From the Department of Genetics and Development, College of Physicians & Surgeons, Columbia University, New York, New York 10032
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ABSTRACT |
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Axin is a negative regulator of embryonic
axis formation in vertebrates, which acts through a Wnt signal
transduction pathway involving the serine/threonine kinase GSK-3 and
Axin, the product of the mouse gene originally called
Fused (1), has been shown to negatively regulate an early
step in vertebrate embryonic axis formation, through its ability to
modulate a Wnt signal transduction pathway (2). The Fused
allele (AxinFu), as well as two other spontaneous
alleles, Kinky (AxinKi) and
Knobbly (AxinKb), caused similar dominant
phenotypes characterized mainly by kinking and shortening of the tail
(1, 3, 4). AxinKi and AxinKb also
caused recessive embryonic lethality at E8-E10. A fourth allele,
AxinTgl, induced by a random transgene insertion,
had no dominant effects but caused recessive lethal embryonic defects
similar to those observed in AxinKi/Ki or
AxinKb/Kb embryos (5). Embryos homozygous for any of
the recessive lethal alleles showed frequent neuroectodermal defects,
including truncation or incomplete closure of the anterior neural
folds, as well as cardiac defects. An intriguing feature of many
homozygous embryos was a duplication of the embryonic axis, suggesting
a role for Axin in embryonic axial development (3, 5,
6).
With the aid of the AxinTgl insertional allele, the
gene was cloned, and the wild type and mutant Axin alleles
were characterized (2, 5, 7). The murine Axin gene is
ubiquitously expressed in wild type embryos and adult tissues, encoding
a major mRNA of ~4 kb1
and a minor 3-kb mRNA. The ~4-kb mRNA is found in two
isoforms that encode proteins of 956 (form 1) and 992 amino acids (form 2). Form 2 is identical to form 1 except for an insertion of 36 amino
acids at position 856, due to alternative splicing. The Axin sequence
revealed two regions of homology to other protein families as follows:
an RGS domain (8, 9) at amino acids 213-338 and a "DIX domain"
(10) at the extreme C terminus. The RGS domains of bona fide RGS
(Regulation of G-protein Signaling) proteins bind to G In both the AxinTgl and AxinKb
alleles, synthesis of the full-length mRNAs is precluded by a
transgene insertion in the former (2, 5) and a retroviral insertion in
the latter (7). As both of these alleles caused axial duplication, it
was suggested that Axin normally plays a negative regulatory
role in the response to an axis-inducing signal in early mouse
embryogenesis. This hypothesis was supported by the ability of Axin
mRNA to block dorsal axis formation, i.e. to
"ventralize," when injected into early Xenopus embryos.
Further analyses revealed that this ability is due to the inhibitory
effect of Axin on a Wnt signaling pathway required for dorsal axis
formation (2). This signaling pathway, which is closely related to the
wingless signaling pathway of Drosophila, includes GSK-3, a
serine/threonine kinase also involved in glycogen metabolism, and
This prediction has been supported by several recent studies, which
showed that Axin binds directly to GSK-3 and So far, the role of the C-terminal third of Axin (beyond the
Recombinant Plasmids--
The bait plasmid pGBT9-Axin-(632-956)
was constructed by inserting the PstI fragment of Axin
cDNA (form 1) into the PstI site of pGBT9 vector
(CLONTECH) to generate a Gal4DB-Axin fusion protein in yeast. This plasmid was sequenced to confirm that the coding sequences for Gal4DB and Axin were in frame. A series of pGBT9-Axin plasmids containing different regions of the Axin cDNA (Fig. 5) were created using convenient restriction enzyme sites. A method to
create unidirectional nested deletions of double-stranded DNA clones
using exonuclease III and mung bean nuclease was also performed to
generate pGBT9-Axin-(632-910) and pGBT9-Axin-(632-836) (Exo-Size Deletion Kit, New England Biolabs). To express N-terminal FLAG-tagged Axin proteins in 293T cell, DNA fragments containing different regions
of FLAG-Axin cDNA were cloned into a mammalian expression vector
containing a CMV promoter (pcDNA3, Invitrogen). The pCMVT7-p36 plasmid contains a full-length PP2Ac cDNA that is
tagged at the N terminus with T7 and inserted into the pCDNA3
vector. The pCMVHA-PR65 plasmid contains a full-length regulatory A
subunit of PP2A tagged with HA at N terminus (29). Three fragments of
Axin cDNA were cloned downstream of the glutathione
S-transferase (GST) gene to generate pGST-Axin-(421-810),
pGST-Axin-(632-810), and pGST-Axin-(632-956) plasmids and to produce
recombinant proteins. The PP2Ac cDNA was inserted into
a vector containing a translation initiation sequence and a FLAG-tag
sequence to create the pBFT4-PP2Ac plasmid for in
vitro transcription and translation. pGADNOT-PP2Ac
contains a full-length mouse PP2Ac cDNA cloned into a
pGADNOT prey vector (30). The pGAD424-Axin-(194-956) plasmid contains
an Axin cDNA fragment encoding amino acids 194-956 inserted
into the pGAD424 prey vector (CLONTECH). The
pGAD-PR65 plasmid contains a cDNA fragment encoding the full-length
regulatory A subunit of PP2A cloned into the pGAD prey vector (31).
Yeast Two-hybrid System--
The pGBT9-Axin-(632-956) plasmid
was co-transformed with an expression library consisting of cDNAs
from a murine macrophage cell line WEHI-3, which were cloned into the
pGADNOT prey vector (30), into the yeast Y190 strain. Yeast
transformants were grown on synthetic medium lacking leucine,
tryptophan, and histidine. The expression of his and
lacZ reporter genes were used to assay for clones encoding
proteins that associate with Axin. The positive colonies were then
grown on synthetic medium lacking only leucine to lose the
pGBT9-Axin-(632-956) plasmid and to test for absence of
In Vitro Binding Assay with GST-Axin--
pGST-Axin-(421-810),
pGST-Axin-(632-810), and pGST-Axin-(632-956) plasmids were used
to express and purify recombinant GST fusion proteins as
described previously (32). PP2Ac RNA was transcribed
in vitro from pBFT4-PP2Ac using T7 RNA
polymerase. PP2Ac protein was translated with reticulocyte
lysate and labeled with [35S]methionine in
vitro (Promega). In vitro binding of GST-Axin and
PP2Ac was essentially as described (32). The labeled
PP2Ac proteins were incubated with GST or GST-Axin proteins
in the association buffer, and protein complexes were precipitated with
glutathione-Sepharose and analyzed by SDS-PAGE and autoradiography.
Transfection--
293T cells were transfected by a calcium
phosphate-mediated transfection method (33) at 24 h after plating
(2.0 × 106 cells per 100-mm dish). Ten mg of each
plasmid DNA was used in each reaction, and sonicated salmon sperm DNA
was used as a supplement to maintain the same DNA concentration in each
transfection. Approximately 48 h after transfection, cells were
lysed for protein expression and binding analyses.
Immunoblot Analysis--
Protein extracts or immunoprecipitated
complexes were subject to immunoblotting as described (34). Anti-FLAG
(Kodak), anti-T7 conjugated with alkaline phosphatase (Novagen), or
anti-Myc (Calbiochem) monoclonal antibodies were used to analyze the
presence of tagged proteins in transfected cells. Except for anti-T7,
bound antibodies were then detected with a goat anti-mouse IgG alkaline
phosphatase-conjugated secondary antibody (American Qualex) and
visualized by the chromogenic substrate reaction.
Co-immunoprecipitation Analysis--
293T cells were lysed in a
buffer containing 50 mM Tris-HCl (pH 8.0), 200 mM NaCl, 20 mM NaF, 20 mM
Identification of Axin-binding Proteins--
A cDNA sequence
encoding a C-terminal segment of Axin (amino acids 632-956, form 1)
was cloned into the pGBT9 bait vector to produce a Gal4-DNA-binding
domain-Axin fusion protein for yeast two-hybrid screening (35). Because
Axin is expressed in most if not all cell types, a murine macrophage
cDNA library cloned in the pGADNOT prey vector was screened to
identify proteins that can interact with Axin. After co-transformation
of pGBT9-Axin-(632-956) with the expression library, 12 colonies out
of approximately 50,000 potential transformants survived on synthetic
medium lacking histidine (Table I,
1o screen). The growth of these colonies implied that
they contained clones encoding proteins that bound to Axin-(632-956)
and also suggested the expression of the his reporter gene
under the control of a Gal4-responsive promoter in these transformants.
The expression of a second Gal4-responsive reporter gene,
The pGADNOT prey plasmids were recovered from these nine transformants.
Only three of them exhibited the ability to bind to Axin upon
co-transformation with pGBT9-Axin-(632-956) in the yeast two-hybrid
system (Table I, 4o screen). Two of the three plasmids
contained a 1.9-kb DNA insert and the other a 1.3-kb insert. DNA
sequence analysis of the two 1.9-kb clones showed that they were
identical and encoded a mouse protein displaying 99% amino acid
identity to the Axin Interacts with PP2Ac in Vitro--
PP2A is a
heterotrimeric enzyme consisting of a catalytic subunit (C)
associated with a 65-kDa regulatory subunit (A) and a third variable
subunit (B) (36-38). In mammals, the closely related Axin Co-immunoprecipitates with PP2Ac--
To examine
the association of Axin and PP2Ac in vivo, T7
epitope-tagged PP2Ac and three different FLAG-tagged Axin
proteins were transiently expressed in 293T cells (Fig.
2, A and B).
PP2Ac was co-immunoprecipitated with FLAG-Axin-(632-956),
which contains a C-terminal polypeptide identical to the one used as
bait in the yeast two-hybrid screen, but not with FLAG-Axin-(194-475), which includes the RGS domain (Fig. 2C). The
FLAG-Axin-(632-992), derived from form 2 of Axin, also co-precipitated
with PP2Ac, suggesting that both isoforms of Axin interact
with PP2Ac regardless of the 36 amino acids insertion (Fig.
2C). Similar results were also obtained when cell lysates
were first immunoprecipitated with anti-FLAG antibody followed by
immunoblotting with anti-T7 antibody (Fig. 2D). These data
agree with the conclusions from yeast two-hybrid and in
vitro biochemical analyses, confirming the ability of Axin and
PP2Ac to interact in vivo.
Axin Directly Associates with the PP2Ac Catalytic
Subunit--
The interaction of Axin and PP2Ac in various
experimental assays raised the question whether Axin binds directly to
the PP2Ac subunit or whether it might associate indirectly
with PP2Ac by binding to the regulatory A subunit, which
itself binds tightly to the catalytic subunit. A co-immunoprecipitated
assay was first performed to test whether the regulatory A subunit was
present in the Axin·PP2Ac complex. The T7-tagged
PP2Ac and HA-tagged PR65 (regulatory A subunit of PP2A)
were transiently expressed together with four different Myc- or
Flag-tagged Axin proteins in 293T cells (Fig.
3, A and B). PR65
only co-precipitated with Axin proteins that contain the PP2A-binding
domain, indicating that PR65 can indeed associate with Axin in
vivo (Fig. 3C). Next, the yeast two-hybrid assay was
used to analyze further the ability of Axin to bind to PR65. Unlike the
catalytic subunit, PR65 failed to show any interaction with Axin in
this assay (Table II). We therefore conclude that Axin can bind directly to the PP2Ac catalytic
subunit and only indirectly to PR65.
Self-interaction of Axin Proteins--
The cloning of a fragment
of Axin by the yeast two-hybrid screen with pGBT9-Axin-(632-956)
(Table I) raised the possibility that Axin may form dimers or high
order complexes with itself. The self-association of Axin was tested
using the co-immunoprecipitated assay (Fig.
4). A Myc epitope-tagged Axin-(811-956),
which could be distinguished from FLAG-tagged Axin proteins, was
transiently expressed in 293T cells (Fig. 4A). The FLAG-Axin
proteins were expressed simultaneously (Fig. 4B) and tested
for co-immunoprecipitation (Fig. 4C). The results confirmed
that Axin can interact with itself through a region located near the
extreme C terminus. The two isoforms of Axin were both capable of
association with each other and with themselves.
Localization of the PP2Ac-binding and Self-binding
Domains of Axin--
To delineate the regions of Axin capable of
mediating self-association and association with PP2Ac, a
series of deletion mutants of Axin was generated for yeast two-hybrid
analysis. This experimental system was used because of its high
sensitivity to detect protein-protein interactions (Fig.
5). Whereas Axin-(632-836) interacted
strongly with PP2Ac, deletion of amino acids 811-836
greatly reduced this interaction, suggesting the C-terminal boundary of
the PP2Ac-binding domain is within this 26-amino acid
region. Similarly, the N-terminal boundary of the
PP2Ac-binding domain was found to reside between amino
acids 632 and 744. The weak interaction between Axin-(632-810) and
PP2Ac detected in the yeast two-hybrid analysis was not
observed in the in vitro biochemical assay (Fig. 1B,
lanes 3 and 4), which may reflect a different
sensitivity of the two assays. Whereas other regions outside this
domain are not required for PP2Ac binding, an N-terminal
region, including the RGS domain, seemed to reduce the ability of
Axin-(194-956) to interact with PP2Ac.
A similar serial deletion mutant analysis revealed that the
self-binding domain of Axin is distinct from the
PP2Ac-binding domain and is located at the extreme C
terminus (Fig. 5). Both isoforms of Axin are equally potent for
self-association (compare Axin-(632-956) and Axin-(632-992)),
suggesting that the self-binding domain is C-terminal to the insertion
in form 2, i.e. within the last 100 amino acids. Moreover,
the DIX domain (amino acids 899-949 of form 1, and 935-985 of
form 2) is required for self-association, because deletion of amino
acids 910-956 completely abolished this activity. In contrast to
PP2Ac-binding, the self-binding of the Axin C terminus was
not affected by the presence of N-terminal sequences
(Axin-(194-956)).
Axin has been shown to serve as a component of a Wnt signal
transduction pathway that involves the phosphorylation of -catenin. Axin has been shown to have distinct binding sites for
GSK-3 and
-catenin and to promote the phosphorylation of
-catenin
and its consequent degradation. This provides an explanation for the
ability of Axin to inhibit signaling through
-catenin. In addition,
a more N-terminal region of Axin binds to adenomatous polyposis coli
(APC), a tumor suppressor protein that also regulates levels of
-catenin. Here, we report the results of a yeast two-hybrid screen
for proteins that interact with the C-terminal third of Axin, a region
in which no binding sites for other proteins have previously been
identified. We found that Axin can bind to the catalytic subunit of the
serine/threonine protein phosphatase 2A through a domain between amino
acids 632 and 836. This interaction was confirmed by in
vitro binding studies as well as by co-immunoprecipitation of
epitope-tagged proteins expressed in cultured cells. Our results
suggest that protein phosphatase 2A might interact with the
Axin·APC·GSK-3·
-catenin complex, where it could modulate the
effect of GSK-3 on
-catenin or other proteins in the complex. We
also identified a region of Axin that may allow it to form dimers or
multimers. Through two-hybrid and co-immunoprecipitation studies, we
demonstrated that the C-terminal 100 amino acids of Axin could bind to
the same region as other Axin molecules.
INTRODUCTION
Top
Abstract
Introduction
References
aI proteins and serve as a
GTPase-activating proteins (reviewed in Ref. 11). However, the Axin RGS
domain differed from the consensus at most of the residues that make important contacts with the Gi
a switch
regions (12), suggesting that it probably has a different function. The
DIX domain is a region of similarity between the N terminus of
Disheveled proteins (Drosophila Dsh and its vertebrate
homologs) and the C terminus of Axin (10). Whereas truncation of a
165-amino acid N-terminal segment of Dsh, including this domain,
abolished its activity in a Drosophila cell culture assay
for Wingless signaling (13), the specific role of this domain remains
obscure. Thus, the sequence of Axin provided few clues as to its function.
-catenin, a protein also involved in cell adhesion (reviewed in Ref.
14). When active, GSK-3 can phosphorylate
-catenin (15), leading to
its degradation through the ubiquitin-dependent proteolysis
system (16). In the presence of certain Wnts, GSK-3 is inhibited
(through an unknown mechanism involving the cytoplasmic protein Dsh),
allowing
-catenin to accumulate in the cytosol and to interact with
transcription factors of the LEF/Tcf family (17, 18).
-Catenin and
LEF/Tcf then translocate to the nucleus, where they bind to DNA and
activate target genes, which, in the early Xenopus embryo,
include the homeobox gene Siamois (19-21). Through
co-injection experiments, Axin was found to inhibit this signaling
pathway at a level downstream of Wnt, Dsh, and GSK-3 but upstream of
-catenin and Siamois. Thus, it was proposed that Axin, directly or
indirectly, stimulated the phosphorylation of
-catenin by GSK-3
(2).
-catenin, through
distinct domains at amino acids 477-561 and 561-630, respectively (22-25). By simultaneously binding GSK-3
and
-catenin, Axin
appears to promote the phosphorylation of
-catenin on
serine/threonine residues. Axin also binds to APC, another protein
implicated in the regulation of
-catenin (26-28), through its RGS
domain (22). The role of APC binding in the function of Axin remains
unclear; a truncated Axin lacking the entire N terminus, including the RGS domain, still promoted the turnover of
-catenin (22) in mammalian cells, whereas an internal deletion of only the RGS domain
abolished the ventralizing ability of Axin in Xenopus
embryos (2).
-catenin binding region) is unclear. To identify proteins that interact with this region of Axin, we performed a yeast two-hybrid screen using the C-terminal 324 amino acids. We have thus identified a
region of Axin that binds to the serine-threonine phosphatase PP2A. Our
results suggest that PP2A may interact with the complex containing
Axin, APC, GSK-3 and
-catenin, where it could serve to antagonize
the effects of the kinase GSK-3. We also identified a C-terminal region
of Axin that can bind to itself, suggesting that the protein may exist
as a dimer or multimer.
EXPERIMENTAL PROCEDURES
-galactosidase activity in the absence of the bait vector. To
recover the pGADNOT plasmids, extracts of plasmid DNA from yeast were
introduced into Escherichia coli JM83 by electroporation. Each of the pGADNOT plasmids was then co-transformed with
pGBT-Axin-(632-956) plasmid into yeast Y190. Transformants were grown
on synthetic medium lacking leucine and tryptophan and tested the
ability to bind Axin in the
-galactosidase filter assay. Inserts of
cDNA clones showing interaction with pGBT9-Axin-(632-956) were sequenced.
-glycerophosphate, 100 mM sodium vanadate, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 2 mg/ml leupeptin, 2 mg/ml antipain, 0.1% Triton X-100, and 0.5% Nonidet P-40. Protein complexes were
immunoprecipitated with monoclonal anti-FLAG or polyclonal
anti-PP2Ac antibody (Promega), followed by immunoblotting analysis.
RESULTS
-galactosidase, was found in 9/12 of these colonies (Table I,
2o screen). All nine of these clones tested negative for
-galactosidase in the absence of the bait vector
pGBT9-Axin-(632-956) (Table I, 3o screen).
Summary of identification of Axin-binding proteins by yeast two-hybrid
screening
medium (1°), followed by the expression of a
second reporter gene (2°),
-galactosidase (
-Gal). The 3°
screen was done by losing the bait pGBT9-Axin-(632-956) plasmid in
those transformants. Finally, pGADNOT prey plasmids were recovered and
cotransformed with the bait vector containing either Gal4DB or
Gal4DB-Axin to reconstitute the expression of the
-galactosidase
reporter gene (4°). +, positive;
, negative; NA, not applicable.
isoform of the catalytic subunit of rat and human
serine/threonine protein phosphatase 2A
(PP2Ac).2 The
1.3-kb clone encoded a C-terminal segment of Axin protein (amino acids
831-956 of form 1). These data suggest that Axin binds to
PP2Ac and also associates with itself through the
C-terminal region.
and
isoforms of PP2Ac are encoded by separate genes but are indistinguishable in function. Axin contains several predicted sites
for Ser/Thr phosphorylation (2), and Ser/Thr phosphorylation of
-catenin is thought to play a critical role in Wnt signaling (14,
15). This suggested that the Axin·PP2Ac interaction might be biologically significant. Therefore, the ability of
PP2Ac to interact physically with Axin was independently
examined by an in vitro binding assay (Fig.
1). Three different recombinant GST-Axin fusion proteins and control GST protein were bacterially expressed and
purified (Fig. 1A) and were incubated with in
vitro synthesized, 35S-labeled PP2Ac.
Analysis of protein complexes precipitated with glutathione-Sepharose
indicated that PP2Ac bound to a C-terminal region of Axin,
amino acids 632-956 (Fig. 1B, lane 5). However, two fusion
proteins lacking the last 146 amino acids, GST-Axin-(421-810) and
GST-Axin-(632-810), failed to bind PP2Ac (Fig. 1B,
lanes 3 and 4).
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Fig. 1.
Binding of Axin to the catalytic subunit of
PP2A (PP2Ac) in vitro. A,
Coomassie Blue staining of purified recombinant glutathione
S-transferase (GST, lane 1) and three
GST-Axin proteins (lanes 2-4). Numbers in
parentheses indicate the corresponding amino acids of Axin
protein (form 1). The migration of protein molecular weight markers is
shown on the left. B, association of Axin with
PP2Ac in vitro. PP2Ac (p36) was
translated in vitro with reticulocyte lysate in the presence
of [35S]methionine and incubated with the recombinant
proteins shown in A. The protein complexes were precipitated
with glutathione-Sepharose and analyzed by SDS-PAGE and
autoradiography. The 1st lane contains
35S-labeled PP2Ac in a quantity equivalent to
20% of the input for each binding reaction, analyzed directly by
SDS-PAGE and autoradiography.
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Fig. 2.
Co-immunoprecipitation of Axin with
PP2Ac. PP2Ac was cloned into a CMV
expression vector encoding a T7 epitope tag (T7-PP2Ac), and
three Axin cDNA fragments were cloned into a similar vector
encoding a FLAG epitope tag. FLAG-Axin-(632-956) contained sequences
from Axin form 1; FLAG-Axin-(632-992) contained sequences from Axin
form 2, with a 36=amino acid insertion at position 856. 293T cells were
either lysed without transfection (293T lanes) or
transfected with T7-PP2Ac alone ( lanes) or
with T7-PP2Ac plus one of the three FLAG-Axin plasmids.
A, cell lysates were immunostained with FLAG antibody,
showing the expression of three FLAG-Axin proteins. B, cell
lysates were immunoblotted with T7 antibody, showing the expression of
T7-PP2Ac. Protein complexes were either immunoprecipitated
(IP) with PP2Ac antibody, separated by SDS-PAGE,
and immunoblotted with FLAG antibody (C), or
immunoprecipitated with FLAG antibody, separated by SDS-PAGE, and
immunoblotted with T7 antibody (D). The FLAG-Axin-(632-956)
and FLAG-Axin-(632-992), but not FLAG-Axin-(194-475), co-precipitated
with T7-PP2Ac from 293T cells. The presence of FLAG-Axin
and T7-PP2Ac proteins was visualized by an alkaline
phosphatase-mediated chromogenic substrate reaction. The
brackets indicate the Axin proteins and
arrowheads the PP2Ac protein.
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Fig. 3.
Co-immunoprecipitation of Axin with the
regulatory A subunit of PP2A. HA-tagged A subunit (PR65) and
T7-tagged PP2Ac were transiently co-expressed with four
different fragments of Axin protein, which were either Myc- or
Flag-tagged, in 293T cells. Lysates from transfected cells were
immunoblotted (IB) with either -HA antibody, to detect
the expression of PR65 (A), or
-Myc and
-Flag
antibodies (arrowheads and arrows, respectively)
to detect the expression of Axin fragments (B). Protein
complexes were immunoprecipitated (IP) with either
-Myc
or
-Flag antibody, followed by immunoblotting with
-HA antibody
(C). Axin-(194-956), -(632-956), and -(632-992) fragments
but not Axin-(194-475) co-precipitated with PR65 as indicated.
M represents the protein molecular weight markers.
The regulatory A subunit of PP2A, PR65, does not interact with Axin
in the yeast two-hybrid system.
-galactosidase (
-Gal) assay;
, only white color
after 12 h of
-galactosidase assay.
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Fig. 4.
Axin self-association detected by
co-immunoprecipitation. A vector encoding Myc-tagged
Axin-(811-956) was transfected into 293T cells alone ( lanes) or together with one of three FLAG-tagged Axin
vectors. Cell lysates from non-transfected cells (293T
lanes) or transfected cells were subject to immunostaining and
immunoprecipitation-immunoblot analyses. Cell lysates were
immunostained with
-Myc antibody, showing the expression of
Myc-tagged Axin-(811-956) (A), or immunostained with
-FLAG antibody, showing the expression of FLAG-tagged Axin proteins
(B). Protein complexes were immunoprecipitated
(IP) with
-FLAG, followed by immunoblotting with
-Myc
(C). FLAG-Axin-(632-956) and -(632-992) but not
-(194-475) were capable of self-association with Myc-Axin-(811-956).
Signal above and below the arrowhead is due to alkaline
phosphatase-conjugated secondary antibody reacting with primary
antibody used in immunoprecipitation.
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Fig. 5.
Determination of PP2Ac binding
and Axin self-binding domains in the yeast two-hybrid (yeast
2-Hyb.) system. The schematic representation indicates
the segments of Axin protein tested for interaction with
PP2Ac or with Axin-(194-965). The -galactosidase filter
assay was performed on Leu
, Trp
plates, and
intensity of color was scored after 5 h at 30 °C. +++, dark
blue color developed in <2 h; +, light blue color developed in >5 h;
, no blue color after >5 h. For the
-galactosidase liquid assay,
values are stated as a percentage of the highest value observed, for
Axin-(476-956). Yeast co-transformed with void bait (pGBT9) and prey
(pGAD424) vectors were used to determine the background level for the
liquid assay. ND, not determined.
DISCUSSION
-catenin by the serine/threonine kinase GSK-3. The involvement of Axin in this
pathway was originally demonstrated based on its ability to inhibit
dorsal axis formation in Xenopus embryos (2). mRNAs encoding certain members of the Wnt family, or several other proteins that function in the signaling pathway downstream from these Wnts, can
induce an ectopic dorsal axis when injected ventrally in the early
embryo (14). Axin appeared to function downstream of GSK-3 and upstream
of
-catenin, as its effect on axis formation could be overcome by
co-injection of
-catenin or the transcription factor Siamois but not
by co-injection of Wnt8, Dsh, or a dominant-negative mutant form of
GSK-3 (2). Recently, it has been demonstrated that Axin binds directly
to both GSK-3 and
-catenin, suggesting a biochemical basis for its
effects on signaling through this pathway (22-25). Axin also binds,
through its RGS domain, to APC, a protein previously shown to bind to
-catenin and promote its degradation (22). In this study, we have
used the yeast two-hybrid system, together with in vitro
binding and in vivo co-immunoprecipitation assays, to
identify two additional protein-binding domains of Axin. A region of
Axin C-terminal to the APC, GSK-3 and
-catenin-binding domains,
between amino acids 632 and 836, can bind to the catalytic subunit of
the Ser/Thr protein phosphatase PP2A, whereas a sequence at the extreme
C terminus of Axin (amino acids 856-956), including the conserved DIX
domain, can bind to the same region of other Axin molecules (Fig.
6). Processes that are regulated by
protein phosphorylation require a protein phosphatase to modulate
the effects of a protein kinase (39). Thus, the ability of Axin to bind
to PP2A suggests that this phosphatase can interact with the
Axin·APC·GSK-3·
-catenin complex, where it might modulate the effect of GSK-3 on
-catenin or other substrates. Potentially, Axin could play a role in other signaling pathways involving PP2A. The
ability of Axin to bind to itself suggests that it may function as a
dimer or higher order multimer.
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Fig. 6.
Schematic representation of Axin protein,
showing the RGS and DIX homology domains, and binding regions for APC,
GSK-3, -catenin (
-Cat),
PP2Ac, and Axin. See text for details and references.
aa, amino acid.
PP2A, one of the four major classes of Ser/Thr protein phosphatases, is
a heterotrimeric enzyme whose core dimer consists of a 36-kDa catalytic
subunit C and a 65-kDa regulatory subunit A. This core dimer can
associate with one of several regulatory B subunits ranging in size
from 54 to 130 kDa. PP2A is widely expressed and has broad substrate
specificity in vitro (36-38). Genetic studies in yeast and
Drosophila, as well as experiments in Xenopus
oocytes and mammalian cells, have implicated PP2A in a wide range of
biological processes, including cell division, transformation, cell
cycle regulation, cell fate determination, and gene expression (40,
41). However, only a few of the specific in vivo substrates
of PP2A have been identified. These are believed to include the
neuronal microtubule-associated protein tau (42), several components of
the mitogen-activated protein kinase cascade (41, 43, 44), the atypical
protein kinase C (45) and Ca2+-calmodulin-dependent protein kinase IV
(46). The function of the regulatory B subunits is not fully
understood, but they are believed to influence substrate specificity
and possibly subcellular localization of PP2A (47, 48). In addition,
several other proteins have been shown to bind to the PP2A catalytic
subunit and regulate its activity or localization, including
viral tumor antigens (49), the translation termination factor eRF1
(50), the Ser/Thr kinase casein kinase 2
(51), and the homeobox
protein Hox11 (52). The ability of Axin to bind to PP2A suggests that Axin might be yet another regulatory protein for PP2A, which could influence any of the various roles of PP2A in the cell. However, the
established role of Axin in the Wnt pathway suggests a novel role for
PP2A in this pathway.
The phosphorylation of -catenin (or Armadillo) by GSK-3 (or
shaggy/zeste-white) appears to be critical step in the
conserved signaling pathway downstream from Drosophila
wingless and certain members of the vertebrate Wnt family (14). Axin
appears to promote this phosphorylation event by simultaneously binding
to GSK-3 and
-catenin (22-25). Wnts, which are believed to bind to
receptors of the Frizzled family, inhibit GSK-3 activity through an
unknown mechanism involving the cytoplasmic protein Dsh (reviewed in
Ref. 10). This explains why these Wnts, Dsh, or dominant-negative GSK-3
can induce a dorsal axis when their mRNAs are injected into Xenopus embryos (reviewed in Ref. 14). However, the failure to identify a Wnt that is expressed in the right time and place to
induce axis formation in the Xenopus embryo has led to the suggestion that some other mechanism may trigger signal transduction through this pathway in the normal amphibian embryo (14, 53, 54).
The ability of Axin to bind to the Ser/Thr phosphatase PP2A raises the
possibility that PP2A might play a role in signal transduction through
-catenin, by opposing the effect of the kinase GSK-3. Binding to
Axin would recruit PP2A to the Axin·APC·GSK-3·
-catenin complex, where it could dephosphorylate Ser/Thr residues on
-catenin (as well as other phosphoproteins in the complex). Recent data show
that the PP2A-binding region of Axin is not required for its
ventralizing effect in the frog embryo (23).3
This indicates that the ability of Axin to negatively
regulate this signaling pathway when overexpressed does not depend on
its ability to bind to PP2A. On the contrary, PP2A might modulate the
tendency of Axin to promote the phosphorylation of
-catenin by
GSK-3. According to this model, the signal initiating embryonic axis
formation might lead to the recruitment of PP2A to the
Axin·APC·GSK-3·
-catenin complex, leading to the
dephosphorylation and stabilization of
-catenin. This model predicts
that a truncated Axin protein lacking the PP2A-binding domain would be
hyperactive, since it could stimulate the GSK-3/
-catenin interaction
without allowing modulation by PP2A. No clear-cut difference in the
ventralizing activity of full-length Axin versus such a
truncated Axin has been observed.2 However, this assay, in
which large quantities of Axin are expressed in the embryo, may be
insensitive to the ability of Axin to bind PP2A (for example, the
amount of Axin expressed may greatly exceed the amount of available
PP2A in the embryo).
Evidence that is consistent with this model comes from studies of
two spontaneous mutant alleles of Axin,
AxinFu and AxinKb. These two
alleles, which have similar dominant phenotypic effects, both produce
abnormally spliced transcripts as a result of retroviral insertions
into nearby regions of the gene. These abnormal transcripts are
predicted to encode proteins with C-terminal truncations, terminating
at residue 720 (AxinFu) or 766 (AxinKb). Whereas these truncated proteins would
retain the binding domains for APC, GSK-3, and -catenin, their
ability to bind PP2A would be either abolished or greatly reduced (Fig.
4). It has been previously concluded that the dominant effects of these
alleles are due to gain-of-function rather than loss-of-function
effects, since a deletion that removes the entire Axin gene
(by definition, a loss-of-function mutation) has no dominant effect
(55). This is consistent with the observation that Axin constructs
encoding proteins with C-terminal truncations at amino acid 724 (23) or
8103 are active in the frog embryo ventralization assay and
therefore do not appear to be loss-of-function mutants. However, the
gain-of-function effects of AxinFu and
AxinKb could be explained if the PP2A-binding region
were a regulatory domain, whose removal resulted in a hyperactive form
of Axin. Interestingly, vestigial tail (vt), a
hypomorphic allele of mouse Wnt-3a, causes a recessive
phenotype characterized by kinking and shortening of the tail (56),
very similar to the dominant phenotype of AxinFu or
AxinKb. Wnt-3a is normally expressed in
the tail bud mesoderm and is required for caudal somitogenesis (57).
Since Wnt-3a is one of the Wnt family members that signal through the
GSK-3/
-catenin pathway (58, 59), Axin is likely to function as a
negative regulator of Wnt-3a signal transduction during tail bud
development. Therefore, a hypermorphic allele of
Axin would be expected to phenocopy a
hypomorphic allele of Wnt-3a.
The self-binding domain of Axin has been localized to a region of ~100 amino acids at the extreme C terminus, which is distinct from the PP2A-binding region. The self-binding region includes the DIX domain, a 51-amino acid region of similarity between Axin and Dsh proteins. This overlap raises the possibility that Dsh, or its vertebrate homologs, might also associate with Axin through the DIX domain. The ability of Axin to bind to itself through the C terminus also suggests that it might normally form dimers or higher order multimers in the cell. Axin is made in two major isoforms, which differ by an insertion of 36 amino acids at position 856; in addition, a smaller protein identical at the C terminus, but missing the N-terminal region, may be encoded by a minor 3-kb mRNA (2). Therefore, hetero- as well as homodimers of Axin may be formed. A different protein with extensive sequence similarity to Axin and apparently similar function has recently been identified (60, 61). The last 100 amino acids of this protein are 72% similar to those of Axin, suggesting that it might be able to dimerize with Axin.
What might be the importance of self-binding for the function of
Axin? Perhaps dimerization of Axin enhances interactions between the
other proteins that bind to Axin, e.g. phosphorylation by
GSK-3 of -catenin or other proteins, or de-phosphorylation by PP2A.
A C-terminal truncated form of Axin lacking this region is active in
the frog embryo ventralization assay (23),3 implying that
self-binding is not absolutely required for the ability of Axin to
inhibit the Wnt signal transduction pathway. However, as discussed
above, this assay may be insensitive to quantitative changes in the
activity of Axin, and other assays may be required to observe the
function of this domain.
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ACKNOWLEDGEMENTS |
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We thank Kimona Alin and Stephen Goff for the expression library and Qin Ye and Howard Worman for advice on yeast two-hybrid screening.
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FOOTNOTES |
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* This work was supported by a fellowship (to W. H.) from the National Kidney Foundation and by grants (to F. C.) from the National Institutes of Health.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF076192.
Present address: Dept. of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115.
§ To whom correspondence should be addressed: Dept. of Genetics and Development, College of Physicians & Surgeons, Columbia University, 701 W. 168th St., New York, NY 10032. Tel.: 212-305-6814; Fax: 212-923-2090, E-mail: fdc3{at}columbia.edu.
The abbreviations used are: kb, kilobase pair(s); APC, adenomatous polyposis coli; PP2A, protein phosphatase 2A; GST, glutathione S-transferase; RGS, regulation of G-protein signaling; CMV, cytomegalovirus; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin.
2 The nucleotide sequence for the mouse PP2Ac gene has been deposited under accession number AF076192.
3 F. Fagotto, E.-H. Jho, L. Zeng, and F. Costantini, unpublished data.
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REFERENCES |
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