COMMUNICATION
Phosphorylation of Axin, a Wnt Signal Negative Regulator, by Glycogen Synthase Kinase-3beta Regulates Its Stability*

Hideki YamamotoDagger , Shosei KishidaDagger §, Michiko KishidaDagger , Satoshi IkedaDagger , Shinji Takada, and Akira KikuchiDagger parallel

From the Dagger  Department of Biochemistry, Hiroshima University School of Medicine, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan, § PRESTO, Japan Science and Technology Corporation, Hiroshima 734-8551, Japan, and the  Center for Molecular and Developmental Biology, Faculty of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan

    ABSTRACT
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Axin forms a complex with glycogen synthase kinase-3beta (GSK-3beta ) and beta -catenin and promotes GSK-3beta -dependent phosphorylation of beta -catenin, thereby stimulating the degradation of beta -catenin. Because GSK-3beta also phosphorylates Axin in the complex, the physiological significance of the phosphorylation of Axin was examined. Treatment of COS cells with LiCl, a GSK-3beta inhibitor, and okadaic acid, a protein phosphatase inhibitor, decreased and increased, respectively, the cellular protein level of Axin. Pulse-chase analyses showed that the phosphorylated form of Axin was more stable than the unphosphorylated form and that an Axin mutant, in which the possible phosphorylation sites for GSK-3beta were mutated, exhibited a shorter half-life than wild type Axin. Dvl-1, which was genetically shown to function upstream of GSK-3beta , inhibited the phosphorylation of Axin by GSK-3beta in vitro. Furthermore, Wnt-3a-containing conditioned medium down-regulated Axin and accumulated beta -catenin in L cells and expression of Dvl-1Delta PDZ, in which the PDZ domain was deleted, suppressed this action of Wnt-3a. These results suggest that the phosphorylation of Axin is important for the regulation of its stability and that Wnt down-regulates Axin through Dvl.

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Genetic and biochemical analyses have revealed that there are components that are structurally and functionally conserved in the Wnt signaling pathway among flies, frogs, and mammals (1-3). In mammals these include Wnt, frizzled, Dvl, GSK-3beta ,1 beta -catenin, and Lef/Tcf, which are homologous to the Drosophila proteins Wg, Dfz2, Dsh (Dishevelled), Shaggy, Armadillo, and Pangolin, respectively. The current model for the Wnt signaling pathway proposes that in the absence of Wnt, GSK-3beta phosphorylates beta -catenin, resulting in the degradation of beta -catenin. In response to Wnt, Dvl antagonizes GSK-3beta activity through an as yet unknown mechanism. This leads to the stabilization and the accumulation of beta -catenin. The accumulated beta -catenin translocates to the nucleus, associates with the transcriptional enhancers of the Lef/Tcf family (4-6), and stimulates gene expression such as Myc (7).

Axin was originally identified as a product of mouse fused gene (8). fused carries recessive mutations that are lethal and that cause a duplication of the embryonic axis (9, 10). Injection of Axin into Xenopus embryos causes strong axis defects, and coexpression of Axin inhibits the Xwnt8-dependent axis duplication (8). Thus, Axin is a negative regulator of the Wnt signaling pathway and inhibits axis formation. We have identified rat Axin (rAxin) and its homolog, Axil (for Axin-like), as GSK-3beta -interacting proteins (11, 12). Conductin has been identified as a beta -catenin-binding protein (13) and is identical to Axil. We have found that both Axin and Axil bind not only to GSK-3beta but also to beta -catenin and that they promote GSK-3beta -dependent phosphorylation of beta -catenin (11, 12). We have also shown that the regulators of G protein signaling (RGS) domain of rAxin directly interacts with APC and that expression of rAxin in COS and SW480 cells stimulates the degradation of beta -catenin (14, 15). Other groups have reported similar results (13, 16-19). Therefore, it appears that Axin family members down-regulate beta -catenin.

Axin enhances GSK-3beta -dependent phosphorylation of APC in addition to beta -catenin in vitro (17), and the phosphorylation of APC increases its binding to beta -catenin (20). Although Axin is also phosphorylated by GSK-3beta directly, the phosphorylation of Axin does not affect its binding to GSK-3beta and beta -catenin in vitro (11). These results indicate that beta -catenin, APC, and Axin form a complex with GSK-3beta and that the phosphorylation occurs efficiently in the complex. However, the physiological significance of the phosphorylation of Axin is not known. Therefore, we examined a role of the phosphorylation of Axin in the Wnt signaling pathway. Here we demonstrate that the phosphorylation of Axin by GSK-3beta regulates its stability, that Dvl inhibits the GSK-3beta -dependent phosphorylation of Axin, and that Wnt-3a down-regulates Axin through Dvl.

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Materials and Chemicals-- Human Dvl-1 cDNA and a synthetic peptide substrate of GSK-3 (GSK peptide 1) were provided by Drs. B. Dallapiccola and G. Novelli (Vergata University, Rome, Italy) (21) and C.W. Turck (University of California, San Francisco, CA) (22), respectively. The anti-Myc antibody was prepared from 9E10 cells. GST-GSK-3beta was purified from Escherichia coli as described (22). GST fusion proteins and MBP fusion proteins were purified from E. coli according to the manufacturer's instructions. L cells stably expressing HA-Dvl-1Delta PDZ (Dvl-1-(Delta 283-336)) were produced by transfecting pCGN/Dvl-1Delta PDZ and pNeo. To prepare Wnt-3a-conditioned medium, L cells were transfected with pGK/Wnt-3a, and a number of stably transfected clones were established (23). The anti-Axin antibody was prepared in rabbits by immunization with a recombinant fragment of rAxin-(1-229). The anti-beta -catenin and anti-GSK-3beta antibodies were purchased from Transduction Laboratories (Lexington, KY). [gamma -32P]ATP, [35S]methionine, and [35S]cysteine were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). Other materials were from commercial sources.

Plasmid Construction-- pBSKS/rAxin, pMAL-c2/rAxin, pEF-BOS-Myc/rAxin, pBJ-Myc/rAxin, pGEX-2T/GSK-3beta , and pBJ-Myc/RalBP1 were constructed as described (11, 14, 15, 22, 24). To construct pMAL-c2/Dvl-1, pBSKS/Dvl-1 was digested with XbaI, and the 2.0-kb fragment encoding Dvl-1 was inserted into the XbaI-cut pMAL-c2. pBSKS/Dvl-1Delta PDZ was constructed as follows. The 0.42-kb fragment encoding Dvl-1-(337-476) with BamHI and PstI sites was synthesized by polymerase chain reaction, digested with BamHI and PstI, and inserted into the BamHI- and PstI-cut pBSKS to generate pBSKS/Dvl-1-(337-476). pBSKS/Dvl-1 was digested with PstI and HindIII, and the 0.6-kb fragment encoding Dvl-1-(477-670) was inserted into the PstI- and HindIII-cut pBSKS/Dvl-1-(337-476) to generate pBSKS/Dvl-1-(337-670). pBSKS/Dvl-1 was digested with XbaI and BamHI, and the 0.85-kb fragment encoding Dvl-1-(1-282) was inserted into the XbaI- and BamHI-cut pBSKS/Dvl-1-(337-670) to generate pBSKS/Dvl-1Delta PDZ. Thus, Dvl-1-(283-336) (the PDZ domain) was deleted in pBSKS/Dvl-1Delta PDZ. To construct pMAL-c2/Dvl-1Delta PDZ, pBSKS/Dvl-1Delta PDZ was digested with XbaI and HindIII, and the 1.9-kb fragment encoding Dvl-1Delta PDZ was inserted into the XbaI- and HindIII-cut pMAL-c2. pMAL-c2/Dvl-1Delta PDZ was digested with HindIII, blunted with Klenow fragment, and digested with XbaI. The 1.9-kb fragment encoding Dvl-1Delta PDZ was inserted into the XbaI- and SmaI-cut pCGN to generate pCGN/Dvl-1Delta PDZ. pBJ/Myc-rAxin322/326/330A was constructed as follows. The 0.65-kb fragment encoding rAxin-(182-401), in which Ser322, Ser326, and Ser330 were mutated to Ala, was synthesized by polymerase chain reaction, digested with ClaI and XbaI, and inserted into the ClaI- and XbaI-cut pEF-BOS-Myc/rAxin-(1-181), which was obtained from pEF-BOS-Myc/rAxin to generate pEF-BOS-Myc/rAxin-(1-401,322/326/330A). To construct pEF-BOS-Myc/rAxin322/326/330A, pEF-BOS-Myc/rAxin was digested with XbaI, and the 1.3-kb fragment encoding rAxin-(402-832) was inserted into the XbaI-cut pEF-BOS-Myc/rAxin-(1-401,322/326/330A). To construct pBJ/Myc-rAxin322/326/330A, pEF-BOS-Myc/rAxin322/326/330A was digested with EcoRI, and the 2.6-kb fragment encoding Myc-rAxin322/326/330A was inserted into the EcoRI-cut pBJ-1.

Phosphorylation of Axin in Intact Cells-- COS cells expressing Myc-rAxin or Myc-rAxin322/326/330A (35-mm-diameter dish) were metabolically labeled with 32Pi (100 µCi/ml) in phosphate-free RPMI for 12 h in the presence or absence of 30 mM LiCl or 100 nM okadaic acid. The cells were lysed, and the lysates were immunoprecipitated with the anti-Myc antibody (11). The immunoprecipitates were probed with the anti-Myc antibody and subjected to autoradiography.

Pulse-Chase Analysis-- COS cells (35-mm-diameter dish) were transfected with pBJ-Myc/rAxin or pBJ/Myc-rAxin322/326/330A. After 48 h, pulse-chase analysis was performed as described (14, 25). Briefly, the cells were pulse-labeled with [35S]methionine and [35S]cysteine (50 µCi/ml) for 1 h at 37 °C. Then the cells were lysed immediately or at the indicated times following incubation with excess unlabeled methionine and cysteine in the presence or absence of 30 mM LiCl or 100 nM okadaic acid. The lysates were immunoprecipitated with the anti-Myc antibody, the precipitates were subjected to autoradiography, and then the densities of the labeled proteins were analyzed with a Fuji BAS 2000 image analyzer.

Kinase Assay-- 90 nM GST-GSK-3beta was incubated with the indicated concentrations of MBP-rAxin and MBP-Dvl-1 in 30 µl of kinase reaction mixture (50 mM Tris/HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, and 50 µM [gamma -32P]ATP (500-1500 cpm/pmol)) for 15 min at 30 °C. The samples were subjected to SDS-polyacrylamide gel electrophoresis followed by autoradiography, and then the radioactivities of the phosphorylated Axin were counted. The kinase activities of GSK-3beta for GSK peptide 1 were measured as described (11, 12, 22).

Down-regulation of Axin by Wnt-3a-- Confluent wild type L cells or L cells expressing HA-Dvl-1Delta PDZ (35-mm-diameter dish) were washed with Dulbecco's modified essential medium twice, and the indicated volume of Wnt-3a-conditioned medium, which was adjusted to a total volume of 700 µl with Dulbecco's modified essential medium, was added to the cells. After stimulation for 6 h, the cells were lysed in 100 µl of lysis buffer (11), and the lysates (20 µg of protein) were probed with the anti-Axin and anti-beta -catenin antibodies.

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Stabilization of Axin by Phosphorylation-- rAxin was phosphorylated by GSK-3beta directly in vitro, and SANDSEQQS330 of rAxin was one of the phosphorylation sites for GSK-3beta (11). First we examined whether rAxin is phosphorylated by GSK-3beta in intact cells. Myc-rAxin was phosphorylated when COS cells were metabolically labeled with 32Pi (Fig. 1A). We tried to express Myc-rAxin322/326/330A, in which Ser322, Ser326, and Ser330 were mutated to Ala, in COS cells, but its protein level was lower than that of Myc-rAxin (wild type) (Fig. 1A, lanes 3 and 4). Consistent with the protein level, the phosphorylation and apparent molecular weight of MycrAxin322/326/330A were reduced in comparison with Myc-rAxin (Fig. 1A, lanes 1 and 2). Therefore, we used LiCl, which is known to be an inhibitor of GSK-3beta (26, 27). It appeared that treatment of COS cells with LiCl decreased the phosphorylation of Myc-rAxin, whereas okadaic acid, a protein phosphatase 1 or 2A inhibitor, increased it (Fig. 1A, lanes 5-7). However, these changes by LiCl and okadaic acid were also correlated with the protein level of Myc-rAxin (Fig. 1A, lanes 8-10). LiCl decreased the protein level of Myc-rAxin in a dose-dependent manner (Fig. 1B). Consistent with the previous observations (28), treatment of COS cells with LiCl resulted in the cytoplasmic accumulation of beta -catenin (Fig. 1B). Okadaic acid prevented the decrease of Myc-rAxin by LiCl (Fig. 1B). LiCl did not affect the protein level of transfected Myc-RalBP1, an effector protein of small GTP-binding protein Ral (29) or endogenous GSK-3beta (Fig. 1C). Therefore, the effect of LiCl that reduces rAxin is not nonspecific. These results suggest that the phosphorylation of rAxin is correlated with its stability.


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Fig. 1.   Stabilization of Axin by phosphorylation. A, phosphorylation of rAxin in intact cells. COS cells expressing Myc-rAxin (wild type, WT) (lanes 1, 3, and 5-10) or Myc-rAxin322/326/330A (SA) (lanes 2 and 4) were metabolically labeled with 32Pi in the absence (lanes 1-4, 5, and 8) or presence of 30 mM LiCl (lanes 6 and 9) or 100 nM okadaic acid (OA) (lanes 7 and 10). The immunoprecipitated Myc-rAxin was subjected to autoradiography (lanes 1, 2, and 5-7) or probed with the anti-Myc antibody (lanes 3, 4, and 8-10). B, effects of LiCl and okadaic acid on the protein levels of rAxin and beta -catenin. COS cells expressing Myc-rAxin were treated with the indicated concentrations of LiCl and okadaic acid. The lysates were probed with the anti-Myc antibody. To examine the protein levels of endogenous beta -catenin, the cytosol fraction of COS cells was prepared and probed with the anti-beta -catenin antibody. C, effects of LiCl on other proteins. COS cells expressing Myc-RalBP1 were treated with the indicated concentrations of LiCl. The lysates were probed with the anti-Myc and anti-GSK-3beta antibodies. The results shown are representative of three independent experiments.

To investigate the stability of Axin by phosphorylation further, pulse-chase analysis was performed. Pulse-labeled Myc-rAxin in COS cells migrated slowly on SDS-polyacrylamide gel electrophoresis in a time-dependent manner (Fig. 2A), suggesting that Myc-rAxin was phosphorylated. Pulse-labeled Myc-rAxin did not exhibit a gel band shift and disappeared at 12 h in COS cells treated with LiCl (Fig. 2A). In contrast, okadaic acid enhanced the band shift and prevented the decay of pulse-labeled Myc-rAxin at 12 h (Fig. 2A). Pulse-labeled Myc-rAxin decreased gradually with a half-life of approximately 8 h, and pulse-labeled Myc-rAxin322/326/330A exhibited a shorter half-life (Fig. 2B). These results indicate that Axin is phosphorylated by GSK-3beta in intact cells and that the phosphorylated form is more stable than the unphosphorylated form.


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Fig. 2.   Pulse-chase analysis. A, effects of LiCl and okadaic acid (OA) on pulse-labeled Myc-rAxin. COS cells expressing Myc-rAxin were pulse-labeled with [35S]methionine and [35S]cysteine for 1 h and lysed at the indicated time in the presence of LiCl or okadaic acid. B, degradation of rAxin mutant. COS cells expressing Myc-rAxin (open circle ) and Myc-rAxin322/326/330A () were pulse-labeled with [35S]methionine and [35S]cysteine, and pulse-chase analysis was carried out. The incorporation of 35S into Myc-rAxin or its mutant was analyzed with a Fuji BAS 2000 image analyzer and expressed as the percentage of the value at time 0. The results shown are the means ± S.E. of four independent experiments.

Inhibition of GSK-3beta -dependent Phosphorylation of Axin by Dvl-- Drosophila Dsh encodes a cytoplasmic protein of unknown biochemical function in the Wg signaling pathway (1-3). In mammals, dvl-1, -2, and -3 genes have been isolated as homologs of Dsh (21, 30, 31). It has been shown that Dsh antagonizes shaggy, a fly homolog of GSK-3beta , in the Wg signaling pathway (2, 3), and that overexpression of Dvl-1 in Chinese hamster ovary cells inhibits GSK-3 activity as measured by the GSK-3-mediated phosphorylation of tau proteins (32). However, little is known about the biochemical pathway leading from Dvl to GSK-3beta . Therefore, we examined whether Dvl-1 affects the phosphorylation of rAxin by GSK-3beta in vitro. MBP-Dvl-1 itself was not phosphorylated by GST-GSK-3beta (data not shown). GST-GSK-3beta phosphorylated MBP-rAxin in a time-dependent manner (11) (Fig. 3A). MBP-Dvl-1 inhibited this phosphorylation of MBP-rAxin (Fig. 3A). This inhibitory activity of MBP-Dvl-1 was dose-dependent, and MBP alone did not inhibit the GST-GSK-3beta -dependent phosphorylation of MBP-rAxin (Fig. 3B). Dvl has the PDZ domain, and disruption of the PDZ domain abolishes its activity in the Wg-Armadillo pathway and in the Xenopus axis induction assay (33, 34). Deletion of the PDZ domain from Dvl-1 (MBP-Dvl-1Delta PDZ) greatly reduced its activity to inhibit the phosphorylation of MBP-rAxin by GST-GSK-3beta (Fig. 3B). Inhibition of the phosphorylation of MBP-rAxin by MBP-Dvl-1 was not recovered even though the amounts of MBP-rAxin increased (Fig. 3C). Lineweaver-Burk plots indicated that the Km and Vmax values of MBP-rAxin for GST-GSK-3beta in the absence of MBP-Dvl-1 were 131 nM and 4.3 nmol/min/mg, respectively, and that those in the presence of MBP-Dvl-1 were 129 nM and 2.5 nmol/min/mg (Fig. 3C). These results suggest that Dvl-1 inhibits the GSK-3beta -dependent phosphorylation of Axin in a noncompetitive manner.


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Fig. 3.   Inhibition of GSK-3beta -dependent phosphorylation of Axin by Dvl. A, time course. 90 nM GST-GSK-3beta and 250 nM MBP-rAxin were incubated with in the presence of 1 µM MBP-Dvl-1 or MBP for the indicated periods. B, dose dependence. GST-GSK-3beta and MBP-rAxin were incubated with the indicated concentrations of MBP-Dvl-1 (), MBP-Dvl-1Delta PDZ (black-square), or MBP (open circle ) for 15 min. C, mode of inhibitory action of Dvl. GST-GSK-3beta was incubated with the indicated concentrations of MBP-rAxin in the presence of 1 µM MBP (open circle ) or 1 µM MBP-Dvl-1 (). Inset, Lineweaver-Burk plot analysis. The results shown are representative of three independent experiments.

This is the first demonstration showing that Dvl inhibits the function of GSK-3beta directly. However, it is not likely that Dvl-1 inhibits GSK-3beta activity itself, because MBP-Dvl-1 did not affect the phosphorylation of synthetic peptide substrate, which is designed from glycogen synthase, by GST-GSK-3beta (data not shown). We have recently found that Dvl-1 directly binds to Axin and that the binding of Dvl-1 to Axin does not affect the interaction of GSK-3beta with Axin.2 It is possible that the binding of Dvl to Axin induces the structural change of the Axin complex; therefore GSK-3beta does not effectively phosphorylate Axin. However, higher concentrations (µM order) of Dvl-1 are required to inhibit the GSK-3beta -dependent phosphorylation of Axin in our in vitro experiments. Therefore, modification of Dvl such as phosphorylation could be necessary to act on the Axin complex in intact cells. These results suggest that Dvl may regulate the stability of Axin.

Down-regulation of Axin by Wnt-3a-- Finally we examined whether Wnt signal regulates the stability of endogenous Axin in intact cells. Although Wnt proteins are secretory, they predominantly bind to the cell surface or extracellular matrix. Small amounts of biologically active Wnt-1 or Wg can be found in culture medium conditioned by cells expressing these proteins (35, 36). The Wg-conditioned medium from Schneider cells increases the level of Armadillo in Drosophila disc cells and inactivates GSK-3 in 10T1/2 fibroblasts (35, 37). Based on assays carried out with mammalian cell lines and Xenopus embryos, the Wnt proteins can be classified into two groups, Wnt-1 and Wnt-5a classes (38-40). The Wnt-1 class includes Wnt-1, Wnt-2, Wnt-3, Wnt-3a, and Wnt-8, which have activities to transform the cells and to accumulate cytoplasmic beta -catenin, whereas the Wnt-5a class includes Wnt-4, Wnt-5a, Wnt-5b, Wnt-7b, and Wnt-11, which do not exhibit the transformation and beta -catenin accumulation activities. Because Wnt-3a displays characteristics similar to those of Wnt-1, we prepared Wnt-3a-containing conditioned medium. In these experiments we used mouse fibroblast L cells, because the changes in the expression level of beta -catenin by Wnt are easily observed due to little expression of cadherin in the cells (15, 23, 41). Furthermore, Western blot analyses with the anti-Axin antibody demonstrated that Axin is most abundant in L cells among various cell lines including SW480, NIH3T3, COS, and Chinese hamster ovary cells (data not shown). Wnt-3a conditioned medium induced the accumulation of beta -catenin in L cells in a dose-dependent manner (Fig. 4A). In contrast, Wnt-3a decreased Axin (Fig. 4A). Control conditioned medium did not affect the amounts of Axin and beta -catenin (data not shown). To examine whether Dvl is involved in this action of Wnt-3a, we established L cells, which express HA-Dvl-1Delta PDZ stably. Wnt-3a-induced increase of beta -catenin and decrease of Axin were suppressed in L cells expressing HA-Dvl-1Delta PDZ (Fig. 4B). These results indicate that Wnt not only accumulates beta -catenin but also down-regulates Axin through Dvl.


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Fig. 4.   Down-regulation of Axin by Wnt-3a. A, effect of Wnt-3a on wild type L cells. L cells were treated with the indicated amounts of Wnt-3a-conditioned medium for 6 h, and the lysates were probed with the anti-Axin and anti-beta -catenin antibodies. B, effect of Wnt-3a on L cells expressing HA-Dvl-1Delta PDZ stably. Wild type L cells (WT) and L cells expressing HA-Dvl-1Delta PDZ (Dvl-1Delta PDZ) were treated with 170 µl of Wnt-3a-conditioned medium for 6 h. The results shown are representative of three independent experiments.

We have recently found that in COS cells Axin interacts with GSK-3beta , beta -catenin, and APC in a high molecular mass complex with a molecular mass of more than 103 kDa on gel filtration column chromatography (15). In L cells beta -catenin is present in the high molecular mass complex in the absence of Wnt-3a, whereas addition of Wnt-3a to L cells increases beta -catenin in a low molecular mass complex with a molecular mass of 200-300 kDa (15). In L cells expressing Axin, Wnt-3a-induced increase of beta -catenin in the low molecular mass complex is not observed (15). These results suggest that a balance between the high and low molecular mass complexes containing beta -catenin is closely regulated and that Axin plays a role in limiting the accumulation of beta -catenin in the low molecular mass complex. Wnt may regulate the assembly of the complex consisting of Axin, APC, beta -catenin, and GSK-3beta and induce the dissociation of beta -catenin from the complex. It is possible that beta -catenin free from the complex is accumulated, binds to different partners such as Lef/Tcf, and transmits the signals. Our results suggest that the Wnt signal could act on the Axin complex through Dvl, resulting in the inhibition of the GSK-3beta -dependent phosphorylation of Axin and the degradation of Axin. Degradation of Axin due to hypophosphorylation may induce the dissociation of beta -catenin from the complex by decreasing the binding of beta -catenin to Axin. Studies to clarify the mechanism of proteolysis of Axin are under way.

    ACKNOWLEDGEMENTS

We are grateful to Drs. B. Dallapiccola, G. Novelli, and C. Turck for reagents. We thank the Research Center for Molecular Medicine, Hiroshima University School of Medicine, for the use of their facilities.

    FOOTNOTES

* This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture, Japan and by grants from the Yamanouchi Foundation for Research on Metabolic Disorders, the Kato Memorial Bioscience Foundation, and the Naito Foundation.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.

parallel 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.

2 Kishida, S., Yamamoto, H., Hino, S., Ikeda, S., Kishida, M., and Kikuchi, A. (1999) Mol. Cell. Biol., in press.

    ABBREVIATIONS

The abbreviations used are: GSK-3beta , glycogen synthase kinase-3beta ; APC, adenomatous polyposis coli; GST, glutathione S-transferase; MBP, maltose-binding protein; HA, hemagglutinin; PDZ, PSD95/Dlg/Zo-1; RalBP1, Ral-binding protein 1; kb, kilobase pair.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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