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Molecular and Cellular Biology, January 2001, p. 330-342, Vol. 21, No. 1
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.1.330-342.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Inhibition of the Wnt Signaling Pathway by Idax, a
Novel Dvl-Binding Protein
Shin-ichiro
Hino,1
Shosei
Kishida,1,2
Tatsuo
Michiue,3
Akimasa
Fukui,4
Ikuo
Sakamoto,1
Shinji
Takada,5,6
Makoto
Asashima,3,4 and
Akira
Kikuchi1,*
Department of Biochemistry, Hiroshima
University School of Medicine, 1-2-3, Kasumi, Minami-ku, Hiroshima
734-8551,1 PRESTO, Japan Science and
Technology Corporation, Hiroshima,2
Crest Project3 and Department of
Life Science (Biology),4 University of Tokyo,
Meguro-ku, Tokyo 153-8902, and Center for Molecular and
Developmental Biology, Faculty of Science, Kyoto University,
Kitashirakawa, Sakyo-ku, Kyoto 606-8502,5
and Kondoh Differentiation Signaling Project,
ERATO, Japan Science and Technology Corporation,
Kyoto,6 Japan
Received 5 June 2000/Returned for modification 6 July 2000/Accepted 12 October 2000
 |
ABSTRACT |
In attempting to clarify the roles of Dvl in the Wnt signaling
pathway, we identified a novel protein which binds to the PDZ domain of
Dvl and named it Idax (for inhibition of the Dvl and Axin complex).
Idax and Axin competed with each other for the binding to Dvl.
Immunocytochemical analyses showed that Idax was localized to the same
place as Dvl in cells and that expression of Axin inhibited the
colocalization of Dvl and Idax. Further, Wnt-induced accumulation of
-catenin and activation of T-cell factor in mammalian cells were
suppressed by expression of Idax. Expression of Idax in
Xenopus embryos induced ventralization with a reduction in
the expression of siamois, a Wnt-inducible gene. Idax
inhibited Wnt- and Dvl- but not
-catenin-induced axis duplication. It is known that Dvl is a positive regulator in the Wnt signaling pathway and that the PDZ domain is important for this activity. Therefore, these results suggest that Idax functions as a negative regulator of the Wnt signaling pathway by directly binding to the PDZ
domain of Dvl.
 |
INTRODUCTION |
Wnt proteins constitute a large
family of cysteine-rich secreted ligands that control development in
organisms ranging from nematode worms to mammals (69). In
vertebrates, the Wnt signaling pathway regulates axis formation, organ
development, and cellular proliferation, morphology, motility, and fate
(7, 9, 18, 42). In the current model, the serine/threonine
kinase glycogen synthase kinase 3
(GSK-3
) targets cytoplasmic
-catenin for degradation in the Axin complex in the absence of Wnt
(20, 75). As a result, cytoplasmic
-catenin levels are
low. Axin has been shown to be important for the degradation of
-catenin (25). It forms a complex with GSK-3
,
-catenin, and adenomatous polyposis coli protein (APC) (2, 13,
20, 22, 28, 52, 71) and promotes GSK-3
-dependent
phosphorylation of
-catenin and APC (13, 19, 20, 71).
Phosphorylated
-catenin forms a complex with Fbw1 (also known as
TrCP or FWD1), a member of the F-box protein family, resulting in
the degradation of
-catenin by the ubiquitin and proteasome pathways
(12, 29). Indeed, Axin inhibits Wnt-dependent
-catenin accumulation and T-cell factor (Tcf) activation
(26, 52). Thus, Axin is a negative regulator of the Wnt
signaling pathway. In addition, Axin is phosphorylated by GSK-3
and
this phosphorylation stabilizes Axin, in contrast to that of
-catenin (70). When Wnt acts on its cell-surface receptor Frizzled, Dvl, a cytoplasmic protein, antagonizes the action of GSK-3
. 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.
Accumulated
-catenin is translocated into the nucleus where it
binds to Tcf (also known as lymphocyte enhancer binding factor
[Lef]), a transcription factor (3, 17, 43), and
stimulates the expression of genes including c-myc,
fra, jun, cyclin D1, and
peroxisome proliferator-activated receptor
(PPAR
) (14, 15, 41, 55, 64).
Three Dvl genes, Dvl-1, -2, and -3,
have been isolated in mammals (31, 48, 61). Expression of
Dvl in cells induces the accumulation of
-catenin and the activation
of Tcf (27, 35, 56). Dvl homologs are conserved in
Drosophila melanogaster (Dishevelled [Dsh]) and
Xenopus laevis (Xenopus dishevelled [Xdsh])
(30, 57, 65). Dsh mediates Wg signaling during
embryogenesis and adult fly development, which in turn determines the
ultimate cell fate in Drosophila (30, 65, 69).
Genetic evidence shows that Dsh acts upstream of shaggy, a GSK-3
homolog, and antagonizes its functions. Expression of Dsh in the
Drosophila imaginal disc cell line clone 8 causes the
accumulation of Armadillo, a
-catenin homolog (72,
73). Expression of Xdsh induces a secondary body axis in
Xenopus embryos, which is similar to the phenotype observed with expression of Wnt and
-catenin (44, 57). Thus, it
is likely that Dvl and Dsh act as positive regulators of the Wnt signaling pathway. In addition to Wg signaling, Dsh mediates the planar
tissue polarity signaling to determine cell polarity by activating Jun
N-terminal kinase (JNK) through the small GTP-binding protein RhoA
(4, 60). Dvl also plays a role in activating JNK in
mammals (38, 45). Although the ligand that is responsible for Drosophila planar tissue polarity signaling has not been
identified, Wnt-11 was shown to stimulate a vertebrate planar tissue
polarity-like pathway (16, 62). Thus, Dvl and Dsh appear
to regulate two distinct downstream pathways.
All Dsh and Dvl family members contain three highly conserved domains
(7, 9, 42): an N-terminal DIX domain which is also found
in the C terminus of Axin; a central PDZ domain which has been shown to
be a protein-protein interaction surface in several proteins; and a DEP
domain which is conserved in proteins that regulate GTP-binding
proteins. The high conservation of these three domains of Dsh and Dvl
reflects their conserved properties. Indeed, several studies
demonstrated the functional significance of the DIX and DEP
domains. The DEP domain of Dsh has been found to be critical for rescue
of the Drosophila Dsh planar polarity defect and for the
activation of JNK but not essential for the Wg pathway (4,
60). The DEP domain of vertebrate Dvl is necessary for JNK
activation in mammals but not for axis formation (38, 45).
The DIX domain is important for axis formation and the Wg and Wnt
signaling pathways (1, 45, 49). In contrast, the findings
concerning the PDZ domain are variable. For example, it was shown
previously that disruption of the PDZ domains of Dsh and Dvl abolishes
their activity in the Wg and Wnt signaling pathways and in the
Xenopus secondary axis formation, suggesting that the PDZ
domain is essential for the Wnt signaling pathway (57,
73). However, it has been recently reported that the PDZ domain
is dispensable for secondary axis formation in Xenopus embryos and that it may be important for maintaining the active conformation of Dvl (49). The inconsistency between these
results could be due to a difference in the degree of deletion in the PDZ domain. Therefore, the roles of the PDZ domain of Dvl in the Wnt
signaling pathway are not clear.
Dvl antagonizes the ability of Axin to induce ventralization in
Xenopus embryos (76). Consistent with these
results, Dvl interacts with Axin (10, 27, 56) and inhibits
GSK-3
-dependent phosphorylation of
-catenin, APC, and Axin in the
Axin complex (27, 70). In addition, it has been shown that
Dvl forms a complex with the Axin-related protein (XARP) in
Xenopus embryos and that the expression of Dvl induces the
displacement of GSK-3 from its complex with XARP (21).
Therefore, Dvl may also induce the dissociation of GSK-3
from Axin.
The DIX and PDZ domains are important for the complex formation between
Dvl and Axin (10, 27, 56). However, how the interaction of
Dvl with Axin is regulated is not known. In our efforts to clarify
further the roles of Dvl in the Wnt signaling pathway, we isolated a
novel protein which binds to the PDZ domain of Dvl by the yeast
two-hybrid screening. We designated this protein Idax (for inhibition
of the Dvl and Axin complex). We show here that Idax inhibits the Wnt-dependent accumulation of
-catenin and activation of Tcf in
mammalian cells. Moreover, we demonstrate that Idax suppresses Wnt- and Dvl-induced but not
-catenin-induced axis
duplication in Xenopus embryos. These results suggest
that Idax acts as a negative regulator of the Wnt signaling pathway by
interacting with Dvl.
 |
MATERIALS AND METHODS |
Materials and chemicals.
pTOPFLASH, pFOPFLASH, and
pUC/EF-1
/
-cateninSA and XBC40 (X
-catenin
expression vector for Xenopus injection) were provided by H. Clevers (University Hospital, Utrecht, The Netherlands), A. Nagafuchi
(Kyoto University, Kyoto, Japan), and D. Kimelman (University of
Washington, Seattle, Wash.), respectively. The cDNA of human Dvl-1, and
the anti-glutathione S-transferase (anti-GST) and the
anti-maltose binding protein (anti-MBP) antibodies were provided by B. Dallapiccola (Vergata University, Rome, Italy) (48) and M. Nakata (Sumitomo Electronics, Yokohama, Japan), respectively. The MBP-
and GST-fused proteins were purified from Escherichia coli
according to the manufacturer's instructions. The anti-Axin and the
anti-Dvl antibodies were prepared in rabbits by immunization with
recombinant proteins of rAxin-(89-216) and Dvl-1-(1-140), respectively
(50, 70). The anti-Myc antibody was prepared from 9E10
cells. L cells stably expressing Idax were generated by selecting with
G418 as described previously (26). Wnt-3a conditioned
medium was generated as already described (54). The
anti-
-catenin antibodies were purchased from Transduction Laboratories (Lexington, Ky.). [
-32P]dCTP and
Cy5-labeled anti-mouse immunoglobulin G (IgG) were obtained from
Amersham Pharmacia Biotech Ltd. (Little Chalfont, Buckinghamshire,
United Kingdom). The Alexa 546-labeled anti-rabbit IgG and the
anti-green fluorescent protein (anti-GFP) antibody were purchased from
Molecular Probes, Inc. (Eugene, Oreg.). Mouse embryo MTN blot was
purchased from Clontech Laboratories, Inc. (Palo Alto, Calif.). Other
materials were from commercial sources.
Plasmid construction.
pBJ-Myc/rAxin, pMAL-c2/rAxin,
pBJ-Myc/Dvl-1, pCGN/Dvl-1, and pGEX-2T/Dvl-1-(1-140) were
constructed as described previously (20, 27, 28).
Standard recombinant DNA techniques were used to construct
the following plasmids: pEF-BOS-HA/Idax, pBSKS/Idax, pMAL-c2/Idax,
pMAL-c2/Idax-(1-108), pMAL-c2/Idax-(109-198),
pBTM116HA/Dvl-1-(251-336), pGEX-2T/Dvl-1-(251-336),
pBSKS/Dvl-1-(337-670), pEF-BOS-Myc/Dvl-1-(224-371), pEF-BOS-Myc/Dvl-1-(337-670), pGEX-4T-1/Dvl-1-(395-670),
pGEX-2T/Dvl-1-(1-250), pEF-BOS-Myc/Dvl-1-(1-378),
pGEX-2T/Dvl-1-(1-378), pEGFP-C3/Dvl-1, pEF-BOS-HA/hTcf-4E,
pBJ-Myc/hTcf-4E, pEF-BOS-Myc/GSK-3
, and pBJ-Myc/
-catenin. 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 the Expand high fidelity PCR system (Roche
Diagnostics GmbH, Manheim, Germany) into the vectors. The entire
PCR products were sequenced, and the structures of all plasmids were
confirmed by restriction analysis.
Immunocytochemistry.
L cells grown on coverslips were fixed
for 20 min in phosphate-buffered saline (PBS) containing 4%
paraformaldehyde. The cells were washed with PBS three times and then
were permeabilized with PBS containing 0.1% Triton X-100 and 2 mg of
bovine serum albumin/ml for 20 min. The cells were washed and incubated
for 1 h with the antihemagglutinin (anti-HA) or the anti-Axin
antibody. After being washed with PBS, they were further incubated for
1 h with Cy5-labeled anti-mouse and Alexa 546-labeled anti-rabbit
immunoglobulin G (IgG). The coverslips were washed with PBS, mounted on
glass slides, and viewed with a confocal laser-scanning microscope
(LSM510; Carl Zeiss).
Interaction of Idax with Dvl.
To determine whether Idax
interacts with Dvl in intact cells, L cells stably expressing HA-Idax
(L-Idax cells) (6-cm-diameter dish) transfected with pBJ-Myc-derived
plasmids were lysed in 200 µl of lysis buffer (20 mM Tris-HCl [pH
7.5], 150 mM NaCl, 20 µg of leupeptin/ml, 20 µg of aprotinin/ml, 1 mM phenylmethylsulfonyl fluoride, 1% Nonidet P-40, and 10% glycerol)
and the lysates were centrifuged at 15,000 × g for 10 min at 4°C. The supernatant (150 µg of protein) was
immunoprecipitated with the anti-Myc antibody, and then the
precipitates were probed with the anti-Myc and anti-HA antibodies. To
examine the interaction of Idax with Dvl-1 using purified proteins in
vitro, 1 µM GST-Dvl-1 mutants was incubated with MBP-Idax or its
deletion mutants (30 pmol) immobilized on amylose resin in 100 µl of
low-salt buffer (20 mM Tris-HCl [pH 7.5] and 1 mM dithiothreitol) for
1 h at 4°C. MBP fusion proteins were precipitated by
centrifugation, and the precipitates were washed successively with
NP-40 buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Nonidet P-40,
and 10% glycerol), LiCl buffer (0.1 M Tris-HCl [pH 7.5] and 0.5 M
LiCl), and low-salt buffer, and then the precipitates were probed with
the anti-GST antibody. To show inhibition by Idax of the binding
between Axin and Dvl-1, the given concentrations of MBP-Idax and 1 µM
MBP-rAxin were incubated with GST-Dvl-1-(1-378) (5 pmol) immobilized
on glutathione Sepharose 4B in 100 µl of low-salt buffer containing
0.5% 3-[(3-cholamidopropyl)dimethylammonio] propanesulfonic acid.
GST-Dvl-1-(1-378) was precipitated by centrifugation, and the
precipitates were probed with the anti-MBP and anti-GST antibodies. In
the reciprocal experiments the given concentrations of MBP-rAxin and 2 µM MBP-Idax were incubated with GST-Dvl-1.
Luciferase assay and
-catenin accumulation in L and 293 cells.
To examine the effects of Idax on Wnt-3a-induced
accumulation of
-catenin in L cells, after L cells stably expressing
Neo (control L cells [L/C cells]) or L-Idax cells (35-mm-diameter dish) were deprived of serum for 6 h, they were treated with the indicated amounts of Wnt-3a conditioned medium. The cells were lysed in
100 µl of NP-40 buffer, and the lysates (20 µg of protein) were
probed with the anti-
-catenin antibody. To measure Wnt-3a-dependent Tcf-4 activity, L/C cells or L-Idax cells (35-mm-diameter dish) were
transfected with pTOPFLASH, which contains optimal Tcf-binding sites,
or pFOPFLASH, which contains mutated Tcf-binding sites (0.5 µg),
pEF-BOS-HA/hTcf-4E (0.1 µg), and pME18S/lacZ (0.5 µg) (26,
32). At 46 h after transfection, the cells were deprived of
serum for 6 h and then were treated with 40 µl of
Wnt-3a-conditioned medium for 6 h. The cells were lysed, and
luciferase activity was measured using a PicaGene (Toyo B-NET Co.,
Ltd., Tokyo, Japan) and lumiphotometer TD4000 (Futaba Medical, Tokyo,
Japan). To standardize the transfection efficiency, pME18S/lacZ
carrying the SR
promoter linked to the coding sequence of the
-galactosidase gene was used as an internal control. To observe Dvl-
and
-catenin-dependent Tcf-4 activation, pCGN/Dvl-1 (0.2 µg),
pUC/EF-1
/
-cateninSA (50 ng), and the indicated
amounts of pEF-BOS-HA/Idax were transfected into 293 cells
(35-mm-diameter dish) with pTOPFLASH or pFOPFLASH (0.5 µg),
pEF-BOS-HA/hTcf-4E (0.1 µg), and pME18S/lacZ (0.5 µg). After
46 h, the cells were lysed and luciferase activity was measured as described.
Xenopus injections and analysis of phenotypes.
Xwnt-8 and Xglobin expression plasmids were constructed as described
previously (71). Myc-tagged Idax, its deletion mutants, and Dvl-1 were individually subcloned into the BglII site of
pSP64T (34). Sense mRNA was obtained by in vitro
transcription of linearized templates using the SP6-mMESSAGE mMACHINE
kit (Ambion, Austin, Tex.). Fertilized eggs were dejellied using 4.5%
cysteine acid, and mRNAs were injected into dorsal or ventral
blastomeres at the four-cell stage. After injection, embryos were
cultured for 3 days (at stage 40 to 41). UV light irradiation was
performed as described previously (53). The phenotypes of
the injected embryos were evaluated by the Dorso-Anterior Index (DAI)
(24). For reverse transcription-PCR (RT-PCR), injected
embryos were incubated at stage 10.5, and then total RNA was isolated.
Oligo(dT)-primed cDNAs were synthesized using 5 µg of total RNA from
10 embryos. PCR analyses (35 cycles) were performed with ExTaq DNA
polymerase (TaKaRa Shuzo Co., Ltd., Ohtsu, Japan). Primers for PCR were
as follows: EF-1
(5'-CAG ATT GGT GCT GGA TAT GC-3' and
5'-ACT GCC TTG ATG ACT CCT AG-3') and siamois (5'-AAG ATA
ACT GGC ATT CCT GAG C-3' and 5'-GGT AGG GCT GTG TAT TTG AAG G-3').
Others.
Yeast two-hybrid screening was carried out as
previously described (20, 71). To obtain a full-length
cDNA of Idax, the clone isolated by the yeast two-hybrid method was
labeled with random primers and [
-32P]dCTP and used to
screen a
ZAP rat brain cDNA library. Northern blot analysis was
performed as already described (40). Protein concentrations were determined with bovine serum albumin as a standard
(5). Rat brain cytosol was prepared as described
previously (46).
Nucleotide sequence accession numbers.
The GenBank accession
numbers for rat Idax cDNA and human Idax cDNA are
AF272158 and AF272159, respectively.
 |
RESULTS |
Identification of Idax.
To identify novel proteins involved in
the Wnt signaling pathway, we screened a rat brain cDNA library by the
yeast two-hybrid method using the PDZ domain of Dvl-1 as bait. Several
clones were found to confer both His+ and LacZ+
phenotypes, and a full-length cDNA of one clone was isolated. This
clone spanned a distance of 1,314 bp and contained an uninterrupted open reading frame of 594 bp, encoding a predicted protein of 198 amino
acids (Fig. 1A). The first ATG was
preceded by stop codons in all three reading frames. The neighboring
sequence of the first ATG was consistent with the translation
initiation start proposed by Kozak (33). Using the rat
cDNA of this Dvl-binding protein as a probe, we isolated the human
cDNA. The predicted amino acid sequence from human cDNA was identical
to that from rat cDNA. By searching several databases, we found mouse
expressed sequence tag clones (AW742348, AW989078, and AW045537) as
highly conserved homologs of this Dvl-interacting protein. However, we
did not find similar sequences in expressed sequence tag clones from
Drosophila or Caenorhabditis elegans. Since this Dvl-interacting protein is a novel protein, we designated it Idax (for
inhibition of the Dvl and Axin complex). The mRNA of Idax was highly expressed in cerebrum, cerebellum, and heart and was slightly expressed in thymus and testis among rat tissues (Fig. 1B).
Idax mRNA was detected during development (embryonic days 7, 11, 15, and 17) of mouse embryos, with the highest expression at
embryonic day 11 (Fig. 1C). The constructs of rat Idax and human Dvl-1
used in this study are shown in Fig. 1D.

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FIG. 1.
Structure and gene expression of Idax. (A) Amino acid
sequence of Idax. (B) Northern blot analysis of Idax in rat tissues.
Total RNA (20 µg per lane) from the indicated rat tissues was probed
with full-length cDNA of rat Idax. The positions of 18S and 28S rRNA
are indicated. (C) Northern blot analysis of Idax in mouse embryos.
Poly(A) RNA (2 µg per lane) from the indicated stages of mouse
embryos was probed with the cDNAs of mouse -actin and rat
Idax-(369-495), which is identical with mouse Idax. E, embryonic day.
(D) Schematic representation of deletion mutants of rat Idax and human
Dvl-1 used in this study.
|
|
Interaction of Idax with Dvl.
Since Idax was isolated as a
protein binding to Dvl by the yeast two-hybrid screening, we examined
whether Idax forms a complex with Dvl in intact cells. To this end, we
prepared L cells stably expressing HA-Idax (L-Idax cells) (Fig.
2A, lane 5). When
Myc-Dvl-1 was expressed in L-Idax cells and the lysates were
immunoprecipitated with the anti-Myc antibody, HA-Idax was detected in
the Myc-Dvl-1 immune complexes (Fig. 2A, lanes 6 and 13). To determine
which region of Dvl is responsible for complex formation with Idax, Myc-Dvl-1-(1-378) or Myc-Dvl-1-(337-670) was expressed in L-Idax cells (Fig. 2A, lanes 7 and 8). HA-Idax was immunoprecipitated with
Myc-Dvl-1-(1-378) but not with Myc-Dvl-1-(337-670) (Fig. 2A, lanes 14 and 15). Further, the region containing the PDZ domain [Myc-Dvl-1-(224-371)] was sufficient for complex formation between Dvl and Idax (Fig. 2B, lane 4). These results indicate that Idax forms
a complex with Dvl in intact cells and that the PDZ domain of Dvl is
important for their interaction. Next, we examined whether HA-Idax
forms a complex with other Wnt signaling molecules in intact cells.
HA-Idax was not immunoprecipitated with Myc-rAxin, Myc-
-catenin,
Myc-Tcf-4, or Myc-GSK-3
in L cells (Fig. 2C, lanes 7 to 11).


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FIG. 2.
Interaction of Idax with Dvl. (A) Interaction of Idax
with Dvl in intact cells. The lysates (20 µg of protein) of wild-type
L cells (lanes 1 to 4) or L-Idax cells (lanes 5 to 8) expressing
Myc-Dvl-1 (lanes 2 and 6), Myc-Dvl-1-(1-378) (lanes 3 and 7), or
Myc-Dvl-1-(337-670) (lanes 4 and 8) were probed with the anti-Myc and
anti-HA antibodies. The same lysates (150 µg of protein) prepared in
lanes 2 to 8 were immunoprecipitated with the anti-Myc antibody, and
the immunoprecipitates were probed with the anti-Myc and anti-HA
antibodies (lanes 9 to 15). IP, immunoprecipitation; Ab, antibody. (B)
Interaction of Idax with the PDZ domain of Dvl. The lysates of L-Idax
cells with (lane 2) or without (lane 1) expression of
Myc-Dvl-1-(224-371) were probed with the anti-Myc and anti-HA
antibodies. The same lysates (150 µg of protein) prepared in lanes 1 and 2 were immunoprecipitated with the anti-Myc antibody, and the
immunoprecipitates were probed with the anti-Myc and anti-HA antibodies
(lanes 3 and 4). (C) Inability of Idax to bind to other Wnt signaling
molecules. The lysates (20 µg of protein) of L-Idax cells expressing
Myc-Dvl-1 (lane 2), Myc-rAxin (lane 3), Myc- -catenin (lane 4),
Myc-Tcf-4 (lane 5), or Myc-GSK-3 (lane 6) were probed with the
anti-Myc and anti-HA antibodies. The same lysates (150 µg of protein)
prepared in lanes 2 to 6 were immunoprecipitated with the anti-Myc
antibody, and the immunoprecipitates were probed with the anti-Myc and
anti-HA antibodies (lanes 7 to 11). (D) Complex formation of Idax with
Dvl in rat brain. Rat brain cytosol (30 µg of protein) was probed
with the anti-Dvl antibody (lane 1). The cytosol (100 µg of protein)
was incubated with MBP-Idax (lane 2) or MBP (lane 3) (30 pmol)
immobilized to amylose resin, and MBP fusion proteins were precipitated
by centrifugation and the precipitates were probed with the anti-Dvl
antibody. (E) Direct interaction of Idax with Dvl. GST-Dvl-1-(1-140),
GST-Dvl-1-(1-250), GST-Dvl-1-(1-378), GST-Dvl-1-PDZ,
GST-Dvl-1-(395-670), and GST (1 µg of protein) were stained with
Coomassie brilliant blue (lanes 1 to 6). GST-Dvl-1-(1-140) (lanes 7 and 13), GST-Dvl-1-(1-250) (lanes 8 and 14), GST-Dvl-1-(1-378) (lanes
9 and 15), GST-Dvl-1-PDZ (lanes 10 and 16), GST-Dvl-1-(395-670)
(lanes 11 and 17), or GST (lane 12) (1 µM) was incubated with
MBP-Idax (lanes 7 to 12) or MBP (lanes 13 to 17) (30 pmol) immobilized
on amylose resin, and MBP fusion proteins were precipitated by
centrifugation and the precipitates were probed with the anti-GST
antibody. (F) The region of Idax which binds to Dvl. MBP-Idax,
MBP-Idax-(1-108), MBP-Idax-(109-198), and MBP (0.5 µg of protein)
were stained with Coomassie brilliant blue (lanes 1 to 4).
GST-Dvl-1-PDZ (lanes 5 to 8) or GST (lanes 9 to 11) (1 µM) was
incubated with MBP-Idax (lanes 5 and 9), MBP-Idax-(1-108) (lanes 6 and
10), MBP-Idax-(109-198) (lanes 7 and 11), or MBP (lanes 8) (30 pmol)
immobilized on amylose resin. MBP fusion proteins were precipitated by
centrifugation, and the precipitates were probed with the anti-GST
antibody. The results shown are representative of three independent
experiments.
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|
To examine whether the interaction of Idax and Dvl is direct, deletion
mutants of GST-fused Dvl-1 (GST-Dvl-1) and of MBP-fused
Idax
(MBP-Idax) were purified from
E. coli (Fig.
2E, lanes 1 to
6; Fig.
2F, lanes 1 to 4). MBP-Idax precipitated endogenous Dvl
from
rat brain cytosol (Fig.
2D). GST-PDZ and GST-Dvl-1-(1-378)
were
precipitated with MBP-Idax immobilized on amylose resin,
but
GST-Dvl-1-(1-140), GST-Dvl-1-(1-250), GST-Dvl-1-(395-670),
and GST
were not (Fig.
2E, lanes 7 to 12). These GST-Dvl-1 mutants
were not
precipitated with MBP (Fig.
2E, lanes 13 to 17). GST-PDZ
was
precipitated with MBP-Idax-(109-198) but not with MBP-Idax-(1-108)
(Fig.
2F, lanes 5 to 11). These results indicate that the C-terminal
region of Idax binds directly to the PDZ domain of Dvl-1. In addition,
Idax also bound to Dvl-3 (data not shown). Since the PDZ domains
of
Dvl-1, -2, and -3 are highly conserved (86% identity), Idax
is
expected to bind to these
Dvls.
Competition of Idax and Axin for their binding to Dvl.
Previously it was shown that Dvl interacts directly with Axin
(27). GST-Dvl-1-(1-378), including the DIX domain and the PDZ domain, bound to MBP-rAxin, but GST-Dvl-1-(1-250) and the PDZ
domain did not, suggesting that both the DIX and PDZ domains of Dvl are
necessary for its interaction with Axin (Fig.
3A). Therefore, we examined whether Idax
inhibits the binding of Dvl to Axin. MBP-Idax, but not MBP, competed
with MBP-rAxin for the binding to GST-Dvl-1-(1-378) in a
dose-dependent manner (Fig. 3B, lanes 1 to 6). In the reciprocal
experiment, MBP-rAxin inhibited the binding of MBP-Idax to
GST-Dvl-1-(1-378) (Fig. 3B, lanes 7 to 12). About 5 µM Idax reduced
the binding of 1 µM Axin to Dvl by 50%, while approximately 1 µM
Axin decreased the binding of 2 µM Idax to Dvl by 50%. These results
suggest that the interaction of Idax with the PDZ domain is sufficient
for interfering with complex formation between Dvl and Axin and that
the affinity of Dvl for Axin is higher than that of Dvl for Idax. We
also examined whether Idax and Axin competed with each other for the
binding to Dvl in intact cells. HA-Idax formed a complex with GFP-fused Dvl-1 (GFP-Dvl-1) in L-Idax cells (Fig. 3C, lanes 2 and 5). When Myc-rAxin was expressed in L-Idax cells, the association of HA-Idax with GFP-Dvl-1 was greatly reduced (Fig. 3C, lanes 3 and 6). These in
vitro and intact cell studies suggest that Idax and Axin mutually inhibit their binding to Dvl.

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FIG. 3.
Competition of Idax and Axin for their binding to Dvl.
(A) Requirement of the DIX and PDZ domains of Dvl for its binding to
Axin. After GST-Dvl-1-(1-250) (lanes 1 and 4), GST-Dvl-1-(1-378)
(lanes 2 and 5), or GST-Dvl-1-PDZ (lanes 3 and 6) (0.5 µM) was
incubated with MBP-rAxin (lanes 1 to 3) or MBP (lanes 4 to 6) (0.5 µM), the GST fusion proteins were precipitated by centrifugation and
the precipitates were probed with the anti-MBP antibody. MBP-rAxin
(lane 7) and MBP (lane 8) (0.5 µg) were stained with Coomassie
brilliant blue. (B) Competition of Idax and Axin for their binding to
Dvl in vitro. MBP-rAxin (1 µM) was incubated with GST-Dvl-1-(1-378)
(5 pmol) immobilized on glutathione Sepharose 4B in the presence of the
indicated concentrations of MBP-Idax (lanes 1 to 5) or MBP (lane 6).
GST-Dvl-1-(1-378) was precipitated by centrifugation, and the
precipitates were probed with the anti-MBP antibody (upper panel). The
amounts of GST-Dvl-1-(1-378) precipitated in each binding assay are
shown (middle panel). In reciprocal experiments, MBP-Idax (2 µM) was
incubated with GST-Dvl-1-(1-378) (5 pmol) immobilized on glutathione
Sepharose 4B in the presence of the indicated concentrations of
MBP-rAxin (lanes 7 to 11) or MBP (lane 12). The results shown in the
upper and middle panels are representative of three independent
experiments. The amounts of MBP-rAxin and MBP-Idax were analyzed using
the NIH Image system and expressed as arbitrary units. The results
shown are the mean ± the standard error of three independent
experiments (lower panel). (C) Inhibition of the binding of Idax to Dvl
by Axin in intact cells. The lysates (20 µg of protein) of L-Idax
cells transfected with empty vector (lane 1), pEGFPC1-Dvl-1 (lane 2),
or pEGFPC1-Dvl-1 and pBJ-Myc/rAxin (lane 3) were probed with the
anti-Myc, anti-GFP, and anti-HA antibodies. The same lysates (150 µg
of protein) were immunoprecipitated with the anti-GFP antibody, and the
immunoprecipitates were probed with the anti-Myc, anti-GFP, and anti-HA
antibodies (lanes 4 to 6). IP, immunoprecipitation; Ab, antibody. The
results shown are representative of three independent experiments.
|
|
Subcellular localization of Idax.
We examined the localization
of HA-Idax, GFP-Dvl-1, and Myc-rAxin in L cells using multiple
immunofluorescence. HA-Idax showed a diffuse cytoplasmic pattern
of expression with fine granules in L-Idax cells (Fig.
4A). Consistent with previous
observations (10, 56), GFP-Dvl-1 was localized to
distinct cytoplasmic vesicles in wild-type L cells (Fig. 4B and D).
When GFP-Dvl-1 was expressed in L-Idax cells, the intracellular
localization of HA-Idax was markedly changed. The localization of
HA-Idax was similar to that of GFP-Dvl-1, and the merged image
demonstrated that the two proteins colocalize (Fig. 4E, F, and G).
Myc-rAxin had a diffuse cytoplasmic pattern of expression with some
areas of particulate or vesicular staining in L cells (Fig. 4H). When Myc-rAxin was coexpressed with GFP-Dvl-1 in L cells, the two proteins were found to colocalize (Fig. 4I, J, and K), consistent with previous
observations (10, 56). These immunocytochemical studies demonstrate that Idax and Axin colocalize with Dvl. When GFP-Dvl-1 was
coexpressed with Myc-rAxin in L-Idax cells, HA-Idax did not exhibit a
vesicular pattern but showed a diffuse cytoplasmic pattern, although Myc-rAxin colocalized with GFP-Dvl-1 (Fig. 4L, M,
N, and O).

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FIG. 4.
Subcellular localization of Idax,
Dvl-1, and rAxin. L-Idax cells (A), wild-type L cells expressing
GFP-Dvl-1 (B, C, and D), L-Idax cells expressing GFP-Dvl-1 (E, F, and
G), wild-type L cells expressing Myc-rAxin (H), wild-type L cells
coexpressing GFP-Dvl-1 and Myc-rAxin (I, J, and K), and L-Idax cells
coexpressing GFP-Dvl-1 and Myc-rAxin (L, M, N, and O) were fixed and
permeabilized. Some of them were directly viewed with a confocal
laser-scanning microscope to detect GFP-Dvl-1 (B, E, I, and L), and
the others were stained with the anti-HA antibody to detect HA-Idax
(A, C, F, and O) or with the anti-Axin antibody to observe
Myc-rAxin (H, J, and M). Note that nuclei were stained nonspecifically
with the anti-HA antibody. Merged pictures of panels B and C, E and F,
I and J, and L and M are shown in panels D, G, K, and N, respectively.
GFP-Dvl-1, Cy5-labeled HA-Idax, and Alexa 546-labeled Myc-rAxin
produced no cross staining. The results shown are representative of
three independent experiments.
|
|
Effects of Idax on
-catenin signaling.
It was shown
previously that Wnt-3a-conditioned medium induces the accumulation of
-catenin and activates Tcf-4 in L cells and that expression of Axin
inhibits these Wnt-3a-dependent responses (26). Therefore,
we examined the roles of Idax in Wnt signaling. Wnt-3a induced the
accumulation of
-catenin in a dose-dependent manner in control L/C
cells. Expression of Idax (L-Idax cells) suppressed this response (Fig.
5A). Similar results were obtained with
three independent clones of L-Idax cells. Consistent with this result,
Wnt-3a-dependent Tcf-4 activation was inhibited in L-Idax cells (Fig.
5B). Dvl-1 and
-catenin activated Tcf-4 in 293 cells (Fig. 5C, lanes
3 and 13), consistent with previous results (52, 56).
Expression of Idax inhibited the Dvl-1-dependent but not the
-cateninSA-dependent activation of Tcf-4 (Fig. 5C).
-cateninSA is a
-catenin mutant which is not degraded
due to the substitution of serine and threonine residues with alanine
in the phosphorylation sites by GSK-3
(75). These
results suggest that Idax acts as a negative regulator of the Wnt
signaling pathway and that it functions between Dvl and
-catenin.

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FIG. 5.
Effects of Idax on -catenin signaling. (A) Inhibition
of Wnt-3a-induced accumulation of -catenin by Idax. After L cells
stably expressing Neo (control L cells [L/C]) (upper panel) and
L-Idax cells (middle panel) were treated with the indicated amounts of
Wnt-3a conditioned medium for 40 min, the cells were lysed and probed
with the anti- -catenin antibody. The results shown in the upper and
middle panels are representative of three independent experiments. The
amounts of -catenin were analyzed using the NIH Image system and
expressed as fold increase compared with the level observed in L/C
cells treated with control medium. The results shown are the mean ± the standard error (SE) of three independent experiments (lower
panel). , L/C cells; , L-Idax cells. (B) Inhibition of
Wnt-3a-induced Tcf activation by Idax. L/C cells (lanes 1 to 4) and
L-Idax cells (lanes 5 to 8) transfected with pTOPFLASH (black bars) or
pFOPFLASH (white bars) were treated with Wnt-3a-conditioned medium
(lanes 3, 4, 7, and 8) or control medium (lanes 1, 2, 5, and 6) for
6 h. Luciferase activity was assayed and expressed as fold
increase compared with the level observed in cells transfected with
pTOPFLASH and treated with control medium. These results represent the
mean ± SE of five independent experiments. (C) Inhibition of Dvl-
and -catenin-dependent Tcf activation by Idax. pCGN-Dvl-1 and
pEF-BOS-HA/Idax (lanes 3 to 12) or
pUC/EF-1 / -cateninSA and pEF-BOS-HA/Idax (lanes 13 to
16) were transfected into 293 cells with pTOPFLASH (black bars) or
pFOPFLASH (white bars). After 46 h, luciferase activity was
assayed and expressed as fold increase compared with the level observed
in cells transfected with pTOPFLASH. These results represent the
mean ± SE of four independent experiments.
|
|
Regulation of axis formation by Idax.
To determine the mode of
action of Idax, we examined the effects of Idax on the Wnt signaling
pathway using Xenopus embryos. The Wnt signaling pathway
regulates axis formation in Xenopus embryos
(44). Dorsal and ventral injection of Xglobin
mRNA into four-cell-stage embryos did not affect normal axis formation
(Fig. 6A, a and b). Dorsal injection of
Idax mRNA into embryos resulted in ventralization phenotypes
such as loss of the head structure (Fig. 6A, c). Embryos injected
ventrally with Idax mRNA developed normally (Fig. 6A, d).
siamois is a homeobox gene which mediates the effects of the
Wnt signaling pathway on axis formation and whose expression is
activated by
-catenin-Tcf (6, 36). Expression of
siamois was suppressed by dorsal but not ventral injection of Idax mRNA (Fig. 6B). When the mRNA of
Idax-(109-198) was injected dorsally, the embryos showed
ventralizing phenotypes (Fig. 6C), while injection of the mRNA of
Idax-(1-108) had no effect (Fig. 6C). Therefore, Idax has
ventralizing activity and the C-terminal region may be sufficient to
regulate the embryonic axis formation, consistent with the observation
that the C-terminal region of Idax has the Dvl-binding site.

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FIG. 6.
Effects of Idax on axis formation in Xenopus
embryos. (A) Ventralizing activity of Idax. (a) Dorsal injection of
Xglobin (1 ng). (b) Ventral injection of Xglobin. (c) Dorsal injection
of Idax (200 pg). (d) Ventral injection of Idax. (B) Inhibition of
siamois expression by Idax. siamois expression in
embryos was detected with RT-PCR analysis. Shown are dorsal injection
with Xglobin (lane 1, 1 ng); dorsal injection with Idax (lane 2, 50 pg;
lane 3, 100 pg; lane 4, 200 pg); and ventral injection with Idax (lane
5, 200 pg). The amounts of cDNA were standardized with EF-1 . no RT,
experiments without RT-PCR. (C) Ventralizing activity of Idax deletion
mutants. Embryos were injected ventrally (V) or dorsally (D) with Idax
(full length) or dorsally with Idax-(1-108) or Idax-(109-198). The
phenotypes were expressed as DAI. DAI 0, completely ventralized; DAI 5, normal.
|
|
Ventral injection of mRNAs of
Xwnt-8,
Dvl, or
X
-catenin has been shown to induce a secondary dorsal
axis (
11,
44,
57,
66) (Fig.
7A, a, c, and e). Coinjection of Xwnt-8
and Idax in
the ventral side repressed secondary axis formation (Fig.
7A,
b). Similar phenotypes were observed upon coexpression of Dvl-1
and
Idax (Fig.
7A, d). In contrast, coinjection of X

-catenin
and Idax
still induced the secondary axis structure (Fig.
7A,
f). The effects of
Idax on secondary axis formation by Xwnt-8,
Dvl, or X

-catenin are
summarized in Fig.
7B. It was shown that
UV light-irradiated embryos
exhibit axial deficiencies (Fig.
7C,
a) (
53). Idax alone
did not affect this phenotype (Fig.
7C,
b). Consistent with previous
observations, Xwnt-8 rescued the
UV-induced axial deficiencies (Fig.
7C, c). Coinjection with Idax
inhibited this activity of Xwnt-8 (Fig.
7C, d). X

-catenin also
rescued the UV-induced axial deficiencies
(Fig.
7C, e), and coinjection
with Idax did not affect
X

-catenin-dependent rescue of axis formation
(Fig.
7C, f). The
average DAI of the embryos is shown in Fig.
7D. Taken together, these
results suggest that Idax negatively
regulates the Wnt signaling
pathway during
Xenopus development
downstream of Wnt and Dvl
and upstream of

-catenin, consistent
with the results observed in
mammalian cells.

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FIG. 7.
Effects of Idax on Wnt signal-dependent axis formation.
(A) Effects of Idax on Wnt signal-dependent secondary axis formation.
Embryos were injected ventrally with Xwnt-8 (40 pg) (a), Xwnt-8 and
Idax (200 pg) (b), Dvl-1 (1 ng) (c), Dvl-1 and Idax (d), X -catenin
(1 ng) (e), or X -catenin and Idax (f). Arrowheads in panel f
indicate secondary axes. (B) The results from panel A are expressed as
percentage of secondary axis formation. Black bars show complete axis
duplication, which includes eyes and cement glands. White bars indicate
incomplete axis duplication characterized by a lack of head structures
but with a distinct branched axis. (C) Effects of Idax on Wnt-dependent
rescue of axis formation. UV-treated embryos were injected with nothing
(a), Idax (200 pg) (b), Xwnt-8 (40 pg) (c), Xwnt-8 and Idax (d),
X -catenin (1 ng) (e), or X -catenin and Idax (f). (D) The results
from panel C are expressed as DAI.
|
|
 |
DISCUSSION |
In this study, we have isolated a novel protein which binds to the
PDZ domain of Dvl directly and named it Idax. Idax suppresses the
Wnt-3a-dependent accumulation of
-catenin and activation of Tcf in L
cells. We have also shown that Idax inhibits axis formation and
siamois expression in Xenopus embryos. Moreover, Idax suppresses Xwnt-8- and Dvl-induced but not
-catenin-induced axis duplication and prevents Xwnt-8-dependent but not
-catenin-dependent rescue of the UV-irradiated
ventralization. These results suggest that Idax acts between
Dvl and
-catenin as a negative regulator of Wnt signaling.
The Wnt signaling pathway plays essential roles in a number of
developmental aspects, including gastrulation and organogenesis. For
instance, Wnt-3 is required for mesoderm formation during gastrulation
and Lef-1 is necessary for hair development at later stages in the
mouse (39, 63, 67). Thus, components involved in the Wnt
signaling pathway should be expressed during gastrulation or later. We
have shown that Idax is expressed at these stages, from embryonic days
7 to 17, suggesting that this gene is likely to be involved in the Wnt
signaling pathway at the appropriate time.
The PDZ domain is conserved in many proteins and generally acts as a
protein-interacting module (8). The PDZ domain can form a
dimer, and a unique amino acid motif, S/T-X-V in the C terminus, is
known to bind to the PDZ domain (58). Idax does not have
this sequence in the C terminus, and the three C-terminal amino acids
are not necessary for the binding of Idax to the PDZ domain of Dvl
(data not shown). Although we have found that the C-terminal half of
Idax is necessary for its interaction with Dvl, the minimal region of
Idax required for binding to Dvl remains to be clarified. Several
proteins have been reported to form a complex with the PDZ domain of
Dvl. These are casein kinase I
(CKI
) (47, 51), CKII
(68), protein phosphatase 2C (PP2C) (59), and
Frat (37). As none of these proteins contain the S/T-X-V
sequence in their C termini, the PDZ domain of Dvl may have different
properties from those of other known PDZ domains regarding
protein-protein interactions.
CKI
and CKII associate with and phosphorylate Dvl (47, 51,
68). Expression of CKI
in Xenopus embryos induces
-catenin accumulation, siamois expression, and secondary
axis formation, whereas GSK-3 blocks the ability of CKI
to rescue
UV-induced axial deficiencies in Xenopus embryos
(47). These results suggest that CKI
functions between
Dvl and GSK-3 and that it regulates the Wnt signaling pathway
positively. However, the molecular mechanism by which CKI
regulates
the Wnt pathway is not known. Although the interaction with CKII
requires the region containing the PDZ domain, whether this interaction
is direct is not known and its significance in the Wnt signaling
pathway is not yet clear. PP2C has been isolated by the yeast
two-hybrid method using the PDZ domain as bait (59).
Expression of PP2C in COS cells dephosphorylates and down-regulates
Axin and stimulates the transcriptional activity of Lef-1
(59). These results suggest that PP2C works as a positive regulator of the Wnt signaling pathway by inhibiting phosphorylation in
the Axin complex. GBP (GSK-3-binding protein) was originally identified
as a Xenopus GSK-3-binding protein that inhibits GSK-3 activity and mimics the effects of Wnt in Xenopus embryos,
and Frat is a mammalian homolog of GBP (74). It has been
suggested that the PDZ domain of Dvl binds to Frat and that Dvl
recruits Frat to the Axin complex, thereby inducing the dissociation of GSK-3 from Axin in response to Wnt (37). Therefore, Frat
also regulates the Wnt signaling pathway positively.
In contrast to the action of CKI
, PP2C, and Frat, Idax functions as
a negative regulator of the Wnt signaling pathway. Several possible
mechanisms for the mode of action of Idax are conceivable. The first is
that Idax competes with CKI
, PP2C, or Frat for the interaction with
Dvl, thereby suppressing their positive regulation of the Wnt pathway.
The second possibility is that Idax relieves the repressive action of
Dvl on Axin by inhibiting their interaction. However, the latter may be
unlikely, because the affinity of Dvl for Idax is lower than that of
Dvl for Axin. Nevertheless, one interesting possibility is that Wnt
activates a protein kinase which phosphorylates Dvl or Idax, thereby
modulating their affinities. Indeed, Dvl is phosphorylated in several
cell lines treated with Wnt-3a (35). As CKI
or CKII
phosphorylates Dvl in vitro, these protein kinases may modify the
affinities of Dvl for Idax and Axin. The third possibility is that Idax
may disrupt the tertiary conformation of Dvl by interacting with the
PDZ domain of Dvl. It has been suggested that the tertiary conformation
of the PDZ domain brings together the DIX and the DEP domains to form a
functional unit (49). We have shown that the region
containing the DIX domain of Dvl is important for its direct
interaction with Axin and for Wnt signaling (27, 45). It
is intriguing to speculate that the PDZ domain is necessary for
maintaining the conformation of the DIX domain of Dvl to bind to Axin
and that Idax causes a conformational change in Dvl, thereby preventing
the binding of Dvl to Axin indirectly.
-catenin is present in a complex including Axin, GSK-3
, APC, Dvl,
and Frat, where its stability is regulated. Inhibition of GSK-3
or
its dissociation from the complex by Dvl-Frat in response to Wnt could
be critical for the disassembly of this complex. The results presented
here may elucidate an additional part in the regulation of the Wnt
signaling pathway. We have recently found Axam as an additional
component in the Wnt signaling pathway (23). Axam is an
Axin-binding protein that relieves the repression by Dvl of Axin's
function, thereby leading to down-regulation of
-catenin. Although
Axam inhibits complex formation between Dvl and Axin, how Idax affects
the function of Axam is not known. The molecular mechanism by which Wnt
regulates the assembly and disassembly of this complicated complex
remains to be clarified.
 |
ACKNOWLEDGMENTS |
We are grateful to H. Clevers, D. Kimelman, B. Dallapiccola, A. Nagafuchi, and M. Nakata for donating plasmids and antibodies. We thank
the Research Center for Molecular Medicine and Research Facilities for
Laboratory Animal Sciences, Hiroshima University School of Medicine,
for the use of their facilities.
This work was supported by grants-in-aid for scientific research (B)
and for scientific research on priority areas (A) from the Ministry of
Education, Science, and Culture, Japan (1999 and 2000), by grants from
the Yamanouchi Foundation for Research on Metabolic Disorders (1999 and
2000), by the Uehara Memorial Foundation (1998), by a Research Grant of
the Princess Takamatsu Cancer Research Fund (1999; 99-23195), and by
the Public Trust Haraguchi Memorial Cancer Research Fund (1999).
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, Hiroshima University School of Medicine, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan. Phone: 81-82-257-5130. Fax: 81-82-257-5134. E-mail:
akikuchi{at}mcai.med.hiroshima-u.ac.jp.
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Molecular and Cellular Biology, January 2001, p. 330-342, Vol. 21, No. 1
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.1.330-342.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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