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SHORT REPORT

Negative-feedback regulation of the Wnt pathway byconductin/axin2 involves insensitivity to upstream signalling

Dominic B. Bernkopf, Michel V. Hadjihannas and Jurgen Behrens*

ABSTRACT

Axin and conductin (also known as axin2) are structurally related

inhibitors of Wnt/b-catenin signalling that promote degradation of b-

catenin. Whereas axin is constitutively expressed, conductin is a

Wnt target gene implicated in Wnt negative-feedback regulation.

Here, we show that axin and conductin differ in their functional

interaction with the upstream Wnt pathway component Dvl.

Conductin shows reduced binding to Dvl2 compared to axin, and

degradation of b-catenin by conductin is only poorly blocked by

Dvl2. We propose that insensitivity to Dvl is an important feature of

the role of conductin as a negative-feedback regulator of Wnt

signalling.

KEY WORDS: Axin, Conductin, Dvl, Negative feedback, Wnt

INTRODUCTIONStabilisation of b-catenin is a key step in the Wnt/b-catenin

signalling pathway, allowing b-catenin to stimulate transcription

of Wnt target genes in conjunction with TCF transcription factors

(Clevers and Nusse, 2012). In the absence of Wnt proteins, b-

catenin is earmarked for ubiquitylation and proteasomal

degradation by phosphorylation mediated by a destruction

complex consisting of the tumour suppressor APC, the kinases

GSK3 and CK1, and the scaffolding proteins axin (also known as

axin1) or conductin (also known as axin2) (Stamos and Weis,

2013). Binding of Wnt ligands to their receptors frizzled and

LRP5 or LRP6 (LRP5/6) leads to membrane recruitment of axin

proteins by the frizzled-associated phosphoprotein dishevelled 1,

2 or 3 (Dvl) ultimately resulting in the inhibition of b-catenin

phosphorylation and degradation (Bilic et al., 2007; Cliffe et al.,

2003; Cselenyi et al., 2008; Smalley et al., 1999; Wu et al., 2009;

Zeng et al., 2008).

Axin molecules form dynamic oligomers by head-to-tail

interaction of their DIX domains (Fiedler et al., 2011; Kishida

et al., 1999). These complexes, often seen as cytoplasmic puncta

upon overexpression, are thought to provide high-avidity interaction

sites for b-catenin and other destruction complex components.

Specific mutations of the DIX domain that abolish the head-to-tail

interaction prevent axin oligomerisation and render axin ineffective

at mediating b-catenin degradation (Fiedler et al., 2011). Dvl also

has a DIX domain through which it binds to axin. Dvl can interfere

with axin function in different ways. Frizzled-associated Dvl

recruits axin to frizzled–LRP5/6 receptor complexes, which causes

inhibition of axin-bound GSK3 mediated by phosphorylated LRP5/

6 receptors. (Cselenyi et al., 2008; Wu et al., 2009; Zeng et al.,

2008). In addition, the DIX–DIX domain interactions of Dvl and

axin disturb self-assembly of axin homopolymers and thereby

interfere with b-catenin degradation (Fiedler et al., 2011; Kishida

et al., 1999; Schwarz-Romond et al., 2007).

Axin and conductin are related proteins that share key

sequence elements (Behrens et al., 1998; Fagotto et al., 1999).

In contrast to axin, which is constitutively expressed, conductin is

a direct Wnt target gene upregulated after activation of the

pathway (Jho et al., 2002; Lustig et al., 2002). Therefore, it is

assumed that conductin acts as a negative-feedback regulator of

Wnt signalling. However, it is unclear how conductin escapes

upstream inhibition upon activation of Wnt signalling to remain

active in b-catenin degradation. In principle, conductin might

accumulate to levels sufficiently high to saturate available

binding sites at Wnt-receptor–Dvl-complexes and might thus

be able to continue degrading b-catenin. Alternatively, or in

addition, conductin might be less susceptible to upstream

inhibition. We provide evidence that favours the latter

mechanism by showing, (1) that Wnt stimulation leads to only

a modest increase in conductin protein compared to axin levels,

and (2) that conductin interacts less efficiently with Dvl2 than

axin making it largely resistant to inhibition mediated by Dvl2.

Thus, functional rather than expression differences determine the

differing roles of axin and conductin as constitutive versus

inducible inhibitors of Wnt signalling.

RESULTS AND DISCUSSIONThe relative amounts of axin and conductin during Wnt signalling

were determined by western blotting of extracts from Wnt3a-

treated MDA-MB-231 cells. In line with previous reports, the

amount of conductin increased after Wnt3a treatment for 6 h,

whereas that of axin decreased (Fig. 1A, lanes 1,2; Lustig et al.,

2002; Yamamoto et al., 1999). Normalisation of the western blot

signals by using serial dilutions of GFP–conductin and GFP–axin

present on the same blots allowed comparison of axin and

conductin levels (Fig. 1A, lanes 3–6 and 7–10). This showed that

conductin levels were lower than axin levels under resting

conditions and, surprisingly, remained substantially lower after

Wnt3a-induced upregulation of conductin and downregulation of

axin (Fig. 1A, bar chart). These results largely exclude strong

accumulation of conductin as a decisive factor for negative-

feedback regulation. We also compared axin and conductin

levels in three colorectal carcinoma cell lines, which exhibit

constitutively high conductin expression due to activation of Wnt

signalling because of mutations of APC (DLD1, SW480) or b-

catenin (HCT116) (Lustig et al., 2002). In all these cell lines, axin

levels exceeded those of conductin (Fig. 1B), confirming that

conductin is only moderately upregulated by Wnt signalling.

Nikolaus-Fiebiger-Center, Chair of Experimental Medicine II, Friedrich-AlexanderUniversitat Erlangen-Nurnberg, Gluckstr. 6, D-91054 Erlangen, Germany.

*Author for correspondence (jbehrens@molmed.uni-erlangen.de)

Received 3 July 2014; Accepted 22 October 2014

� 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 33–39 doi:10.1242/jcs.159145

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We next compared the potency of axin and conductin indegrading b-catenin. Transfection of axin in SW480 cells led to a

stronger reduction of endogenous b-catenin than transfection ofconductin (Fig. 1C,D) over a range of different expression levels(Fig. 1E). Notably, axin was present in puncta, whereas conductin

was more diffusely distributed, possibly explaining its loweractivity in degrading b-catenin (Fig. 1C). Similarly, transientlyexpressed b-catenin was strongly reduced by coexpression of axin

but less by coexpression of conductin (Fig. 1F).To compare the individual contribution of endogenous axin

and conductin to the inhibition of Wnt signalling, we knockeddown their expression in MDA-MB-231 cells using small

interfering RNAs (siRNAs) and monitored nuclear b-cateninlevels by immunofluorescence staining. Depletion of axin orconductin led to an increase in b-catenin staining intensity in the

absence of exogenous Wnt ligands (Fig. 2A), suggesting that bothfactors are active in degrading b-catenin. Of note, MDA-MB-231cells express endogenous Wnts (Bafico et al., 2004) that might

stimulate the pathway to a certain extent, but they are apparentlynot sufficient to fully block axin proteins. Wnt3a treatmentincreased b-catenin intensity, similar to treatment with the GSK3inhibitor BIO, whereas knockdown of b-catenin reduced staining.

Importantly, knockdown of conductin further increased theWnt3a-induced b-catenin staining intensity, whereas

knockdown of axin did not (Fig. 2A). The Wnt3a-inducedincrease of dephosphorylated b-catenin (ABC) was augmentedby knockdown of conductin, but not axin, in three different cell

lines, at similar knockdown rates (Fig. 2B–D). Moreover,knockdown of conductin led to a stronger increase of Wnt3a-induced TCF/b-catenin reporter activity than knockdown of axin

(Fig. 2D). Thus, despite its lower expression level and activityconductin appears to retain negative regulatory activity undersustained Wnt activation, whereas axin activity is more stronglyinhibited.

Recruitment of axin and conductin proteins to frizzledreceptors by Dvl is widely considered the initial step towardsinhibition of the b-catenin destruction complex (MacDonald

et al., 2009; Metcalfe and Bienz, 2011). We therefore testedwhether axin and conductin might differ in their interaction withDvl. For this, recruitment of axin and conductin by CAAX-tagged

Dvl2, a membrane-associated form of Dvl2, was determined bycell fractionation (Fig. 3A; Smalley et al., 1999). Axin wasequally distributed between membrane and cytoplasmic fractions,and coexpression of Dvl2–CAAX increased the proportion of

Fig. 1. Expression levels and b-catenin degradation capacity of axin and conductin. (A,B) Western blotting for conductin (Cdt), axin and GFP in lysates ofMDA-MB-231 cells treated with Wnt3a (+) for 6 h or left untreated (–) (A, lanes 1,2), in lysates of colorectal cancer cells HCT116, SW480 and DLD1 (B, lanes 1–3), and in serially diluted lysates of HEK293Tcells transfected with human GFP–conductin or GFP–axin (lanes 3–6 and 7–10 in A, and lanes 4–6 and 7–9 in B).b-actin was used as a loading control. Relative levels of axin (black) and conductin (grey) were determined by densitometric measurements and normalisation tolevels of overexpressed proteins based on the anti-GFP antibody blot. Results are mean6s.e.m. of five independent experiments. (C) Immunofluorescencestaining of b-catenin (red) in SW480 cells transfected with GFP–conductin or GFP–axin (green). Dotted lines mark transfected cells. Scale bar: 20 mm.(D) Quantification of b-catenin fluorescence intensities of three independent experiments as in C. Ut, untransfected. Results are mean6s.e.m. (n520);*P,0.001 (Student’s t-test). (E) Blotting of GFP versus b-catenin intensities of individual cells from C. (F) Western blotting for Flag and GFP in lysates of SW480cells transfected with the indicated plasmids. Numbers below the Flag blot show densitometric measurements of Flag–b-catenin normalised to coexpressedGFP (empty vector). 2, Flag-only vector.

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axin in the membrane fraction (Fig. 3A, lanes 1–4). Membranerecruitment of axin was strongly reduced when its DIX domainwas mutated (construct axinM3; Fiedler et al., 2011)

demonstrating dependence on the specific DIX–DIX-mediatedinteraction with Dvl2 (Fig. 3A, lanes 5–8). In contrast to axin,conductin was mainly present in the cytoplasmic fraction and

only poorly recruited to the membrane by Dvl2–CAAX (Fig. 3A,lanes 9–12). Importantly, replacement of the DIX domain ofconductin with that of axin, generating CdtAxinDIX, resulted inincreased membrane recruitment of this protein by Dvl2–CAAX

(Fig. 3A, lanes 13–16). These results show that conductinexhibits a weaker interaction with Dvl2 than axin, and that thisis determined by the DIX domains of axin and conductin. The

punctate pattern of GFP–axin in the cytoplasm was lesspredominant upon coexpression of Dvl2–CAAX, resulting incolocalisation of both proteins at the plasma membrane (Fig. 3B,

upper panels). In contrast, GFP–axinCdtDIX (axin containing theDIX domain of conductin) did not become associated with themembrane upon coexpression of Dvl2–CAAX (Fig. 3B, lower

panels).Like axin, Dvl2 localises in cytoplasmic puncta, and axin

colocalises with Dvl2 in such puncta (Fig. 3C). The DIX domainmutant axinM3 does not form puncta (Fiedler et al., 2011) but,

surprisingly, abolished formation of Dvl2 puncta (Fig. 3C). TheM3 mutation impairs the head interaction surface of the axin DIXdomain preventing its homopolymerisation, but leaves the tail

interaction surface intact for interaction with Dvl2 (Fiedler et al.,2011). We propose that this results in inhibition of Dvl2polymerisation, leading to smaller complexes no longer visible

as puncta (Fig. 3C, see scheme). Conductin alone had a diffuse

staining pattern (Fig. 1C), and colocalised with Dvl2 in puncta,albeit with a lower efficiency than axin, as indicated by itsremaining partially diffuse staining pattern (Fig. 3C).

ConductinM3 remained diffuse in the presence of Dvl2 but, incontrast to axinM3, did not abolish the formation of Dvl2 puncta.We propose that conductinM3 integrates to a lower extent in Dvl2

polymers than axinM3 and therefore fails to disrupt thesepolymers. These data support differences in the strength of theaxin and conductin interaction with Dvl2.

We next analysed whether b-catenin degradation mediated by

axin and conductin is differently inhibited by Dvl2. As a readout,we used immunofluorescence staining of endogenous b-catenin inSW480 cells (Fig. 4A). Axin efficiently degraded b-catenin,

whereas conductin was less efficient, resulting in residual b-catenin staining (Fig. 4A,a,f). Coexpression of Dvl2–CAAX,but not of the DIX domain mutant Dvl2M2–CAAX, reduced

the level of axin-mediated b-catenin degradation (Fig. 4A,b,c).Importantly, Dvl2–CAAX only weakly affected degradationmediated by conductin (Fig. 4A,g). Axin containing the DIX

domain of conductin (GFP–axinCdtDIX) became largely resistantto inhibition by Dvl2–CAAX (Fig. 4A,d,e), whereas conductincontaining the DIX domain of axin (GFP–CdtAxinDIX) becamemore sensitive to inhibition by Dvl2–CAAX (Fig. 4A,h,i). These

results are quantified in Fig. 4B,C. Similarly, degradation oftransiently expressed b-catenin upon coexpression of axin wasmore efficiently inhibited by Dvl2–CAAX than degradation

by conductin. Conversely, b-catenin degradation mediated byAxinCdtDIX was unaffected by Dvl2–CAAX, whereas degradationmediated by CdtAxinDIX was blocked by Dvl2–CAAX (Fig. 4D,E).

Taken together, these results show that the different capability of

Fig. 2. Impact of depletion of axinand conductin on Wnt signalling.(A) MDA-MB-231 cells transfectedwith the indicated siRNAs weretreated with Wnt3a for 6 h (+Wnt) orleft untreated. b-catenin staining inDAPI-stained nuclei was quantified inindividual cells. Results aremean6s.e.m. of three independentexperiments (n.1200); *P,0.05(ANOVA). (B–D) Western blotting fordephosphorylated (active) b-catenin(ABC), axin and conductin in lysatesfrom MDA-MB-231 (B), U2OS(C; upper three panels, hypotoniclysates; lower two panels, Triton X-100 lysates) and HEK293T cells(D) transfected with the indicatedsiRNAs, and incubated with or withoutWnt3a for 6 h. b-actin was used as aloading control. The bar chart in Dshows fold changes of TCF/b-catenin-dependent reporter activity.Results are mean6s.e.m. of threeindependent experiments (n56);*P,0.001 (Student’s t-test).

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Dvl2 to interfere with axin and conductin depends on the respectiveDIX–DIX domain interactions. A previous bioinformatical

analysis has predicted that there are ten amino acids that are

likely to be required for specificity of DIX domain interactions(Ehebauer and Arias, 2009). Four of these amino acids differ

between axin and conductin and, therefore, might mediate

Fig. 3. Differential association ofaxin and conductin with Dvl aremediated by DIX–DIX domaininteractions. (A) Western blotting forGFP and HA in membrane (M) andcytoplasmic (C) fractions ofHEK293T cells transfected withindicated plasmids. Tubulin and pan-cadherin blots show purity offractions. *, unspecific band.(B) Immunofluorescence staining ofHEK293T cells co-transfected withGFP–axin or GFP–axinCdtDIX (green)together with either a HA-only vector(+HA) or HA–Dvl2–CAAX vector(red). The boxed regions aremagnified on the right. Scale bar:20 mm. Cells with uniform membranerecruitment of the GFP construct inthe presence of Dvl2-CAAX werequantified. Results are mean6s.e.m.of three independent experiments(n5600). (C) Immunofluorescencestaining of indicated Flag constructs(red) in U2OS cells coexpressingCFP–Dvl2 (green). Scale bar: 20 mm.Quantification of cells with diffuseCFP–Dvl2 localisation. Results aremean6s.e.m. of three independentexperiments (n5400). Schemes onthe right show interaction models ofDIX domains of axin and conductin(red) and Dvl2 (green). ‘X’ indicatesdefective DIX heads owing to theM3 mutation.

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Fig. 4. See next page for legend.

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differences in their interaction with Dvl (supplementary material

Fig. S1). Like Dvl2, Dvl1 and Dvl3 inhibited b-catenin degradationmediated by axin but not degradation mediated by AxinCdtDIX

(Fig. 4F). In line with the differences in inhibition of axin versusconductin mediated by Dvl2, knockdown of conductin but not of

axin further increased Dvl2-stimulated activity of the TCF/b-catenin dependent reporter (Fig. 4G).

Our results shed light on the molecular basis of negative-

feedback regulation in Wnt signalling by showing that the amountof conductin in Wnt-stimulated cells remained lower than that ofaxin and that conductin is relatively insensitive to Dvl2. This

rules out mere overproduction of conductin as the basis ofnegative pathway regulation and favours the idea that the capacityof conductin to act as a negative-feedback regulator is based

mainly on its reduced responsiveness towards upstreamsignalling. It remains to be determined whether the qualitativerather than quantitative feedback mode revealed here has specificconsequences for the regulation of Wnt signalling. Replacement

of axin by knock-in of conductin/axin2 cDNA leads to viablemice, suggesting that compensatory mechanisms can neutralisefunctional differences between axin and conductin in vivo (Chia

and Costantini, 2005).The separation of tasks between axin and conductin as

constitutive and inducible negative regulators of Wnt signalling,

respectively, is remarkable and contrasts with feedback modes inother pathways where negative regulators act in both constitutiveand inducible modes (e.g. patched in hedgehog signalling).

Although the reasons for this are not clear, we found thatconductin was less active in b-catenin degradation than axin. It isconceivable that a gradual suppression of signalling by conductinis favoured over an abrupt block by axin to allow for temporal

and spatial fine tuning of Wnt pathway activity.

MATERIALS AND METHODSCell culture, transfections and treatmentsCells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with

10% fetal bovine serum and 1% penicillin-streptomycin at 37 C under

10% CO2. siRNAs against axin (Tanneberger et al., 2011), b-catenin

and conductin (Hadjihannas et al., 2006) were transfected using

Oligofectamine (Invitrogen), and plasmids were transfected using

polyethylenimine (Sigma) (HEK293T, U2OS cells) or Lipofectamine

2000 (Invitrogen) (SW480 cells). Cells were treated with Wnt3a-

conditioned medium (Willert et al., 2003) at 48 h after siRNA

transfection. (29Z,39E)-6-Bromoindirubin-39-oxime (BIO) was obtained

from Sigma. TCF/b-catenin-dependent pBAR reporter activity (Major

et al., 2007) was determined in HEK293T cells.

Molecular biologyTo generate GFP–axinCdtDIX and GFP–CdtAxinDIX, amino acids 719–832

of rat axin and amino acids 700–840 of mouse conductin were

exchanged. The M2 mutant of Dvl2 and M3 mutants of axin and

conductin (Schwarz-Romond et al., 2007 and Fiedler et al., 2011,

respectively) were generated using the Quikchange site-directed

mutagenesis kit (Stratagene). Expression plasmids for Dvl1 and Dvl3

were provided by D. Sussman (University of Maryland, Baltimore, MD),

Flag–axin (rat) by A. Kikuchi (Osaka University, Japan), GFP–axin

(human) by M. Bienz (MRC, Cambridge, UK) and HA–Dvl2-CAAX by

T. Dale (Cardiff University, UK).

Cell lysis, fractionation and western blottingWhole-cell extracts were prepared in Triton X-100-based lysis buffer or

hypotonic buffer (experiments in Fig. 2C, upper panels, Lustig et al., 2002)

or Passive Lysis Buffer (Promega) (experiments in Fig. 2D). Cytoplasmic

and membrane fractions were obtained using the ProteoJETTM Membrane

Protein Extraction Kit (Fermentas). Western blotting was performed as

described previously (Tanneberger et al., 2011). Primary antibodies were:

mouse anti-b-actin, rabbit anti-Flag, rabbit anti-HA, rabbit anti-PanCadherin

(all from Sigma), rabbit anti-axin (Cell Signaling), mouse anti-conductin (C/

G7; Lustig et al., 2002), mouse anti-active-b-catenin (ABC; Millipore),

mouse anti-GFP (Roche) and rat anti-a-tubulin (Serotec) antibodies.

Densitometric analysis was performed with AIDA 2D Densitometry

software (raytest). For comparison of axin and conductin amounts in

Fig. 1A,B, signal ratios obtained with anti-axin and anti-conductin

antibodies were normalised to those obtained with anti-GFP antibodies.

Immunofluorescence microscopyMethanol-fixed cells were stained as described previously (Hadjihannas

et al., 2006). Images were acquired on an Axioplan2 microscope using

Metamorph (Zeiss). For intensity measurements, images were acquired at

constant exposure times, and background-free intensities of the nuclear

regions (Fig. 2) or of whole cells (Figs 1, 4) were determined.

StatisticsP-values were determined using unpaired, two-tailed Student’s t-tests

(Fig. 1D; Fig. 2D and Fig. 4B,F,G) or significance was tested after

ANOVA by post-hoc testing based on the Bonferroni method (Fig. 2A).

AcknowledgementsWe thank A. Kikuchi, M. Bienz, T. Dale, and D. Sussman for reagents, and M.Bruckner for technical assistance.

Competing interestsThe authors declare no competing interests.

Author contributionsD.B.B. and M.V.H. designed and performed experiments, D.B.B., M.V.H., and J.B.analysed data, and D.B.B. and J.B. wrote the manuscript.

FundingThis study was supported by Emerging Fields Initiative funding of the Friedrich-Alexander Universitat Erlangen-Nurnberg; and the DeutscheForschungsgemeinschaft [grant number KFO257 to J.B.].

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.159145/-/DC1

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