Casein Kinase I
Enhances the Binding of Dvl-1 to Frat-1
and Is Essential for Wnt-3a-induced Accumulation of
-Catenin*
Shin-ichiro
Hino
,
Tatsuo
Michiue§,
Makoto
Asashima§, and
Akira
Kikuchi
¶
From the
Department of Biochemistry, Graduate School
of Biomedical Sciences, Hiroshima University, 1-2-3, Kasumi,
Minami-ku, Hiroshima 734-8551, Japan and the § Sorst Project
and Department of Life Science (Biology), University of Tokyo, 3-8-1, Komaba, Meguro-ku, Tokyo 153-8902, Japan
Received for publication, December 30, 2002, and in revised form, January 19, 2003
 |
ABSTRACT |
We demonstrate that Dvl-1, casein kinase I
(CKI
), and Frat-1 activate the Wnt signaling pathway cooperatively.
The amino acid region 228-250 of Dvl-1 was necessary for its binding
to Frat-1, and the interaction of Dvl-1 with Frat-1 was enhanced by
CKI
. Coexpression of Dvl-1 and Frat-1 caused accumulation of
-catenin synergistically in L cells. Both proteins also activated the transcriptional activity of T-cell factor-4 (Tcf-4) synergistically in human embryonic kidney 293 cells, but coexpression of
Dvl-1-(
228-250), which lacks the amino acid region 228-250 from
Dvl-1, and Frat-1 did not. Dvl-1, but not Dvl-1-(
228-250), acted
synergistically with CKI
to activate Tcf-4. Depletion of CKI
by
double-stranded RNA interference in HeLa S3 cells led to the inhibition
of Wnt-3a-induced phosphorylation of Dvl and the binding of Dvl-1 to
Frat-1. Furthermore, depletion of CKI
reduced the Wnt-3a-induced
accumulation of
-catenin, although it did not affect the basal level
of
-catenin. These results indicate that
CKI
-dependent phosphorylation of Dvl enhances the
formation of a complex of Dvl-1 with Frat-1 and that this complex leads
to the activation of the Wnt signaling pathway.
 |
INTRODUCTION |
Wnt proteins constitute a large family of cysteine-rich secreted
ligands that control development in organisms ranging from nematode
worms to mammals (1). In vertebrates, the Wnt signaling pathway
regulates axis formation, organ development, and cellular proliferation, morphology, motility, and fate (2, 3). The protein level
of free cytoplasmic
-catenin is controlled by the Wnt signal. In
unstimulated cells, cytoplasmic
-catenin is destabilized by a
multiprotein complex containing Axin (or its homolog Axil/conductin), GSK-3
,1 and APC (4-8).
Interaction of GSK-3
with Axin in the complex facilitates efficient
phosphorylation of
-catenin by GSK-3
. Phosphorylated
-catenin
forms a complex with Fbw1 (
TrCP/FWD1), which resides in an E3
ubiquitin ligase complex (9, 10). Ubiquitination of
-catenin induces
its rapid proteosomal degradation (11).
When Wnt binds to the Frizzled/LRP co-receptor at the cell surface, a
cytoplasmic protein, Dvl, antagonizes GSK-3
-dependent phosphorylation of
-catenin. Once the phosphorylation of
-catenin is reduced, it dissociates from the Axin complex, and
-catenin is no
longer degraded, resulting in its accumulation in the cytoplasm. Stabilized
-catenin is translocated into the nucleus, where it binds
to transcriptional factors such as the Tcf/lymphoid enhancer binding
factor and thereby stimulates the transcription of Wnt target genes (1,
12). Thus, the Wnt signal stabilizes
-catenin, thereby
regulating the expression of various genes. However, the precise
molecular mechanism by which the signal is transmitted to stabilize
-catenin after Wnt binds to the Frizzled/LRP co-receptor is unknown.
Dvl is a cytoplasmic phosphoprotein that acts downstream of Frizzled
and is a key protein mediating the Wnt signal (1-3). Three Dvl genes,
Dvl-1, -2, and -3, have been isolated
in mammals. Dvl homologs are conserved in Drosophila
(Dishevelled, abbreviated Dsh) and Xenopus
(Xenopus dishevelled, abbreviated Xdsh). All Dvl and Dsh
family members contain the following three highly conserved domains: an
N-terminal DIX domain; a central PDZ domain; and a DEP domain.
Expression of Dvl in cells induces the accumulation of
-catenin and
the activation of Tcf (13, 14). Although it is not known at present
whether Dvl binds directly to the Frizzled/LRP co-receptor or whether
intermediary proteins are involved in the signal transmission between
Frizzled and Dvl, Dvl appears to bind to Axin and inhibit
GSK-3
-dependent phosphorylation of
-catenin, APC, and
Axin (13-17). Furthermore, Dvl has been shown to bind to CKI
and
Frat-1 (18-21).
CKI comprises a large family of related gene products, namely
,
,
,
, and
(22). They all share at least 50% amino acid
identity within the protein kinase catalytic domain. Different CKI
family members generally show different tissue distributions and
subcellular localization and have distinct roles (22, 23). As for
regulation of the Wnt signaling pathway by CKI, seemingly conflicting
findings have been reported. CKI
forms a complex with Dvl and Axin,
and CKI
and Dvl-1 activate Tcf-4 cooperatively in mammalian cells
(18-20,24). Overexpression of CKI
in Xenopus embryos
induces expression of siamois, a Wnt-response gene, and axis
duplication (18, 19, 25). These results suggest that CKI
regulates
the Wnt signaling pathway positively. It has also been shown that
CKI
primes phosphorylation of
-catenin by GSK-3
and induces
the degradation of
-catenin (26-28). Furthermore, disruption of
CKI
stabilizes
-catenin in mammalian HEK-293T cells and
Drosophila Schneider cells (26, 27). These results suggest
that CKI
functions as a negative regulator of the Wnt signaling
pathway. One possible explanation for these different findings may be
that distinct CKI isoforms have opposite roles in the Wnt signaling pathway.
Frat is yet another Dvl-binding protein. Frat was originally isolated
on the basis of its tumor-promoting activity in human lymphocytes (29)
and shares three conserved regions with Xenopus GBP, which
binds to GSK-3 and activates the Wnt signaling pathway (30). The Frat
family consists of three members: Frat-1, -2, and -3. It has been shown
that different sites of Frat-1 interact with GSK-3 and Dvl-1 and that
Wnt-1 disintegrates the complex formation of Frat-1, Dvl-1, and Axin,
resulting in the activation of the Wnt signaling pathway (21). However,
how these three proteins, Dvl-1, CKI
, and Frat-1, functionally
interact with one another to regulate the Wnt signaling pathway is not known.
Here we demonstrate that CKI
enhances the binding of Dvl-1 and
Frat-1 and that the interaction of Dvl-1 with Frat-1 is important for
the activation of the Wnt signaling pathway. Furthermore, we
demonstrate that depletion of CKI
, but not CKI
, is essential for
Wnt-3a-induced
-catenin accumulation by the use of ds RNAi.
 |
EXPERIMENTAL PROCEDURES |
Materials and Chemicals--
pRSETB/human CKI
(hCKI
) and
pRSETB/hCKI
kinase negative (KN), pTOPFLASH and pFOPFLASH,
pcDNA3/hFrat-1, pcDNA3-FLAG/rAxin, pPGK/Wnt-3a, and HeLa S3
cells were provided by Drs. D. M. Virshup (University of Utah,
Salt Lake City, UT), H. Clevers (University Hospital, Utrecht, The
Netherlands), S. Tanaka (Kyusyu University, Fukuoka, Japan), K. Miyazono (Tokyo University, Tokyo, Japan), S. Takada (Kyoto University,
Kyoto, Japan), and K. Matsumoto (Nagoya University, Nagoya, Japan),
respectively. Recombinant baculoviruses expressing GST-fused Frat-1
(GST-Frat-1) (wild type) were generated by Dr. Y. Matsuura (Research
Institute of Microbial Diseases, Osaka University, Suita, Japan).
GST-Frat-1 was purified from Spodoptera frugiperda 9 cells.
MBP and His6 fusion proteins were purified from
Escherichia coli according to the supplier's instructions. L cells stably expressing hDvl-1 or hDvl-1-(
228-250) were generated by selection with G418 as described (31). Wnt-3a-conditioned medium was
produced as described previously (32). COS and L, HeLa S3, and HEK-293
cells were grown in Dulbecco's modified Eagle's medium supplemented
with 10% calf serum and 10% fetal bovine serum, respectively. The
anti-Myc antibody was prepared from 9E10 cells. The anti-GST, anti-MBP,
and anti-Dvl antibodies were prepared in rabbits by immunization with
recombinant GST, MBP, and Dvl-1-(1-140) proteins, respectively. The
anti-FLAG and polyclonal anti-
-catenin antibodies, the monoclonal
anti-
-catenin, anti-GSK-3
, the anti-CKI
antibodies, and the
anti-CKI
antibody were purchased from Sigma, Transduction
Laboratories (Lexington, KY), and Santa Cruz Biotechnology, respectively. Cy5-labeled anti-mouse IgG was obtained from Amersham Biosciences. The Alexa 546-labeled anti-rabbit or mouse IgG and the
anti-GFP antibody were from Molecular Probes, Inc. (Eugene, OR). Other
materials were from commercial sources.
Plasmid Construction--
pCGN/hDvl-1 (wild type),
pCGN/hDvl-1-(1-519), pCGN/hDvl-1-(140-670), pMAL-c2/hDvl-1 (wild
type), pSP64T-Myc/mDvl-1 (wild type), pEF-BOS-HA/hTcf-4E, pCGN/hCKI
,
pCGN/hCKI
(KN), pEGFP-C1/hCKI
, pEGFP-C1/hCKI
(KN), and
pSP64T-Myc/hCKI
were constructed as described (5, 7, 14, 20).
Standard recombinant DNA techniques were used to construct the
following plasmids: pCGN/hFrat-1 (wild type); pCGN/hFrat-1-(1-185);
pEF-BOS-Myc/hFrat-1; pVIKS/hFrat-1; pEGFP-C1/hFrat-1;
pEF-BOS-HA/hDvl-1-(1-250); pCGN/hDvl-1-(
141-227); pCGN/hDvl-1-(
228-250); pCGN/hDvl-1-(
251-336);
pCGN/hDvl-1-(337-670); pEF-BOS-Myc/hDvl-1 (wild type);
pEF-BOS-Myc/hDvl-1-(
228-250); pMAL-c2/hDvl-1-(
228-250); and
pSP64T-Myc/hDvl-1-(
228-250). In these plasmids, some plasmid
constructions were done by digesting the original plasmids with
restriction enzymes and inserting the fragments into the vectors. Other
constructions were done by inserting the fragments generated by PCR
into the vectors. The entire PCR products were sequenced, and the
structures of all plasmids were confirmed by restriction enzyme analysis.
Complex Formation of Frat-1 with Dvl-1--
To determine whether
Frat-1 forms a complex with Dvl-1 in intact cells, COS cells
(60-mm-diameter dishes) transfected with pCGN-, pEGFP-,
pcDNA3-FLAG-, or pEF-BOS-Myc-derived plasmids were lysed in 200 µl of the lysis buffer (20 mM Tris/HCl, pH 7.5, 137 mM NaCl, 1% Nonidet P-40, 10% glycerol, 25 mM
-glycerophosphate, 5 mM sodium orthovanadate, 5 mM NaF, 5 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and 20 µg/ml aprotinin), and the lysates were
centrifuged at 15, 000 × g for 10 min at 4 °C. The supernatant (20 µg of protein) was probed with the anti-Myc, anti-HA, anti-GFP, anti-FLAG, or anti-GSK-3
antibody to detect the protein expression levels. The same lysates (200 µg of protein) were
immunoprecipitated with the anti-Myc antibody, and then the
immunoprecipitates were probed with the same antibodies.
To examine the direct interaction of Frat-1 with phosphorylated Dvl-1
using purified proteins in vitro, MBP-Dvl-1 and
MBP-Dvl-1-(
228-250) (2.6 µg of protein) immobilized on amylose
resin were incubated with or without His6-CKI
(0.3 µg
of protein) in 15 µl of kinase reaction mixture (50 mM
Tris/HCl, pH 7.5, 10 mM MgCl2, 1 mM
dithiothreitol, and 50 µM ATP) for 30 min at 30 °C.
Phosphorylated Dvl-1 (20 pmol) was incubated with GST-Frat-1 (wild
type) (30 pmol) in 100 µl of reaction mixture (20 mM
Tris/HCl, pH 7.5, and 1 mM dithiothreitol) for 1 h at
4 °C. After MBP fusion proteins were precipitated by centrifugation,
the precipitates were probed with the anti-GST antibody.
Immunocytochemistry--
L cells grown on coverslips were fixed
for 20 min in PBS containing 4% paraformaldehyde. The cells were
washed with PBS three times and then permeabilized with PBS containing
0.1% Triton X-100 and 2 mg/ml bovine serum albumin for 2 h. The
cells were washed and incubated with the anti-HA and polyclonal
anti-
-catenin antibodies for 1 h. After being washed with PBS,
they were further incubated for 1 h with Alexa 546-labeled mouse
IgG, Cy5-labeled anti-mouse IgG, or Alexa 546-labeled anti-rabbit IgG.
The coverslips were washed with PBS, mounted on glass slides, and
viewed with a confocal laser-scanning microscope (LSM510, Carl-Zeiss,
Jena, Germany). All procedures were carried out at room temperature.
Luciferase Assay--
To observe Tcf-4 activity, the indicated
amounts of pCGN/Dvl-1, pCGN/hDvl-1-(
228-250), pCGN/hFrat-1,
pCGN/hFrat-1-(1-185), pCGN/hCKI
, pCGN/hCKI
(KN), or pPGK/Wnt-3a
were transfected into HEK-293 cells (35-mm-diameter dishes) with
pTOPFLASH (0.5 µg), pEF-BOS-HA/hTcf-4E (0.1 µg), and pME18S/lacZ
(0.5 µg) (31, 33). Forty-six hours after the transfection, the cells
were lysed, and the luciferase activity was measured using a PicaGene
(Toyo B-NET Co., Ltd., Tokyo, Japan) and a lumiphotometer TD4000
(Futaba Medical, Tokyo, Japan). To standardize the transfection
efficiency, pME18S/lacZ carrying the SR
promotor linked to
the coding sequence of the
-galactosidase gene was used as an
internal control.
Xenopus Injections and Analyses of Phenotypes--
Myc-tagged
Dvl-1, Myc-Dvl-1-(
228-250), and HA-CKI
cDNA were subcloned
into pSP64T (34). Sense mRNA was obtained by in vitro
transcription of linearized templates using the SP6-mMESSAGE mMACHINE
kit (Ambion, Austin, TX). Fertilized eggs were dejellied using 4.5%
L-cysteine hydrochloride monohydrate, and mRNAs were injected into ventral blastomeres at the four-cell stage. After injection, embryos were cultured for 3 days (stage 40-41).
RNA Interference--
Two RNA interferences specific to human
CKI
(sense) and human CKI
(sense),
5'-CCAGGCAUCCCCAGUUGCUTT-3' and
5'-UGGCCAAGAAGUACCGGGATT-3', respectively, were synthesized, and
double-stranded RNA oligonucleotides were annealed in vitro
before transfection. Transfection was done with Oligofectamine
(Invitrogen) on HeLa S3 cells (35-mm-diameter dishes). Ninety-six hours
after the transfection, the cells were treated with Wnt-3a-conditioned
medium or control medium for 1 h. Then the cells were washed in
cold PBS and homogenized at 4 °C in 200 µl of PBS containing 25 mM
-glycerophosphate, 5 mM sodium
orthovanadate, 5 mM NaF, 20 µg/ml leupeptin, 20 µg/ml
aprotinin, and 5 mM phenylmethylsulfonyl fluoride. The
homogenates were centrifuged at 100,000 × g for 30 min
at 4 °C, and the supernatant was used as the cytosolic extract.
Aliquots (10 µl) of the cytosolic extract were probed with the
anti-Dvl, anti-CKI
, anti-CKI
, anti-GSK-3
, and monoclonal
anti-
-catenin antibodies.
 |
RESULTS |
Enhancement of the Interaction of Dvl-1 with Frat-1 by
CKI
--
The constructions of Frat-1 and Dvl-1 used in this study
are shown in Fig. 1. Although we showed
in a previous report that CKI
forms a complex with and
phosphorylates Dvl-1 (20), the physiological significance of the
phosphorylation of Dvl-1 by CKI
remained unclear. Because the
phosphorylation of Dvl-2 by CKII enhances the interaction of Dvl-2 with
-arrestin1 (35), we tried to identify protein(s) that associate with
Dvl-1 phosphorylated by CKI
. It has been shown that CKI
stimulates the binding of GBP to Dvl in Xenopus extracts
in vitro (24). Human Frat-1 contains three regions that are
well conserved with the corresponding regions in Xenopus GBP
(30), and it binds to Dvl-1 (21). Therefore, we examined whether CKI
enhances the binding of Dvl-1 to Frat-1. HA-Dvl-1 and Myc-Frat-1 were
expressed with GFP-CKI
or its kinase negative form, GFP-CKI
KN, in
COS cells. GFP-CKI
, but not GFP-CKI
KN, induced a mobility shift
of HA-Dvl-1 on an SDS-PAGE gel, reflecting the phosphorylation of Dvl-1
(20) (Fig. 2A,
lanes 2-4). When the lysates expressing HA-Dvl-1 and
Myc-Frat-1 were immunoprecipitated with the anti-Myc antibody, a small
amount of HA-Dvl-1 was detected in the Myc-Frat-1 immune complexes
(Fig. 2A, lane 14). Expression of GFP-CKI
, but
not GFP-CKI
KN, greatly enhanced the formation of the complex between
HA-Dvl-1 and Myc-Frat-1 (Fig. 2A, lanes 15 and
16).

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Fig. 2.
Enhancement of the interaction of Dvl-1 with
Frat-1 by CKI . A, interaction of the deletion
mutants of Dvl-1 with Frat-1 in intact cells. Lysates (20 µg of
protein) of COS cells expressing the indicated proteins were probed
with the anti-HA, anti-GFP, or anti-Myc antibody (lanes
1-13 and 26-38). The same lysates (200 µg of
protein) were immunoprecipitated with the anti-Myc antibody, and then
the immunoprecipitates were probed with the same antibodies
(lanes 14-25 and 39-50). WT, wild
type; KN, kinase negative form; IP,
immunoprecipitation; Ab, antibody. B, sequence
alignment of Dvl. The region of residues 228-250 of human Dvl-1 is
aligned to mouse Dvl-1, Xenopus dsh, and
Drosophila Dsh. C, complex formation of GSK-3 ,
Dvl-1, Frat-1, and CKI in intact cells. Lysates (20 µg of protein)
of COS cells expressing the indicated proteins were probed with the
anti-Myc, anti-GFP, anti-GSK-3 , or anti-HA antibody (lanes
1-5). The same lysates (200 µg of protein) were
immunoprecipitated with the anti-Myc antibody, and then the
immunoprecipitates were probed with the same antibodies (lanes
6-9). D, direct interaction of Frat-1 with
phosphorylated Dvl-1. After MBP-Dvl-1 (lanes 1-3) and
MBP-Dvl-1-( 228-250) (lanes 4-6) immobilized on amylose
resin were incubated with (lanes 2, 3,
5, and 6) or without (lanes 1 and
4) His6-CKI in the presence (lanes
1, 2, 4, and 5) or absence
(lanes 3 and 6) of ATP, the samples were further
incubated with GST-Frat-1. MBP fusion proteins were precipitated by
centrifugation, and the precipitates were probed with the anti-MBP and
anti-GST antibodies. MBP-Dvl-1 (lane 7),
MBP-Dvl-1-( 228-250) (lane 8), and GST-Frat-1 (lane
9) (1 µg of protein) were stained with Coomassie Brilliant Blue.
The arrowheads indicate MBP-Dvl-1 (WT),
MBP-Dvl-1-( 228-250), and GST-Frat-1, and the other bands are their
degradation products. The results shown are representative of four
independent experiments.
|
|
To clarify which region of Dvl-1 is necessary for the interaction with
Frat-1, various deletion mutants of HA-Dvl-1 were expressed with
Myc-Frat-1 and GFP-CKI
(Fig. 2A, lanes 5-13
and 27-38). Among these deletion mutants, HA-Dvl-1-(1-250)
and HA-Dvl-1-(337-670) did not form a complex with Myc-Frat-1
irrespective of the expression of GFP-CKI
(Fig. 2A,
lanes 17-19 and 48-50), but HA-Dvl-1-(1-519) and HA-Dvl-1-(140-670) associated with Myc-Frat-1 in a manner dependent on their phosphorylation by GFP-CKI
(Fig. 2A,
lanes 20-25). Therefore, the amino acid region 141-336 of
Dvl-1 may be important for the interaction with Frat-1. These results
are consistent with the previous observations that mouse
Dvl-1-(201-375) interacts with Frat-1 (21). Furthermore, we deleted
three amino acid regions, 141-227, 228-250, and 251-336 (PDZ
domain), from HA-Dvl-1. HA-Dvl-1-(
141-227) and
HA-Dvl-1-(
251-336) formed a complex with Myc-Frat-1 in a manner
dependent on CKI
, but HA-Dvl-1-(
228-250) did not (Fig.
2A, lanes 27-35 and 39-47). These
results clearly indicate that CKI
-dependent
phosphorylation of Dvl-1 enhances the interaction of Dvl-1 with Frat-1
and that amino acid region 228-250 of Dvl-1 is necessary for the
interaction with Frat-1. As Dvl-1-(1-250) does not contain the
CKI
-binding region (20), the reason for the failure of this Dvl-1
mutant to bind to Frat-1 might be that CKI
does not phosphorylate
this mutant. The amino acid region 228-250 of Dvl-1 is evolutionarily
conserved (Fig. 2B), suggesting that this region is
functionally important.
The C-terminal region of GBP binds to GSK-3 (30), and Frat-1-(186-279)
indeed interacted with GSK-3
in intact cells (data not shown). When
coexpressed with GFP-CKI
, significant amounts of HA-Frat-1 were
observed in the Myc-Dvl-1 immune complex in COS cells (Fig.
2C, lanes 2, 3, 6, and
7). Furthermore, endogenous GSK-3
was also observed in
the Myc-Dvl-1 immune complex when GFP-CKI
was coexpressed (Fig.
2C, lane 6). GFP-CKI
enhanced the formation of
the complex of Myc-Dvl-1 with the N-terminal region of Frat-1
(HA-Frat-1-(1-185)), but endogenous GSK-3
was not observed in this
complex (Fig. 2C, lanes 4, 5,
8, and 9). These results suggest that CKI
enhances the formation of the complex of GSK-3
with Dvl-1 through
Frat-1.
GST-Frat-1 bound to MBP-fused Dvl-1 (MBP-Dvl-1) when they were
incubated with His6-CKI
and ATP, although they did not
interact with each other in the absence of His6-CKI
(Fig. 2D, lanes 1 and 2). Incubation
with His6-CKI
in the absence of ATP resulted in a
detectable interaction of MBP-Dvl-1 with GST-Frat-1 (Fig. 2D, lane 3). Therefore, the binding of CKI
to
Dvl-1 may induce a conformational change of Dvl-1, resulting in the
interaction of Dvl-1 with Frat-1. GST-Frat-1 did not bind to
MBP-Dvl-1-(
228-250) irrespective of incubation with
His6-CKI
and ATP (Fig. 2D, lanes 4-6). Previously we showed that phosphorylation of GST-Dvl-1 by CKI
in vitro exhibits a mobility shift (20). Although we
do not know the reasons for the undetectable mobility shift of
MBP-Dvl-1 and MBP-Dvl-1-(
228-250), we confirmed that CKI
indeed
phosphorylates them by autoradiography (data not shown). CKI
did not
phosphorylate MBP-Dvl-1-(228-250) (data not shown). Taken together,
Frat-1 prefers Dvl-1 phosphorylated by CKI
, and the amino acid
region 228-250 of Dvl-1 is necessary for the direct binding of Dvl-1
to Frat-1.
Subcellular Localization of Dvl-1 and Frat-1--
The importance
of the amino acid region 228-250 of Dvl-1 for the binding to Frat-1
was confirmed by the immunocytochemical assay. When GFP-Frat-1 was
expressed alone in L cells, Frat-1 was distributed throughout the
cytoplasm (Fig. 3A). HA-Dvl-1
was observed as small particles, consistent with previous reports (13,
14). Coexpression with HA-Dvl-1 changed the localization of GFP-Frat-1
dramatically, and these two proteins were found to colocalize (Fig. 3,
B-D). HA-Dvl-1-(
228-250) was detected as small
particles, like HA-Dvl-1, indicating that the amino acid region
228-250 of Dvl-1 is not essential for the localization of Dvl-1.
However, HA-Dvl-1-(
228-250) did not affect the distribution of
GFP-Frat-1 (Fig. 3, E-G). These results support the
findings that the region 228-250 of Dvl-1 is important for its binding to Frat-1 in intact cells.

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Fig. 3.
Subcellular localization of Dvl-1 and
Frat-1. GFP-Frat-1 alone (A), HA-Dvl-1 and GFP-Frat-1
(B-D), or HA-Dvl-1-( 228-250) and GFP-Frat-1
(E-G) were expressed in wild-type L cells. Some of
the cell cultures were directly viewed with a confocal laser-scanning
microscope to detect GFP-Frat-1 (A, C, and
F), and the others were stained with the anti-HA antibody to
detect HA-Dvl-1 (B) or HA-Dvl-1-( 228-250)
(E). Merged images of B and C, and of
E and F are shown in D and
G, respectively. GFP-Frat-1 and Alexa 546-labeled HA-Dvl or
HA-Dvl-1-( 228-250) produced no cross staining. The results shown
are representative of three independent experiments.
|
|
Synergistic Effects by Dvl-1 and Frat-1 on
-Catenin Accumulation
and Tcf-4 Activation--
Transient overexpression of Dvl-1 and
Dvl-1-(
228-250) in L cells induced the nuclear accumulation of
-catenin (Fig. 4, A-D, arrows), indicating that the amino acid region 228-250 of
Dvl-1 is not essential for the ability of Dvl-1 to activate the Wnt signal canonical pathway. However, accumulation of
-catenin was not
observed in L cells stably expressing Dvl-1 (L/Dvl cells) or
Dvl-1-(
228-250) (L/Dvl-(
228-250) cells) (Fig. 4, F,
H, J, and L, the cells not indicated
by arrows). This finding suggests that a low expression
level of Dvl-1 or Dvl-1-(
228-250) in L cells is not sufficient for
the stabilization of
-catenin. Although expression of Frat-1 in
wild-type L cells did not induce the accumulation of
-catenin (data
not shown), its expression in L/Dvl cells increased the level of
-catenin in the nucleus (Fig. 4, E and F,
arrows). However, expression of Frat-1 in
L/Dvl-(
228-250) did not induce the nuclear accumulation of
-catenin (Fig. 4, G and H, arrows). Furthermore, expression of CKI
in L/Dvl cells, but not in
L/Dvl-(
228-250) cells, resulted in the accumulation of
-catenin
(Fig. 4, I-L, arrows). Taken together with our
previous observations (20), these results suggest that not only CKI
but also Frat-1 act synergistically with Dvl-1 to induce the
accumulation of
-catenin and that the amino acid region 228-250 of
Dvl-1 is important for the functional interaction of Dvl-1 with Frat-1
or CKI
.

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Fig. 4.
Synergistic effects of Dvl-1 and Frat-1 on
the accumulation of -catenin. HA-Dvl-1
(A and B) or HA-Dvl-1-( 228-250) (C
and D) was transiently expressed in wild-type L cells
(L/WT). GFP-Frat-1 was transiently expressed in L/Dvl cells
(E and F) or L/Dvl-( 228-250) cells
(G and H). GFP-CKI was transiently expressed
in L/Dvl cells (I and J) or L/Dvl-( 228-250)
cells (K and L). Some of the cells were directly
viewed with a confocal laser-scanning microscope to detect GFP-Frat-1
(E and G) and GFP-CKI (I and
K), and others were stained with the anti-HA antibody to
detect HA-Dvl-1 and HA-Dvl-1-( 228-250) (A and
C) or with the polyclonal anti- -catenin antibody to
observe endogenous  catenin (B, D,
F, H, J, and L).
Arrows indicate L/WT cells expressing HA-Dvl-1 (A
and B) or HA-Dvl-1-( 228-250) (C and
D), L/Dvl cells expressing GFP-Frat-1 (E and
F) or GFP-CKI (I and J), or
L/Dvl-( 228-250) cells expressing GFP-Frat-1 (G and
H) or GFP-CKI (K and L). The
results shown are representative of three independent
experiments.
|
|
We also examined the effects of the combination of Dvl-1, Frat-1, and
CKI
on the activation of Tcf-4 by the use of Top-fos-Luc as a reporter gene (33). Expression of either HA-Dvl-1 or
HA-Dvl-1-(
228-250) alone in HEK-293 cells activated Tcf-4 in a
dose-dependent manner, although HA-Dvl-1-(
228-250) was
slightly less active than HA-Dvl-1 (Fig.
5A). Frat-1 alone increased
the activity of Tcf-4 slightly (Fig. 5B). Dvl-1 promoted the
ability of Frat-1 to stimulate Tcf-4, whereas Dvl-1-(
228-250) did
not affect the ability of Frat-1 to stimulate Tcf-4 (Fig.
5B). CKI
alone increased the activity of Tcf-4 only
slightly (Fig. 5C). Although Dvl-1 greatly promoted the
ability of CKI
to stimulate Tcf-4, Dvl-1-(
228-250) did not act
synergistically with CKI
(Fig. 5C). Dvl-1, Frat-1, or
CKI
did not activate Tcf-4 for FOP-fos-Luc, in which the
Tcf-4-binding elements are mutated (data not shown). These results
suggest that Dvl-1 acts synergistically with Frat-1 or CKI
to
activate Tcf-4 and that the amino acid region 228-250 of Dvl-1 is
necessary for the synergistic activation of Tcf-4 by Dvl-1 and Frat-1
or CKI
.

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Fig. 5.
Requirement for amino acid region 228-250 of
Dvl-1 for the synergistic activation of Tcf-4 by Dvl-1 and Frat-1 or
Dvl-1 and CKI . A, the indicated amounts of
pCGN/hDvl-1 or pCGN/hDvl-1-( 228-250) were transfected into HEK-293
cells. , pCGN/hDvl-1; , pCGN/hDvl-1-( 228-250). B,
the indicated amounts of pCGN/hFrat-1 were transfected into HEK-293
cells with pCGN/hDvl-1 (0.2 µg) or pCGN/hDvl-1-( 228-250) (0.2 µg). , pCGN/hFrat-1 alone; , pCGN/hFrat-1 and pCGN/hDvl-1; ,
pCGN/hFrat-1 and pCGN/hDvl-1-( 228-250). C, the indicated
amounts of pCGN/hCKI were transfected into HEK-293 cells with
pCGN/hDvl-1 (0.2 µg) or pCGN/hDvl-1-( 228-250) (0.2 µg). ,
pCGN/hCKI alone; , pCGN/hCKI and pCGN/hDvl-1; ,
pCGN/hCKI and pCGN/hDvl-1-( 228-250). The luciferase activities
were assayed and expressed as fold increase compared with the level in
cells transfected with TOP-fos-Luc and pEF-BOS-HA/hTcf-4E
alone.
|
|
To confirm further that the amino acid region 228-250 of Dvl-1 is
important in the synergistic effects of Dvl-1 and CKI
, they were
expressed in Xenopus embryos. Ventral injection of a high
dose (1 ng) of Dvl-1 or Dvl-1-(
228-250)
mRNA into embryos resulted in dorsalization of the phenotype,
causing effects such as axis duplication, with a similar efficiency
(Fig. 6, A and B),
but ventral injection of embryos with a low dose (50 pg) of Dvl-1 or Dvl-1-(
228-250) mRNA did not
cause significant abnormalities (Fig. 6B). Coinjection of
low doses of Dvl-1 and CKI
mRNA markedly induced axis duplication (Fig. 6, A and B).
However, coinjection of low doses of Dvl-1-(
228-250) and
CKI
mRNA did not result in significant axis
duplication (Fig. 6, A and B). These results demonstrate that the amino acid region 228-250 of Dvl-1 is necessary for the synergistic effects of CKI
and Dvl-1 on axis formation in
Xenopus embryos, consistent with the results observed in
mammalian cells.

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Fig. 6.
Requirement of amino acid region 228-250 of
Dvl-1 for the synergistic effects of Dvl-1 and CKI
on axis duplication in Xenopus embryos.
A, embryos were injected ventrally with the mRNAs of
CKI and Dvl-1 or
Dvl-1-( 228-250). a, Dvl-1 mRNA
(1 ng); b, Dvl-1-( 228-250) mRNA (1 ng);
c, CKI mRNA (50 pg); d,
CKI mRNA (50 pg) and Dvl-1 mRNA (50 pg); e, CKI mRNA (50 pg) and
Dvl-1-( 228-250) mRNA (50 pg). WT, wild
type; 228-250, Dvl-1-( 228-250). B, the
results in A were expressed as the percentage of secondary
axis formation. Black bars indicate complete axis
duplication, including eyes and cement glands. White bars
indicate incomplete axis duplication characterized by a lack of head
structures but with a distinct branched axis.
|
|
Functional Relationships among Dvl-1, Frat-1, and CKI
--
In
Figs. 4-6 we found that Dvl-1 and Frat-1 or Dvl-1 and CKI
activate
the Wnt signaling pathway synergistically. CKI
also enhanced the
ability of Frat-1 to activate Tcf-4 in HEK-293 cells (Fig.
7A, lanes 3,
5, and 7). Therefore, we next examined the functional relationships among these three proteins, Dvl-1, Frat-1, and
CKI
. Frat-1-(1-185) inhibited the Dvl-1-and
CKI
-dependent activation of Tcf-4, suggesting that
Frat-1-(1-185) functions as a dominant negative form (Fig.
7A, lanes 8 and 9). Although CKI
enhanced the synergistic effects of Dvl-1 and Frat-1 on the activation
of Tcf-4, CKI
KN suppressed them (Fig. 7A, lanes
10, 11, and 12).

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Fig. 7.
Functional relationships among Dvl-1, Frat-1,
and CKI . A, dominant negative
effects of Frat-1-(1-185) and CKI KN on the activation of Tcf-4.
HEK-293 cells were transfected with pCGN/hDvl-1, pCGN/hFrat-1,
pCGN/hFrat-1-(1-185), pCGN/hCKI , or pCGN/hCKI (KN) (0.2 µg
each) as indicated. The luciferase activities were assayed and
expressed as fold increase compared with the level in cells transfected
with pTOPFLASH and pEF-BOS-HA/hTcf-4E alone (lane 1).
B, inhibition of Dvl-1- and Wnt-3a-dependent
Tcf-4 activation by Frat-1-(1-185). HEK-293 cells were transfected
with pCGN/hDvl-1, pPGK/Wnt-3a, pCGN/hFrat-1, or pCGN/hFrat-1-(1-185)
(0.2 µg each) as indicated. The luciferase activities were assayed
and expressed as fold increase compared with the level observed in
cells transfected with TOP-fos-Luc and
pEF-BOS-HA/hTcf-4E alone (lane 1). The results represent the
mean ± S.E. of four independent experiments.
|
|
Wnt-3a and Dvl-1 synergistically activated Tcf-4 (Fig. 7B,
lanes 2, 3, and 6), and Frat-1
enhanced the synergistic effect of Wnt-3a and Dvl-1, and
Frat-1-(1-185) inhibited it (Fig. 7B, lanes
6-8). These results support the idea that the binding of Dvl-1
and Frat-1 is important for the efficient activation of the Wnt
signaling pathway and that CKI
modulates their binding.
Inhibition of the Complex Formation between Dvl-1 and Axin by
Frat-1--
We examined whether the binding of Dvl-1 and Frat-1
affects the Axin complex in the presence of CKI
. Myc-Dvl-1 and
Myc-Dvl-1-(
228-250) formed a complex with FLAG-rAxin with similar
efficiencies (Fig. 8, lanes 12 and 13). When HA-Frat-1 was further expressed, the amount of
FLAG-rAxin immunoprecipitated with Myc-Dvl-1 was reduced, whereas that
with Myc-Dvl-1-(
228-250) was unaffected (Fig. 8, lanes
10 and 11). The level of GSK-3
in the immune
complexes seemed unchanged (Fig. 8, lanes 10 and
11). These results demonstrate that
CKI
-dependent binding of Dvl-1 and Frat-1 inhibits the
complex formation of Dvl-1 with Axin and suggest that it promotes the disintegration of the
-catenin destruction complex.

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Fig. 8.
Inhibition of the complex formation between
Dvl-1 and Axin by Frat-1. Lysates (20 µg of protein) of COS
cells expressing the indicated proteins were probed with the anti-FLAG,
anti-Myc, anti-GFP, anti-GSK-3 , or anti-HA antibody (lanes
1-7). The same lysates (200 µg of protein) were
immunoprecipitated with the anti-Myc antibody, and then the
immunoprecipitates were probed with the same antibodies (lanes
8-13). The results shown are representative of three independent
experiments.
|
|
Inhibition of the Wnt Signaling Pathway by Depletion of CKI
Expression--
It has been shown that CKI
is essential for
-catenin degradation in mammalian cells (26). Therefore, we depleted
the endogenous CKI
in HeLa S3 cells via ds RNAi (36) to ask whether
CKI
is involved in the Wnt-dependent accumulation of
-catenin. A ds RNAi oligo for CKI
reduced the protein level of
CKI
but not that of CKI
, and, conversely, a ds RNAi oligo for
CKI
reduced the protein level of CKI
but not that of CKI
(Fig.
9A, third and
fourth panels from the top). Single-stranded
sense oligos for CKI
and CKI
did not affect the protein levels of
CKI
or CKI
(data not shown). These oligos did not alter the
protein level of GSK-3
(Fig. 9A, bottom
panel).

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Fig. 9.
Inhibition of the Wnt signaling pathway by
depletion of CKI expression.
A, inhibition of Wnt-3a-induced accumulation of -catenin
by depletion of CKI expression. HeLa S3 cells were transfected with
a ds RNAi oligo specific for human CKI (lanes 2 and
5) or a ds RNAi oligo specific for human CKI (lanes
3 and 6). Untransfected cells were used as a control
(lanes 1 and 4). The cells were treated with
Wnt-3a-conditioned medium (+) (lanes 4-6) or control medium
( ) (lanes 1-3) for 1 h. Aliquots (10 µl) of the
cytosolic extract were probed with the anti-Dvl-1, anti-CKI ,
anti-CKI , anti-GSK-3 , or monoclonal anti- -catenin antibody.
, CKI ds RNAi; , CKI ds RNAi. B, inhibition of
Wnt-3a-dependent phosphorylation of Dvl by CKI-7. HeLa S3
cells were treated with Wnt-3a-conditioned medium (lanes 2 and 3) or control medium (lane 1) in the presence
(lane 3) or absence (lanes 1 and 2) of
100 µM CKI-7. Aliquots (20 µl) of the cytosolic extract
were probed with the anti-Dvl antibodies. C, interference of
the interaction of Dvl-1 with Frat-1 by depletion of CKI expression.
HeLa S3 cells were transfected with a single-stranded sense oligo
(a-e), a ds RNAi oligo specific for human CKI
(f-h), or a ds RNAi oligo specific for human CKI
(i-k), and 3 days later the cells were further transfected
with pCGN/hDvl-1 (a), or pEGFP-C1/hFrat-1 (b), or
pCGN/hDvl-1 and pEGFP-C1/hFrat-1 (c-k). GFP-Frat-1 was
directly visualized with a confocal laser-scanning microscope (b,
d, g, and j), and HA-Dvl-1 was stained with the anti-HA
antibody (a, c, f, and i). Merged images of
c and d, f and g, and
i and j are shown in e, h,
and k, respectively. The results shown are
representative of three independent experiments.
|
|
A decrease of CKI
, but not CKI
, inhibited the
Wnt-3a-dependent mobility shift of endogenous Dvl (Fig.
9A, second panel from the top). The
Wnt-3a-dependent mobility shift of Dvl-1 was inhibited by
treatment with CKI-7, a CKI inhibitor (18, 37) (Fig. 9B), or
with alkaline phosphatase (data not shown). Therefore, the slowly
migrating Dvl band appears to be a phosphorylated form of Dvl. These
results indicate that CKI
is necessary for the Wnt-3a-dependent phosphorylation of Dvl-1.
When HA-Dvl-1 or GFP-Frat-1 was expressed alone in HeLa S3 cells,
HA-Dvl-1 was observed as small particles, and GFP-Frat-1 was diffusely
distributed in the cytoplasm (Fig. 9C, a and
b) as well as in L cells (see Fig. 3). When HA-Dvl-1 and
GFP-Frat-1 were coexpressed in HeLa S3 cells, the two proteins were
colocalized with each other (Fig. 9C, c-e).
However, HA-Dvl-1 did not change the subcellular distribution of
GFP-Frat-1 in the cells when the expression of CKI
was depleted via
ds RNAi (Fig. 9C, f-h). Depletion of CKI
did
not affect colocalization of HA-Dvl-1 and GFP-Frat-1 (Fig.
9C, i-k). These results are in agreement with
the observations that CKI
enhances the binding of Dvl-1 and
Frat-1.
Furthermore, CKI
ds RNAi did not affect the protein level of
-catenin in the absence of Wnt-3a but did inhibit Wnt-3a-induced
-catenin accumulation significantly (Fig. 9A, first
panel). A decrease of CKI
led to an accumulation of
-catenin
in the absence of Wnt-3a, consistent with the previously reported
observations (26), but did not affect Wnt-3a-induced
-catenin
accumulation (Fig. 9A, first panel). These
results clearly show that CKI
, but not CKI
, is essential for the
Wnt-3a-induced accumulation of
-catenin in HeLa S3 cells.
 |
DISCUSSION |
Complex Formation between Dvl-1 and Frat-1 and Its Enhancement by
CKI
--
In this study, we demonstrated that CKI
enhances the
interaction of Dvl-1 with Frat-1 in intact mammalian cells. The amino acid region 228-250 of Dvl-1 was necessary for the binding of Dvl-1 to
Frat-1. This region is conserved evolutionarily, supporting the idea
that it is important for the functions of Dvl. Although there is GBP, a
Frat homolog, in Xenopus, no gene similar to Frat/GBP has
been found in Drosophila. Therefore, a different protein may act on this region in Drosophila. Although multiple sites of
Dvl-1 were phosphorylated by CKI
(20), the amino acid region
228-250 was not phosphorylated. Because 17% of the 670 amino acids in human Dvl-1 are serines and threonines, it is difficult to precisely map the phosphorylation sites. Although we have not yet identified the
sites of phosphorylation of Dvl-1 by CKI
, phosphorylation may result
in conformational changes of Dvl-1, rendering the region, including
amino acids 228-250, able to associate with Frat-1. Mutations of each
of the putative phosphorylation sites by CKII in Drosophila
Dsh do not affect the ability of the mutant proteins to rescue
dsh mutant animals (38). Therefore, studies of compound phosphorylation mutants may be required to identify the most important phosphorylation sites.
It has been shown that Wnt induces the phosphorylation of Dvl (39). Our
results using ds RNAi for CKI
have shown that CKI
is required for
the Wnt-3a-induced phosphorylation of Dvl-1 in HeLa S3 cells and that
CKI
is necessary for the interaction of Dvl-1 with Frat-1. Depletion
of CKI
did not affect the Wnt-3a-induced phosphorylation of Dvl-1
and the interaction of Dvl-1 with Frat-1. Taken together, these
findings suggest that, when Wnt binds to the Frizzled/LRP co-receptor,
Dvl is phosphorylated by CKI
but not by CKI
, resulting in
enhancement of the binding of Dvl-1 and Frat-1.
Molecular Mechanism by Which Complex Formation between Dvl-1 and
Frat-1 Activates the Wnt Signaling Pathway--
We demonstrated that
Dvl-1-(
228-250) does not act synergistically with either Frat-1 or
CKI
to activate the Wnt signaling pathway. Frat-1-(1-185) inhibited
the synergistic activation of Tcf-4 by Dvl-1 and CKI
or by Dvl-1 and
Wnt-3a. Because Frat-1-(1-185) binds not to GSK-3
but to Dvl-1,
these results support the idea that the binding of Dvl-1 and Frat-1
induces the accumulation of
-catenin and the activation of Tcf-4.
The stability of
-catenin is regulated in a destruction complex that
includes
-catenin, Axin, APC, GSK-3
, Dvl-1, CKI
, and PP2A (4,
21, 26, 40, 41). It has been demonstrated that the expression of Wnt-1
in COS cells promotes the disintegration of this complex, resulting in
the dissociation of Frat from the Dvl and Axin complex (21) and that
the expression of CKI
in HEK-293 cells decreases the association
between PP2A and Axin (25). Therefore, the Wnt signal appears to
destabilize the
-catenin degradation complex. Consistent with these
observations, we showed that the binding of Dvl-1 and Frat-1 in COS
cells decreases the interaction of Dvl-1 with Axin.
How the Wnt signal stabilizes
-catenin by causing the disintegration
of its degradation complex remains unclear.
CKI
-dependent interaction of Dvl-1 with Frat-1 may cause
a conformational change of the complex that phosphorylates
-catenin.
Dvl-1 forms a complex with GSK-3
via Axin or Frat-1. The level of
GSK-3
associated with Dvl-1 remained the same in the presence or
absence of Axin when Frat-1 was present in the complex. Therefore, it
is likely that GSK-3
maintains its association with Dvl-1 after the
disintegration of the complex. Because Wnt reduces the phosphorylation
of
-catenin by GSK-3
(26), one possibility is that a
conformational change of the degradation complex leads to the
dissociation of GSK-3
from Axin and recruits it to Frat-1, thereby
reducing the phosphorylation of
-catenin by GSK-3
.
Involvement of CKI
as a Positive Regulator in the Wnt Signaling
Pathway--
Several reports have shown that CKI binds to Dvl and
stabilizes
-catenin (18-20,42), whereas other reports have
demonstrated that CKI phosphorylates
-catenin and promotes its
degradation (26-28). One means by which CKI could play dual positive
and negative roles in the Wnt signaling pathway might be that some CKI
isoforms perform positive functions while others perform negative
functions. Indeed, it has been shown that depletion by ds RNAi of the
expression of CKI
, but not of CKI
, stabilizes the basal level of
-catenin in HEK-293 cells (26). The expression of CKI
, but not of
CKI
, induces axis duplication in Xenopus embryos (19).
However, arguing against these observations, it has shown that
depletion of either CKI
or CKI
stabilizes Armadillo in
Drosophila cells (27) and that expression of either CKI
or CKI
induces axis duplication in Xenopus embryos (42).
Furthermore, it has also been demonstrated that Diversin recruits
CKI
to the
-catenin destruction complex, thereby inducing the
down-regulation of
-catenin (43). Therefore, we performed a loss of
function study using the ds RNAi methods in mammalian cells to clarify
the roles of CKI
and CKI
in the Wnt-dependent
accumulation of
-catenin.
Our results support the hypothesis of CKI-isoform-specific differences.
Depletion of CKI
expression by ds RNAi in HeLa S3 cells did not
affect the basal level of
-catenin but reduced Wnt-3a-induced
accumulation of
-catenin. Wnt-3a-dependent
phosphorylation of Dvl-1 and the interaction of Dvl-1 with Frat-1 were
reduced in the cells in which CKI
was depleted. Furthermore,
reduction of CKI
increased the basal level of
-catenin but did
not affect Wnt-3a-induced accumulation of
-catenin. These results
strongly suggest that CKI
and CKI
have opposite actions on the
Wnt signaling pathway in HeLa S3 cells. Of course, we can not rule out
the possibility that the positive and negative roles of CKI
in the
Wnt signaling pathway may be dependent on the abundance of various
substrates and interacting proteins. Further studies will be necessary
to clarify the dual roles of CKI
in the Wnt signaling pathway.
 |
ACKNOWLEDGEMENTS |
We are grateful to Drs. D. M. Virshup,
H. Clevers, S. Tanaka, K. Miyazono, S. Takada, Y. Matsuura, and K. Matsumoto for donating plasmids,
viruses, and cells.
 |
FOOTNOTES |
*
This work was supported by Grants-in-Aid for Scientific
Research and for Scientific Research on priority areas from the
Ministry of Education, Science, and Culture, Japan (2001, 2002).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@hiroshima-u.ac.jp.
Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M213265200
 |
ABBREVIATIONS |
The abbreviations used are:
GSK-3
, glycogen
synthase kinase-3
;
GBP, GSK-3-binding protein;
APC, adenomatous
polyposis coli gene product;
LRP, low density lipoprotein-related
protein;
Tcf, T-cell factor;
Dsh, Dishevelled;
CKI, casein kinase I;
HEK-293, human embryonic kidney;
ds RNAi, double-stranded RNA
interference;
KN, kinase negative;
GST, glutathione
S-transferase;
MBP, maltose-binding protein;
PBS, phosphate-buffered saline;
HA, hemagglutinin A;
GFP, green fluorescent
protein;
PP2A, protein phosphatase 2A.
 |
REFERENCES |
1.
|
Wodarz, A.,
and Nusse, R.
(1998)
Annu. Rev. Cell Dev. Biol.
14,
59-88[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Miller, J. R.,
Hocking, A. M.,
Brown, J. D.,
and Moon, R. T.
(1999)
Oncogene
18,
7860-7872[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Seidensticker, M. J.,
and Behrens, J.
(2000)
Biochim. Biophys. Acta
1495,
168-182[Medline]
[Order article via Infotrieve]
|
4.
|
Kikuchi, A.
(1999)
Cell. Signal.
11,
777-788[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Ikeda, S.,
Kishida, S.,
Yamamoto, H.,
Murai, H.,
Koyama, S.,
and Kikuchi, A.
(1998)
EMBO J.
17,
1371-1384[Abstract/Free Full Text]
|
6.
|
Yamamoto, H.,
Kishida, S.,
Uochi, T.,
Ikeda, S.,
Koyama, S.,
Asashima, M.,
and Kikuchi, A.
(1998)
Mol. Cell. Biol.
18,
2867-2875[Abstract/Free Full Text]
|
7.
|
Kishida, S.,
Yamamoto, H.,
Ikeda, S.,
Kishida, M.,
Sakamoto, I.,
Koyama, S.,
and Kikuchi, A.
(1998)
J. Biol. Chem.
273,
10823-10826[Abstract/Free Full Text]
|
8.
|
Behrens, J.,
Jerchow, B.-A.,
Würtele, M.,
Grimm, J.,
Asbrand, C.,
Wirtz, R.,
Kühl, M.,
Wedlich, D.,
and Birchmeier, W.
(1998)
Science
280,
596-599[Abstract/Free Full Text]
|
9.
|
Kitagawa, M.,
Hatakeyama, S.,
Shirane, M.,
Matsumoto, M.,
Ishida, N.,
Hattori, K.,
Nakamichi, I.,
Kikuchi, A.,
Nakayama, K.-I.,
and Nakayama, K.
(1999)
EMBO J.
18,
2401-2410[Abstract/Free Full Text]
|
10.
|
Hart, M.,
Concordet, J.-P.,
Lassot, I.,
Albert, I.,
de los Santos, R.,
Durand, H.,
Perret, C.,
Rubinfeld, B.,
Margottin, F.,
Benarous, R.,
and Polakis, P.
(1999)
Curr. Biol.
9,
207-210[CrossRef][Medline]
[Order article via Infotrieve]
|
11.
|
Aberle, H.,
Bauer, A.,
Stappert, J.,
Kispert, A.,
and Kemler, R.
(1997)
EMBO J.
16,
3797-3804[Abstract/Free Full Text]
|
12.
|
Bienz, M.,
and Clevers, H.
(2000)
Cell
103,
311-320[Medline]
[Order article via Infotrieve]
|
13.
|
Smalley, M. J.,
Sara, E.,
Paterson, H.,
Naylor, S.,
Cook, D.,
Jayatilake, H.,
Fryer, L. G.,
Hutchinson, L.,
Fry, M. J.,
and Dale, T. C.
(1999)
EMBO J.
18,
2823-2835[Abstract/Free Full Text]
|
14.
|
Kishida, S.,
Yamamoto, H.,
Hino, S.-I.,
Ikeda, S.,
Kishida, M.,
and Kikuchi, A.
(1999)
Mol. Cell. Biol.
19,
4414-4422[Abstract/Free Full Text]
|
15.
|
Fagotto, F.,
Jho, E.-H.,
Zeng, L.,
Kurth, T.,
Joos, T.,
Kaufmann, C.,
and Costantini, F.
(1999)
J. Cell Biol.
145,
741-756[Abstract/Free Full Text]
|
16.
|
Yamamoto, H.,
Kishida, S.,
Kishida, M.,
Ikeda, S.,
Takada, S.,
and Kikuchi, A.
(1999)
J. Biol. Chem.
274,
10681-10684[Abstract/Free Full Text]
|
17.
|
Kadoya, T.,
Kishida, S.,
Fukui, A.,
Hinoi, T.,
Michiue, T.,
Asashima, M.,
and Kikuchi, A.
(2000)
J. Biol. Chem.
275,
37030-37037[Abstract/Free Full Text]
|
18.
|
Peters, J. M.,
McKay, R. M.,
McKay, J. P.,
and Graff, J. M.
(1999)
Nature
401,
345-350[CrossRef][Medline]
[Order article via Infotrieve]
|
19.
|
Sakanaka, C.,
Leong, P.,
Xu, L.,
Harrison, S. D.,
and Williams, L. T.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
12548-12552[Abstract/Free Full Text]
|
20.
|
Kishida, M.,
Hino, S.-I.,
Michiue, T.,
Yamamoto, H.,
Kishida, S.,
Fukui, A.,
Asashima, M.,
and Kikuchi, A.
(2001)
J. Biol. Chem.
276,
33147-33155[Abstract/Free Full Text]
|
21.
|
Li, L.,
Yuan, H.,
Weaver, C. D.,
Mao, J.,
Farr III, G. H.,
Sussman, D. J.,
Jonkers, J.,
Kimelman, D.,
and Wu, D.
(1999)
EMBO J.
18,
4233-4240[Abstract/Free Full Text]
|
22.
|
Gross, S. D.,
and Anderson, R. A.
(1998)
Cell. Signal.
10,
699-711[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Kloss, B.,
Price, J. L.,
Saez, L.,
Blau, J.,
Rothenfluh, A.,
Wesley, C. S.,
and Young, M. W.
(1998)
Cell
94,
97-107[Medline]
[Order article via Infotrieve]
|
24.
|
Lee, E.,
Salic, A.,
and Kirschner, M. W.
(2001)
J. Cell Biol.
154,
983-993[Abstract/Free Full Text]
|
25.
|
Gao, Z.-H.,
Seeling, J. M.,
Hill, V.,
Yochum, A.,
and Virshup, D. M.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
1182-1187[Abstract/Free Full Text]
|
26.
|
Liu, C.,
Li, Y.,
Semenov, M.,
Han, C.,
Baeg, G.-H.,
Tan, Y.,
Zhang, Z.,
Lin, X.,
and He, X.
(2002)
Cell
108,
837-847[Medline]
[Order article via Infotrieve]
|
27.
|
Yanagawa, S.-I.,
Matsuda, Y.,
Lee, J.-S.,
Matsubayashi, H.,
Sese, S.,
Kadowaki, T.,
and Ishimoto, A.
(2002)
EMBO J.
21,
1733-1742[Abstract/Free Full Text]
|
28.
|
Amit, S.,
Hatzubai, A.,
Birman, Y.,
Andersen, J. S.,
Ben-Shushan, E.,
Mann, M.,
Ben-Neriah, Y.,
and Alkalay, I.
(2002)
Genes Dev.
16,
1066-1076[Abstract/Free Full Text]
|
29.
|
Jonkers, J.,
Korswagen, H. C.,
Acton, D.,
Breuer, M.,
and Berns, A.
(1997)
EMBO J.
16,
441-450[Abstract/Free Full Text]
|
30.
|
Yost, C.,
Farr, G. H., III.,
Pierce, S. B.,
Ferkey, D. M.,
Chen, M. M.,
and Kimelman, D.
(1998)
Cell
93,
1031-1041[Medline]
[Order article via Infotrieve]
|
31.
|
Kishida, M.,
Koyama, S.,
Kishida, S.,
Matsubara, K.,
Nakashima, S.,
Higano, K.,
Takada, R.,
Takada, S.,
and Kikuchi, A.
(1999)
Oncogene
18,
979-985[CrossRef][Medline]
[Order article via Infotrieve]
|
32.
|
Shibamoto, S.,
Higano, K.,
Takada, R.,
Ito, F.,
Takeichi, M.,
and Takada, S.
(1998)
Genes Cells
3,
659-670[Abstract/Free Full Text]
|
33.
|
Korinek, V.,
Barker, N.,
Morin, P. J.,
van Wichen, D.,
de Weger, R.,
Kinzler, K. W.,
Vogelstein, B.,
and Clevers, H.
(1997)
Science
275,
1784-1787[Abstract/Free Full Text]
|
34.
|
Kreig, P. A.,
and Melton, D. A.
(1984)
Nucleic Acids Res.
12,
7057-7070[Abstract]
|
35.
|
Chen, W.,
Hu, L. A.,
Semenov, M. V.,
Yanagawa, S.,
Kikuchi, A.,
Lefkowitz, R. J.,
and Miller, W. E.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
14889-14894[Abstract/Free Full Text]
|
36.
|
Elbashir, S. M.,
Harborth, J.,
Lendeckel, W.,
Yalcin, A.,
Weber, K.,
and Tuschl, T.
(2001)
Nature
411,
494-498[CrossRef][Medline]
[Order article via Infotrieve]
|
37.
|
Chijiwa, T.,
Hagiwara, M.,
and Hidaka, H.
(1989)
J. Biol. Chem.
264,
4924-4927[Abstract/Free Full Text]
|
38.
|
Penton, A.,
Wodarz, A.,
and Nusse, R.
(2002)
Genetics
161,
747-762[Abstract/Free Full Text]
|
39.
|
Lee, J.-S.,
Ishimoto, A.,
and Yanagawa, S.-I.
(1999)
J. Biol. Chem.
274,
21464-21470[Abstract/Free Full Text]
|
40.
|
Li, X.,
Yost, H. J.,
Virshup, D. M.,
and Seeling, J. M.
(2001)
EMBO J.
20,
4122-4131[Abstract/Free Full Text]
|
41.
|
Yamamoto, H.,
Hinoi, T.,
Michiue, T.,
Fukui, A.,
Usui, H.,
Janssens, V.,
Van Hoof, C.,
Goris, J.,
Asashima, M.,
and Kikuchi, A.
(2001)
J. Biol. Chem.
276,
26875-26882[Abstract/Free Full Text]
|
42.
|
McKay, R. M.,
Peters, J. M.,
and Graff, J. M.
(2001)
Dev. Biol.
235,
388-396[CrossRef][Medline]
[Order article via Infotrieve]
|
43.
|
Schwarz-Romond, T.,
Asbrand, C.,
Bakkers, J.,
Kühl, M.,
Schaeffer, H.-J.,
Huelsken, J.,
Behrens, J.,
Hammerschmidt, M.,
and Birchmeier, W.
(2002)
Genes Dev.
16,
2073-2084[Abstract/Free Full Text]
|
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