Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling

ARTICLES NATURE |Vol 461 |1 October 2009a b c dCRE (forskolin)e fSTF (Wnt3a CM)siRNA:Axin 1Axin 2PARP1TubulinsiRNA:Axin 1FlagTubulinPARP1PARP2PARP1/2TNKS1/2pGL2siRNA:p-β-cateninβ-cateninAxin 1Axin 2Actin1A2A1B2B1A+2A1B+2B1A/B+2A/BpGL2Flag–TNKS2siRNA:Axin 1TubulinsiRNA:Wnt3a CM – + + + +β-cateninTubulinpGL21A2A1A+2A1B2B1B+2BpGL2pGL2g TNKS2 TNKS2(M1054V) h i–DOX +DOX –DOX +DOXpGL2TNKS1/2SW480pGL2TNKS1/2pGL2HEK293TNKS1/2pGL2TNKS1/2SW480TNKS2 (Fig. 3i and Supplementary Fig. 3i), did not affect the proteinlevels of axin and TNKS (Fig. 3j). Collectively, these results indicatethat TNKS1 and TNKS2 are the cellular efficacy targets of XAV939.Using additional siRNAs, we further demonstrated that co-depletionof TNKS1 and TNKS2 increases b-catenin phosphorylation, decreasesb-catenin abundance, and inhibits the transcription of b-catenin targetgenes in SW480 cells (Fig. 3b and Supplementary Fig. 3c, d). Notably,depletion of TNKS1 or TNKS2 alone did not increase axin 1/2protein levels (Fig. 3b), indicating that TNKS1 and TNKS2 functionredundantly in regulating axin protein levels. Co-depletion of TNKS1and TNKS2 also phenocopied the pharmacological effect of XAV939 inHEK293 and DLD-1 cells (Fig. 3c, d and Supplementary Fig. 3e). Inaddition, combined XAV939 and TNKS1/2 siRNA treatment increasedaxin protein levels even further (Supplementary Fig. 3f).We examined whether regulation of axin and Wnt signalling byTNKS is evolutionarily conserved. We found that a double-strandedRNA (dsRNA) targeting Drosophila TNKS increased protein levels,but not mRNA levels, of exogenously expressed Drosophila axin in S2cells (Fig. 3e and Supplementary Fig. 3g), and specifically inhibited aWnt reporter (P , 0.001) (Fig. 3f). We also showed that treatment ofzebrafish embryos with XAV939, but not its inactive analogueLDW643, significantly decreased the signal from a b-catenin/LEF1reporter (TOP–GFP; green fluorescent protein) (SupplementaryFig. 4a) and inhibited the mRNA expression of the b-catenin targetgene axin2 (Supplementary Fig. 4c). Furthermore, concomitantknockdown of two zebrafish TNKS genes (tnks1a and tnks1b) withmorpholinos specifically decreased TOP–GFP expression (SupplementaryFig. 4b). Together, these findings indicate that the regulatoryfunction of TNKS in Wnt signalling is evolutionarily conserved.DMSOXAV939LDW643GST–TNKS2SAM+PARP1B2B1B+2BHEK293Figure 3 | Tankyrase modulates axin protein levels. a, b, Simultaneousdepletion of TNKS1 and TNKS2 phenocopies XAV939 by increasing axinprotein levels and decreasing b-catenin protein levels in SW480 cells. Forboth TNKS1 and TNKS2, two independent siRNAs were generated fromunique target sequences, labelled A and B (1A, 2A, 1B and 2B). c, Codepletionof TNKS1 and TNKS2 increases the protein level of axin 1 (upperpanel) and blocks Wnt3a-induced b-catenin accumulation (lower panel) inHEK293 cells. d, Co-depletion of TNKS1 and TNKS2 inhibits STF reporter,but not CRE reporter, in HEK293 cells (n 5 4). e, Knockdown of TNKSincreases the protein level (upper panel), but not the mRNA level (lowerpanel), of haemagglutinin-conjugated Drosophila axin (Axin–33HA) inDrosophila S2 cells. dsRNA against white was used as control (n 5 4).616Percentage luciferase activity120100806040200siRNA:pGL21A+2A1B+2BHEK293β-cateninIC 50 (µM)dsRNA:Axin–3×HATubulinAxin–3×HAmRNA expression–765h43210dsRNA: –PARP1 PARP2 TNKS1 TNKS2XAV939 2.194 0.114 0.011 0.004LDW643 >18.75 >18.75 >18.75 >18.75ABT-888 0.0083 0.011 14.97 6.519IWR-1-endo >18.75 >18.75 0.131 0.056IWR-1-exo >18.75 >18.75 1.897 0.78©2009 Macmillan Publishers Limited. All rights reservedwhiteTNKSwhite TNKSjS2Axin 2TNKSTubulinWnt signalling is required for regenerative processes in adultzebrafish, including fin regeneration 15,16 . Inks genes are expressed incaudal fin tissue, and no transcriptional changes are detectable afterinjury (Supplementary Fig. 4d, panel iii). XAV939, but not LDW643,inhibited fin regeneration (Supplementary Fig. 4d, panels i and ii) andWnt-dependent activation of axin2 transcription (SupplementaryFig. 4d, panel iii). This phenotype is remarkably similar to whatwas observed for IWR-1, an axin stabilizer recently identified 17 , whichwe have found to also inhibit TNKS (as described in detail below).The fact that two structurally unrelated TNKS inhibitors blockWnt-dependent fin regeneration indicates that TNKS inhibition canantagonize Wnt signalling in vivo. However, we did not observeobvious early developmental defects associated with Wnt inhibition.Given that axin accumulation requires several hours, it is possible thatthe kinetics of axin stabilization are too slow to have an impact onearly developmental phenotypes in this model system. Alternatively,TNKS function in Wnt pathway modulation may be cell-type- ororgan-specific (for example, tail fin).TNKS1 and TNKS2 modify their substrates through the additionof several ADP-ribose units, referred to as poly-ADP-ribosylation(PARsylation) 18 . To determine whether the PARsylation activity ofTNKS is essential for regulating axin protein levels, we performedsiRNA-rescue experiments. In transient transfection experiments,TNKS overexpression at high levels induces b-catenin stabilizationin a PARP-domain-independent fashion. However, this is an overexpressionartefact probably mediated by sequestration of endogenousaxin by overexpressed TNKS (see Supplementary Fig. 6 fordetailed discussion). We therefore used lentiviral transduction at alow multiplicity of infection for the rescue experiments, in which theDMSORelative luciferase activity700600500400300200100No ligandLigand0dsRNA white TNKS white TNKSXAV939LDW643WinglessS2ABT-888SW480*IWR-1-endoIWR-1-exoBMP*TNKS1TNKS2f, Depletion of TNKS specifically inhibits Wnt reporter (LEF–Luc), but notBMP reporter (BRE–Luc), in S2 cells (*P , 0.001, n 5 4). g, Doxycycline(DOX)-induced expression of wild-type TNKS2, but not catalyticallyinactive mutant TNKS2(M1054V), rescues TNKS1/2 siRNA-inducedaccumulation of axin 1 in HEK293 cells. h, XAV939 inhibits auto-PARsylation of TNKS2. Recombinant TNKS2 protein used in thisexperiment contains the sterile alpha motif (SAM) domain and the PARPdomain. i, Compound activity in PARP1/2 and TNKS1/2 biochemical assays.j, Effects of compounds (5 mM, 24 h) on the protein levels of axin 2 andTNKS1/2 in SW480 cells. A background band that migrated right below theTNKS1 band is indicated by an asterisk.