From the Department of Biochemistry, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan
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ABSTRACT |
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The regulators of G protein signaling (RGS)
domain of Axin, a negative regulator of the Wnt signaling pathway, made
a complex with full-length adenomatous polyposis coli (APC) in COS,
293, and L cells but not with truncated APC in SW480 or DLD-1 cells. The RGS domain directly interacted with the region containing the
20-amino acid repeats but not with that containing the 15-amino acid
repeats of APC, although both regions are known to bind to -catenin.
In the region containing seven 20-amino acid repeats, the region
containing the latter five repeats bound to the RGS domain of Axin.
Axin and
-catenin simultaneously interacted with APC. Furthermore,
Axin stimulated the degradation of
-catenin in COS cells. Taken
together with our recent observations that Axin directly interacts with
glycogen synthase kinase-3
(GSK-3
) and
-catenin and that it
promotes GSK-3
-dependent phosphorylation of
-catenin,
these results suggest that Axin, APC, GSK-3
, and
-catenin make a
tetrameric complex, resulting in the regulation of the stabilization of
-catenin.
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INTRODUCTION |
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Axin, which is a product of the mouse Fused locus, has
been identified as a negative regulator of the Wnt signaling pathway (1). Fused is a mutation that causes dominant skeletal and neurological defects and recessive lethal embryonic defects including neuroectodermal abnormalities (2-4). Because dorsal injection of wild
type Axin in Xenopus embryos blocks axis formation and coinjection of Axin inhibits Wnt8-, Dsh-, and kinase-negative GSK-31-induced axis
duplication (1), Axin could exert its effects on axis formation by
inhibiting the Wnt signaling pathway. However, the molecular mechanism
by which Axin regulates axis formation has not been shown. We have
recently identified rat Axin (rAxin) as a GSK-3
-interacting protein
(5). rAxin is phosphorylated by GSK-3
, directly binds to not only
GSK-3
but also
-catenin, and promotes
GSK-3
-dependent phosphorylation of
-catenin (5). Because the phosphorylation of
-catenin by GSK-3
is essential for
the down-regulation of
-catenin (6, 7), our results suggest that
rAxin may induce the degradation of
-catenin. These actions of rAxin
are consistent with the observation that Axin inhibits dorsal axis
formation in Xenopus embryos, because the accumulation of
-catenin induces the axis duplication (8).
It has been shown that besides the phosphorylation by GSK-3, the
down-regulation of
-catenin requires APC, which is a tumor suppressor linked to FAP and to the initiation of sporadic human colorectal cancer (9). The middle portion of APC contains three successive 15-amino acid (aa) repeats followed by seven related but
distinct 20-aa repeats. Both types of repeats are able to bind
independently to
-catenin (10-12). In FAP and colorectal cancers,
most patients carry APC mutations that result in the expression of
truncated proteins (9). Almost all mutant proteins lack the C-terminal
half including most of the 20-aa repeats but retain the 15-aa repeats.
Colorectal carcinoma cells with mutant APC contain large amounts of
monomeric
-catenin (13). The accumulated
-catenin translocates to
the nucleus, and this translocation involves the association of
-catenin with the transcription enhancers of the lymphocyte enhancer
binding factor/T cell factor family (14, 15). Because the APC mutants
retain the
-catenin-binding activity, the interaction of APC with
-catenin is not sufficient for the down-regulation of
-catenin.
How APC down-regulates
-catenin and the relationship between APC and
Axin in the degradation of
-catenin are not clear.
In addition to GSK-3- and
-catenin-binding sites, rAxin has a
domain that is homologous to RGS, and this domain is called the RGS
domain (1, 5). RGS has been originally identified as a protein that
binds to the GTP- but not GDP-bound form of G
and
stimulates GTP hydrolysis of G
(16). It has been shown
that
RGS, a mutant of Axin in which the RGS domain is deleted, acts
as a potent dorsalizer, producing a secondary axis and that Axin blocks
the axis-inducing activity of
RGS (1). These results indicate that
RGS acts through a dominant-negative mechanism to inhibit an
endogenous Axin activity and that it competes for binding to a protein
with which Axin normally interacts. Therefore, the RGS domain may have
an activity to transmit the signal by interacting with other
protein(s). Here we report that the RGS domain of rAxin directly
interacts with the region containing the 20-aa repeats of APC and that
rAxin stimulates the down-regulation of
-catenin. Taken together
with our recent observations (5), these results indicate that Axin
directly binds to APC,
-catenin, and GSK-3
and that it regulates
the stabilization of
-catenin.
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EXPERIMENTAL PROCEDURES |
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Materials and Chemicals--
APC cDNA, 293 cells, L cells,
and SW480 and DLD-1 cells were kindly supplied from Drs. T. Akiyama
(Osaka University, Suita, Japan), K. Morishita (Daiichi Pharmaceutical
Co. Ltd., Tokyo, Japan), A. Nagafuchi and Sh. Tsukita (Kyoto
University, Kyoto, Japan), and E. Tahara (Hiroshima University,
Hiroshima, Japan), respectively. GST and MBP fusion proteins were
purified from Escherichia coli according to the
manufacturer's instructions. The anti-APC (Ab-1) and -catenin
antibodies were purchased from Oncogene Science Inc. (Cambridge, MA)
and Transduction Laboratories (Lexington, KY), respectively.
[35S]Methionine and [35S]cysteine were
purchased from Amersham Inc. (Buckinghamshire, United Kingdom). Other
materials and chemicals were from commercial sources.
Plasmid Constructions--
pEF-BOS-Myc/rAxin (full-length),
pBSKS/rAxin (full-length), pBJ-Myc/rAxin-(1-229),
pEF-BOS-Myc/rAxin-(1-713), pBJ-Myc/rAxin-(298-713), pEF-BOS/Myc-rAxin-(298-506), pBJ-Myc/rAxin-(713-832),
pGEX-2T/-catenin, and pMAL-c2/rAxin-(298-506) were constructed as
described (5). To construct pGEX-2T/RGS, the RGS cDNA fragment
encoding rAxin-(89-216) was synthesized by polymerase chain reaction
and inserted into pGEX-2T. To construct pMAL-c2 containing APC mutants,
pMKITneo/APC was digested with various restriction enzymes, and the APC
cDNA fragments were inserted into pMAL-c2. These procedures will be described in detail elsewhere. To construct pMAL-c2/rAxin
(full-length), pBSKS/rAxin was digested with SmaI and
EcoRV, and the rAxin cDNA fragment was inserted into
pMALc-2, which was digested with XbaI and blunted with
Klenow fragment. To construct pGEX-2T/rAxin-(1-529), pBSKS/rAxin was
digested with SmaI and PvuII, and this fragment was inserted into SmaI cut pGEX-2T. To construct
pCGN/
-catenin, pBSSK/
-catenin was digested with XhoI,
blunted with Klenow fragment, and digested with XbaI. The
-catenin cDNA fragment was inserted into pCGN.
Interaction of APC with rAxin--
COS cells (10-cm diameter
dish) transfected with pBJ- and pEF-BOS-derived plasmids were lysed as
described (17-19). rAxin and its deletion mutants were tagged with Myc
epitope at their N termini. The lysates (160-800 µg of protein) were
immunoprecipitated with the anti-Myc antibody, then the precipitates
were probed with the anti-APC and -catenin antibodies. When the
interaction of the RGS domain of rAxin with APC was examined in
vitro, 1 µM GST-RGS was incubated with the lysates
(200 µg of protein) of COS, 293, L, SW480, and DLD-1 cells for 2 h at 4 °C. GST-RGS were precipitated with glutathione-Sepharose 4B,
and the precipitates were probed with the anti-APC antibody.
Kinetics of the Binding of rAxin, -Catenin, and
APC--
Various deletion mutants of MBP-APC (0.5-10 pmol)
immobilized on the amylose resin were incubated with various
concentrations of GST-RGS, GST-rAxin-(1-529), and GST-
-catenin in
100 µl of reaction mixture (20 mM Tris/HCl (pH 7.5) and 1 mM dithiothreitol) for 2 h at 4 °C. MBP fusion
proteins were precipitated by centrifugation, and the precipitates were
probed with the anti-GST antibody. When the effect of rAxin on the
interaction of APC with
-catenin was examined, 50 nM
GST-
-catenin was incubated with 250 nM
MBP-APC-(959-1338) in the presence of various concentrations of
MBP-rAxin-(298-506) or MBP-rAxin (full-length) in 100 µl of reaction
mixture for 2 h at 4 °C. GST-
-catenin was precipitated by
glutathione-Sepharose 4B, and the precipitates were probed with the
anti-MBP antibody. Where specified, the relative intensities of the
precipitated GST and MBP fusion proteins were quantitated by
densitometric tracing of the stained sheets using an NIH image
program.
Pulse-Chase Analysis of -Catenin--
COS cells (60-70%
confluent on a 35-mm diameter dish) were transfected with
pCGN/
-catenin alone or with pCGN/
-catenin and pEF-BOS-Myc/rAxin
(full-length). After 60 h, pulse-chase analysis was performed as
described (13). Briefly, the cells were pulse-labeled with
[35S]methionine and [35S]cysteine (50 µCi/ml) for 30 min at 37 °C. Then the cells were lysed immediately
or at the indicated times following incubation with excess unlabeled
methionine and cysteine. The lysates were immunoprecipitated with the
anti-HA antibody, and the precipitates were probed with the anti-HA
antibody and analyzed with a Fuji BAS 2000 image analyzer.
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RESULTS AND DISCUSSION |
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Complex Formation of the RGS Domain of rAxin with APC in Intact
Cells--
We have recently found that rAxin directly binds to
-catenin (5). Because it has been shown that
-catenin directly
binds to APC (10-12), we examined whether rAxin makes a complex with APC through
-catenin. Various deletion mutants of Myc-rAxin
expressed in COS cells were immunoprecipitated with the anti-Myc
antibody. Consistent with our recent observations (5),
-catenin was coprecipitated with Myc-rAxin (full-length), Myc-rAxin-(1-713), Myc-rAxin-(298-713), and Myc-rAxin-(298-506) (Fig.
1A). Among them, APC was
detected in the Myc-rAxin (full-length) and Myc-rAxin-(1-713) immune
complexes, but not in the Myc-rAxin-(298-713) and Myc-rAxin-(298-506) immune complexes (Fig. 1A). Unexpectedly, APC but not
-catenin was detected in the Myc-rAxin-(1-229) immune complex.
Neither
-catenin nor APC was coprecipitated with
Myc-rAxin-(713-832). Because rAxin-(1-229) contains the RGS domain,
we examined whether the RGS domain itself (amino acids 89-216) makes a
complex with APC. APC in COS, 293, and L cells was coprecipitated with
GST-RGS (Fig. 1B). It is known that APC is truncated at
amino acids 1337 and 1427 in SW480 and DLD-1 cells, respectively, and
that these truncated forms of APC fail to down-regulate
-catenin (9, 13). These APC mutants in SW480 and DLD-1 cells were not coprecipitated with GST-RGS (Fig. 1B). Consistent with the previous
observations (10, 11), both full-length and truncated APC were
coprecipitated with GST-
-catenin (Fig. 1B). These results
suggest that the RGS domain of rAxin makes a complex with the
C-terminal half of APC in intact cells. Taken together with our
observations (5), rAxin has distinct binding sites for APC,
-catenin, and GSK-3
. It is notable that the APC mutants in
colorectal carcinoma cell lines such as SW480 and DLD-1 cells do not
associate with rAxin.
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Direct Interaction of rAxin with APC--
To examine whether the
RGS domain of rAxin directly interacts with APC, various deletion
mutants of APC were purified as MBP fusion proteins (Fig.
2A). GST-RGS bound to
MBP-APC-(1211-2075), which contains seven 20-aa repeats, in a
dose-dependent manner (Fig. 2B). The
Kd value was calculated to be 115 nM. However, GST-RGS did not bind to MBP-APC-(959-1338) which contains three 15-aa repeats and the first 20-aa repeat (Fig. 2B).
These results show that the RGS domain of rAxin directly interacts with the region containing the 20-aa repeats of APC. To characterize the
interaction of APC with rAxin further, MBP-APC-(1211-1787), which
contains the former four 20-aa repeats, and MBP-APC-(1788-2075), which
contains the latter three 20-aa repeats, were purified. Both GST-RGS
and GST--catenin bound to MBP-APC-(1211-1787), but they bound to
MBP-APC-(1788-2075) less efficiently (Fig. 2C). Furthermore, GST-
-catenin bound to both MBP-APC-(1211-1495) and MBP-APC-(1475-1787), whereas GST-RGS bound to MBP-APC-(1475-1787) but
not to MBP-APC-(1211-1495) (Fig. 2C). Therefore, the RGS
domain does not interact with the region of APC containing the 15-aa repeats and the first and the second 20-aa repeats, which binds to
-catenin. These results are consistent with the observations that
-catenin but not the RGS domain of rAxin associated with the APC
mutants in SW480 and DLD-1 cells.
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Effect of rAxin on the Interaction of APC with -Catenin--
We
have found that rAxin-(298-506) directly binds to
-catenin-(175-423), which contains armadillo repeats 2-7 (5). APC interacts with the armadillo repeats 2-10 of
-catenin (12). Therefore, we next examined whether rAxin and APC share the binding site on
-catenin. GST-
-catenin bound to MBP-rAxin-(298-506) and
MBP-APC-(959-1338) in a dose-dependent manner, and their
Kd values were calculated to be 227 nM
and 273 nM, respectively (data not shown).
MBP-rAxin-(298-506) inhibited the binding of MBP-APC-(959-1338) to
GST-
-catenin in a dose-dependent manner (Fig.
3A). MBP-rAxin (full-length)
also inhibited their binding although the inhibitory efficiency was
less than MBP-rAxin-(298-506) (Fig. 3A). Furthermore, we
examined the effect of rAxin-(1-529), which contains the binding sites
for APC and
-catenin, on the interaction of
-catenin with MBP-APC-(1211-1787), which binds to both
-catenin and rAxin. Although GST-rAxin-(1-529) bound to MBP-APC-(1211-1787) in a
dose-dependent manner, it did not affect significantly the
interaction of GST-
-catenin with MBP-APC-(1211-1787) (Fig.
3B). These results are consistent with the results that in
APC-(1211-1787)
-catenin prefers APC-(1211-1495) to
APC-(1475-1787); inversely the RGS domain binds to APC-(1475-1787) but not to APC-(1211-1495). Taken together, although the
-catenin-binding sites of rAxin and APC do not simultaneously bind
to
-catenin,
-catenin does not compete with rAxin for the binding
to APC when they are full-length proteins. Furthermore, since rAxin has
distinct binding sites for APC and
-catenin, these three proteins
could make a complex.
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Down-regulation of -Catenin by rAxin--
To investigate
whether rAxin regulates the stabilization of
-catenin, pulse-chase
analysis in COS cells expressing HA-
-catenin was performed. Although
equivalent amounts of HA-
-catenin were immunoprecipitated with the
anti-HA antibody from the lysates of COS cells expressing
HA-
-catenin alone and coexpressing HA-
-catenin and Myc-rAxin as
assessed by immunoblot analysis (data not shown), pulse-labeled.
HA-
-catenin gradually decreased with a half-life of 4 h (Fig.
4). When Myc-rAxin was cotransfected,
HA-
-catenin exhibited a shorter half-life (Fig. 4). These results
indicate that rAxin has an activity to stimulate the down-regulation of
-catenin.
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ACKNOWLEDGEMENTS |
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We thank Drs. T. Akiyama, K. Morishita, A. Nagafuchi, Sh. Tsukita, and E. Tahara for their plasmids and cell lines. We wish to thank the Research Center for Molecular Medicine, Hiroshima University School of Medicine, for the use of their facilities.
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FOOTNOTES |
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* This work was supported by grants-in-aid for scientific research and for scientific research on priority areas from the Ministry of Education, Science, and Culture of Japan (1997) and by grants from the Yamanouchi Foundation for Research on Metabolic Disorders (1997), the Kato Memorial Bioscience Foundation (1997), and the Naito Foundation (1997).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 81-82-257-5130;
Fax: 81-82-257-5134; E-mail: akikuchi{at}mcai.med.hiroshima-u.ac.jp.
1
The abbreviations used are: GSK-3, glycogen
synthase kinase-3
; APC, adenomatous polyposis coli; FAP, familial
adenomatous polyposis; aa, amino acid(s); RGS, regulators of G protein
signaling; G protein, GTP-binding protein; GST, glutathione
S-transferase; MBP, maltose-binding protein; HA,
hemagglutinin.
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REFERENCES |
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