Functional Domains of Axin
IMPORTANCE OF THE C TERMINUS AS AN OLIGOMERIZATION DOMAIN*

Chie SakanakaDagger and Lewis T. WilliamsDagger §

From the Dagger  Cardiovascular Research Institute, University of California, San Francisco, California 94143-0130 and § Chiron Corporation, Emeryville, California 94608

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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To understand the mechanism of how Axin acts as an inhibitory molecule in the Wnt pathway, we generated a series of mutated forms of Axin. From the binding experiments, we defined the domains of Axin that bind glycogen synthase kinase-3beta (GSK-3beta ) and beta -catenin. We also examined the ability of each Axin mutant to inhibit lymphoid enhancer factor-1 (Lef-1) reporter activity in a cell line expressing high levels of beta -catenin. Axin mutants that did not bind GSK-3beta or beta -catenin were ineffective in suppressing Lef-1 reporter activity. Binding GSK-3beta and beta -catenin was not sufficient for this inhibitory effect of Axin. Axin mutants with C-terminal truncations lacked the ability to inhibit Lef-1 reporter activity, even though they bound GSK-3beta and beta -catenin. The C-terminal region was required for binding to Axin itself. Substitution of the C-terminal region with an unrelated dimerizing molecule, the retinoid X receptor restored its inhibitory effect on Lef-1-dependent transcription. The oligomerization of Axin through its C terminus is important for its function in regulation of beta -catenin-mediated response.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The Wnt signaling pathway controls developmental processes in both invertebrates and vertebrates (1, 2). Wnt inhibits the activity of glycogen synthase kinase-3beta (GSK-3beta )1 by an unknown mechanism, and this inhibition leads to the accumulation of cytoplasmic beta -catenin. The protein level of cytoplasmic beta -catenin is post-transcriptionally regulated by GSK-3beta (3, 4). When GSK-3beta activity is inhibited, cytoplasmic beta -catenin is stabilized, which allows it to accumulate. This accumulated cytoplasmic beta -catenin interacts with the T-cell factor/Lef-1 family of transcription factors and then enters the nucleus to enhance T-cell factor/Lef-1-dependent transcription (5, 6). The Wnt pathway is also implicated in cancer. Wnt-1 is overexpressed in certain mouse mammary tumors (7). Mutations in beta -catenin and adenomatous polyposis coli (APC) genes are found in several human cancer cell lines (8-13).

Axin is a molecule recently identified as a negative regulator of the Wnt pathway (14). In mouse, homozygous loss of function mutations in the Axin locus cause an axial duplication of embryos along with the other developmental defects. In Xenopus, overexpression of Axin inhibits ectopic axis formation by Wnt in addition to normal axis formation. This inhibitory effect of Axin was reversed by co-microinjection of the downstream molecule of the Wnt pathway, beta -catenin. These findings indicate that Axin inhibits the Wnt signaling pathway, which regulates embryonic axis formation in vertebrates.

Recently we showed that Axin inhibits T-cell factor/Lef-1-dependent transcriptional activity stimulated by either Wnt or beta -catenin (15). We and others (16-18) also showed that Axin directly interacts with GSK-3beta and beta -catenin and acts as a bridge between these molecules. It was proposed that the bridging effect of Axin explains its ability to inhibit the Wnt pathway by enhancing beta -catenin phosphorylation by GSK-3beta (16). In this study we showed that the bridging of GSK-3beta and beta -catenin is necessary but not sufficient for the inhibitory effect of Axin on Lef-1-dependent transcriptional activity. We also showed that oligomerization of Axin through its C terminus is important for its function to inhibit the Wnt pathway.

    EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
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Cell Culture and Transfection-- COS-7 cells and SW480 cells were grown in Dulbecco's modified Eagle's medium, 10% fetal calf serum. Transfections were performed by LipofectAMINETM (Life Technologies, Inc.) following the protocol of the manufacturer.

Plasmid Constructions-- All the mouse Axin expression constructs were c-Myc epitope tagged (EQKLISEEDLNEED) and subcloned into pcDNA3.1 vector (Invitrogen). Full-length Axin and was generated as described previously (15). Other truncated mutant constructs were made by restriction enzyme digestion and re-ligation using the adapters, or polymerase chain reaction as follows: Delta N-Axin, digested at Asp1 site (deleted amino acids 126-299), Delta RGS-Axin, digested at first and second PstI sites (deleted amino acids 251-351), Delta NM-Axin, digested at BbrP1 site (deleted amino acids 126-604), Delta MC-Axin, digested at BbrP1 site (deleted amino acids 604-956), Delta M-Axin, digested at Bfr1 and BbrP1 sites (deleted amino acids 332-602), Delta CM-Axin, digested at BbrP1 and EcoRI sites (deleted amino acids 603-809), Delta C-Axin, digested at EcoRI site (deleted amino acids 803-956), and Delta CC-Axin was made by ligating polymerase chain reaction product (primers: 5'-TCA GCTTCGGAATTCTGTCCAGCCTTC-3' and 5'-TTCCTTTTGCGGCCGCTCATCAGA TATCAGCACGGCCCCTCACCAG-3') at EcoRI site in Axin and NotI site in pcDNA3.1 vector (deleted amino acids 892-956). Glutathione S-transferase (GST) fusion proteins (GST-beta -catenin and GST-Delta N-Axin) were constructed in pGEX-5X-1 vector (Amersham Pharmacia Biotech), expressed in Escherichia coli BL21 cells, and purified on glutathione-Sepharose 4B. The mouse full-length beta -catenin cDNA was a generous gift from Dr. A. Nagafuchi (Dept. of Cell Biol., Faculty of Med., Kyoto University, Japan). The retinoid X receptor (RXR) and the ecdyson receptor (EcR) were derived from pVgRXR (Invitrogen) and fused to the Delta C-Axin at EcoRI and NotI sites using the adaptors to generate Delta C-RXR-Axin and Delta C-EcR-Axin.

In Vitro Translation and in Vitro Binding-- Axin constructs in pcDNA3.1 vector were in vitro translated by TNT T7 coupled rabbit reticulocyte system (Promega). In vitro binding experiments were performed essentially as described previously (15). For GSK-3beta and Axin binding, partially purified GSK-3beta (Upstate Biotechnology) was immobilized to Dynabeads (Dynal) through anti-GSK-3beta antibody (Transduction Laboratory), incubated with in vitro translated, [35S]methionine-labeled Axin (20 µl each) in binding buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1% Triton X-100, 0.3 M NaCl, and 1 mM phenylmethylsulfonyl fluoride). For beta -catenin and Axin binding, GST-beta -catenin immobilized to glutathione-Sepharose 4B was mixed with in vitro translated Axin (20 µl each) in binding buffer. For Axin-Axin binding, GST-Delta N-Axin immobilized to glutathione-Sepharose 4B was mixed with in vitro translated Axin. After incubating at 4 °C for 4 h, the mixtures were extensively washed and in vitro translated Axin bound to immobilized protein was analyzed by SDS-PAGE and autoradiography.

Immunoprecipitation and Immunoblotting-- 48 h after transfection, cells were lysed in lysis buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1% Triton X-100, 0.15 M NaCl, and 1 mM phenylmethylsulfonyl fluoride). Cell lysates were incubated with antibodies as indicated at 4 °C overnight, then with Dynabeads coupled to sheep anti-mouse IgG1 (Dynal) for 1 h. Immunocomplexes were washed extensively with lysis buffer and analyzed by immunoblotting. Enhanced chemiluminescence reagents (Amersham) were used for detection of the immunoblots.

Lef-1 Reporter Gene Assay-- Lef-1 reporter gene assay was performed as described previously (15). Briefly, SW480 cells were seeded at 1 × 105 cells/well in 12-well culture plate, then transfected on the next day by LipofectAMINE 48 h before the assay. Luciferase activity was measured by luciferase assay system (Promega) and beta -galactosidase activity was measured by beta -Gal Assay System II (CLONTECH) using Luminometer (Monolight 2010, Analytical Luminescence Laboratory). pCG-Lef-1 and pGL3-fos-7LEF-luciferase were kindly provided by Dr. R. Grosschedl (University of California, San Francisco). pTK-beta -galactosidase was obtained from Promega.

    RESULTS AND DISCUSSION
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Interactions of Axin Mutants with GSK-3beta -- Axin was recently identified as a GSK-3beta -binding protein by yeast two-hybrid screening (15, 16). To determine the binding domain for GSK-3beta in Axin, we made mutant constructs in which small regions of Axin were deleted (Fig. 1). First, we checked the interaction of GSK-3beta and in vitro translated Axin constructs (Fig. 2A). We found that the GSK-3beta binding domain mapped to the middle part of Axin as reported previously (16-18). The RGS domain and the C terminus region were not required for binding to GSK-3beta . The association was also assessed in COS-7 cells. Axin mutants were expressed and immunoprecipitated; the binding of GSK-3beta was assessed by immunoblotting (Fig. 3A). Consistent with the in vitro results, the middle region of Axin confers GSK-3beta binding.


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Fig. 1.   Schematic representation of the Axin deletion mutants and corresponding activities. Constructs were generated as described under "Experimental Procedures." For binding activities, + represents strong binding, and - represents comparatively weak binding.


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Fig. 2.   In vitro binding of GSK-3beta or beta -catenin with Axin. In vitro translated, 35S-labeled Axin mutants bound to GSK-3beta (A) or beta -catenin (B) immobilized to Sepharose were analyzed by 10% SDS-PAGE and autoradiography (upper panels). 5 µl each of the in vitro translated Axin mutants were analyzed by SDS-PAGE (10%) and autoradiography (lower panels).


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Fig. 3.   In vivo binding of GSK-3beta with Axin or beta -catenin. Axin mutants tagged with c-Myc epitope were transiently transfected in COS-7 cells. Cell lysates were immunoprecipitated either with anti c-Myc antibody (Pharmingen) (A) or with anti beta -catenin antibody (Zymed Laboratories Inc.) (B) and blotted with GSK-3beta antibody (Transduction Laboratory). The expressions of Axin mutants were confirmed by immunoblotting with c-Myc antibody.

Interaction of Axin with beta -Catenin-- Next, we examined the interaction of beta -catenin and Axin deletion mutants. From the in vitro binding experiments using recombinant beta -catenin and in vitro translated Axin mutants, the beta -catenin binding domain in Axin overlapped with the GSK-3beta binding domain but extended further toward the C terminus (Fig. 2B). Neither the RGS domain nor the DIX domain was required for beta -catenin binding (Fig. 1).

Bridging Effect of Axin between GSK-3beta and beta -Catenin-- In a previous study (15), we showed that Axin is a bridging molecule between GSK-3beta and beta -catenin. For example in COS-7 cells or 293 cells, beta -catenin and GSK-3beta do not form a complex unless Axin is exogeneously expressed. We measured this complex formation by detecting GSK-3beta in the beta -catenin immunocomplex by immunoblotting. Among Axin mutants, FL-, Delta RGS-, Delta N-, Delta CM-, Delta C-, and Delta CC-Axin provide this bridging function (Fig. 3B). These data suggested that the bridging function is mediated through the central region of Axin.

Inhibition of Lef-1 Transcriptional Activity in Colon Cancer Cells-- Using a Lef-1 reporter gene assay, we have shown that Axin inhibits Lef-1 transcriptional activity in SW480 cells, a colon cancer cell line that carries an APC mutation (15). In this cell line, cytosolic beta -catenin is stabilized and accumulates, causing an increase in Lef-1 transcriptional activity (12, 13). We examined the inhibitory effect of Axin deletion mutants in SW480 cells. FL-, Delta RGS-, and Delta N-Axin showed inhibitory effects in the Lef-1 reporter gene assay, and Axin mutants deficient in the bridging function (Delta NM-, Delta MC-, and Delta M-) did not show any inhibition (Fig. 4). This result suggests that Axin binding to both GSK-3beta and beta -catenin is necessary for inhibition of Lef-1 reporter gene transcription. This is consistent with the hypothesis that a major effect of Axin is to facilitate GSK-3beta mediated phosphorylation of beta -catenin (16). However, Delta CM-, Delta C-, and Delta CC-Axin also lacked the inhibitory effect, even though those mutants can act as bridging molecules between GSK-3beta and beta -catenin (Fig. 3B). Therefore the bridging effect is not sufficient for Axin to suppress Lef-1-dependent transcription. An additional function of Axin must be provided by its C-terminal region to allow inhibition of Lef-1-dependent transcription.


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Fig. 4.   Lef-1 reporter gene assay in SW480 cells. Axin mutants (0.3 µg each) were transfected with other plasmids as follows: pCG-Lef-1 (0.02 µg), pGL3-fos-7LEF-luciferase (0.2 µg), and pTK-beta -galactosidase (0.03 µg). The ratio of luciferase activity to beta -galactosidase activity varied less than 10% among samples. Each bar represents mean ± S.E. of the relative Lef-1 reporter gene activity compared with pcDNA3.1 vector transfected sample. Similar results were obtained in four separate transfections.

The C Terminus Region of Axin Is Required for Its Oligomerization-- Despite their bridging function, Axin mutants with C-terminal deletions did not inhibit Lef-1 reporter activity in SW480 cells (Fig. 4). The C terminus of Axin contains a DIX domain, which shares loose homology with Dishevelled (14, 19). We tested whether Axin binds Dishevelled by overexpressing each protein in COS-7 cells but could not detect any interaction (data not shown). We then examined whether Axin binds to itself using in vitro translated protein. In this assay, recombinant GST-Delta N-Axin bound to in vitro translated FL-Axin but not to Delta MC-, Delta CM-, Delta C-, and Delta CC-Axin (Fig. 5). These data indicate that the C-terminal half of Axin contains a domain that mediates oligomerization. Although binding sites for GSK-3beta and beta -catenin in Axin overlap, C-terminally deleted Axin mutants, as well as FL-Axin, could bind both molecules simultaneously in vitro (data not shown). Because Delta CM- and Delta CC-Axin did not bind to Delta N-Axin, both CM region (amino acids 603-809) and DIX domain appeared to be crucial for oligomerization.


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Fig. 5.   In vitro binding of Axin with Axin. In vitro translated 35S-labeled Axin mutants bound to GST-Delta N-Axin were analyzed by 10% SDS-PAGE and autoradiography (A). 5 µl each of the in vitro translated Axin mutants were subjected to SDS-PAGE (10%) and autoradiography (B).

The Dimerized Delta C-Axin Restored Its Inhibitory Effect on Lef-1 Reporter Gene Activity-- To test whether oligomerization of Axin is important for its function, we fused Delta C-Axin to the RXR, which is known to form a homodimer in vivo (20). Delta C-RXR-Axin was able to inhibit Lef-1 reporter gene activity in SW480 cells (Fig. 6.). Delta C-Axin fused to EcR, which does not form a homodimer (21), lacked the inhibitory effect on Lef-1 reporter gene assay. Both Delta C-RXR-Axin and Delta C-EcR-Axin could bind GSK-3beta and beta -catenin in vivo (data not shown). This result suggest that oligomerization of Axin as well as bridging of GSK-3beta and beta -catenin is necessary for the function of Axin.


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Fig. 6.   Lef-1 reporter gene assay in SW480 cells with Delta C-Axin constructs. Delta C-Axin constructs (Delta -Axin, Delta C-RXR-Axin and Delta C-EcR-Axin) or pcDNA3.1 vector were transfected (0.3 µg each) with pCG-Lef-1 (0.02 µg), pGL3-fos-7LEF-luciferase (0.2 µg), and pTK-beta -galactosidase (0.03 µg). Luciferase activity and beta -galactosidase activity were measured and shown as in Fig. 4.

Concluding Discussion-- In this study, we identified binding domains of GSK-3beta and beta -catenin in Axin. We showed that these domains of Axin are important for its inhibition of Lef-1-dependent transcription in colon cancer cells. In Xenopus experiments, Delta RGS-Axin acts as a dominant-negative molecule, which activates the Wnt pathway (14). It was also shown that the RGS domain in Axin binds to APC (17, 22). From our results, the RGS domain is not required for binding to GSK-3beta or beta -catenin and is not necessary for inhibition of Lef-1 reporter activity in colon cancer cells. It has been suggested that Axin acts as a negative regulatory molecule in the Wnt pathway by presenting beta -catenin to GSK-3beta as a substrate, which mediates degradation of beta -catenin (16). Yet our data using colon cancer cells expressing high levels of beta -catenin are not consistent with this simplistic model, because we found Axin mutants that link beta -catenin and GSK-3beta but do not inhibit Lef-1 reporter activity. beta -catenin is already stabilized independent of GSK-3beta activity in those cancer cells. Our results suggest that besides scaffolding a kinase and a substrate Axin has some other effects that appear to depend on the C terminus of the molecule. There are mutant mice of Axin that have a dominant kinked tail phenotype (23). In those mice, Axin gene products are likely to have a deletion at the C terminus as a result of transposon insertion. This also suggests that the C terminus of Axin has some functional importance. We showed that the C terminus region of Axin is involved in the oligomerization of Axin. The chimeric Delta C-Axin construct fused to the RXR, which mediates dimerization inhibited Lef-1 reporter activity. These data suggest that both oligomerization and bridging of GSK-3beta with beta -catenin are necessary for the function of Axin to inhibit Lef-1-dependent transcription.

    ACKNOWLEDGEMENTS

We thank Drs. R. Grosschedl and S. Hsu for the Lef-1 reporter assay, Dr. A. Nagafuchi for mouse beta -catenin cDNA, Dr. K. Ramer for helpful suggestions throughout this work and critical reading of the manuscript, Dr. T. Iwatsubo for comments on the manuscript, and B. Cheung for assistance with the preparation of this paper.

    FOOTNOTES

* This work was supported by an unrestricted award from the Howard Hughes Medical Institute.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.: 510-923-3553; Fax: 510-923-3744; E-mail: rusty_williams{at}cc.chiron.com.

    ABBREVIATIONS

The abbreviations used are: GSK-3beta , glycogen synthase kinase-3beta ; Lef-1, lymphoid enhancer factor-1; APC, adenomatous polyposis coli; RGS, regulator of G-protein signaling; GST, glutathione S-transferase; RXR, retinoid X receptor; EcR, ecdyson receptor; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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