Institut für Molekularbiologie, Universität Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland
* Author for correspondence (e-mail: basler{at}molbio.unizh.ch)
Accepted 8 June 2004
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SUMMARY |
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Key words: Drosophila, Disease, Colorectal cancer, Wnt signalling, ß-catenin
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Introduction |
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Here, we set out to characterize the interaction of ß-catenin and Lgs. We report the identification of two amino acids of ß-catenin that play an essential role in Lgs binding. This presumed binding site is specific for Lgs and is not required for APC, E-cadherin or TCF4. We show that Armadillo (Arm), the Drosophila homologue of ß-catenin, depends on these amino acid residues for mediating Wnt/Wg signalling in vivo, but not for establishing functional adherens junctions. Together, our results indicate that the ß-catenin/Lgs interaction may provide an attractive target for therapeutic intervention.
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Materials and methods |
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A subset of the ß-catenin mutations were also introduced into Arm. For simplicity, we use the amino acid numbering of ß-catenin for both ß-catenin and Arm throughout the text. ß-Catenin D162, E163, D164 and K435 would correspond to Arm D170, E171, D172 and K443.
Yeast two-hybrid assays
The yeast two-hybrid system as described previously
(Bartel and Fields, 1997) was
used. Interactions between proteins were measured using the quantitative
`Liquid Culture Assay Using ONPG as Substrate'
(Clontech, 2001
).
Transgenes
For embryonic experiments arm transgenes were expressed from
UAS-constructs under control of the daughterless-Gal4 driver
(Wodarz et al., 1995). Three
independent lines were established and tested for the
armS10-wt, armS10-D164A and
armS10-K435E constructs, and two independent lines for
arm-wt,
arm-D164A and
arm-K435E; in all cases, different integrations of the same
construct yielded similar effects. For rescue experiments full-length
arm transgenes were driven by the tubulin
1
(tub) promoter (Basler and Struhl,
1994
). All arm-coding regions used contain a Myc
epitope in their C-terminal region at the same position as the
armS10 construct used by Pai et al.
(Pai et al., 1997
).
Germline clones
To obtain arm germline clones, second and third instar larvae
generated from a cross between arm2a9 FRT101/FM7;
tub-arm[-wt or -D164A]/+ virgins with ovoD1
FRT101/Y; hs-flp[F38]/hs-flp[F38] were
heat-shocked at 38°C for 1.5 hours. After hatching, the fertile females
will produce only progeny from arm2a9 mutant germlines.
Females bearing the tub-arm-wt transgene were crossed to y w
males and laid embryos that all contain maternal tub-arm-wt product
(otherwise no eggs would be generated). Only 25% of these embryos will inherit
neither the rescuing transgene nor the paternal arm+
allele, and these embryos resemble zygotic arm2a9 embryos.
The observed number for such embryos was 90 out of 385. Females that were
arm2a9/arm2a9;
tub-arm-D164A/+ were crossed with y w males and
laid embryos that all contain maternal tub-arm-D164A product
(otherwise no eggs would be generated). Three classes of embryos are expected
(50% class I, 25% class II, 25% class III): while all embryos are maternally
mutant for arm2a9, 50% of them (class I) are zygotically
arm+, and hence rescued (from the paternal X chromosome),
and the other 50% (classes II and III) are also zygotically mutant for
arm2a9. Half of these (class II), however, inherit the
tub-arm-D164A transgene, and show a slightly weaker segment polarity
phenotype (Fig. 5C) compared
with the other half (class III) that does not
(Fig. 5D). The observed numbers
for these three classes were 33, 12 and 14, respectively. For the generation
of lgs germline clones see Kramps et al.
(Kramps et al., 2002).
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Results |
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In addition to its role in Wnt signalling, ß-catenin is also a
component of the cadherin-based cell adhesion system, linking the
transmembrane protein E-cadherin to -catenin, thereby connecting
adherens junctions to the cytoskeleton (reviewed by
Pokutta and Weis, 2002
).
ß-Catenin is also part of its own degradation complex consisting of APC,
Axin and GSK3ß. In order to evaluate the specificity of the mutations
that disrupt ß-catenin/human LGS1 binding, mutations D162A and D164A were
tested for their effect on the interactions between ß-catenin and APC,
E-cadherin, TCF4 and
-catenin. As a control, we also included in this
analysis E163A, which had virtually no effect on the ß-catenin/human LGS1
interaction even though it also reduces the negative charge at this region of
the protein. None of the three mutations affected the binding of
ß-catenin to APC, E-cadherin or TCF4. Binding to
-catenin,
however, was reduced approximately twofold by D162A and D164A
(Fig. 1F). This was unexpected,
as the region comprising amino acids 120-151 of ß-catenin has been shown
to be necessary and sufficient for binding to
-catenin
(Aberle et al., 1994
). Amino
acids D162, E163 and D164 form an acidic knob in repeat 1 of ß-catenin
(Fig. 1B, white arrow), on the
side opposite to the basic groove. From these results we conclude: (1) that
ß-catenin binds human LGS1 and APC/E-cadherin/TCF4 on opposite sides; and
(2) that the binding to human LGS1 but not to APC/E-cadherin/TCF4 is disrupted
by the mutations D162A and D164A.
Arm-D164A fails to rescue armadillo null mutant animals
Armadillo (Arm) is the Drosophila homologue of ß-catenin. The
two proteins show high sequence similarity, especially in the Arm repeat
region (Peifer and Wieschaus,
1990; Peifer et al.,
1994
). To investigate whether a mutant form of Arm, which can no
longer bind Lgs, has impaired transcriptional activity in Drosophila,
we first analyzed whether ß-catenin and Arm use equivalent sites for
binding human and Drosophila Lgs. The D164A mutation - and as a
negative control the E163A mutation - were introduced into Arm and found to
affect the Arm/Lgs interaction to the same extent as the corresponding
mutations in ß-catenin (not shown). We then tested whether Arm-D164A can
substitute for the wild-type form of Arm in vivo by performing a rescue assay
with the arm2a9 allele, which has a frameshift mutation in
Arm repeat 3 (Fig. 1A) and is
the strongest arm allele known. Hemizygous arm2a9
males die as embryos but can be rescued by
tubulin
1-promoter-driven arm-wt or
arm-E163A transgenes to adulthood with no obvious phenotypes
(Table 1). By contrast,
arm2a9 males die as embryos or early larvae when these
transgenes contain the D164A mutation or K435E, which affects TCF/Pan binding
(Graham et al., 2000
). We
interpret these results to indicate that the wild-type function of Arm depends
crucially on its ability to bind Lgs and Pan.
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Reduced expression levels of Wg targets in arm-D164A cells
To assess the role of the D164 site in the transcriptional function of Arm,
we analyzed Wg target gene expression in arm mutant clones in third
instar wing imaginal discs. There Wg is expressed at the dorsoventral boundary
in a narrow stripe of cells and regulates the expression of a number of genes,
among them Distalless (Dll), which is expressed in a broad band of
cells on both sides of the wing margin
(Diaz-Benjumea and Cohen,
1995; Zecca et al.,
1996
). We used the strong arm allele
arm2a9 to induce mutant clones in the second larval
instar. Dll expression was lost in these clones 48 hours later
(Fig. 3B). Ubiquitous
expression of the tubulin
1-arm-wt transgene fully
restored Dll expression in such clones
(Fig. 3C). By contrast,
arm2a9 clones showed severely reduced Dll expression when
the tubulin
1-arm-D164A transgene was used
(Fig. 3D). Thus, Arm-D164A is
severely impaired in transducing the Wg signal, suggesting that Arm needs to
bind Lgs to efficiently upregulate Dll expression in response to larval
Wg.
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Arm-D164A can restore adherens junctions of arm mutant cells
We noticed that arm2a9 clones are not only smaller than
their twin-spots but that their shape is round compared with the irregular
outline of normal clones (Fig.
3B). On the contrary, arm clones that express
arm-wt or arm-D164A transgenes are similar in size to their
twin-spots and have an irregular shape
(Fig. 3C,D). These differences
hint at an adherens junction defect of arm null mutant cells, as
ß-catenin is required at these sites to link E-cadherin and
-catenin, and cells with defective adherens junctions or different
E-cadherin levels sort out from neighbouring cells
(Dahmann and Basler, 2000
;
Uemura et al., 1996
). In order
to visualize adherens junctions we stained wing discs with an antibody
directed against Drosophila E-cadherin
(Uemura et al., 1996
). Indeed,
E-cadherin distribution is diffuse in arm null clones
(Fig. 4A). As arm-wt
and arm-D164A transgenes rescue the abnormal distribution of
dE-cadherin in arm cells (Fig.
4B,C), and no longer cause aggregation into non-intermingling cell
groups, their products appear to restore the function of adherens junction.
This suggests that Arm-D164A can confer ß-catenin function at adherens
junctions, and hence is able to sufficiently tether E-cadherin and
-catenin.
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Discussion |
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Binding site
By means of site-directed mutagenesis we assayed the role of conspicuous
ß-catenin residues in the binding to human LGS1. Two amino acids, D162
and D164, were identified that are both necessary for human LGS1 binding.
Because substitutions of these residues with other amino acids did not affect
the binding of several other proteins to ß-catenin, we interpret their
role as contact sites for human LGS1, rather than a structural function
enhancing stability and/or three-dimensional conformation of ß-catenin.
This conclusion, however, will need to be confirmed by determining the crystal
structure of the ß-catenin/human LGS1 complex.
Specificity
We showed that neither D162 nor D164 is required for binding to APC,
E-cadherin or TCF4. Substitutions of these amino acids did reduce binding to
-catenin twofold, but our in vivo data suggest that this reduction does
not prevent the assembly of adherens junctions. The specificity of the
ß-catenin/human LGS1 interaction vis-à-vis that of ß-catenin
and APC, E-cadherin or TCF4 is consistent with their respective locations on
the surface of ß-catenin. While crystallographic studies showed that APC,
E-cadherin and TCF4 all bind to a common, extended surface within the groove
of ß-catenin formed by Arm repeats 3-10 (reviewed by
Daniels et al., 2001
), our
analysis indicates that human LGS1 binds an acidic knob in Arm repeat 1. This
knob is not only located more N terminally, it is also situated on the side of
ß-catenin, which is opposite to the groove
(Fig. 1B). The spatial
separation of these binding sites is in agreement with their separable
functions observed in our yeast binding assays, as well as with previous GST
pull-down assays, in which we observed simultaneous binding of TCF4 and human
LGS1 to ß-catenin (Kramps et al.,
2002
).
In vivo significance
To assess the role of D162 and D164 in Wg transduction, we subjected mutant
forms of Arm to various assays designed to reveal their in vivo function.
Simple rescue and overexpression experiments showed that transgenic Arm-D164A
cannot substitute for endogenous Arm, and that the D164A mutation
significantly reduces the constitutive signalling activity associated with
N-terminal deletions of Arm. When tested in more advanced assays, we find that
D164 is required by wing disc cells to maintain Wg target gene expression and
by developing embryos for segmentation. Together, these experiments support
the conclusion that Arm signalling function relies on its capability to bind
to Lgs throughout development.
Although it is straightforward to interpret our results as a qualitative indication for the significance of the Arm/Lgs interaction, it is more difficult to assess their outcome in a quantitative manner. For example, the apparent residual expression of Dll in Arm-D164A cells may reflect perdurance of wild-type Arm or Dll proteins, but it could also indicate that a fraction of the Wg signal can be transmitted despite the D164A mutation. This latter scenario could in turn be attributed to some residual binding of Arm to Lgs, but it could also be explained by a partial redundancy of Lgs function. Lgs may be required for efficient Arm-mediated activation of Wg targets, but some activation may also occur in its absence. Consistent with this latter view, we have observed that animals lacking maternal and zygotic lgs product exhibit phenotypes equivalent to animals in which the sole source of Arm is the D164A transgene, yet neither of the two phenotypes are quite as severe as that of wg-null mutants.
Possible relevance for human cancer
The Wnt pathway is highly conserved between Drosophila and
vertebrates. The human homologues of Lgs (LGS1/BCL9) and Pygo (PYGO1 and
PYGO2) can rescue lgs and pygo mutant flies, respectively
(Kramps et al., 2002). This
suggests that these proteins have the same function in vertebrates and in
Drosophila. It is possible therefore, that our in vivo data can be
extrapolated to Wnt signalling in mammals.
Mutations in APC occur in more than 80% of inherited and sporadic
colorectal cancers (Kinzler and
Vogelstein, 1996). These mutations lead to accumulation of free
ß-catenin and as a result to overexpression of Wnt target genes. A
chemical compound that interferes with the formation of the nuclear
TCF/ß-catenin/Lgs/Pygo complex should in theory halt the progression of
cancer. Such an anti-cancer drug must be highly specific though, as it should
only disrupt the nuclear ß-catenin complex, but neither the cytoplasmic
ß-catenin/APC/Axin complex nor the ß-catenin/E-cadherin complex at
the cell membrane. APC, Axin and E-cadherin functions should not be
compromised, as all three of them have tumour suppressor roles (reviewed by
Giles et al., 2003
). This is
not the case, however, for TCF and Lgs. Crystal structure data indicates that
APC, Axin, E-cadherin and TCF4 partly use of the same contact sites of
ß-catenin for their binding (Eklof
Spink et al., 2001
; Graham et
al., 2000
; Graham et al.,
2001
; Huber and Weis,
2001
; Xing et al.,
2003
). Therefore, designing an inhibitor that specifically
disrupts the ß-catenin/TCF interaction is a difficult task
(Daniels et al., 2001
;
Lepourcelet et al., 2004
). On
the contrary, our mapping and specificity results indicate that the
ß-catenin/Lgs interaction site could be targeted without interfering with
the binding of ß-catenin to APC and E-cadherin. Moreover, our analysis
shows that genetic disruption of the Arm/Lgs interaction leads to severely
reduced Wg signalling, suggesting that the protein-protein interaction between
ß-catenin and Lgs may provide an attractive target for therapeutic
intervention.
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ACKNOWLEDGMENTS |
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