Download In the prevailing view Conducitn is usually considered to act as a

© 2014. Published by The Company of Biologists Ltd.
Negative feedback regulation of the Wnt pathway by conductin/axin2 involves
insensitivity to upstream signalling
Dominic B. Bernkopf, Michel V. Hadjihannas, and Jürgen Behrens
Nikolaus-Fiebiger-Center, Chair of Experimental Medicine II,
Friedrich-Alexander Universität Erlangen-Nürnberg,
[email protected]
Keywords: axin / conductin / Dvl / negative feedback / Wnt
Journal of Cell Science
Accepted manuscript
Glückstr. 6, D-91054 Erlangen, Germany
1
JCS Advance Online Article. Posted on 6 November 2014
Abstract
Axin and conductin/axin2 are structurally related inhibitors of Wnt/-catenin signalling that
promote degradation of -catenin. Whereas axin is constitutively expressed, conductin is a
Wnt target gene implicated in 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 -
Journal of Cell Science
Accepted manuscript
catenin by conductin is only poorly blocked by Dvl2. We propose that insensitivity to Dvl is
an important feature of conductin´s role as a negative feedback regulator of Wnt signalling.
Introduction
Stabilisation of -catenin is a key step in the Wnt/-catenin signalling pathway allowing catenin to stimulate transcription of Wnt target genes in conjunction with TCF transcription
factors (Clevers and Nusse, 2012). In the absence of Wnts, -catenin is earmarked for
ubiquitination and proteasomal degradation by phosphorylation in a destruction complex
consisting of the tumor suppressor APC, the kinases GSK3 and CK1 and the scaffolding
proteins axin or conductin/axin2 (Stamos and Weis, 2013). Binding of Wnt ligands to their
receptors frizzled and LRP5/6 leads to membrane recruitment of axin proteins by the frizzledassociated phosphoprotein Dvl (dishevelled) ultimately resulting in the inhibition of -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 -catenin and
other destruction complex components. Specific mutations of the DIX domain that abolish the
head-to-tail interaction dissolve axin oligomers and render axin ineffective in -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. Recruitment of axin by Dvl leads to
inhibition of GSK3 activity 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 -catenin degradation
(Fiedler et al., 2011; Kishida et al., 1999; Schwarz-Romond et al., 2007).
2
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 by
activated Wnt signalling to remain active in -catenin degradation. In principle, conductin
might accumulate to levels sufficiently high to saturate available binding sites at Wnt
Journal of Cell Science
Accepted manuscript
receptor/Dvl complexes and might thus be able to continue degrading -catenin.
Alternatively, or in addition, conductin might be less susceptible to upstream inhibition. We
provide evidence that favours the latter mechanism by showing (i) that Wnt stimulation leads
to only a modest increase in conductin protein compared to axin levels, and (ii) that conductin
interacts less efficiently with Dvl2 than axin making it largely resistant to inhibition by Dvl2.
Thus, functional rather than expression differences determine the differential roles of axin and
conductin as constitutive versus inducible inhibitors of Wnt signalling.
Results and discussion
The relative amounts of axin and conductin under 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 hrs whereas that of
axin decreased (Fig. 1A, lanes 1,2; (Lustig et al., 2002; Yamamoto et al., 1999)).
Normalisation of the Western blot signals on serial dilutions of GFP-Conductin and GFPAxin 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 significantly 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 by mutations of APC (DLD1, SW480) or -catenin (HCT116) (Lustig et al., 2002).
In all cell lines axin levels exceeded those of conductin (Fig. 1B) confirming that conductin is
only moderately upregulated by Wnt signalling.
3
We next compared the potency of axin and conductin in degrading -catenin. Transfection of
axin in SW480 cells led to a stronger reduction of endogenous -catenin than transfection of
conductin (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
lower activity in degrading -catenin (Fig. 1C). Similarly, transiently expressed -catenin was
strongly reduced by coexpression of axin but less by coexpression of conductin (Fig. 1F).
Journal of Cell Science
Accepted manuscript
To compare the individual contribution of endogenous axin and conductin to the inhibition of
Wnt signalling, we knocked down their expression in MDA-MB-231 cells using siRNAs and
monitored nuclear -catenin levels by immunofluorescence staining. Depletion of axin or
conductin led to an increase in -catenin staining intensity in the absence of exogenous Wnt
ligands (Fig. 2A) suggesting that both factors are active in degrading -catenin. Of note,
MDA-MB-231 cells express endogenous Wnts (Bafico et al., 2004) which might stimulate the
pathway to a certain extent but are apparently not sufficient to fully block axin proteins.
Wnt3a treatment increased -catenin intensity, similar to treatment with the GSK3 inhibitor
BIO, whereas knockdown of -catenin reduced staining. Importantly, knockdown of
conductin further increased the Wnt3a-induced -catenin staining intensity, whereas
knockdown of axin did not (Fig. 2A). The Wnt3a-induced increase of dephosphorylated catenin (ABC) was augmented by 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/-catenin reporter activity than knockdown of axin
(Fig. 2D). Thus, despite its lower expression level and activity conductin appears to retain
negative regulatory activity under sustained Wnt activation whereas axin activity is more
strongly inhibited.
Recruitment of axin/conductin proteins to frizzled receptors by Dvl is widely considered the
initial step towards inhibition of the -catenin destruction complex (MacDonald et al., 2009;
Metcalfe and Bienz, 2011). We therefore tested whether axin and conductin might differ in
their interaction with Dvl. For this, recruitment of axin and conductin by CAAX-tagged Dvl2,
a membrane-associated modification of Dvl2, was determined by cell fractionation (Fig. 3A;
(Smalley et al., 1999)). Axin was equally distributed between membrane and cytoplasmic
fractions, and coexpression of Dvl2-CAAX increased the presence of axin in the membrane
4
fraction (Fig. 3A, lanes 1-4). Membrane recruitment of axin was strongly reduced when its
DIX domain was mutated (construct axinM3 (Fiedler et al., 2011)) demonstrating dependence
on the specific DIX-DIX mediated interaction 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 of conductin with that of axin generating CdtAxinDIX resulted in increased membrane
recruitment of this protein by Dvl2-CAAX (Fig. 3A, lanes 13-16). These results show that
conductin exhibits weaker interaction with Dvl2 than axin which is determined by the DIX
Accepted manuscript
domains of axin and conductin. The punctate pattern of GFP-Axin in the cytoplasm was
partially resolved by Dvl2-CAAX resulting in colocalisation of both proteins at the plasma
membrane (Fig. 3B, upper panels). In contrast, GFP-AxinCdtDIX containing the DIX domain of
conductin did not become membrane associated by 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 domain mutant axinM3 did not form puncta (Fiedler et al., 2011)
Journal of Cell Science
but, surprisingly, abolished puncta formation by Dvl2 (Fig. 3C). The M3 mutation impairs the
head interaction surface of the axin DIX domain preventing its homopolymerisation, but
leaves the tail interaction surface intact for interaction with Dvl2 (Fiedler et al., 2011). We
propose that this results in blunting of Dvl2 polymers leading to smaller complexes no longer
visible as puncta (see scheme). Conductin alone shows a diffuse staining pattern (cf. Fig. 1C),
and colocalised with Dvl2 in puncta, albeit with a lower efficiency than axin as indicated by
its remaining partially diffuse staining pattern (Fig. 3C). ConductinM3 remained diffuse in the
presence of Dvl2 but in contrast to axinM3 did not dissolve Dvl2 puncta. We propose that
conductinM3 integrates to a lower extent in Dvl2 polymers than axinM3 and therefore fails to
disrupt these polymers. These data support differences in the strength of interaction of axin
and conductin with Dvl2.
We next analysed whether -catenin degradation by axin and conductin is differently
inhibited by Dvl2. As readout we used immunofluorescence staining of endogenous -catenin
in SW480 cells (Fig. 4A). Axin efficiently degraded -catenin whereas conductin was less
efficient resulting in residual -catenin staining (Fig. 4A, a,f). Coexpression of Dvl2-CAAX
but not of the DIX domain mutant Dvl2M2-CAAX reduced -catenin degradation by axin
(Fig. 4A, b,c). Importantly, Dvl2-CAAX only weakly affected degradation by conductin (Fig.
5
4A, g). Axin containing the DIX domain of conductin (GFP-AxinCdtDIX) became largely
resistant to inhibition by Dvl2-CAAX (Fig. 4A, d,e), whereas conductin containing the DIX
domain of axin (GFP-CdtAxinDIX) became more sensitive to inhibition by Dvl2-CAAX (Fig.
4A, h,i). These results are quantified in Fig. 4B,C. Similarly, degradation of transiently
expressed -catenin by coexpressed axin was more efficiently inhibited by Dvl2-CAAX than
degradation by conductin. Conversely, -catenin degradation by AxinCdtDIX was unaffected by
Dvl2-CAAX whereas degradation by CdtAxinDIX was blocked by Dvl2-CAAX (Fig. 4D,E).
Journal of Cell Science
Accepted manuscript
Together these results show that the different capability of Dvl2 to interfere with axin and
conductin depends on the respective DIX-DIX domain interactions. A bioinformatical
analysis predicted ten amino acids 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 may mediate differential interaction with Dvl (Fig. S1). Like Dvl2, Dvl1 and
Dvl3 inhibited -catenin degradation by axin but not degradation by AxinCdtDIX (Fig. 4F). In
line with differential inhibition of axin versus conductin by Dvl2, knockdown of conductin
but not of axin further increased Dvl2 stimulated activity of the TCF/-catenin dependent
reporter (Fig. 4G).
Our results shed light on the molecular basis of negative feedback regulation in Wnt
signalling by showing that the amounts of conductin in Wnt stimulated cells remained lower
than those of axin and that conductin is relatively insensitive to Dvl2. This rules out mere
overproduction of conductin as the basis of negative pathway regulation and favours the idea
that conductin´s capacity as a negative feedback regulator is based mainly on its reduced
responsiveness towards upstream signalling. It remains to be determined whether the
qualitative rather than quantitative feedback mode revealed here has specific consequences for
the regulation of Wnt signalling. Replacement of axin by knock-in of the conductin/axin2
cDNA generated viable mice suggesting that compensatory mechanism can neutralise
functional 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 in
other pathways in which negative regulators act in both, constitutive and inducible modes,
e.g. patched in hedgehog signalling. While the reasons for this are not clear, we found that
conductin was less active in -catenin degradation than axin. It is conceivable that a gradual
6
suppression of signalling by conductin is favoured over an abrupt block by axin to allow for
temporal and spatial fine tuning of Wnt pathway activity.
Materials and Methods
Cell culture, transfections, and treatments
Cells were grown in DMEM /10% fetal bovine serum /1% penicillin/streptomycin at
Journal of Cell Science
Accepted manuscript
37°C/10% CO2. siRNAs against axin (Tanneberger et al., 2011), -catenin and conductin
(Hadjihannas et al., 2006) were transfected with Oligofectamine (Invitrogen), and plasmids
with polyethylenimine (Sigma) (HEK293T, U2OS cells) or Lipofectamine 2000 (Invitrogen)
(SW480 cells). Cells were treated with Wnt3a-conditioned medium (Willert et al., 2003) 48h
after siRNA transfection. (2’Z,3’E)-6-Bromoindirubin-3′-oxime (BIO) was obtained from
Sigma. TCF/-catenin dependent pBAR reporter activity (Major et al., 2007) was determined
in HEK293T cells.
Molecular biology
To 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 were generated using the Quickchange Site-Directed mutagenesis kit
(Stratagene) according to (Schwarz-Romond et al., 2007) and (Fiedler et al., 2011),
respectively.
Cell lysis, fractionation and Western blotting
Whole cell extracts were prepared in Triton X-100-based lysis buffer or hypotonic buffer
(Fig. 2C upper panels, (Lustig et al., 2002)) or Passive Lysis Buffer (Promega) (Fig. 2D).
Cytoplasmic and membrane fractions were obtained using the ProteoJET™ Membrane
Protein Extraction Kit (Fermentas). Western blotting was performed as described
(Tanneberger et al., 2011). Primary antibodies: mouse anti--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--catenin (ABC; Millipore),
mouse anti-GFP (Roche) and rat anti--tubulin (Serotec). Densitometric analysis was
performed with AIDA 2D Densitometry (raytest). For comparison of axin and conductin
7
amounts in Fig. 1A and B signal ratios obtained with anti-axin and anti-conductin antibodies
were normalised to those obtained with anti-GFP antibodies.
Immunofluorescence microscopy
Methanol-fixed cells were stained as described (Hadjihannas et al., 2006). Images were
acquired on an Axioplan2 microscope using Metamorph (Zeiss). For intensitiy measurements,
images were acquired at constant exposure times, and background-free intensities of the
Journal of Cell Science
Accepted manuscript
nuclear regions (Fig. 2) or of whole cells (Fig. 1,4) were determined.
Statistics
p-Values were determined using unpaired, two-tailed t-tests (Fig. 1D, 2D and 4B,F,G) or
significance was tested after ANOVA by post hoc testing based on the Bonferroni method
(Fig. 2A).
Acknowledgements
We thank A. Kikuchi, M. Bienz, T. Dale, and D. Sussman for reagents and M. Brückner for
technical assistance. This study was supported by EFI funding of the Friedrich-Alexander
Universität Erlangen-Nürnberg and the KFO257 of the DFG to J.B.
Author Contribution
D.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.
Competing interests
The authors declare no competing interests.
8
References
Bafico, A., Liu, G., Goldin, L., Harris, V. and Aaronson, S. A. (2004). An
autocrine mechanism for constitutive Wnt pathway activation in human cancer cells.
Cancer Cell 6, 497-506.
Journal of Cell Science
Accepted manuscript
Behrens, J., Jerchow, B. A., Wurtele, M., Grimm, J., Asbrand, C., Wirtz,
R., Kuhl, M., Wedlich, D. and Birchmeier, W. (1998). Functional interaction of an
axin homolog, conductin, with beta-catenin, APC, and GSK3beta. Science 280, 5969.
Bilic, J., Huang, Y. L., Davidson, G., Zimmermann, T., Cruciat, C. M.,
Bienz, M. and Niehrs, C. (2007). Wnt induces LRP6 signalosomes and promotes
dishevelled-dependent LRP6 phosphorylation. Science 316, 1619-22.
Chia, I. V. and Costantini, F. (2005). Mouse axin and axin2/conductin
proteins are functionally equivalent in vivo. Mol Cell Biol 25, 4371-6.
Clevers, H. and Nusse, R. (2012). Wnt/beta-catenin signaling and disease.
Cell 149, 1192-205.
Cliffe, A., Hamada, F. and Bienz, M. (2003). A role of Dishevelled in
relocating Axin to the plasma membrane during wingless signaling. Curr Biol 13, 9606.
Cselenyi, C. S., Jernigan, K. K., Tahinci, E., Thorne, C. A., Lee, L. A. and
Lee, E. (2008). LRP6 transduces a canonical Wnt signal independently of Axin
degradation by inhibiting GSK3's phosphorylation of beta-catenin. Proc Natl Acad Sci
U S A 105, 8032-7.
Ehebauer, M. T. and Arias, A. M. (2009). The structural and functional
determinants of the Axin and Dishevelled DIX domains. BMC Struct Biol 9, 70.
Fagotto, F., Jho, E., Zeng, L., Kurth, T., Joos, T., Kaufmann, C. and
Costantini, F. (1999). Domains of axin involved in protein-protein interactions, Wnt
pathway inhibition, and intracellular localization. J Cell Biol 145, 741-56.
Fiedler, M., Mendoza-Topaz, C., Rutherford, T. J., Mieszczanek, J. and
Bienz, M. (2011). Dishevelled interacts with the DIX domain polymerization interface
of Axin to interfere with its function in down-regulating beta-catenin. Proc Natl Acad
Sci U S A 108, 1937-42.
Hadjihannas, M. V., Bruckner, M., Jerchow, B., Birchmeier, W., Dietmaier,
W. and Behrens, J. (2006). Aberrant Wnt/beta-catenin signaling can induce
chromosomal instability in colon cancer. Proc Natl Acad Sci U S A 103, 10747-52.
Jho, E. H., Zhang, T., Domon, C., Joo, C. K., Freund, J. N. and Costantini,
F. (2002). Wnt/beta-catenin/Tcf signaling induces the transcription of Axin2, a
negative regulator of the signaling pathway. Mol Cell Biol 22, 1172-83.
9
Kishida, S., Yamamoto, H., Hino, S., Ikeda, S., Kishida, M. and Kikuchi, A.
(1999). DIX domains of Dvl and axin are necessary for protein interactions and their
ability to regulate beta-catenin stability. Mol Cell Biol 19, 4414-22.
Lustig, B., Jerchow, B., Sachs, M., Weiler, S., Pietsch, T., Karsten, U., van
de Wetering, M., Clevers, H., Schlag, P. M., Birchmeier, W. et al. (2002). Negative
feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal
and liver tumors. Mol Cell Biol 22, 1184-93.
Journal of Cell Science
Accepted manuscript
MacDonald, B. T., Tamai, K. and He, X. (2009). Wnt/beta-catenin signaling:
components, mechanisms, and diseases. Dev Cell 17, 9-26.
Metcalfe, C. and Bienz, M. (2011). Inhibition of GSK3 by Wnt signalling--two
contrasting models. J Cell Sci 124, 3537-44.
Schwarz-Romond, T., Fiedler, M., Shibata, N., Butler, P. J., Kikuchi, A.,
Higuchi, Y. and Bienz, M. (2007). The DIX domain of Dishevelled confers Wnt
signaling by dynamic polymerization. Nat Struct Mol Biol 14, 484-92.
Smalley, M. J., Sara, E., Paterson, H., Naylor, S., Cook, D., Jayatilake, H.,
Fryer, L. G., Hutchinson, L., Fry, M. J. and Dale, T. C. (1999). Interaction of axin
and Dvl-2 proteins regulates Dvl-2-stimulated TCF-dependent transcription. Embo J
18, 2823-35.
Stamos, J. L. and Weis, W. I. (2013). The beta-catenin destruction complex.
Cold Spring Harb Perspect Biol 5, a007898.
Tanneberger, K., Pfister, A. S., Kriz, V., Bryja, V., Schambony, A. and
Behrens, J. (2011). Structural and functional characterization of the Wnt inhibitor
APC membrane recruitment 1 (Amer1). J Biol Chem 286, 19204-14.
Willert, K., Brown, J. D., Danenberg, E., Duncan, A. W., Weissman, I. L.,
Reya, T., Yates, J. R., 3rd and Nusse, R. (2003). Wnt proteins are lipid-modified
and can act as stem cell growth factors. Nature 423, 448-52.
Wu, G., Huang, H., Garcia Abreu, J. and He, X. (2009). Inhibition of GSK3
phosphorylation of beta-catenin via phosphorylated PPPSPXS motifs of Wnt
coreceptor LRP6. PLoS ONE 4, e4926.
Yamamoto, H., Kishida, S., Kishida, M., Ikeda, S., Takada, S. and Kikuchi,
A. (1999). Phosphorylation of axin, a Wnt signal negative regulator, by glycogen
synthase kinase-3beta regulates its stability. J Biol Chem 274, 10681-4.
Zeng, X., Huang, H., Tamai, K., Zhang, X., Harada, Y., Yokota, C., Almeida,
K., Wang, J., Doble, B., Woodgett, J. et al. (2008). Initiation of Wnt signaling:
control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled
and axin functions. Development 135, 367-75.
10
Figure Legends
Fig. 1. Expression levels and -catenin degradation capacity of axin and conductin
(A, B) Western blotting for conductin (Cdt), axin and GFP in lysates of MDA-MB-231 cells
treated with Wnt3a for 6h 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 HEK293T cells
transfected with human GFP-Conductin or GFP-Axin (lanes 3-6 and 7-10 in A, and lanes 4-6
Journal of Cell Science
Accepted manuscript
and 7-9 in B). Loading control: -actin. Relative levels of axin (black) and conductin (grey)
were determined by densitometric measurements and normalisation to levels of overexpressed
proteins based on the anti-GFP blot. Error bars: s.e.m. of five independent experiments.
(C) Immunofluorescence staining of -catenin (red) in SW480 cells transfected with GFPConductin or GFP-Axin (green). Dotted lines mark transfected cells. Scale bar: 20µm.
(D) Quantification of -catenin fluorescence intensities of three independent experiments as
in (C). Ut, untransfected. Error bars: s.e.m. (n=20); * p<0.001 (t-test).
(E) Blotting of GFP versus -catenin intensities of individual cells from (C).
(F) Western blotting for Flag and GFP in lysates of SW480 cells transfected with indicated
plasmids. Numbers below Flag blot show densitometric measurements of Flag--catenin
normalised to empty GFP. (-), empty vector.
Fig. 2. Impact of depletion of axin and conductin on Wnt signalling
(A) MDA-MB-231 cells transfected with indicated siRNAs were treated with Wnt3a for 6 hrs
(+Wnt) or left untreated. -Catenin staining at DAPI stained nuclei was quantified in
individual cells. Error bars: s.e.m. of three independent experiments (n>1200); * p<0.05
(ANOVA).
(B-D) Western blotting for dephosphorylated (active) -catenin (ABC), axin and conductin in
lysates from MDA-MB-231 (B), U2OS (C; upper three panels: hypotonic lysates, lower two
panels: Triton X-100 lysates) and HEK293T cells (D) transfected with indicated siRNAs, and
incubated with or without Wnt3a for 6 hrs. Loading control: -actin. Bar chart in (D) shows
fold changes of TCF/-catenin dependent reporter activity. Error bars: s.e.m. of three
independent experiments (n=6); * p<0.001 (t-test).
11
Fig. 3. Differential association of axin and conductin with Dvl mediated by DIX-DIX
domain interactions
(A) Western blotting for GFP and HA in membrane (M) and cytoplasmic (C) fractions of
HEK293T cells transfected with indicated plasmids. Tubulin and PanCadherin blots show
purity of fractions. *, unspecific band.
(B) Immunofluorescence staining of HEK293T cells cotransfected with GFP-Axin or GFPAxinCdtDIX (green) together with either empty HA or HA-Dvl2-CAAX (red). Inlayed boxes
are magnified on the right. Scale bar: 20µm. Cells with uniform membrane recruitment of the
Journal of Cell Science
Accepted manuscript
GFP construct in the presence of Dvl2-CAAX were quantified. Error bars: s.e.m. of three
independent experiments (n=600).
(C) Immunofluorescence staining of indicated Flag constructs (red) in U2OS cells
coexpressing CFP-Dvl2 (green). Scale bar: 20µm. Quantification of cells with diffuse CFPDvl2 localisation. Error bars: s.e.m. of three independent experiments (n=400). Schemes on
the right show interaction models of DIX domains of axin and conductin (red) and Dvl2
(green). “X” indicates defective DIX heads due to M3 mutation.
Fig. 4. Differential inhibition of axin and conductin by Dvl via DIX domains
(A) Immunofluorescence staining of -catenin (red) in SW480 cells transfected with GFPAxin (a-c), GFP-AxinCdtDIX (d,e), GFP-Conductin (Cdt) (f,g) or GFP-CdtAxinDIX (h,i) (green)
together with either empty HA vector (-), HA-Dvl2-CAAX or HA-Dvl2M2-CAAX. Dotted
lines mark transfected cells. Scale bar: 20µm.
(B) Quantification of -catenin fluorescence intensities from (A). Error bars: s.e.m. of three
independent experiments (n>65); * p<0.01, ** p<0.001 (t-test).
(C) Blotting of GFP intensities versus -catenin intensities of individual cells from (A).
(D, E) Western blotting for Flag, GFP and HA in lysates from SW480 cells transfected with
indicated plasmids. Numbers below Flag blots show densitometric measurements of Flag-catenin normalised to empty GFP. (-), empty vector .
(F) Quantification of -catenin fluorescence intensities of SW480 cells expressing indicated
plasmids. (-), empty vector. Error bars: s.e.m. of three independent experiments (n>65); **
p<0.001 (t-test).
(G) Fold changes of TCF/-catenin dependent reporter activity in HEK293T cells transfected
with indicated siRNAs together with empty vector or HA-Dvl2. Error bars: s.e.m. of three
independent experiments (n=6); * p<0.01 (t-test).
12
Journal of Cell Science
Accepted manuscript
Journal of Cell Science
Accepted manuscript
Journal of Cell Science
Accepted manuscript
Journal of Cell Science
Accepted manuscript