From the Department of Biochemistry, Hiroshima
University School of Medicine, 1-2-3, Kasumi, Minami-ku, Hiroshima
734-8551, Japan, § PRESTO, Japan Science and Technology
Corporation, Hiroshima 734-8551, Japan, and the ¶ Center for
Molecular and Developmental Biology, Faculty of Science, Kyoto
University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan
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ABSTRACT |
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Axin forms a complex with glycogen synthase
kinase-3 Genetic and biochemical analyses have revealed that there are
components that are structurally and functionally conserved in the Wnt
signaling pathway among flies, frogs, and mammals (1-3). In mammals
these include Wnt, frizzled, Dvl,
GSK-3 Axin was originally identified as a product of mouse fused
gene (8). fused carries recessive mutations that are lethal and that cause a duplication of the embryonic axis (9, 10). Injection
of Axin into Xenopus embryos causes strong axis defects, and
coexpression of Axin inhibits the Xwnt8-dependent axis
duplication (8). Thus, Axin is a negative regulator of the Wnt
signaling pathway and inhibits axis formation. We have identified rat
Axin (rAxin) and its homolog, Axil (for
Axin-like), as GSK-3 Axin enhances GSK-3 Materials and Chemicals--
Human Dvl-1 cDNA and a
synthetic peptide substrate of GSK-3 (GSK peptide 1) were provided by
Drs. B. Dallapiccola and G. Novelli (Vergata University, Rome, Italy)
(21) and C.W. Turck (University of California, San Francisco, CA) (22),
respectively. The anti-Myc antibody was prepared from 9E10 cells.
GST-GSK-3 Plasmid Construction--
pBSKS/rAxin, pMAL-c2/rAxin,
pEF-BOS-Myc/rAxin, pBJ-Myc/rAxin, pGEX-2T/GSK-3 Phosphorylation of Axin in Intact Cells--
COS cells
expressing Myc-rAxin or Myc-rAxin322/326/330A
(35-mm-diameter dish) were metabolically labeled with
32Pi (100 µCi/ml) in phosphate-free RPMI for
12 h in the presence or absence of 30 mM LiCl or 100 nM okadaic acid. The cells were lysed, and the lysates were
immunoprecipitated with the anti-Myc antibody (11). The
immunoprecipitates were probed with the anti-Myc antibody and subjected
to autoradiography.
Pulse-Chase Analysis--
COS cells (35-mm-diameter dish)
were transfected with pBJ-Myc/rAxin or
pBJ/Myc-rAxin322/326/330A. After 48 h, pulse-chase
analysis was performed as described (14, 25). Briefly, the cells were
pulse-labeled with [35S]methionine and
[35S]cysteine (50 µCi/ml) for 1 h at 37 °C.
Then the cells were lysed immediately or at the indicated times
following incubation with excess unlabeled methionine and cysteine in
the presence or absence of 30 mM LiCl or 100 nM
okadaic acid. The lysates were immunoprecipitated with the anti-Myc
antibody, the precipitates were subjected to autoradiography, and then
the densities of the labeled proteins were analyzed with a Fuji BAS
2000 image analyzer.
Kinase Assay--
90 nM GST-GSK-3 Down-regulation of Axin by Wnt-3a--
Confluent wild type L
cells or L cells expressing HA-Dvl-1 Stabilization of Axin by Phosphorylation--
rAxin was
phosphorylated by GSK-3
To investigate the stability of Axin by phosphorylation further,
pulse-chase analysis was performed. Pulse-labeled Myc-rAxin in COS
cells migrated slowly on SDS-polyacrylamide gel electrophoresis in a
time-dependent manner (Fig.
2A), suggesting that Myc-rAxin was phosphorylated. Pulse-labeled Myc-rAxin did not exhibit a gel band
shift and disappeared at 12 h in COS cells treated with LiCl (Fig.
2A). In contrast, okadaic acid enhanced the band shift and
prevented the decay of pulse-labeled Myc-rAxin at 12 h (Fig. 2A). Pulse-labeled Myc-rAxin decreased gradually with a
half-life of approximately 8 h, and pulse-labeled
Myc-rAxin322/326/330A exhibited a shorter half-life (Fig.
2B). These results indicate that Axin is phosphorylated by
GSK-3 Inhibition of GSK-3
This is the first demonstration showing that Dvl inhibits the function
of GSK-3 Down-regulation of Axin by Wnt-3a--
Finally we examined whether
Wnt signal regulates the stability of endogenous Axin in intact cells.
Although Wnt proteins are secretory, they predominantly bind to the
cell surface or extracellular matrix. Small amounts of biologically
active Wnt-1 or Wg can be found in culture medium conditioned by cells
expressing these proteins (35, 36). The Wg-conditioned medium from
Schneider cells increases the level of Armadillo in
Drosophila disc cells and inactivates GSK-3 in 10T1/2
fibroblasts (35, 37). Based on assays carried out with mammalian cell
lines and Xenopus embryos, the Wnt proteins can be
classified into two groups, Wnt-1 and Wnt-5a classes (38-40). The
Wnt-1 class includes Wnt-1, Wnt-2, Wnt-3, Wnt-3a, and Wnt-8, which have
activities to transform the cells and to accumulate cytoplasmic
We have recently found that in COS cells Axin interacts with
GSK-3 (GSK-3
) and
-catenin and promotes
GSK-3
-dependent phosphorylation of
-catenin, thereby
stimulating the degradation of
-catenin. Because GSK-3
also
phosphorylates Axin in the complex, the physiological significance of
the phosphorylation of Axin was examined. Treatment of COS cells with
LiCl, a GSK-3
inhibitor, and okadaic acid, a protein phosphatase
inhibitor, decreased and increased, respectively, the cellular protein
level of Axin. Pulse-chase analyses showed that the phosphorylated form
of Axin was more stable than the unphosphorylated form and that an Axin
mutant, in which the possible phosphorylation sites for GSK-3
were
mutated, exhibited a shorter half-life than wild type Axin. Dvl-1,
which was genetically shown to function upstream of GSK-3
, inhibited
the phosphorylation of Axin by GSK-3
in vitro.
Furthermore, Wnt-3a-containing conditioned medium down-regulated Axin
and accumulated
-catenin in L cells and expression of
Dvl-1
PDZ, in which the PDZ domain was deleted,
suppressed this action of Wnt-3a. These results suggest that the
phosphorylation of Axin is important for the regulation of its
stability and that Wnt down-regulates Axin through Dvl.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
,1
-catenin, and
Lef/Tcf, which are homologous to the Drosophila proteins Wg,
Dfz2, Dsh (Dishevelled), Shaggy, Armadillo, and Pangolin, respectively.
The current model for the Wnt signaling pathway proposes that in the
absence of Wnt, GSK-3
phosphorylates
-catenin, resulting in the
degradation of
-catenin. In response to Wnt, Dvl antagonizes
GSK-3
activity through an as yet unknown mechanism. This leads to
the stabilization and the accumulation of
-catenin. The accumulated
-catenin translocates to the nucleus, associates with the
transcriptional enhancers of the Lef/Tcf family (4-6), and stimulates
gene expression such as Myc (7).
-interacting proteins
(11, 12). Conductin has been identified as a
-catenin-binding protein (13) and is identical to Axil. We have found that both Axin and
Axil bind not only to GSK-3
but also to
-catenin and that they
promote GSK-3
-dependent phosphorylation of
-catenin (11, 12). We have also shown that the regulators of G protein signaling
(RGS) domain of rAxin directly interacts with APC and that expression
of rAxin in COS and SW480 cells stimulates the degradation of
-catenin (14, 15). Other groups have reported similar results (13,
16-19). Therefore, it appears that Axin family members down-regulate
-catenin.
-dependent phosphorylation of APC in
addition to
-catenin in vitro (17), and the
phosphorylation of APC increases its binding to
-catenin (20).
Although Axin is also phosphorylated by GSK-3
directly, the
phosphorylation of Axin does not affect its binding to GSK-3
and
-catenin in vitro (11). These results indicate that
-catenin, APC, and Axin form a complex with GSK-3
and that the
phosphorylation occurs efficiently in the complex. However, the
physiological significance of the phosphorylation of Axin is not known.
Therefore, we examined a role of the phosphorylation of Axin in the Wnt
signaling pathway. Here we demonstrate that the phosphorylation of Axin
by GSK-3
regulates its stability, that Dvl inhibits the
GSK-3
-dependent phosphorylation of Axin, and that Wnt-3a
down-regulates Axin through Dvl.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
was purified from Escherichia coli as described
(22). GST fusion proteins and MBP fusion proteins were purified from
E. coli according to the manufacturer's instructions. L
cells stably expressing HA-Dvl-1
PDZ
(Dvl-1-(
283-336)) were produced by transfecting
pCGN/Dvl-1
PDZ and pNeo. To prepare Wnt-3a-conditioned
medium, L cells were transfected with pGK/Wnt-3a, and a number of
stably transfected clones were established (23). The anti-Axin antibody
was prepared in rabbits by immunization with a recombinant fragment of
rAxin-(1-229). The anti-
-catenin and anti-GSK-3
antibodies were
purchased from Transduction Laboratories (Lexington, KY).
[
-32P]ATP, [35S]methionine, and
[35S]cysteine were purchased from Amersham Pharmacia
Biotech (Buckinghamshire, UK). Other materials were from commercial sources.
, and pBJ-Myc/RalBP1
were constructed as described (11, 14, 15, 22, 24). To construct
pMAL-c2/Dvl-1, pBSKS/Dvl-1 was digested with XbaI, and the
2.0-kb fragment encoding Dvl-1 was inserted into the
XbaI-cut pMAL-c2. pBSKS/Dvl-1
PDZ was
constructed as follows. The 0.42-kb fragment encoding Dvl-1-(337-476) with BamHI and PstI sites was synthesized by
polymerase chain reaction, digested with BamHI and
PstI, and inserted into the BamHI- and
PstI-cut pBSKS to generate pBSKS/Dvl-1-(337-476).
pBSKS/Dvl-1 was digested with PstI and HindIII,
and the 0.6-kb fragment encoding Dvl-1-(477-670) was inserted into the
PstI- and HindIII-cut pBSKS/Dvl-1-(337-476) to
generate pBSKS/Dvl-1-(337-670). pBSKS/Dvl-1 was digested with XbaI and BamHI, and the 0.85-kb fragment encoding
Dvl-1-(1-282) was inserted into the XbaI- and
BamHI-cut pBSKS/Dvl-1-(337-670) to generate
pBSKS/Dvl-1
PDZ. Thus, Dvl-1-(283-336) (the PDZ domain)
was deleted in pBSKS/Dvl-1
PDZ. To construct
pMAL-c2/Dvl-1
PDZ, pBSKS/Dvl-1
PDZ was
digested with XbaI and HindIII, and the 1.9-kb
fragment encoding Dvl-1
PDZ was inserted into the
XbaI- and HindIII-cut pMAL-c2.
pMAL-c2/Dvl-1
PDZ was digested with HindIII,
blunted with Klenow fragment, and digested with XbaI. The
1.9-kb fragment encoding Dvl-1
PDZ was inserted into the
XbaI- and SmaI-cut pCGN to generate
pCGN/Dvl-1
PDZ. pBJ/Myc-rAxin322/326/330A was
constructed as follows. The 0.65-kb fragment encoding rAxin-(182-401), in which Ser322, Ser326, and Ser330
were mutated to Ala, was synthesized by polymerase chain reaction, digested with ClaI and XbaI, and inserted into
the ClaI- and XbaI-cut pEF-BOS-Myc/rAxin-(1-181), which was obtained from
pEF-BOS-Myc/rAxin to generate
pEF-BOS-Myc/rAxin-(1-401,322/326/330A). To construct pEF-BOS-Myc/rAxin322/326/330A, pEF-BOS-Myc/rAxin was
digested with XbaI, and the 1.3-kb fragment encoding rAxin-(402-832) was inserted into the XbaI-cut
pEF-BOS-Myc/rAxin-(1-401,322/326/330A). To construct
pBJ/Myc-rAxin322/326/330A,
pEF-BOS-Myc/rAxin322/326/330A was digested with
EcoRI, and the 2.6-kb fragment encoding
Myc-rAxin322/326/330A was inserted into the
EcoRI-cut pBJ-1.
was incubated
with the indicated concentrations of MBP-rAxin and MBP-Dvl-1 in 30 µl
of kinase reaction mixture (50 mM Tris/HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, and 50 µM [
-32P]ATP (500-1500 cpm/pmol))
for 15 min at 30 °C. The samples were subjected to
SDS-polyacrylamide gel electrophoresis followed by autoradiography, and
then the radioactivities of the phosphorylated Axin were counted. The
kinase activities of GSK-3
for GSK peptide 1 were measured as
described (11, 12, 22).
PDZ (35-mm-diameter
dish) were washed with Dulbecco's modified essential medium twice, and
the indicated volume of Wnt-3a-conditioned medium, which was adjusted
to a total volume of 700 µl with Dulbecco's modified essential
medium, was added to the cells. After stimulation for 6 h, the
cells were lysed in 100 µl of lysis buffer (11), and the lysates (20 µg of protein) were probed with the anti-Axin and anti-
-catenin antibodies.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
directly in vitro, and
SANDSEQQS330 of rAxin
was one of the phosphorylation sites for GSK-3
(11). First we
examined whether rAxin is phosphorylated by GSK-3
in intact cells.
Myc-rAxin was phosphorylated when COS cells were metabolically labeled
with 32Pi (Fig.
1A). We tried to express
Myc-rAxin322/326/330A, in which Ser322,
Ser326, and Ser330 were mutated to Ala, in COS
cells, but its protein level was lower than that of Myc-rAxin (wild
type) (Fig. 1A, lanes 3 and 4).
Consistent with the protein level, the phosphorylation and apparent molecular weight of MycrAxin322/326/330A were
reduced in comparison with Myc-rAxin (Fig. 1A, lanes
1 and 2). Therefore, we used LiCl, which is known to be
an inhibitor of GSK-3
(26, 27). It appeared that treatment of COS
cells with LiCl decreased the phosphorylation of Myc-rAxin, whereas okadaic acid, a protein phosphatase 1 or 2A inhibitor, increased it
(Fig. 1A, lanes 5-7). However, these changes by
LiCl and okadaic acid were also correlated with the protein level of
Myc-rAxin (Fig. 1A, lanes 8-10). LiCl decreased
the protein level of Myc-rAxin in a dose-dependent manner
(Fig. 1B). Consistent with the previous observations (28),
treatment of COS cells with LiCl resulted in the cytoplasmic
accumulation of
-catenin (Fig. 1B). Okadaic acid
prevented the decrease of Myc-rAxin by LiCl (Fig. 1B). LiCl did not affect the protein level of transfected Myc-RalBP1, an effector
protein of small GTP-binding protein Ral (29) or endogenous GSK-3
(Fig. 1C). Therefore, the effect of LiCl that reduces rAxin is not nonspecific. These results suggest that the phosphorylation of
rAxin is correlated with its stability.
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Fig. 1.
Stabilization of Axin by
phosphorylation. A, phosphorylation of rAxin in intact
cells. COS cells expressing Myc-rAxin (wild type, WT)
(lanes 1, 3, and 5-10) or
Myc-rAxin322/326/330A (SA) (lanes 2 and 4) were metabolically labeled with
32Pi in the absence (lanes 1-4,
5, and 8) or presence of 30 mM LiCl
(lanes 6 and 9) or 100 nM okadaic
acid (OA) (lanes 7 and 10). The
immunoprecipitated Myc-rAxin was subjected to autoradiography
(lanes 1, 2, and 5-7) or probed with
the anti-Myc antibody (lanes 3, 4, and
8-10). B, effects of LiCl and okadaic acid on
the protein levels of rAxin and -catenin. COS cells expressing
Myc-rAxin were treated with the indicated concentrations of LiCl and
okadaic acid. The lysates were probed with the anti-Myc antibody. To
examine the protein levels of endogenous
-catenin, the cytosol
fraction of COS cells was prepared and probed with the anti-
-catenin
antibody. C, effects of LiCl on other proteins. COS cells
expressing Myc-RalBP1 were treated with the indicated concentrations of
LiCl. The lysates were probed with the anti-Myc and anti-GSK-3
antibodies. The results shown are representative of three independent
experiments.
in intact cells and that the phosphorylated form is more
stable than the unphosphorylated form.
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Fig. 2.
Pulse-chase analysis. A,
effects of LiCl and okadaic acid (OA) on pulse-labeled
Myc-rAxin. COS cells expressing Myc-rAxin were pulse-labeled with
[35S]methionine and [35S]cysteine for
1 h and lysed at the indicated time in the presence of LiCl or
okadaic acid. B, degradation of rAxin mutant. COS cells
expressing Myc-rAxin ( ) and Myc-rAxin322/326/330A (
)
were pulse-labeled with [35S]methionine and
[35S]cysteine, and pulse-chase analysis was carried out.
The incorporation of 35S into Myc-rAxin or its mutant was
analyzed with a Fuji BAS 2000 image analyzer and expressed as the
percentage of the value at time 0. The results shown are the means ± S.E. of four independent experiments.
-dependent Phosphorylation
of Axin by Dvl--
Drosophila Dsh encodes a cytoplasmic
protein of unknown biochemical function in the Wg signaling pathway
(1-3). In mammals, dvl-1, -2, and -3 genes have been isolated as homologs of Dsh (21, 30, 31). It has been
shown that Dsh antagonizes shaggy, a fly homolog of GSK-3
, in the Wg
signaling pathway (2, 3), and that overexpression of Dvl-1 in Chinese
hamster ovary cells inhibits GSK-3 activity as measured by the
GSK-3-mediated phosphorylation of tau proteins (32). However, little is
known about the biochemical pathway leading from Dvl to GSK-3
.
Therefore, we examined whether Dvl-1 affects the phosphorylation of
rAxin by GSK-3
in vitro. MBP-Dvl-1 itself was not
phosphorylated by GST-GSK-3
(data not shown). GST-GSK-3
phosphorylated MBP-rAxin in a time-dependent manner (11)
(Fig. 3A). MBP-Dvl-1 inhibited
this phosphorylation of MBP-rAxin (Fig. 3A). This inhibitory
activity of MBP-Dvl-1 was dose-dependent, and MBP alone did
not inhibit the GST-GSK-3
-dependent phosphorylation of
MBP-rAxin (Fig. 3B). Dvl has the PDZ domain, and disruption
of the PDZ domain abolishes its activity in the Wg-Armadillo pathway
and in the Xenopus axis induction assay (33, 34). Deletion
of the PDZ domain from Dvl-1 (MBP-Dvl-1
PDZ) greatly
reduced its activity to inhibit the phosphorylation of MBP-rAxin by
GST-GSK-3
(Fig. 3B). Inhibition of the phosphorylation of
MBP-rAxin by MBP-Dvl-1 was not recovered even though the amounts of
MBP-rAxin increased (Fig. 3C). Lineweaver-Burk plots
indicated that the Km and
Vmax values of MBP-rAxin for GST-GSK-3
in the
absence of MBP-Dvl-1 were 131 nM and 4.3 nmol/min/mg,
respectively, and that those in the presence of MBP-Dvl-1 were 129 nM and 2.5 nmol/min/mg (Fig. 3C). These results
suggest that Dvl-1 inhibits the GSK-3
-dependent
phosphorylation of Axin in a noncompetitive manner.
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Fig. 3.
Inhibition of
GSK-3 -dependent phosphorylation of
Axin by Dvl. A, time course. 90 nM
GST-GSK-3
and 250 nM MBP-rAxin were incubated with in
the presence of 1 µM MBP-Dvl-1 or MBP for the indicated
periods. B, dose dependence. GST-GSK-3
and MBP-rAxin were
incubated with the indicated concentrations of MBP-Dvl-1 (
),
MBP-Dvl-1
PDZ (
), or MBP (
) for 15 min.
C, mode of inhibitory action of Dvl. GST-GSK-3
was
incubated with the indicated concentrations of MBP-rAxin in the
presence of 1 µM MBP (
) or 1 µM
MBP-Dvl-1 (
). Inset, Lineweaver-Burk plot analysis. The
results shown are representative of three independent
experiments.
directly. However, it is not likely that Dvl-1 inhibits
GSK-3
activity itself, because MBP-Dvl-1 did not affect the
phosphorylation of synthetic peptide substrate, which is designed from
glycogen synthase, by GST-GSK-3
(data not shown). We have recently
found that Dvl-1 directly binds to Axin and that the binding of Dvl-1
to Axin does not affect the interaction of GSK-3
with
Axin.2 It is possible that
the binding of Dvl to Axin induces the structural change of the Axin
complex; therefore GSK-3
does not effectively phosphorylate Axin.
However, higher concentrations (µM order) of Dvl-1 are
required to inhibit the GSK-3
-dependent phosphorylation of Axin in our in vitro experiments. Therefore, modification
of Dvl such as phosphorylation could be necessary to act on the Axin complex in intact cells. These results suggest that Dvl may regulate the stability of Axin.
-catenin, whereas the Wnt-5a class includes Wnt-4, Wnt-5a, Wnt-5b,
Wnt-7b, and Wnt-11, which do not exhibit the transformation and
-catenin accumulation activities. Because Wnt-3a displays
characteristics similar to those of Wnt-1, we prepared
Wnt-3a-containing conditioned medium. In these experiments we used
mouse fibroblast L cells, because the changes in the expression level
of
-catenin by Wnt are easily observed due to little expression of
cadherin in the cells (15, 23, 41). Furthermore, Western blot analyses
with the anti-Axin antibody demonstrated that Axin is most abundant in
L cells among various cell lines including SW480, NIH3T3, COS, and
Chinese hamster ovary cells (data not shown). Wnt-3a conditioned medium
induced the accumulation of
-catenin in L cells in a
dose-dependent manner (Fig.
4A). In contrast, Wnt-3a
decreased Axin (Fig. 4A). Control conditioned medium did not
affect the amounts of Axin and
-catenin (data not shown). To examine
whether Dvl is involved in this action of Wnt-3a, we established L
cells, which express HA-Dvl-1
PDZ stably. Wnt-3a-induced
increase of
-catenin and decrease of Axin were suppressed in L cells
expressing HA-Dvl-1
PDZ (Fig. 4B). These
results indicate that Wnt not only accumulates
-catenin but also
down-regulates Axin through Dvl.
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Fig. 4.
Down-regulation of Axin by Wnt-3a.
A, effect of Wnt-3a on wild type L cells. L cells were
treated with the indicated amounts of Wnt-3a-conditioned medium for
6 h, and the lysates were probed with the anti-Axin and
anti- -catenin antibodies. B, effect of Wnt-3a on L cells
expressing HA-Dvl-1
PDZ stably. Wild type L cells
(WT) and L cells expressing HA-Dvl-1
PDZ
(Dvl-1
PDZ) were treated with 170 µl of
Wnt-3a-conditioned medium for 6 h. The results shown are
representative of three independent experiments.
,
-catenin, and APC in a high molecular mass complex with a
molecular mass of more than 103 kDa on gel filtration
column chromatography (15). In L cells
-catenin is present in the
high molecular mass complex in the absence of Wnt-3a, whereas addition
of Wnt-3a to L cells increases
-catenin in a low molecular mass
complex with a molecular mass of 200-300 kDa (15). In L cells
expressing Axin, Wnt-3a-induced increase of
-catenin in the low
molecular mass complex is not observed (15). These results suggest that
a balance between the high and low molecular mass complexes containing
-catenin is closely regulated and that Axin plays a role in limiting
the accumulation of
-catenin in the low molecular mass complex. Wnt may regulate the assembly of the complex consisting of Axin, APC,
-catenin, and GSK-3
and induce the dissociation of
-catenin from the complex. It is possible that
-catenin free from the complex
is accumulated, binds to different partners such as Lef/Tcf, and
transmits the signals. Our results suggest that the Wnt signal could
act on the Axin complex through Dvl, resulting in the inhibition of the
GSK-3
-dependent phosphorylation of Axin and the
degradation of Axin. Degradation of Axin due to hypophosphorylation may
induce the dissociation of
-catenin from the complex by decreasing
the binding of
-catenin to Axin. Studies to clarify the mechanism of
proteolysis of Axin are under way.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. B. Dallapiccola, G. Novelli, and C. Turck for reagents. We thank the Research Center for Molecular Medicine, Hiroshima University School of Medicine, for the use of their facilities.
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FOOTNOTES |
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* This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture, Japan and by grants from the Yamanouchi Foundation for Research on Metabolic Disorders, the Kato Memorial Bioscience Foundation, and the Naito Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
81-82-257-5130; Fax: 81-82-257-5134; E-mail:
akikuchi{at}mcai.med.hiroshima-u.ac.jp.
2 Kishida, S., Yamamoto, H., Hino, S., Ikeda, S., Kishida, M., and Kikuchi, A. (1999) Mol. Cell. Biol., in press.
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ABBREVIATIONS |
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The abbreviations used are:
GSK-3, glycogen
synthase kinase-3
;
APC, adenomatous polyposis coli;
GST, glutathione
S-transferase;
MBP, maltose-binding protein;
HA, hemagglutinin;
PDZ, PSD95/Dlg/Zo-1;
RalBP1, Ral-binding protein 1;
kb, kilobase pair.
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REFERENCES |
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