Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling

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NATURE | Vol 461 | 1 October 2009ARTICLESexpression of TNKS is markedly lower than in the transient transfectionexperiments (data not shown). Doxycycline-induced expressionof wild-type TNKS2, but not the catalytically inactiveTNKS2(M1054V) mutant 19 , rescued the effect of TNKS1/2 siRNAson axin 1 protein expression (Fig. 3g). Similar results were obtainedwith TNKS1 (Supplementary Fig. 3h). These data indicate that thecatalytic activity of both tankyrases is required for the regulation ofaxin protein levels. Consistent with this finding, we showed thatXAV939, but not LDW643, inhibits auto-PARsylation of TNKS2 invitro (Fig. 3h). In biochemical activity assays, XAV939 stronglyinhibited TNKS1 and TNKS2, with half-maximal inhibitory concentrationvalues of 0.011 and 0.004 mM, respectively, but displayed muchweaker effects on PARP1 and PARP2 (Fig. 3i). Auto-PARsylation ofTNKS has been reported to promote its own degradation through theubiquitin-proteasome pathway 20 . We found that XAV939 treatmentled to a significant increase in TNKS protein levels (Fig. 3j), indicatingthat XAV939 also inhibits TNKS auto-PARsylation in vivo.A recent study described two compound series (IWR-1, -2, andIWR-3, -4, -5) that increase axin protein levels and inhibit Wnt signalling17 . It was shown that IWR-1-endo (also referred to as IWR-1)increased axin protein levels, whereas its close analogue IWR-1-exohad much weaker activity. Although the authors suggested that IWR-1might stabilize axin by binding to a region in the carboxy-terminal halfof axin, we examined whether IWR-1 compounds may in fact stabilizeaxin by inhibiting TNKS. Notably, IWR-1-endo strongly inhibitedTNKS1 and TNKS2 in biochemical assays, with IWR-1-exo beingapproximately tenfold less active (Fig. 3i and Supplementary Fig. 3i),consistent with their potency in axin stabilization assays (Fig. 3j). Incontrast, neither of these two compounds inhibited PARP1/2 (Fig. 3i).Consistent with the activity of IWR-1-endo in TNKS1/2 biochemicalassays, IWR-1-endo, but not IWR-1-exo, significantly stabilized endogenousTNKS1, TNKS2 and axin 2 (Fig. 3j and Supplementary Fig. 3j),indicating that IWR-1-endo inhibited auto-PARsylation of TNKS invivo. These results indicate that IWR-1-endo stabilizes axin at least inpart through TNKS inhibition. Together, our data demonstrate thatthe PARsylation activity of TNKS1/2, but not PARP1/2, is required forthe regulation of axin protein levels.TNKS interacts with axin to regulate axin levelsWe next explored how TNKS regulates axin protein levels. Using coimmunoprecipitationexperiments, we found that endogenousTNKS1 and TNKS2 associate with axin 2 in SW480 cells (Fig. 4a).In addition, we detected a strong interaction between axin 1/2 andTNKS1/2 in the yeast two-hybrid assay (Fig. 4b and data not shown).The ankyrin repeat domain of TNKS1 was required and sufficient forthe interaction with axin 1 (Supplementary Fig. 6a). Strikingly, asmall amino-terminal region of axin 1 (amino acids 19–30), whichencompasses the most conserved stretch of amino acids within axin(Fig. 4c), was both required and sufficient to interact with TNKS1(Fig. 4b). The specific interaction of axin 1 with TNKS1 through thisdomain, which we named tankyrase-binding domain (TBD), wasfurther substantiated by glutathione S-transferase (GST) pull-downand co-immunoprecipitation assays (Fig. 4d, e).We next assessed the functional consequences of disrupting thephysical interaction between axin and TNKS. If binding between axinand TNKS promoted axin degradation, deletion of the TBD shouldresult in the stabilization of axin. Indeed, whereas cells expressingwild-type GFP–axin 1 demonstrated low basal protein levels that werestrongly increased in response to XAV939 treatment, cells expressingGFP–axin 1(D19–30) already exhibited high basal protein levels thatdid not further respond to compound treatment (Fig. 4f andSupplementary Fig. 5). Importantly, restoring the TNKS1–axin 1interaction by fusing the heterologous TNKS binding domain ofeither IRAP (insulin-regulated aminopeptidase, also known asLNPEP) or TRF1 (telomeric repeat binding factor, also known asTERF1) to GFP–axin 1(D19–30) fully restored its response toXAV939 (Fig. 4f). We further hypothesized that overexpression ofthe N-terminal domain of axin may compete with endogenous axinfor the binding of TNKS and thus stabilize axin. Indeed, overexpressionof GFP–axin 1N (amino acids 1–87), but not the mutant with theapGL2pGL2AXIN2siRNA:IP: Axin 2 IgG Axin 2WB: TNKSWB: axin 2SW480TNKS1TNKS2β-galactosidasebassaycTNKS1 GSK3β VectorRGS GSK3β β-cateninDIXWT+ + –19 301 863 Human (axin 1) ASFTEDAPRPPVPGEEGELVSN88+ +/– +/–Mouse ASFTEDAPRPPVPGEEGELVSN215+ - –Xenopus GSFTEDAPRPPVPGEEGELITΔN35– + –Zebrafish SSFTEDAPRPPVPGEEGDLVSΔN65– + –Drosophila DDNECSGPRPPVPGEESRVKKΔN88– + –Sea urchin SGGSQLSERPPVPGEETEDRGΔN100– + –ΔC150+ + –Human (axin 2) SSFREDAPRPPVPGEEGETPPΔ19-30– + –19-30+ – –GFP–axind e f g 1NGFP–axin 1 + – + –GFP–axin 1IRAP TRF1– + – +constructs:WT Δ19–30 Δ19–30 Δ19–30 DOXGFP–axin 1(Δ19–30)Flag–TNKS1 – – + +Axin 1IP: FlagGFP–axin 1WB: GFPTubulinTNKSGFPGFPGFP–axin 1GSTTCLFlagFlag–TNKS1TubulinGFP10% inputGSTGST–axin 1NGST–axin 1N(Δ19–30)GST–axin 1(19–30)DMSOXAV939DMSOXAV939DMSOXAV939DMSOXAV939GFP–axin 1N(Δ19–30)– + – +HEK293 HEK293 SW480 HEK293Figure 4 | Tankyrase physically and functionally interacts with axin. a, Coimmunoprecipitationof endogenous axin 2 and TNKS in SW480 cells.b, Binding between axin 1 fragments and TNKS1 in the yeast two-hybridassay (1, strong binding; 1/2, weak binding; 2, no binding). The N88fragment retains partial self-activation activity. c, The TBDs of axin proteinsare evolutionarily conserved. Highly conserved amino acid residues aredepicted in red. d, The N-terminal fragment of axin 1 (amino acids 1–87)©2009 Macmillan Publishers Limited. All rights reservedbinds to TNKS1 in a GST pull-down assay. e, The TBD is required for theinteraction between axin 1 and TNKS in a co-immunoprecipitation assay.f, The TBD is required for XAV939-induced axin 1 protein accumulation inSW480 cells. The expression of GFP–axin 1 was under the control of themetallothionein promoter to ensure low transcription. g, DOX-inducedoverexpression of GFP–axin 1N (amino acids 1–87) leads to accumulation ofendogenous axin 1 in HEK293 cells.617
ARTICLES NATURE |Vol 461 |1 October 2009deleted TBD, substantially increased endogenous axin 1 protein levelswhile not affecting its mRNA expression (Fig. 4g and data not shown).Together, these findings demonstrate that the physical interactionbetween axin and TNKS, which is mediated by the evolutionarilyconserved TBD, is critical for regulating axin protein levels.Axin is degraded through PARsylation and ubiquitinationThe increase in axin protein levels in response to XAV939 treatmentcould be due to modulation of translation or protein stability.Consistent with the latter possibility, XAV939 treatment prolongedthe half-life of endogenous axin 2 in SW480 cells (Fig. 5a). The degradationof axin is probably mediated by the ubiquitin-proteasomepathway, because the polyubiquitination of axin 1 increased significantlyafter addition of the proteasome inhibitor MG132 (Fig. 5b). Incontrast, co-treatment of XAV939 with MG132 significantly diminishedaxin 1 and axin 2 polyubiquitination (Fig. 5b and SupplementaryFig. 7a), indicating that XAV939 may stabilize axin by preventingits polyubiquitination.Auto-PARsylation of TNKS or TNKS-mediated TRF1 PARsylationleads to ubiquitination and degradation of TNKS or TRF1, respectively20,21 . Together with our findings, this raised the possibility thatabcWB: axin 1TCLDMSO XAV9390 1 2 3 1 2 3 (h)IP: IgG UbDMSOMG132MG132+XAV939DMSOMG132MG132+XAV939WB: UbWB: axin 1WB: tubulinSW480GST–axin 1(1–280)XAV939TNKS2TNKS2GST–axin 1(1–280)SW480––+––+–++–+++–++–+Axin 2Poly-Ub-axin 1+++++++––+––deIP:XAV939WB: PARWB: GFPControlGFPGFP–axin 1GFP–axin 1– – +Axin 1Wash off with:Pre-treat with XAV939 – + + + +IP: axin 2TCLWB: UbWB: PARWB: axin 2SW480DMSOXAV939MG132MG132+XAV939WB: tubulin1 2 3 4 5SW480GFP–axin 1GFP–axin 1Poly-Ubaxin2Axin 2Axin 2Figure 5 | XAV939 stabilizes axin protein and inhibits ubiquitination ofaxin. a, XAV939 (1 mM) stabilizes axin 2 in SW480 cells as indicated by apulse-chase analysis. b, Ubiquitination of axin is inhibited by XAV939(1 mM). The position of axin 1 is labelled with an arrow. Slow-migratingpolyubiquitinated axin 1 conjugates are indicated. c, In vitro PARsylation ofGST–axin 1 (amino acids 1–280) by TNKS2. d, XAV939 (1 mM, 6 h) inhibitsin vivo PARsylation of axin 1. e, Post-translational modification of axin 2 in acompound wash-off experiment. SW480 cells pretreated with XAV939 werewashed and incubated with medium supplemented with the indicatedcompound for 1 h, and analysed by the immunoprecipitation assay. Theposition of axin 2 is indicated by the arrows. IP, immunoprecipitation; TCL,total cell lysates.618©2009 Macmillan Publishers Limited. All rights reservedaxin degradation may be facilitated through direct PARsylation byTNKS. Indeed, TNKS2 was able to PARsylate an axin 1 fragment(amino acids 1–280) containing the TBD in vitro (Fig. 5c), whichwas completely inhibited by XAV939 treatment (Fig. 5c). Using anantibody that specifically recognizes the PAR modification, we alsoobserved that exogenously expressed GFP–axin 1 was PARsylated incells (Fig. 5d). In addition, the PARsylation signal was stronglyreduced in the presence of XAV939 (Fig. 5d), indicating that axinPARsylation is mediated by TNKS in vivo. To enhance the detectionof endogenous axin 2 ubiquitination and PARsylation, we pretreatedcells with XAV939 to increase endogenous axin 2 levels. Axin 2 wasrapidly degraded within 1 h after XAV939 was washed off (Fig. 5e). Asexpected, treatment of MG132 blocked axin 2 degradation andstrongly increased its polyubiquitination (Fig. 5e and SupplementaryFig. 7b). Treatment with XAV939 and MG132, however,completely blocked the ubiquitination of axin 2 (Fig. 5e and SupplementaryFig. 7b). Interestingly, an anti-PAR antibody reactivesignal that co-migrated with axin 2 was detected when cells weretreated with MG132 alone and disappeared when cells were alsotreated with XAV939 (Fig. 5e). Together, these findings are consistentwith the hypothesis that TNKS promotes the ubiquitination anddegradation of axin, which may be mediated, at least in part, throughthe direct PARsylation of axin.XAV939 inhibits growth of DLD-1 cancer cellsBecause XAV939 inhibited b-catenin signalling even in APC-deficientcells, we examined whether this compound could inhibit the proliferationof APC-deficient colorectal cancer cells using b-catenin-dependentDLD-1 cells and b-catenin-independent RKO cells (SupplementaryFig. 8a). Under low serum growth conditions, XAV939, but not theinactive analogue LDW643, significantly inhibited colony formation ofDLD-1 cells (Fig. 6a). Importantly, XAV939 did not affect colonyformationinRKOcells(Fig.6b).TNKS1 was proposed to be required for the resolution of sistertelomere association or assembly of bipolar spindles, and TNKS1knockdown was reported to cause strong mitotic arrest 22,23 .However, using XAV939 treatment or individual/combinatorialTNKS1/TNKS2 siRNA knockdown, we did not observe any overtmitotic arrest phenotype (Supplementary Fig. 8b and data notshown), demonstrating that XAV939 does not inhibit the proliferationof DLD-1 cells through an antimitotic function.If the antiproliferative effect of XAV939 on DLD-1 cells wasinstead mediated by an increase in axin protein levels, knockdownof AXIN1/2 may rescue the growth inhibitory effects of compoundtreatment. Indeed, siRNA-mediated depletion of axin 1 and axin 2proteins completely abolished the antiproliferative effect of XAV939(Fig. 6c), indicating that the growth inhibitory effects of XAV939 inDLD-1 cells are due to axin-dependent inhibition of Wnt signalling.DiscussionDespite the fact that the Wnt pathway is an attractive target for anticancertherapy, there are few druggable targets in this pathway togenerate small molecule Wnt inhibitors. Using a chemical geneticsapproach, we have discovered tankyrases as novel targets for Wntinhibition and described a novel mechanism to promote b-catenindegradation through inhibition of tankyrases and stabilization ofaxin (Supplementary Fig. 9). This finding may pave the way formechanism-based treatments of Wnt-dependent cancers.Our data indicate that tankyrases are physiological regulators of axinprotein homeostasis and Wnt signalling. Although the precise mechanisticdetails remain unclear, tankyrases seem to promote the ubiquitinationof axin, possibly through direct PARsylation of axin. Notably,TNKS-mediated PARsylation seems to also stimulate ubiquitinmediatedproteolysis of TRF1 and TNKS itself 20,21 . These findingsindicate that coupling of these biochemical processes may be a moregeneral regulatory mechanism, akin to the widespread occurrence ofphosphodegrons that couple phosphorylation to ubiquitination 24,25 .
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