© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 3892-3902 doi:10.1242/jcs.195685
RESEARCH ARTICLE
Essential role of the Dishevelled DEP domain in a Wnt-dependent
human-cell-based complementation assay
ABSTRACT
Dishevelled (DVL) assembles Wnt signalosomes through dynamic
head-to-tail polymerisation by means of its DIX domain. It thus
transduces Wnt signals to cytoplasmic effectors including β-catenin,
to control cell fates during normal development, tissue homeostasis
and also in cancer. To date, most functional studies of Dishevelled
relied on its Wnt-independent signalling activity resulting from
overexpression, which is sufficient to trigger polymerisation,
bypassing the requirement for Wnt signals. Here, we generate a
human cell line devoid of endogenous Dishevelled (DVL1– DVL3),
which lacks Wnt signal transduction to β-catenin. However, Wnt
responses can be restored by DVL2 stably re-expressed at nearendogenous levels. Using this assay to test mutant DVL2, we show
that its DEP domain is essential, whereas its PDZ domain is
dispensable, for signalling to β-catenin. Our results imply two
mutually exclusive functions of the DEP domain in Wnt signal
transduction – binding to Frizzled to recruit Dishevelled to the
receptor complex, and dimerising to cross-link DIX domain polymers
for signalosome assembly. Our assay avoids the caveats associated
with overexpressing Dishevelled, and provides a powerful tool for
rigorous functional tests of this pivotal human signalling protein.
KEY WORDS: Dishevelled, DEP domain, PDZ domain, Frizzled
INTRODUCTION
The Wnt/β-catenin cascade is an ancient signalling pathway that
specifies numerous cell fates during embryonic development and
tissue homeostasis of animals, and its aberrant activation leads to
various cancers, most notably colorectal cancer (Clevers and Nusse,
2012). Its key effector, β-catenin, is continuously destabilised by the
Axin degradasome, which also contains the APC tumour suppressor
and two serine-threonine protein kinases (glycogen synthases
kinase 3, GSK3; casein kinase 1α, CK1α) that phosphorylate the
N-terminus of β-catenin to earmark it for proteasomal degradation
(MacDonald et al., 2009). This process is inhibited when a Wnt
protein couples a cognate Frizzled receptor (FZD1–FZD10 in
humans; Fz1–Fz4 in flies) with its low-density lipoprotein receptorrelated protein 5/6 co-receptor (typically LRP6) (Cong et al., 2004;
Tamai et al., 2000), which triggers recruitment of Dishevelled
1
MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis
2
Crick Avenue, Cambridge CB2 0QH, UK. Leslie Dan Faculty of Pharmacy, Room
901, University of Toronto, 144 College Street, Toronto, Ontario, Canada M5S 3M2.
*Authors for correspondence ([email protected];
[email protected])
M.B., 0000-0002-7170-8706
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is properly attributed.
Received 22 July 2016; Accepted 30 August 2016
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(DVL) to the plasma membrane. DVL thus assembles a
signalosome that engages the Axin degradasome to block the
enzymatic activity of GSK3 within this complex (Metcalfe and
Bienz, 2011; Piao et al., 2008; Wu et al., 2009). This allows
β-catenin to accumulate and to bind to TCF/LEF factors in the
nucleus, to operate context-dependent transcriptional switches that
determine specific cell fates during normal development and in
neoplasia (Mosimann et al., 2009).
DVL is an ancient signalling hub protein found in all animals
and in humans. It functions as a pivot in transducing Wnt signals
either to β-catenin to effect nuclear Wnt responses, or to various
alternative cytoplasmic effectors that elicit ‘non-canonical’ Wnt
responses, typically in post-mitotic cells, specifying cellular
features such as planar cell polarity (PCP) (Angers and Moon,
2009; Wynshaw-Boris, 2012). Animals encode between one and
three DVL paralogs (DVL1–DVL3 in humans), typically with
redundant functions. Each paralog has three structured domains –
DIX (Dishevelled and Axin) at its N-terminus, PDZ (Post-synaptic
density protein-95, Disc large tumour suppressor, Zonula
occludens-1) and DEP (Dishevelled, Egl-10 and Pleckstrin) –
separated from each other by long flexible linkers. Various
functions and ligands have been ascribed to these domains,
typically based on overexpression assays.
The DIX domain has a unique activity in undergoing dynamic
and reversible head-to-tail polymerisation resulting in meta-stable
filaments, which enables Dishevelled to assemble signalosomes
(Schwarz-Romond et al., 2007a). These are dynamic protein
assemblies that are detectable in cells by immunofluorescence as
distinct membrane-associated or cytoplasmic puncta (SchwarzRomond et al., 2005). DIX-dependent polymerisation causes a
dramatic increase in the local concentration of Dishevelled, which
increases its avidity for low-affinity binding partners, thus allowing
Dishevelled to interact efficiently with signalling effectors (Bienz,
2014). One of these is Axin whose own DIX domain mediates its
co-polymerisation with Dishevelled through direct heterotypic
DIX–DIX interactions (Fiedler et al., 2011). Polymerised
Dishevelled also promotes the phosphorylation of the cytoplasmic
tail of LRP6 (Bilic et al., 2007), to generate phosphorylated
PPPSPxS/T motifs, which serve as high-affinity docking sites for
Axin (Tamai et al., 2004) and inhibit GSK3 by binding to its
catalytic pocket (Stamos et al., 2014), thereby stabilising β-catenin
and transducing Wnt signals to the nucleus. Dishevelled
signalosomes are also likely to transduce non-canonical Wnt
signals, given that these responses seem to be contingent on
membrane-associated Dishevelled puncta (e.g. Axelrod, 2001; Park
et al., 2005). We have recently discovered that the signalling activity
of Dishevelled also depends on its DEP domain, which dimerises,
probably to crosslink DIX polymers to catalyse signalosome
assembly (Gammons et al., 2016).
The PDZ and DEP domains have both been implicated in the
binding to Frizzled receptors. Frizzled proteins are distantly related
Journal of Cell Science
Melissa V. Gammons1,*, Trevor J. Rutherford1, Zachary Steinhart2, Stephane Angers2 and Mariann Bienz1,*
to the G-protein coupled receptor (GPCR) family (forming their
fourth class, class F; Venkatakrishnan et al., 2013), and their
cytoplasmic tail contains a highly conserved KTxxxW motif that is
essential for Wnt signal transduction to β-catenin (Umbhauer et al.,
2000), and also for non-canonical Wnt responses (Wu et al., 2008).
Evidence from biophysical in vitro binding assays based on
recombinant protein suggested that this motif can interact directly
with the PDZ (Wong et al., 2003) or the DEP domain (Tauriello
et al., 2012). Cell-based assays provided evidence for a functional
interaction between Frizzled and DEP but not PDZ (Pan et al., 2004;
Tauriello et al., 2012; Gammons et al., 2016). However, definitive
proof for the function of these domains in Frizzled binding is still
missing as the signalling by Dishevelled in these overexpression
assays does not depend on physiological Wnt signals.
The reason for this is that overexpression of Dishevelled alone is
sufficient to trigger its polymerisation (Schwarz-Romond et al.,
2007a), which initiates signal transduction to β-catenin through
Axin co-polymerisation (Fiedler et al., 2011; Schwarz-Romond
et al., 2007b). Thus, these activities of overexpressed Dishevelled
neither require Wnt nor its recruitment to Frizzled, although they
depend at least partially on the LRP6 co-receptor, both in
mammalian cells and in flies (Metcalfe et al., 2010). Indeed, it is
well-established that Dishevelled overexpression not only activates
signalling through β-catenin, but also interferes with PCP and other
non-canonical signalling responses both in vertebrate and
invertebrate tissues (e.g. Boutros et al., 1998; Park et al., 2005;
Penton et al., 2002). This imposes a caveat on the conclusions that
can be derived from these overexpression studies, in particular those
regarding the functional interaction of Dishevelled with Frizzled
upon Wnt signalling – the crucial step initiating Wnt signal
transduction.
Here, we have developed a complementation assay based on
human embryonic kidney (HEK293T) cells whose three DVL
paralogs were deleted by CRISPR–Cas9 technology (DVL triple-
Journal of Cell Science (2016) 129, 3892-3902 doi:10.1242/jcs.195685
knockout, TKO). These DVL TKO cells are unable to stabilise βcatenin in response to Wnt and to activate β-catenin-dependent
transcription, but these responses can be restored by re-expression of
DVL2 at near-endogenous levels. Using this assay to test various
mutants of DVL2, we demonstrate that its DEP-dependent binding to
Frizzled is essential for Wnt-dependent signalling to β-catenin,
whereas its PDZ domain is dispensable for β-catenin responses. This
establishes two mutually exclusive functions of the DEP domain,
switching between binding to Frizzled and cross-linking Dishevelled
polymers for signalosome assembly. Our complementation assay
opens up further studies of the function of this pivotal signalling
protein, and might also be potentially adaptable for the study of noncanonical Wnt responses.
RESULTS
DVL TKO cells fail to respond to Wnt
We generated DVL TKO HEK293T cells using CRISPR–Cas9
technology, selecting individual cell clones that fail to express any
of the three DVL paralog proteins, as judged by western blotting
(Fig. 1A), and confirmed by sequencing of target sites. As expected,
Wnt3a-treated DVL TKO cells (unlike their parental controls) failed
to accumulate non-phosphorylated (‘active’) β-catenin (ABC) and
to stimulate β-catenin-dependent transcription, as judged by
SuperTOP luciferase reporter assays (Fig. 1B), demonstrating that
DVL function is essential for these β-catenin responses. We also
confirmed that these TKO cells exhibit increased levels of several
FZD paralogs expressed by them (Fig. 1B), corroborating recent
findings that Dishevelled promotes the destabilisation of Frizzled by
RNF43 and/or ZNRF3, a pair of paralogous membrane-spanning E3
ubiquitin ligases that clear Frizzled from the plasma membrane in
the absence of Wnt signalling (Jiang et al., 2015). Furthermore,
phosphorylation of LRP6 S1490 (within the membrane-proximal
PPPSP motif ) is reduced to background levels in the TKO cells
(Fig. 1B), confirming the key role of Dishevelled in conferring this
Fig. 1. Dishevelled TKO cells fail to respond to Wnt3a. (A) Western blot analysis of lysates from DVL TKO or parental control cells (wt), probed with
antibodies as indicated. (B) Graph shows SuperTOP assays in DVL TKO or control cells, with or without 6 h of incubation in Wnt3a-conditioned medium (WCM).
Above, corresponding western blots (with or without 2 h incubation in WCM). ABC, activated β-catenin. (C) Graph shows SuperTOP assays in DVL TKO or control
cells (as in B), transiently transfected with LRP6–GFP or SNAP–FZD5 (lysed 24 h after transfection). Above, corresponding western blot. (D) Proliferation of DVL
TKO or control cells. n=3 independent experiments, statistical test two-way ANOVA with repeated measures, *P<0.05. Error bars represent s.e.m. of three
independent experiments.
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RESEARCH ARTICLE
Wnt-dependent phosphorylation of LRP6 (Bilic et al., 2007; Zeng
et al., 2008).
As Dishevelled signals through LRP6 (Metcalfe et al., 2010), we
wondered whether LRP6 overexpression might restore β-catenin
responses in DVL TKO cells. This was the case; overexpression of
LRP6–GFP, but not of SNAP–FZD5, stimulated β-catenindependent transcription in DVL TKO cells, regardless of Wnt3a
(Fig. 1C), consistent with previous epistasis experiments (Cong
et al., 2004). Note that there was very little S1490 phosphorylation
in these experiments despite the high levels of LRP6
overexpression, corroborating the important role of Dishevelled in
promoting this phosphorylation. However, the low level of
Dishevelled-independent S1490 phosphorylation ( probably
resulting from clustering of the overexpressed LRP6; Metcalfe
and Bienz, 2011) seemed to be sufficient for signalling to β-catenin,
possibly because this phosphorylation within PPPSP primes for
CK1-mediated phosphorylation of the +2 serine or threonine
residue flanking these motifs (Davidson et al., 2005; Zeng et al.,
2005), which might suffice for blocking GSK3. Conversely,
overexpressed DVL2–GFP (giving rise to cytoplasmic puncta) is
severely compromised in its ability to signal in LRP6 KO cells,
regardless of Wnt stimulation (Fig. S1). These results corroborate
the epistatic relationship between Dishevelled and LRP6 in human
cells, establishing LRP6 as a key effector of Dishevelled during
signal transduction to β-catenin.
Finally, we also noticed that DVL TKO cells proliferate more
slowly than their parental control (Fig. 1D), consistent with the
previously reported roles of Wnt signalling and Dishevelled in
promoting cell cycle progression (Kikuchi et al., 2010; Niehrs and
Acebron, 2012).
Low levels of re-expressed DVL2 restores Wnt-dependent
β-catenin responses
We chose DVL2 to restore Wnt/β-catenin responses in DVL TKO
cells as this is the predominant DVL paralog in many mammalian
cells (e.g. Lee et al., 2008). Dvl2 is also crucial in murine
development although its function is redundant with Dvl3 in
several tissues (Etheridge et al., 2008; Hamblet et al., 2002),
whereas Dvl1 is dispensable for normal development (Lijam et al.,
1997). Re-supplying DVL2–GFP by transient transfection of DVL
Journal of Cell Science (2016) 129, 3892-3902 doi:10.1242/jcs.195685
TKO resulted in high levels of β-catenin-dependent transcription,
which was not further increased by Wnt stimulation (Fig. 2A),
indicating efficient Wnt-independent signalling activity of this
transgene. This is accompanied by cytoplasmic DVL2–GFP
puncta that are visible virtually in every transfected cell (see
below).
By contrast, β-catenin-dependent transcription is clearly Wnt3adependent if DVL TKO cells are supplied with low levels of DVL2–
GFP by means of the pBABEpuro vector, which produces ∼10×
induction of the β-catenin-dependent signalling activity,
corresponding to ∼50% of the transcriptional response mounted
by the parental HEK293T cells (Fig. 2B). This implies that the resupplied DVL2–GFP signals as efficiently as endogenous DVL: the
level of DVL2–GFP in the DVL TKO cells is roughly the same as
that of the endogenous DVL2 in the parental cells (Fig. 2B), which,
in addition, also express DVL1 and DVL3 (Fig. 1A). Importantly,
DVL2–GFP is completely diffuse in the absence of Wnt3a, and the
only puncta that can be detected occasionally in these cells are
associated with the nuclear envelope, as previously observed in
HEK293T cells expressing an inducible DVL2–GFP (Smalley
et al., 2005), probably reflecting DVL2–GFP depositions at
microtubule-organising centres. However, short pulses of Wnt3a
produce a small number of membrane-associated puncta in a
fraction of the cells (Fig. 2C, lower panels), reminiscent of the Wntdependent membrane-associated DVL puncta observed in zebrafish
embryos (Hagemann et al., 2014).
The rate of proliferation of the DVL TKO line stably
expressing DVL2–GFP is indistinguishable from that of its
parental wild-type (wt) control (Fig. 2D), indicating that the
DVL2–GFP rescue transgene restores normal proliferation in the
DVL TKO cells. Notably, live-cell imaging of transiently
transfected COS-7 cells revealed that DVL2–GFP continues to
accumulate to high levels in these cells after transient transfection
until they inevitably arrest, without ever progressing through
mitosis (Movie 1). It is thus likely that the growth of the stably
transfected DVL TKO cells depends on their ability to limit the
expression of the DVL2–GFP rescue transgene. There seems to
be negative selective pressure to ensure that the cellular
concentration of DVL2–GFP is kept to near-physiological
levels (see also below).
Fig. 2. Re-expressed DVL2 restores Wnt responses in DVL TKO cells. (A,B) Graph shows SuperTOP assays in DVL TKO or control cells (A) transiently
or (B) stably transfected with DVL2–GFP, n=3 independent experiments (as in Fig. 1C). Above, corresponding western blots. (C) Confocal images of
representative DVL TKO cells transiently or stably expressing DVL2–GFP (fixed 18 h after transfection), as indicated (OE, overexpression). Lower panels show
examples of cytoplasmic and plasma membrane-associated puncta (arrowhead) at higher magnification. Scale bar, 10 µm. (D) Proliferation assay of DVL TKO
cells, with and without stable expression of DVL2–GFP. n=3 independent experiments, two-way ANOVA with repeated measures, *P<0.05. Error bars represent s.
e.m. of three independent experiments.
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The PDZ domain is dispensable for Wnt signal transduction
to β-catenin
An early biochemical study provided evidence that a peptide
spanning the KTxxxW motif from Xenopus Fz7 interacted with the
purified PDZ domain from Dvl1 (Wong et al., 2003), implicating
this domain in the recruitment of Dishevelled to the receptor
complex in the plasma membrane. However, overexpressed PDZ
fails to be recruited to FZD in cell-based assays (Pan et al., 2004;
Tauriello et al., 2012; Gammons et al., 2016), questioning the
physiological role of this in vitro interaction. To determine the role
of the PDZ domain during Wnt-dependent signalling by
Dishevelled, we established stable DVL TKO lines expressing
various DVL2–GFP mutants at physiological levels, to assess their
rescue activity (henceforth called Rescuestable assay). For
comparison, we also tested these mutants after transient
transfection of these cells, i.e. under conditions of overexpression
(henceforth called RescueOE assay). Described below is the use of
the β-catenin-dependent reporter to assess the rescue activity of
DVL2 as, of the five read-outs monitored (Fig. 1), the transcription
assay proved to be the most quantitative and sensitive.
To test whether the PDZ domain is required for DVL rescue
activity, we designed two point mutations in its ligand-binding cleft
(V334E and D331A R338A, called DARA; see below) that are
expected to block ligand access, based on structural information on
this cleft and its interaction with cognate physiological ligands
(Cheyette et al., 2002; Lee et al., 2015). To confirm that these
mutations affect ligand binding, we used a sensitive in vitro binding
assay based on nuclear magnetic resonance (NMR) spectroscopy,
which is capable of detecting low-affinity binding in the high
micromolar Kd range. We purified 15N-labelled minimal wt or
mutant PDZ domains of DVL2 after bacterial expression, and
acquired 1H–15N correlation spectra, alone or after incubation with a
well-validated C-terminal peptide from Dvl1 (DVL-C; Lee et al.,
2015). As expected, this peptide induced exchange broadening or
shift perturbation of numerous cross peaks in the spectrum of the wt
domain (Fig. 3A), as previously observed (Lee et al., 2015).
Projection of these affected residues onto the solution structure of
the Dvl1 PDZ domain in complex with its DVL peptide ligand
(2MX6; Lee et al., 2015) confirmed that these residues are located
predominantly in the αB/βB peptide-binding groove (Fig. 3B). In
contrast, we only observed weak exchange broadening and shift
perturbation of a subset of these cross peaks in the case of the DARA
mutant domain upon incubation with excess DVL-C peptide (Fig.
S2), confirming that the ligand binding of this mutant is severely
compromised, as previously shown (Lee et al., 2015). However, the
V334E mutant domain exhibited no exchange broadening or shift
perturbation whatsoever upon incubation with peptide (Fig. 3C),
which demonstrates that the V334E point mutation completely
blocks ligand binding to the DVL2 PDZ domain. In both cases, the
spectra show a pattern of peak dispersion similar to that of the wt
domain (Fig. 3B; Fig. S2), confirming that the overall structural fold
of the PDZ domain is not affected by any of these point mutations.
We also tested a Lipoyl (Lip)-tagged peptide spanning the KTxxxW
motif from FZD5 (GKTLESWRRFTS) for binding to Lip–PDZ, but
failed to see any spectral changes (Fig. 3D). An equally negative
result was obtained with an equivalent Lip-tagged peptide from
FZD8 (GKTLESWRALCTR; data not shown), or with the peptide
from Xenopus Fz7 (GKTLQSWRRFYH; Fig. S2) previously tested
for interaction with Dvl1 PDZ (Wong et al., 2003). Our failure to
detect any interaction of these Frizzled peptides with DVL2 PDZ
argues against these cytoplasmic tail sequences of Frizzled being
direct ligands for the PDZ domain.
Journal of Cell Science (2016) 129, 3892-3902 doi:10.1242/jcs.195685
Having confirmed that the ligand binding of DVL2 PDZ is
blocked by V334E and severely reduced by DARA, we proceeded to
test whether these point mutations affect the rescue activity of DVL2.
As a benchmark, we included a DIX domain mutant that blocks
Dishevelled polymerisation (M2M4; Fig. 4A) (Schwarz-Romond
et al., 2007a). As expected, M2M4 was completely inactive in
RescueOE assays, despite its relatively high expression levels;
however, V334E and DARA responded to Wnt in the same way as
wt DVL2–GFP (Fig. 4B). Essentially the same results were obtained
in Rescuestable assays in which M2M4 provided no signalling activity
whatsoever, whereas V334E and DARA restored full activity, like wt
DVL2–GFP (Fig. 4C). To rule out that these rescue activities reflect
residual ligand-binding activity of these cleft mutants, we also tested
a deletion mutant of DVL2–GFP that lacks its entire PDZ domain
(Δ244–353, ΔPDZ) in our complementation assays. Indeed, the Wnt
response of ΔPDZ proved indistinguishable from that of wt DVL2–
GFP (Fig. 4D), confirming our results with the PDZ point mutants.
These experiments establish that the PDZ domain does not play an
essential role in the Wnt signal transduction to β-catenin.
DEP-dependent DVL2 binding to Frizzled is essential for Wnt
signal transduction
DEP-dependent dimerisation of DVL2 is essential for signalosome
assembly, as was revealed by point mutations in the DEP domain that
block its dimerisation (including G436P and E499G; Gammons
et al., 2016). A different set of DEP mutations block the binding of
Dishevelled to Frizzled, but these did not affect its signalling activity
in functional assays based on overexpression, as expected given that
these assays bypass the need for Wnt stimulation. We thus selected
two of our DEP mutants that block recruitment of the minimal DEP
domain to FZD5 (L445E, K446M; Gammons et al., 2016) and
generated stable lines expressing these mutants, for complementation
assays in DVL TKO cells. Both mutants bear single amino acid
substitutions in the Frizzled-binding ‘finger’ of the DEP domain
(Fig. 5A; see also Fig. 6A); K446M corresponds to the dsh1 mutation
in Drosophila that causes PCP defects in flies (Axelrod et al., 1998;
Perrimon and Mahowald, 1987). We then performed RescueOE and
Rescuestable assays to compare their rescue activities with those of
G436P and E499G that block DEP-dependent dimerisation.
As expected, both dimerisation mutants proved to be completely
inactive in both rescue assays, regardless of expression levels
(Fig. 5B,C). In contrast, L445E and K446M provided substantial
rescue activity in RescueOE assays (Fig. 5B); however, neither
mutant exhibited any activity in Rescuestable assays, despite being
expressed at substantially higher levels than wt DVL2–GFP –
neither mutant was able to induce stabilization of β-catenin, nor βcatenin-dependent transcription, in response to Wnt3a (Fig. 5C).
This indicates that, in our physiological complementation assay, the
activity of DVL2 to transduce the Wnt signal to β-catenin crucially
depends on its DEP-dependent recruitment to Frizzled.
To confirm that the L445E and K446M mutations block the
binding of DVL2 to Frizzled, we tested their effects on full-length
DVL2–GFP in a recruitment assay based on co-overexpression with
SNAP–FZD5 in HEK293T cells (see also Tauriello et al., 2012),
alongside the other mutations used in this study. Of all seven
mutations tested, L445E and K446M are the only ones that block
recruitment of DVL2–GFP to SNAP–FZD5, whereas the other
mutations (including V334E and DARA) retain efficient recruitment
to membrane-associated SNAP–FZD5, indistinguishably from wt
DVL2–GFP (Fig. S3). Thus, the DEP domain is essential for the
recruitment of Dishevelled to Frizzled, whereas the PDZ and DIX
domain are dispensable.
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Fig. 3. PDZ cleft mutation blocks DVL-C peptide binding to PDZ. (A) Overlay for HSQC spectra of 100 µM wt 15N-PDZ alone (red) or with 300 µM DVL-C
(blue); line broadening and chemical shift perturbation induced by DVL-C peptide affect predominantly PDZ cleft residues (labelled). (B) Heat-map of line
broadening [red, >mean+1 standard deviation (1σ) fractional reduction of peak height] and chemical shift perturbation (orange, >mean+1σ |Δδ1H|+|Δδ15N/5|)
projected onto the solution structure (2MX6; Lee et al., 2015) of Dvl1 PDZ (in surface representation) in complex with DVL-C peptide (in stick representation).
(C) Spectral overlay as in A, of 100 µM 15N-PDZ-V334E alone (red) or with 300 µM DVL-C (blue). (D) Overlay for BEST-TROSY spectra of 100 µM wt 15N-PDZ
alone (red) or with 300 µM Lip–FZD5 (blue). Spectral overlays in C,D indicate lack of interaction in both cases.
DEP tetramerisation is dispensable for Wnt signal
transduction
Mutation of L445 not only blocks DEP recruitment to FZD5, but
also DEP-dependent tetramerisation, which is a consequence of
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DEP dimerisation by domain swapping (Gammons et al., 2016);
this leucine residue is located at the tip of an exposed loop of the
DEP domain (called ‘DEP finger’; turquoise, Fig. 6A), which is
required for recruitment to Frizzled. However, this loop can change
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RESEARCH ARTICLE
Fig. 4. The PDZ domain is dispensable for Wnt signal transduction to
β-catenin. (A) Cartoon of DVL, with DIX and PDZ mutations indicated.
(B,C) Graphs show SuperTOP assays in DVL TKO or control cells transiently
(B) or stably (C) transfected with wt or mutant DVL2–GFP, with or without 6 h of
incubation in WCM. Above, corresponding western blots. AP, treatment
with alkaline phosphatase. (D) Graph shows SuperTOP assays of DVL TKO
cells stably transfected with wt or ΔPDZ (Δ244–353) DVL2–GFP as in C. Error
bars represent mean±s.e.m., n=3 independent experiments. Right panel,
corresponding western blots.
conformation, by extending to swap the N-terminal α-helix of DEP
with that of a second DEP monomer (Fig. S4), thus forming a βsheet that connects two DEP monomers. Some of the residues in this
β-sheet have hydrophobic side-chains (including the hydrophobic
triad M443, L445, I447) that mediate association of two dimers into
a tetramer. Each of these residues also functions in the monomer to
mediate DEP binding to Frizzled (Gammons et al., 2016). This
complicates the interpretation of mutations in these residues, e.g. of
L445E, which is defective for both Frizzled binding and
tetramerisation.
To resolve this, and to determine whether DEP tetramerisation is
required for Wnt-dependent signalling in DVL TKO cells, we aimed
to substitute residues that are involved selectively in tetramerisation
but not in Frizzled binding. The best candidate is I447 whose side
chain contributes two key hydrophobic interactions to the DEP
tetramerisation interface (owing to its inherent symmetry; Fig. S4),
but which occupies a relatively peripheral position within the
Frizzled-binding DEP finger (Fig. 6A, red). We thus generated three
relatively conservative substitutions of I447, altering it to a residue
with a small hydrophilic (I447T) or large hydrophobic side-chain
(I447M or I447F), aiming to retain Frizzled binding. Indeed, each of
these mutations blocked tetramerisation of purified recombinant
DEP domain in size-exclusion chromatography with multi-angle
light scattering (SEC-MALS) assays while retaining at least partial
Journal of Cell Science (2016) 129, 3892-3902 doi:10.1242/jcs.195685
dimerisation (Fig. 6B). Conversely, the tetramerisation of K446M is
normal (not shown), as expected, given that the positively-charged
side chain of K446 remains fully solvent-exposed in the tetramer
and thus cannot participate in DEP tetramerisation (Fig. S4).
Next, we introduced these I447 substitutions into DEP–GFP, to
test Frizzled binding in two assays based on wt HEK293T cells, as
previously described (Pan et al., 2004; Tauriello et al., 2012;
Gammons et al., 2016). Indeed, all three mutants were recruited as
efficiently as wt DEP–GFP to plasma-membrane-associated
SNAP–FZD5 (Fig. 6C), and they also retained full activity in
blocking the Wnt-dependent signalling activity of endogenous
Dishevelled by interfering with its recruitment to Frizzled (Fig. 6D).
By comparison, K446M and L445E were inactive in both assays
(Fig. 6C,D), reconfirming that these two mutations block Frizzled
binding (see also Fig. S3), as previously shown (Gammons et al.,
2016). We conclude that DEP tetramerisation can be blocked
selectively without affecting Frizzled binding by all three I447
substitutions tested.
We thus introduced these isoleucine substitutions into DVL2–
GFP, for complementation assays in DVL TKO cells. We found that
each was fully active in both types of rescue assays, regardless of
their expression levels (Fig. 6E,F), in contrast to L445E and K446M
whose Wnt-independent signalling activities were somewhat
attenuated in RescueOE assays (Fig. 6E), and reduced to
background levels in Rescuestable assays (Fig. 6F), as shown
above. This reinforces our conclusion that the DEP-dependent
binding of DVL2 to Frizzled is crucial for signal transduction to βcatenin; however, signalling can evidently proceed normally
without DEP-dependent tetramerisation.
Frizzled binding but not dimerisation is required for Frizzled
downregulation by Dishevelled
In the absence of Wnt signalling, Dishevelled promotes the
downregulation of Frizzled and its clearance from the plasma
membrane, and this depends on its DEP domain (Jiang et al., 2015).
To identify the molecular property of the DEP domain required for
this downregulation, we tested our panel of DVL2–GFP mutants for
their ability to restore low levels of Frizzled in DVL TKO cells,
assessing the levels of endogenous FZD6 and FZD7 in the various
stable cell lines. This revealed that the only mutants unable to restore
downregulation of Frizzled were K446M and L445E (Fig. 7), i.e.
the only two mutants whose recruitment to membrane-associated
FZD5 is blocked (Fig. 6C; Fig. S3). By contrast, the dimerisationdefective DEP mutants are both proficient in restoring low levels of
FZD6 and FZD7, like the PDZ cleft mutants, the polymerizationdeficient DIX mutants, and a mutant whose YHEL motif
downstream of the DEP domain was substituted to AHEA (Yu
et al., 2007, 2010) to block DVL2 binding to the clathrin adaptor
AP2µ (Fig. 7). We conclude that the downregulation of Frizzled by
Dishevelled depends purely on the DEP-dependent interaction
between the two proteins, but neither on the competence of
Dishevelled to signal (i.e. on its DEP-dependent dimerisation and
DIX-dependent polymerisation) nor on its ability to bind to the
AP2μ clathrin adaptor.
Throughout our study, we noted a striking inverse correlation
between the signalling activities of the DVL2–GFP mutants and
their expression levels in stable DVL TKO cells lines, whereby the
expression levels of all signalling-competent mutants are limited to
the low physiological levels seen for wt DVL2–GFP (Fig. 7; see
also Fig. 2B). By contrast, the expression levels of the signallingdefective mutants are boosted to high levels (Fig. 7), suggesting that
these mutants are subject to positive selective pressure during the
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Journal of Cell Science (2016) 129, 3892-3902 doi:10.1242/jcs.195685
establishment of stable cell lines. If so, this implies that these
mutants might retain residual function.
DISCUSSION
We have developed a complementation assay in a human cell line
lacking all three DVL paralogs, to test the function of wt and mutant
DVL2 stably expressed at near-endogenous level. This DVL TKO
line is unable to respond to Wnt stimulation, but Wnt signal
transduction to β-catenin is fully restored by a DVL2 transgene
whose rescue activity is strictly Wnt-dependent. This assay has
enabled us to demonstrate that the DEP domain is essential for Wnt
signalling to β-catenin, whereas the PDZ domain is dispensable for
this response. This fundamental function of the DEP domain in
mediating activation of β-catenin was overlooked in previous
functional assays as these relied on Dishevelled overexpression,
which bypass the requirement for Wnt-activated receptor signalling.
Its essential role in mediating Dishevelled recruitment to Frizzled
was uncovered by our assay for the first time.
Undoubtedly, the binding of Dishevelled to Frizzled receptor is
key to its function in Wnt signal transduction. This was initially
thought to be mediated by the PDZ domain, based on an in vitro
interaction of purified domain with a peptide spanning
the functionally relevant KTxxxW motif derived from the
cytoplasmic tail of Frizzled (Wong et al., 2003). However, in the
context of full-length Frizzled, these peptides are likely to form an αhelix adjacent to the seventh transmembrane domain and along the
lipid bilayer, as in most known GPCRs structures (Venkatakrishnan
et al., 2013) including the close Frizzled relative Smoothened (Wang
et al., 2013). The notion of this peptide adopting a helical structure is
further supported by experimental evidence on micelle-binding
peptides from FZD1 (Gayen et al., 2013). If so, this is incompatible
with recognition by PDZ domains; these domains require their
ligands to adopt a β-strand-like conformation to form a β-sheet with a
β-strand in their cognate binding clefts (Ye and Zhang, 2013) in a socalled β-augmentation, also seen in DVL-PDZ–ligand binding
(Cheyette et al., 2002; Lee et al., 2015; Ye and Zhang, 2013; Zhang
et al., 2009). Furthermore, although the DVL PDZ domain has an
unusually versatile binding cleft, accommodating a bewildering
range of sequence-unrelated C-terminal and internal peptides
(Zhang et al., 2009), the only well-documented cases of PDZ
3898
structures in complex with physiologically relevant ligands
represent C-terminal peptides of the negative Wnt regulator
Dapper (Cheyette et al., 2002) and of Dvl1 itself (Lee et al.,
2015). Indeed, we were unable to detect any binding between
KTxxxW-spanning peptides from three different Frizzled paralogs
(including FZD5) and the DVL2 PDZ domain in our NMR binding
assay (Fig. 3D; Fig. S2). Thus, the cumulative structural and
biophysical evidence argues strongly against the notion that the
Dishevelled PDZ domain binds directly to Frizzled, consistent with
our findings that this domain is also dispensable for Wnt-dependent
signal transduction to β-catenin. In other words, the physiological
role of the Dishevelled PDZ domain is yet to be demonstrated; its
main function might be to downregulate signalling through binding
to negative Wnt regulators such as Dapper and Naked (Cheyette
et al., 2002) or to the C-terminus of Dishevelled itself (Lee et al.,
2015), or to transduce non-canonical Wnt signals, as indicated by
functional studies in flies (Axelrod et al., 1998).
Instead, it seems far more plausible that Frizzled binding is
achieved by the DEP domain. Evidence for this first emerged from
biophysical studies revealing a direct interaction between purified
DEP domain and peptides derived from the cytoplasmic face of
Frizzled, including its C-terminal tail (Tauriello et al., 2012).
Furthermore, the DEP domain is required for Dishevelled
translocation to membrane-associated Frizzled, and also blocks
Wnt signal transduction by endogenous Dishevelled upon
overexpression (Cong et al., 2004; Pan et al., 2004; Tauriello
et al., 2012; Strutt et al., 2012; Gammons et al., 2016). Indeed, the
single-most important result of our current study is the demonstration
that the DEP-dependent recruitment of Dishevelled to Frizzled is
essential for Wnt signal transduction to β-catenin in human cells,
based on the K446M mutant whose sole defect is in Frizzled
binding. In previous overexpression assays, this mutant retained
efficient signalling activity (Boutros et al., 1998; Li et al., 1999;
Moriguchi et al., 1999; Tauriello et al., 2012; Gammons et al., 2016);
however, it proved to be completely inactive in our physiological
complementation assay. This result provides powerful support for
the notion that the DEP domain harbours a fundamental function in
Wnt signal transduction – the binding of Dishevelled to Frizzled.
A distinct yet equally important function residing in the DEP
domain is its ability to dimerise, uncovered by a separate set of point
Journal of Cell Science
Fig. 5. The DEP domain is essential for Wnt signal
transduction to β-catenin. (A) Cartoon of DVL, with
DEP mutations indicated. (B,C) Graphs show
SuperTOP assays in DVL TKO or control cells
transiently (B) or stably (C) transfected with wt or mutant
DVL2–GFP, as in Fig. 3B,C. Error bars represent
mean±s.e.m., n=3 independent experiments. Above,
corresponding western blots.
RESEARCH ARTICLE
Journal of Cell Science (2016) 129, 3892-3902 doi:10.1242/jcs.195685
mutations that block dimerisation (Gammons et al., 2016). DEPdependent dimerisation triggers signalosome assembly, probably by
cross-linking DIX-dependent Dishevelled polymers, and is essential
Fig. 7. DEP dimerisation is dispensable for Frizzled downregulation.
Western blots of lysates of DVL TKO cells stably transfected with wt or mutant
DVL2–GFP, probed with antibodies as indicated on the left.
for both Wnt-dependent and Wnt-independent signalling by
Dishevelled. Therefore, unlike the DEP-dependent binding to
Frizzled, this dimerisation function is detectable in overexpression
assays, explaining previous evidence for the fundamental
importance of the DEP domain in Wnt signalling (e.g. Axelrod
et al., 1998; Li et al., 1999; Moriguchi et al., 1999; Rothbacher et al.,
2000; Penton et al., 2002; Wang et al., 2006; Strutt et al., 2012). DEP
dimerisation is not required for Frizzled binding, and structural
analysis revealed that these two molecular interactions are mutually
exclusive and depend on distinct conformations of the DEP domain.
Indeed, the residues required for Frizzled interaction are masked in
the DEP dimer, and engage in DEP tetramerisation, which is an
inevitable consequence of DEP dimerisation (Gammons et al.,
2016). In our current study, we have succeeded in generating mutants
that block tetramerisation without affecting Frizzled binding, but
these provided full rescue activity in our complementation assay.
Although the tetramerisation by the DEP domain could ensure the
unidirectionality of signalling by blocking the re-binding of
Dishevelled to Frizzed after catalysing signalosome formation, this
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Journal of Cell Science
Fig. 6. DEP tetramerisation is not essential for Wnt
signal transduction. (A) Ribbon representation of DEP
monomer structure (Protein Data Bank ID number 1FSH),
with hydrophobic residues in the tetramerisation interface (in
stick) labelled (see also Fig. S4). Turquoise, DEP finger
residues required for Frizzled binding (including M443,
mutated in Drosophila dsh1). (B) SEC-MALS of purified wt
and mutant DEP domains. Elution profiles of unfractionated
Lip–DEP417-511 (grey) revealing multiple species of
monomers, dimers and tetramers (*). Tetramerisation
mutants (colours) elute as monomers and dimers only.
(C) Graph shows quantitation of FZD5-dependent
recruitment of wt or mutant DEP–GFP to the plasma
membrane (100 cells scored in each case). Above,
representative images of HEK293T cells showing strong,
weak and no recruitment of DEP–GFP. (D) Graph shows
SuperTOP assays in transiently transfected HEK293T cells
(as in Fig. 1C), showing blocking of endogenous Wnt
response by overexpressed wt or mutant DEP–GFP (or GFP
as internal control). Above, corresponding western blots.
(E,F) Graphs show SuperTOP assays in DVL TKO or
control cells transiently (E) or stably (F) transfected with wt or
mutant DVL2–GFP, as in Fig. 3B,C; Error bars represent
mean±s.e.m., n=3 independent experiments. Above,
corresponding western blots.
function is clearly non-essential in our assay and might only be
detectable in more sensitive functional tests.
Our complementation assay also uncovered a Wnt-independent
function of the DEP domain in downregulating the steady-state
levels of Frizzled, probably through ubiquitin E3 ligases RNF43
and/or ZNRF3 (Jiang et al., 2015), which is independent of its
ability to dimerise. This function also seems to be independent of a
direct interaction between Dishevelled and the AP2µ clathrin
adaptor (Fig. 7), arguing against a direct role of Dishevelled
in coupling ubiquitylated Frizzled with clathrin-dependent
endocytosis in the absence of Wnt. Indeed, its DEP-dependent
binding to Frizzled seems to suffice for this function, suggesting
that the role of Dishevelled in this process is that of an adaptor
between Frizzled and RNF43 and/or ZNRF3.
One of our most striking observations was that the expression
levels of wt and signalling-competent DVL2 mutants were kept low,
to a level similar to that of endogenous DVL, indicating that these
levels are subject to negative selective pressure. This can be
explained by our finding that cell cycle progression is blocked if
DVL2 is left to accumulate to high levels, although we have not
investigated the molecular basis for this phenomenon. Conversely,
the expression levels of our inactive DVL2 mutants were universally
high, possibly as a result of positive selective pressure; clearly,
absence of Dishevelled slows down the rate of cell proliferation
(Fig. 1D), and the process of isolating stably transfected cell lines
might favour cells whose proliferation rate is slightly enhanced if
their rescue transgene retained residual function. It is possible that
our point mutations do not fully inactivate DVL2, which could also
explain why dsh1 (corresponding to K446M) sustains development
to viable flies without a detectable defect in signalling to Drosophila
β-catenin (Axelrod et al., 1998; Perrimon and Mahowald, 1987).
Likewise, CRISPR–Cas9-engineered dimerisation mutations of the
endogenous fly dsh gene (equivalent to G436P and E499G) are
viable (J. Mieszczanek, MRC Laboratory of Molecular Biology,
Cambridge, UK and M.G., unpublished observations). It thus seems
that normal development in flies requires only a fraction of the wt
function of its Dishevelled DEP domain.
Our complementation assay also allowed us to establish the
epistatic relationship between Dishevelled and LRP6. Initially,
epistasis analysis in Drosophila suggested that Dishevelled
functions below LRP6 (Wehrli et al., 2000), but this was
subsequently revised to the converse (Metcalfe et al., 2010),
reconciling the fly data with results from vertebrate systems showing
that the Wnt-dependent phosphorylation of LRP6 depends on
Dishevelled (Bilic et al., 2007). Indeed, the signalling activity of
DVL2 was found to be much reduced in LRP6-depleted cells
(Metcalfe et al., 2010). We now use null-mutant human cells to
establish that the overexpressed LRP6 is equally active whether or
not Dishevelled is present, whereas overexpressed DVL2 barely
signals in the complete absence of LRP6. This identifies LRP6 as an
essential signalling effector of Dishevelled, transducing the Wnt
signal from Dishevelled to β-catenin.
In summary, our complementation assay provides a test system
for the Wnt-dependent signalling activity of Dishevelled, which
enabled us for the first time to demonstrate the essential role of the
DEP domain in mediating Frizzled binding by Dishevelled for its
activity in Wnt signal transduction to β-catenin. It also provides an
assay system for other functions of Dishevelled, such as the
downregulation of Frizzled and the progression of the cell cycle,
which we have not further pursued. Indeed, it might be adaptable to
the study of non-canonical functions of Dishevelled although this
might require the supply of additional co-factors, the use of different
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Journal of Cell Science (2016) 129, 3892-3902 doi:10.1242/jcs.195685
cell types or even organoids. It might thus pave the way for the
development of similar assay systems that combine loss-of-function
with stable re-expression of rescue transgenes at physiological
levels, yielding definitive functional insights.
MATERIALS AND METHODS
Plasmids and antibodies
The following plasmids were used: human DVL2–GFP ( pEGFP-N1);
SNAP–FZD5 (Koo et al., 2012); LRP6–GFP (Metcalfe et al., 2010);
PX330-spCas9 (Addgene, #43330) for DVL TKO; PX458-spCas9-GFP
(Addgene, #48138) for LRP6 KO. DVL2–GFP was subcloned into pBabe
PURO (Addgene) to generate stable cell lines. DVL2 mutants were
generated by standard procedures and verified by sequencing. The following
antibodies were used: anti-tubulin (1:5000; T4026, Sigma), anti-GFP
(1:5000; G1544, Sigma), anti-actin (1:5000; ab8227, Abcam), anti-FZD3
(1:2500; ab75233, Abcam), anti-FZD6 (1:5000; ab128916, Abcam), antiFZD7 (1:1000; ab64636, Abcam), anti-FZD8 (1:3000; ab40012, Abcam),
anti-SNAP (1:5000; P9310S, NEB), anti-DVL1 (1:1000; sc8025, Santa
Cruz), anti-DVL3 (1:1000; sc26506, Santa Cruz), anti-DVL2 (1:5000;
3216, Cell Signaling), anti-ABC (1:5000; 8814, Cell Signaling), anti-pLRP6 S1490 (1:2500; 2568, Cell Signaling).
Isolation of stable cell lines
To generate DVL TKO cells, HEK293T cells (Chiu et al., 2016; not
authenticated) were transfected with plasmids encoding Cas9 and guide
RNAs (Ran et al., 2013) targeting genomic loci of DVL (DVL1/2, 5′-CTACATTGGCTCCATCATGA; DVL3, 5′-ACCATGCTTCAATGGCCGGG) or LRP6 (LRP6, 5′-CGATTGGTTGATGCTACAAA); clones were
screened by immunoblotting, and deletions were confirmed by genomic
DNA sequencing. To generate stable DVL2-expressing cell lines, DVL
TKO cells were transfected with 200 ng DVL2–GFP pBabePURO:PEI
(1:3), and selected with 2 µg/ml puromycin.
Cell-based assays
HEK293T cells were cultured and transfected as described (Mund et al.,
2015), and single confocal images were acquired at identical settings with a
Zeiss confocal microscope. For SuperTOP assays (Veeman et al., 2003),
HEK293T cells were lysed 16–24 h after transfection, and analysed with
the Dual-Glo Luciferase Reporter Assay (Promega) according to the
manufacturer’s protocol. Experiments were repeated three times, and values
were normalised to Renilla luciferase (shown as mean±s.e.m. relative to
vector controls). Wnt3a-conditioned media (WCM) were generated from
L-cells (ATCC CRL-2647) according to manufacturer’s specification.
Alkaline phosphatase treatments (Roche), used in some experiments to
dephosphorylate DVL, were done for 30 min at 37°C. For proliferation
assays, 3×104 cells were seeded, and cells were counted every 24 h (in
triplicate, three independent experiments); results were analysed by twoway ANOVA with repeated measures, and are shown as mean±s.e.m.
Protein purification and biophysics
DVL2 DEP417-511, DVL2 PDZ265-361, FZD5 (GKTLESWRRFTS) and
FZD8 peptide sequences (GKTLESWRALCTR) were tagged N-terminally
with 6×His and Lip followed by a linker (ENLYFQS) encoding a cleavage
site for tobacco etch virus (TEV) protease. Lip–Fz7 (encoding
GKTLQSWRRFYH; Wong et al., 2003) was generated similarly, except
that the TEV linker sequence was flanked by serine–glycine on both sides.
Proteins were expressed in E. coli BL21-CodonPlus(DE3)-RIL cells
(Stratagene) at 37°C, grown overnight at 24°C, and induced with
isopropyl β-D-1-thiogalactopyranoside at OD 0.8. Lip–DEP, Lip–PDZ
and Lip–FZDs were purified on NiNTA beads (Qiagen) essentially as
described (Fiedler et al., 2011). SEC-MALS was performed in phosphatebuffered saline, using a GE Superdex S-200 10/300 analytical column, and
analysed as described (Madrzak et al., 2015).
NMR spectroscopy
Minimal wt and mutant 15N-labelled PDZ domains were purified essentially
as described (Fiedler et al., 2011), and used for NMR spectroscopy after
Journal of Cell Science
RESEARCH ARTICLE
cleaving off the Lip tag (with TEV protease overnight at 4°C). NMR spectra
were acquired on a Bruker Avance-III spectrometer operating at 600 MHz
1
H frequency, and equipped with a cryogenic inverse 5 mm probe at a
sample temperature of 298 K. All samples were prepared in aqueous
phosphate buffer at physiological ionic strength ( pH 6.7). Backbone
resonance frequencies were obtained for 500 µM 13C,15N-labelled protein,
using unmodified Bruker pulse programmes for HNCACB, CBCA(CO)NH,
HN(CA)CO and HNCO. BEST-TROSY (Favier and Brutscher, 2011) or
fast-HSQC 1H-15N correlation spectra were acquired for purified 15N-PDZ
with 128 complex and 1024 points (1.1 and 0.7 Hz/point in the processed
data) in t1 and t2, respectively, and 8 transients per t1 point. In each case,
100 µM PDZ protein was incubated with 3× molar excess of DVL-C
(SEFFVDVM; Lee et al., 2015; kindly provided by Jie Zheng, Department
of Ophthalmology, University of California, Los Angeles, USA) or Lip–
FZD peptides (as indicated in Fig. 3 and Fig. S2).
Acknowledgements
We thank Jie Zheng for DVL-C peptide, Christopher Johnson for assistance with
SEC-MALS data collection and analysis, Miha Renko for advice in designing PDZ
mutations, and the flow cytometry team for cell sorting.
Competing interests
The authors declare no competing or financial interests.
Author contributions
M.V.G. and M.B. conceived and supervised the study, M.V.G. performed most of the
experiments, T.J.R. contributed the NMR data, Z.S. and S.A. contributed the DVL
TKO cell line, and M.B. wrote the manuscript with input from all authors.
Funding
This work was supported by the Medical Research Council [grant number
MC_U105192713 to M.B.], Cancer Research UK [grant number C7379/A15291,
to M.B.], and the Canadian Institutes of Health Research [grant number 273548 to
S.A.]. S.A. is a Canada Research Chair in Functional Architecture of Signal
Transduction. Deposited in PMC for immediate release.
Supplementary information
Supplementary information available online at
http://jcs.biologists.org/lookup/doi/10.1242/jcs.195685.supplemental
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