNF146 but not RNF146R163A. (d) PARsylated TNKS2 enhances autoubiquitination of RNF146 but not RNF146R163A. The experiment was performed as described in (c), and western blot analysis was performed using anti-GST antibody. (e) XAV939 significantly reduced autoubiquitination of exogenously expressed RNF146 in HEK293 cells.

a

db

c

26

182

115

82644937

E2: Ubc

H1

Ubc

H5a

- Ubc

H2

Ubc

H3

Ubc

H5b

Ubc

H5c

Ubc

H6

Ubc

H7

Ubc

H8

Ubc

H9

Ubc

H10

Ubc

H12

Ubc

H13

-Ubc

H1

Ub-HA

GST-RNF146

e

IP:Ubiquitin

WB:HA

TCL

poly-Ub-RNF146

RNF146-HA

DM

SO

XAV

939

WB:HA

Ub-HA

182

115

82

64

49

His-TNKS2 His-TNKS2-PAR

GST-RNF146: W

T

WT

R16

3A

R16

3A

- - --

-

- - -

182

1168264

182

1168264

Ub-HA

MDM2 GST

GST-MDM2

GST-SMURF1- HECT

PAR

PAR

PAR

GST

182

115

8264493726

GST-RNF146

GST-RNF146R163A

His

-TN

KS

2

His

-TN

KS

2

His

-TN

KS

2-PA

R

His

-TN

KS

2-PA

R

- -

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Figure S6 CASC3 is degraded by tankyrase and RNF146. (a) Inhibition of tankyrase by XAV939 increases the protein level of GFP-CASC3 in SW480 cells. (b) Depletion of RNF146 or co-depletion of TNKS1 and TNKS2 increases the protein level of GFP-CASC3 in SW480 cells. (c) The TBD domain of CASC3 is evolutionary conserved. (d) Deletion of

TBD abolishes the interaction between TNKS1and CASC3 in a co-immunoprecipitation assay. (e) The TBD domain is required for XAV939-induced stabilization of CASC3 in SW480 cells. (f) Deletion of TBD abolishes tankyrase and RNF146-dependent degradation of CASC3.

b

e

a c

df

Human TVTGERQSGDGQESTE

Mouse TVTGERQSGDGQESTE

Xenopus AVTGERQSGDGQESTE

Drosophila NLAGERQSGDGQESTE

GFP-CASC3

DM

SO

XAV

939

182

116

6449

Tubulin

GFP-CASC3

Tubulin

182

116

6449

siRNA: pGL2

TNK

S1+

2R

NF1

46-B

82

CACS3:

GFP-CASC3

FLAG-TNKS1

GFP-CASC3

WT

∆TB

D CACS3:

GFP-CASC3

Tubulin

182

116

64

49

WT ∆TBD

DM

SO

XAV

939

DM

SO

XAV

939

GFP-CASC3

CACS3: WT ∆TBD

pGL2

TNK

S1+

2R

NF1

46-B

pGL2

TNK

S1+

2R

NF1

46-B

siRNA:

Tubulin

182

116

64

49

GFP

IP:FLAGWB:GFP

WB:FLAG

182

116

116

182Input:FLAG

116

GFP GFP

GFP

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Figure S7 Full scans

WB: HA

WB: PAR

WB: GST

Fig. 2d

Fig. 2g

Fig. 1b

WB: Tubulin

64

WB: β-catenin

11582

6449

WB: HA

Fig. 2f

WB: HA

6449

WB: FLAG

115182

82

WB: PAR

115182

82

WB: axin1

115182

WB: TNKS

115182

82

WB: HA

6449

WB: Tubulin

6449

Fig. 3d

WB: Tubulin

6449

WB: Ub

115182

82

Fig. 4c

WB: HA

115182

8264

WB: GST

115182

8264

Fig. 5c and d

WB: GFP

115182

82

64

WB: Tubulin

182

82

6449

Fig. 5e

WB: GFP

115182

82

64

Fig. 5h

WB:GFP

115182

8264

WB:Tubulin

6449

Fig. 1d

WB: HA

8264

WB: Tubulin

6482

Fig. 2b

WB: axin1

115182

WB: TNKS

115182

82

WB: HA

WB: Tubulin

64

49

64

Fig. 2c

6449

WB: HA

6449

82

Fig. 3a

WB: axin1

115182

82

WB: PAR

115182

82

Fig. 3b

WB: axin1

115182

82

WB: axin1

115182

82

WB: axin1

115182

82

115182

82

WB: TNKS

Fig. 3c

WB: TNKS

115182

82

Fig. 3d

115182

82

WB: axin2 WB: axin2

115182

82

WB: Ub

115182

82

Fig. 4a

WB: HA

220100

50

WB: GST

220100

50

Fig. 4b

WB: HA

115

182

8264

WB: GST

115

182

8264

Fig. 4d

115182

82

64

WB:TNKS

Fig. 1d

115182

6482115

WB: axin1 WB: TNKS WB: Tubulin

Fig. 5g

WB:GFP

115182

82

64

1158264

WB:FLAG

1158264

Fig. 3e

115182

82

S35 labeled

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Table S1 Quantitative data for all 70 identified proteins (ProteinProphet-derived False Positive Rate <1%) with fold changes for DOX (TMT129) over control (No DOX, TMT131). The following information is provided: Representative IPI accession number, Entrez gene name, ProteinProphet-derived protein probability, Number of total spectra matched to the protein; Number of identified peptides unique to this protein in this dataset and considered for protein quantitation; Number of spectra matched to unique peptides; Log10 fold changes and associated p-values for DOX (TMT 129) with respect to No DOX (TMT131). P-values are arbitrarily set to 1 for non-significant single peptide quantitations.

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