 |
INTRODUCTION |
The Wnt signaling pathway controls developmental processes in both
invertebrates and vertebrates (1, 2). Wnt inhibits the activity of
glycogen synthase kinase-3
(GSK-3
)1 by an unknown
mechanism, and this inhibition leads to the accumulation of cytoplasmic
-catenin. The protein level of cytoplasmic
-catenin is
post-transcriptionally regulated by GSK-3
(3, 4). When GSK-3
activity is inhibited, cytoplasmic
-catenin is stabilized, which
allows it to accumulate. This accumulated cytoplasmic
-catenin interacts with the T-cell factor/Lef-1 family of transcription factors
and then enters the nucleus to enhance T-cell
factor/Lef-1-dependent transcription (5, 6). The Wnt
pathway is also implicated in cancer. Wnt-1 is overexpressed in certain
mouse mammary tumors (7). Mutations in
-catenin and adenomatous
polyposis coli (APC) genes are found in several human cancer cell lines
(8-13).
Axin is a molecule recently identified as a negative regulator of the
Wnt pathway (14). In mouse, homozygous loss of function mutations in
the Axin locus cause an axial duplication of embryos along with the
other developmental defects. In Xenopus, overexpression of
Axin inhibits ectopic axis formation by Wnt in addition to normal axis
formation. This inhibitory effect of Axin was reversed by
co-microinjection of the downstream molecule of the Wnt pathway,
-catenin. These findings indicate that Axin inhibits the Wnt signaling pathway, which regulates embryonic axis formation in vertebrates.
Recently we showed that Axin inhibits T-cell
factor/Lef-1-dependent transcriptional activity stimulated
by either Wnt or
-catenin (15). We and others (16-18) also showed
that Axin directly interacts with GSK-3
and
-catenin and acts as
a bridge between these molecules. It was proposed that the bridging
effect of Axin explains its ability to inhibit the Wnt pathway by
enhancing
-catenin phosphorylation by GSK-3
(16). In this study
we showed that the bridging of GSK-3
and
-catenin is necessary
but not sufficient for the inhibitory effect of Axin on
Lef-1-dependent transcriptional activity. We also showed
that oligomerization of Axin through its C terminus is important for
its function to inhibit the Wnt pathway.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Transfection--
COS-7 cells and SW480 cells
were grown in Dulbecco's modified Eagle's medium, 10% fetal calf
serum. Transfections were performed by LipofectAMINETM
(Life Technologies, Inc.) following the protocol of the manufacturer.
Plasmid Constructions--
All the mouse Axin expression
constructs were c-Myc epitope tagged (EQKLISEEDLNEED) and subcloned
into pcDNA3.1 vector (Invitrogen). Full-length Axin and was
generated as described previously (15). Other truncated mutant
constructs were made by restriction enzyme digestion and re-ligation
using the adapters, or polymerase chain reaction as follows:
N-Axin,
digested at Asp1 site (deleted amino acids 126-299),
RGS-Axin, digested at first and second PstI sites (deleted amino acids 251-351),
NM-Axin, digested at
BbrP1 site (deleted amino acids 126-604),
MC-Axin,
digested at BbrP1 site (deleted amino acids 604-956),
M-Axin, digested at Bfr1 and BbrP1 sites
(deleted amino acids 332-602),
CM-Axin, digested at
BbrP1 and EcoRI sites (deleted amino acids
603-809),
C-Axin, digested at EcoRI site (deleted amino
acids 803-956), and
CC-Axin was made by ligating polymerase chain
reaction product (primers: 5'-TCA GCTTCGGAATTCTGTCCAGCCTTC-3' and
5'-TTCCTTTTGCGGCCGCTCATCAGA TATCAGCACGGCCCCTCACCAG-3') at
EcoRI site in Axin and NotI site in pcDNA3.1
vector (deleted amino acids 892-956). Glutathione
S-transferase (GST) fusion proteins (GST-
-catenin and
GST-
N-Axin) were constructed in pGEX-5X-1 vector (Amersham Pharmacia
Biotech), expressed in Escherichia coli BL21 cells, and
purified on glutathione-Sepharose 4B. The mouse full-length
-catenin
cDNA was a generous gift from Dr. A. Nagafuchi (Dept. of Cell
Biol., Faculty of Med., Kyoto University, Japan). The retinoid X
receptor (RXR) and the ecdyson receptor (EcR) were derived from pVgRXR
(Invitrogen) and fused to the
C-Axin at EcoRI and
NotI sites using the adaptors to generate
C-RXR-Axin and
C-EcR-Axin.
In Vitro Translation and in Vitro Binding--
Axin constructs
in pcDNA3.1 vector were in vitro translated by TNT T7
coupled rabbit reticulocyte system (Promega). In vitro binding experiments were performed essentially as described previously (15). For GSK-3
and Axin binding, partially purified GSK-3
(Upstate Biotechnology) was immobilized to Dynabeads (Dynal) through anti-GSK-3
antibody (Transduction Laboratory), incubated with in vitro translated, [35S]methionine-labeled
Axin (20 µl each) in binding buffer (20 mM Tris-HCl, pH
7.5, 1 mM EDTA, 0.1% Triton X-100, 0.3 M
NaCl, and 1 mM phenylmethylsulfonyl fluoride). For
-catenin and Axin binding, GST-
-catenin immobilized to
glutathione-Sepharose 4B was mixed with in vitro translated
Axin (20 µl each) in binding buffer. For Axin-Axin binding,
GST-
N-Axin immobilized to glutathione-Sepharose 4B was mixed with
in vitro translated Axin. After incubating at 4 °C for
4 h, the mixtures were extensively washed and in vitro translated Axin bound to immobilized protein was analyzed by SDS-PAGE and autoradiography.
Immunoprecipitation and Immunoblotting--
48 h after
transfection, cells were lysed in lysis buffer (20 mM
Tris-HCl, pH 7.5, 1 mM EDTA, 0.1% Triton X-100, 0.15 M NaCl, and 1 mM phenylmethylsulfonyl
fluoride). Cell lysates were incubated with antibodies as indicated at
4 °C overnight, then with Dynabeads coupled to sheep anti-mouse IgG1
(Dynal) for 1 h. Immunocomplexes were washed extensively with
lysis buffer and analyzed by immunoblotting. Enhanced chemiluminescence
reagents (Amersham) were used for detection of the immunoblots.
Lef-1 Reporter Gene Assay--
Lef-1 reporter gene assay was
performed as described previously (15). Briefly, SW480 cells were
seeded at 1 × 105 cells/well in 12-well culture
plate, then transfected on the next day by LipofectAMINE 48 h
before the assay. Luciferase activity was measured by luciferase assay
system (Promega) and
-galactosidase activity was measured by
-Gal
Assay System II (CLONTECH) using Luminometer
(Monolight 2010, Analytical Luminescence Laboratory). pCG-Lef-1 and
pGL3-fos-7LEF-luciferase were kindly provided by Dr. R. Grosschedl
(University of California, San Francisco). pTK-
-galactosidase was
obtained from Promega.
 |
RESULTS AND DISCUSSION |
Interactions of Axin Mutants with GSK-3
--
Axin was recently
identified as a GSK-3
-binding protein by yeast two-hybrid screening
(15, 16). To determine the binding domain for GSK-3
in Axin, we made
mutant constructs in which small regions of Axin were deleted (Fig.
1). First, we checked the interaction of
GSK-3
and in vitro translated Axin constructs (Fig.
2A). We found that the
GSK-3
binding domain mapped to the middle part of Axin as reported
previously (16-18). The RGS domain and the C terminus region were not
required for binding to GSK-3
. The association was also assessed in
COS-7 cells. Axin mutants were expressed and immunoprecipitated; the
binding of GSK-3
was assessed by immunoblotting (Fig.
3A). Consistent with the
in vitro results, the middle region of Axin confers GSK-3
binding.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 1.
Schematic representation of the Axin deletion
mutants and corresponding activities. Constructs were generated as
described under "Experimental Procedures." For binding activities, + represents strong binding, and represents comparatively weak
binding.
|
|

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 2.
In vitro binding of
GSK-3 or -catenin
with Axin. In vitro translated, 35S-labeled
Axin mutants bound to GSK-3 (A) or -catenin
(B) immobilized to Sepharose were analyzed by 10% SDS-PAGE
and autoradiography (upper panels). 5 µl each of the
in vitro translated Axin mutants were analyzed by SDS-PAGE
(10%) and autoradiography (lower panels).
|
|

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 3.
In vivo binding of GSK-3
with Axin or -catenin. Axin mutants
tagged with c-Myc epitope were transiently transfected in COS-7 cells.
Cell lysates were immunoprecipitated either with anti c-Myc antibody
(Pharmingen) (A) or with anti -catenin antibody
(Zymed Laboratories Inc.) (B) and blotted
with GSK-3 antibody (Transduction Laboratory). The expressions of
Axin mutants were confirmed by immunoblotting with c-Myc
antibody.
|
|
Interaction of Axin with
-Catenin--
Next, we examined the
interaction of
-catenin and Axin deletion mutants. From the in
vitro binding experiments using recombinant
-catenin and
in vitro translated Axin mutants, the
-catenin binding
domain in Axin overlapped with the GSK-3
binding domain but extended
further toward the C terminus (Fig. 2B). Neither the RGS
domain nor the DIX domain was required for
-catenin binding (Fig.
1).
Bridging Effect of Axin between GSK-3
and
-Catenin--
In a
previous study (15), we showed that Axin is a bridging molecule between
GSK-3
and
-catenin. For example in COS-7 cells or 293 cells,
-catenin and GSK-3
do not form a complex unless Axin is
exogeneously expressed. We measured this complex formation by detecting
GSK-3
in the
-catenin immunocomplex by immunoblotting. Among Axin
mutants, FL-,
RGS-,
N-,
CM-,
C-, and
CC-Axin provide
this bridging function (Fig. 3B). These data suggested that
the bridging function is mediated through the central region of Axin.
Inhibition of Lef-1 Transcriptional Activity in Colon Cancer
Cells--
Using a Lef-1 reporter gene assay, we have shown that Axin
inhibits Lef-1 transcriptional activity in SW480 cells, a colon cancer
cell line that carries an APC mutation (15). In this cell line,
cytosolic
-catenin is stabilized and accumulates, causing an
increase in Lef-1 transcriptional activity (12, 13). We examined the
inhibitory effect of Axin deletion mutants in SW480 cells. FL-,
RGS-, and
N-Axin showed inhibitory effects in the Lef-1 reporter
gene assay, and Axin mutants deficient in the bridging function
(
NM-,
MC-, and
M-) did not show any inhibition (Fig.
4). This result suggests that Axin
binding to both GSK-3
and
-catenin is necessary for inhibition of
Lef-1 reporter gene transcription. This is consistent with the
hypothesis that a major effect of Axin is to facilitate GSK-3
mediated phosphorylation of
-catenin (16). However,
CM-,
C-,
and
CC-Axin also lacked the inhibitory effect, even though those
mutants can act as bridging molecules between GSK-3
and
-catenin
(Fig. 3B). Therefore the bridging effect is not sufficient
for Axin to suppress Lef-1-dependent transcription. An
additional function of Axin must be provided by its C-terminal region
to allow inhibition of Lef-1-dependent transcription.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 4.
Lef-1 reporter gene assay in SW480
cells. Axin mutants (0.3 µg each) were transfected with other
plasmids as follows: pCG-Lef-1 (0.02 µg), pGL3-fos-7LEF-luciferase
(0.2 µg), and pTK- -galactosidase (0.03 µg). The ratio of
luciferase activity to -galactosidase activity varied less than 10%
among samples. Each bar represents mean ± S.E. of the relative
Lef-1 reporter gene activity compared with pcDNA3.1 vector
transfected sample. Similar results were obtained in four separate
transfections.
|
|
The C Terminus Region of Axin Is Required for Its
Oligomerization--
Despite their bridging function, Axin mutants
with C-terminal deletions did not inhibit Lef-1 reporter activity in
SW480 cells (Fig. 4). The C terminus of Axin contains a DIX domain,
which shares loose homology with Dishevelled (14, 19). We tested whether Axin binds Dishevelled by overexpressing each protein in COS-7
cells but could not detect any interaction (data not shown). We then
examined whether Axin binds to itself using in vitro
translated protein. In this assay, recombinant GST-
N-Axin bound to
in vitro translated FL-Axin but not to
MC-,
CM-,
C-, and
CC-Axin (Fig. 5). These
data indicate that the C-terminal half of Axin contains a domain that
mediates oligomerization. Although binding sites for GSK-3
and
-catenin in Axin overlap, C-terminally deleted Axin mutants, as well
as FL-Axin, could bind both molecules simultaneously in
vitro (data not shown). Because
CM- and
CC-Axin did not bind
to
N-Axin, both CM region (amino acids 603-809) and DIX domain
appeared to be crucial for oligomerization.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 5.
In vitro binding of Axin with Axin.
In vitro translated 35S-labeled Axin mutants
bound to GST- N-Axin were analyzed by 10% SDS-PAGE and
autoradiography (A). 5 µl each of the in vitro
translated Axin mutants were subjected to SDS-PAGE (10%) and
autoradiography (B).
|
|
The Dimerized
C-Axin Restored Its Inhibitory Effect on Lef-1
Reporter Gene Activity--
To test whether oligomerization of Axin is
important for its function, we fused
C-Axin to the RXR, which is
known to form a homodimer in vivo (20).
C-RXR-Axin was
able to inhibit Lef-1 reporter gene activity in SW480 cells (Fig.
6.).
C-Axin fused to EcR, which does
not form a homodimer (21), lacked the inhibitory effect on Lef-1
reporter gene assay. Both
C-RXR-Axin and
C-EcR-Axin could bind
GSK-3
and
-catenin in vivo (data not shown). This result suggest that oligomerization of Axin as well as bridging of
GSK-3
and
-catenin is necessary for the function of Axin.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 6.
Lef-1 reporter gene assay in SW480 cells
with C-Axin constructs. C-Axin
constructs ( -Axin, C-RXR-Axin and C-EcR-Axin) or pcDNA3.1
vector were transfected (0.3 µg each) with pCG-Lef-1 (0.02 µg),
pGL3-fos-7LEF-luciferase (0.2 µg), and pTK- -galactosidase (0.03 µg). Luciferase activity and -galactosidase activity were measured
and shown as in Fig. 4.
|
|
Concluding Discussion--
In this study, we identified binding
domains of GSK-3
and
-catenin in Axin. We showed that these
domains of Axin are important for its inhibition of
Lef-1-dependent transcription in colon cancer cells. In
Xenopus experiments,
RGS-Axin acts as a dominant-negative molecule, which activates the Wnt pathway (14). It was also shown that
the RGS domain in Axin binds to APC (17, 22). From our results, the RGS
domain is not required for binding to GSK-3
or
-catenin and is
not necessary for inhibition of Lef-1 reporter activity in colon cancer
cells. It has been suggested that Axin acts as a negative regulatory
molecule in the Wnt pathway by presenting
-catenin to GSK-3
as a
substrate, which mediates degradation of
-catenin (16). Yet our data
using colon cancer cells expressing high levels of
-catenin are not
consistent with this simplistic model, because we found Axin mutants
that link
-catenin and GSK-3
but do not inhibit Lef-1 reporter
activity.
-catenin is already stabilized independent of GSK-3
activity in those cancer cells. Our results suggest that besides
scaffolding a kinase and a substrate Axin has some other effects that
appear to depend on the C terminus of the molecule. There are mutant
mice of Axin that have a dominant kinked tail phenotype (23). In those
mice, Axin gene products are likely to have a deletion at the C
terminus as a result of transposon insertion. This also suggests that
the C terminus of Axin has some functional importance. We showed that
the C terminus region of Axin is involved in the oligomerization of
Axin. The chimeric
C-Axin construct fused to the RXR, which mediates
dimerization inhibited Lef-1 reporter activity. These data suggest that
both oligomerization and bridging of GSK-3
with
-catenin are
necessary for the function of Axin to inhibit
Lef-1-dependent transcription.