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J Biol Chem, Vol. 274, Issue 39, 27682-27688, September 24, 1999


Axin Directly Interacts with Plakoglobin and Regulates Its Stability*

Shinya KodamaDagger , Satoshi IkedaDagger , Toshimasa Asahara§, Michiko KishidaDagger , and Akira KikuchiDagger

From the Departments of Dagger  Biochemistry and § Second Surgery, Hiroshima University School of Medicine, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plakoglobin is homologous to beta -catenin. Axin, a Wnt signal negative regulator, enhances glycogen synthase kinase (GSK)-3beta -dependent phosphorylation of beta -catenin and stimulates the degradation of beta -catenin. Therefore, we examined the effect of Axin on plakoglobin stability. Axin formed a complex with plakoglobin in COS cells and SW480 cells. Axin directly bound to plakoglobin, and this binding was inhibited by beta -catenin. Axin promoted GSK-3beta -dependent phosphorylation of plakoglobin. Furthermore, overexpression of Axin down-regulated the level of plakoglobin in SW480 cells. These results suggest that Axin regulates the stability of plakoglobin by enhancing its phosphorylation by GSK-3beta and that Axin may act on beta -catenin and plakoglobin in similar manners.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell adhesion plays a central role in various biological processes, including motility, growth, and differentiation. The most direct effect of adhesion is on morphogenesis, i.e. the assembly of the individual cells into highly ordered tissues and organs through cell-cell junctions (1-3). There are two types of cell-cell junctions: desmosomes and adherens junctions (4, 5). Desmosomes have two transmembrane components, desmoglein and desmocollin, which are members of the cadherin family (6). Plakoglobin has been shown to interact with the cytoplasmic regions of desmoglein and desmocollin (7-11). The transmembrane components of adherens junctions are members of the classical cadherin family. The cytoplasmic region of cadherin forms a complex with three proteins, alpha -, beta -, and gamma -catenins (4, 12); gamma -catenin is identical to plakoglobin (13, 14). The cytoplasmic components of adherens junctions and desmosomes are necessary for linking to actin filaments and intermediate filaments, respectively (4, 12). beta -Catenin and plakoglobin are closely related and are part of the gene family that includes the Drosophila protein armadillo (15). It has been shown that beta -catenin and plakoglobin bind directly to cadherin and alpha -catenin (16-19). The N-terminal regions of beta -catenin and plakoglobin contain their alpha -catenin-binding sites. The central region, which has multiple copies of the armadillo motif, is involved in the association with cadherin, and in the case of plakoglobin, with desmoglein and desmocollin. Thus, plakoglobin is a common plaque component of both types of cell-cell junctions and is essential for sorting out of desmosomes and adherens junctions. Disruption of plakoglobin in mice causes embryonic death at embryonic days 12-16 because of severe defects in heart structure, resulting in ventricle burst and blood flooding the pericardium (20, 21). beta -Catenin knockout mutations are lethal in early mouse embryos, causing specific defects in the embryonic ectoderm cell layer (22). Therefore, beta -catenin and plakoglobin do not appear to compensate for each other in embryos.

In addition to their roles in cell-cell adhesion, beta -catenin and plakoglobin are components in the Wnt signaling pathway (19, 23-25). Wnt genes encode secreted glycoproteins required for a large number of developmental processes (26). In the fly, the Wnt-1 homolog, wingless, is required for the patterning of each segment, and for pattern formation and cell proliferation in imaginal discs (27). Multiple components of the wingless signaling pathway have been identified and characterized genetically (23-25). They include Dfrizzled2, dishevelled, shaggy, armadillo, and pangolin. Vertebrate homologs of these genes have also been identified (23-25). Armadillo is homologous to both beta -catenin and plakoglobin. Wingless expression in Drosophila embryos promotes the posttranslational accumulation of armadillo (28). Rapid accumulation and stabilization of armadillo have also been demonstrated in cultured Drosophila cells incubated with soluble wingless (29). Similarly, in mammalian cells, Wnt-1 expression leads to increasing steady state level of beta -catenin and plakoglobin (30-32). In Xenopus laevis, overexpression of beta -catenin or plakoglobin leads to ectopic axis formation, and this effect is also obtained with overexpression of Xenopus Wnt (33-35). These results suggest that beta -catenin and plakoglobin may have similar functions in the Wnt signaling pathway.

Recent biochemical studies in mammals have revealed the molecular mechanism by which Wnt regulates the stabilization of beta -catenin (23-25). In the absence of Wnt, GSK-3beta ,1 a shaggy homolog, phosphorylates beta -catenin, and the phosphorylated beta -catenin is ubiquitinated, resulting in the degradation of beta -catenin by proteasomes (36-38). Wnt binds to its receptor Frizzled, a Dfrizzled2 homolog, and inactivates GSK-3beta , probably through Dvl, a dishevelled homolog, although the mechanism is not clear (39). This leads to the stabilization of beta -catenin, and accumulated beta -catenin translocates into the nucleus where it binds to and activates the transcriptional factor Tcf/Lef, a pangolin homolog (40, 41), resulting in the expression of target genes including c-myc, c-jun, fra, and cyclin D1 (42-44). We have identified rat Axin (rAxin) and its homolog, Axil (for Axin-like), as GSK-3beta -interacting proteins (45, 46). Conductin has been identified as a beta -catenin-binding protein (47) and is identical to Axil. Both Axin and Axil bind not only to GSK-3beta but also to beta -catenin and APC (45-49) and promote GSK-3beta -dependent phosphorylation of beta -catenin and APC (45, 46, 49). Axin enhances a complex formation of phosphorylated beta -catenin with beta TrCP/FWD (50), resulting in stimulating the degradation of beta -catenin (47, 49, 51, 52). Furthermore, Dvl also binds to Axin (53, 54) and inhibits GSK-3beta -dependent phosphorylation of beta -catenin, APC, and Axin (53, 55). Thus, Axin functions as a scaffold protein and regulates the stability of beta -catenin.

Although it has been shown that the expression of Wnt-1 also stabilizes plakoglobin in several cell lines (30, 32), how the stability of plakoglobin is regulated is not fully understood. These results prompted us to examine whether Axin is involved in the regulation of the stability of plakoglobin. Here we demonstrate that Axin directly binds to plakoglobin and enhances GSK-3beta -dependent phosphorylation of plakoglobin. Further, we show that Axin degrades plakoglobin. These results suggest that Axin regulates the stability of plakoglobin via a mechanism like that by which Wnt regulates beta -catenin.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials and Chemicals-- Plakoglobin cDNA, SW480 cells, and the anti-HA and anti-MBP antibodies were kindly supplied by Drs. W. W. Franke (German Cancer Research Center, Heidelberg, Germany), E. Tahara (Hiroshima University, Hiroshima, Japan), Q. Hu (Chiron Corp., Emeryville, CA), and M. Nakata (Sumitomo Electronics, Yokohama, Japan), respectively. The anti-Myc antibody was generated from 9E10 cells. GST and MBP fusion proteins were purified from Escherichia coli according to the manufacturer's instructions. SW480 cells stably expressing Myc-rAxin were made as described (53). The anti-plakoglobin and anti-beta -catenin antibodies were purchased from Transduction Laboratories (Lexington, KY). [gamma -32P]ATP was obtained from Amersham Pharmacia Biotech. Other materials were from commercial sources.

Plasmid Constructions-- pBJ-Myc/rAxin (full length), pMAL-c2/rAxin (full length), pBSKS/rAxin (full length), pMAL-c2/rAxin-(298-832), pUC19/rAxin-(298-832), pEF-BOS-Myc/rAxin-(298-506), pMAL-c2/rAxin-(298-506), pEF-BOS-Myc/rAxin-(508-832), pBJ-Myc/rAxin-(713-832), pMAL-c2/rAxin-(713-832), pGEX-2T/beta -catenin, pGEX-2T/GSK-3beta , and pEGFP-c1/hEpsin were constructed as described (45, 51, 52, 56). To construct pGAD10/plakoglobin, the fragment encoding plakoglobin (full length) was synthesized by polymerase chain reaction and inserted into BamHI-cut pGAD10. To construct pGEX-2T/plakoglobin (full length), pGAD10/plakoglobin was digested with BamHI. The 2.2-kilobase pair fragment was inserted into pGEX-2T that had been digested with BamHI. To construct pGEX-2T/plakoglobin-(1-121), pGAD10/plakoglobin was digested with BamHI and PvuII, and the appropriate fragment was inserted into pGEX-2T that had been digested with BamHI and SmaI. To construct pBSKS/plakoglobin-(185-399), pGAD10/plakoglobin was digested with PvuII, and the appropriate fragment was inserted into the SmaI-cut pBSKS. To construct pGEX-2T/plakoglobin-(185-399), pBSKS/plakoglobin-(185-399) was digested with BamHI and EcoRV, and the appropriate fragment was inserted into pGEX-2T that had been digested with BamHI and SmaI. To construct pBSKS/plakoglobin-(399-744), pGAD10/plakoglobin was digested with PvuII and BamHI, and the appropriate fragment was inserted into pBSKS that had been digested with SmaI and BamHI. To construct pGEX-4T-1/plakoglobin-(399-744), pBSKS/plakoglobin-(399-744) was digested with NotI and EcoRI, and the appropriate fragment was inserted into pGEX-4T-1 that had been digested with NotI and EcoRI. To construct pEF-BOS-HA/plakoglobin (full length), pGAD10/plakoglobin was digested with BamHI, and the appropriate fragment was inserted into pEF-BOS-HA that had been digested with BamHI. To construct pGEX-2T/rAxin-(1-229), pBSKS/rAxin (full length) was digested with SacI and SmaI and blunted with T4 polymerase. The fragment encoding rAxin-(1-229) was inserted into the SmaI-cut pGEX-2T. To construct pMAL-c2/rAxin-(1-229), pGEX-2T/rAxin-(1-229) was digested with EcoRI and blunted with Klenow fragment and then digested with BamHI. The fragment encoding rAxin-(1-229) was inserted into pMAL-c2 that had been digested with HindIII and blunted with Klenow fragment and then digested with BamHI. To construct pBJ-Myc/rAxin-(1-353), pBSKS/rAxin (full length) was digested with SmaI and BamHI and blunted with Klenow fragment. The fragment encoding rAxin-(1-353) was inserted into pBJ-Myc that had been digested with XbaI and blunted with Klenow fragment. To construct pEGFP-c3/rAxin-(298-832), pUC19/rAxin-(298-832) was digested with SacI, and the appropriate fragment was inserted into pEGFP-c3 that had been digested with SacI.

Interaction of Plakoglobin with rAxin in Intact Cells-- COS cells transiently transfected with pBJ- and pEF-BOS-derived plasmids or SW480 cells stably expressing Myc-rAxin were lysed in the lysis buffer (20 mM Tris/HCl, pH 8.0, 1% Nonidet P-40, 137 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 40 mM beta -glycerophosphate, and 1 mM sodium orthovanadate). rAxin and its deletion mutants were tagged with Myc epitope at their N termini. Plakoglobin was tagged with HA epitope at its N terminus. The lysates (200-800 µg of protein) were immunoprecipitated with the anti-Myc antibody, and the precipitates were probed with the anti-Myc, anti-HA, and anti-plakoglobin antibodies.

Interaction of Plakoglobin with rAxin in Vitro-- For determination of the region of plakoglobin that binds to rAxin, various deletion mutants of GST-plakoglobin (each at 250 nM) were incubated with 500 nM MBP-rAxin (full length) for 1 h at 4 °C in 50 µl of reaction mixture (20 mM Tris/HCl, pH 7.5, and 1 mM dithiothreitol). GST-plakoglobin deletion mutants were precipitated with glutathione-Sepharose 4B, and then the precipitates were probed with the anti-MBP antibody. To examine the region of rAxin that binds to plakoglobin, various deletion mutants of MBP-rAxin (7.5 pmol of each) immobilized on amylose resin were incubated with 500 nM GST-plakoglobin (full length) for 1 h at 4 °C in 50 µl of reaction mixture. MBP-rAxin deletion mutants were precipitated by centrifugation, and then the precipitates were probed with the anti-plakoglobin antibody.

Kinetics of the Binding of rAxin, beta -Catenin, and Plakoglobin-- To determine the Kd value of the binding of plakoglobin to rAxin, MBP-rAxin (full length) (5 pmol) immobilized on amylose resin was incubated with various concentrations of GST-plakoglobin (full length) for 1 h at 4 °C in 40 µl of reaction mixture. To show the inhibition of the binding of plakoglobin to rAxin by beta -catenin, MBP-rAxin (full length) (3 pmol) immobilized on amylose resin was incubated with 500 nM GST-plakoglobin (full length) and various concentrations of GST-beta -catenin (full length) for 1 h at 4 °C in 40 µl of reaction mixture. MBP-rAxin was precipitated by centrifugation, and then the precipitates were probed with the anti-beta -catenin and anti-plakoglobin antibodies. The relative intensities of precipitated GST-plakoglobin and GST-beta -catenin were quantified by densitometric tracing of the stained immunoblots using the NIH image program.

Phosphorylation of Plakoglobin by GSK-3beta -- After 1.2 µM GST-plakoglobin and 180 nM GST-GSK-3beta were incubated with 100 nM MBP-rAxin 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 (500-2000 cpm/pmol)) for 30 min at 30 °C, the samples were subjected to SDS-polyacrylamide gel electrophoresis followed by autoradiography. Where specified, the radioactivities of the phosphorylated plakoglobin were counted.

Degradation of Plakoglobin by rAxin in SW480 Cells-- SW480 cells were grown on glass coverslips and transfected with pEGFP-c3/rAxin-(298-832) by TransFast Reagent (Promega Corp., Madison, WI). At 48 h after the transfection, the cells were fixed for 20 min in PBS containing 4% paraformaldehyde. After washing with PBS three times, the cells were permeabilized with PBS containing 0.2% Triton X-100 and 2 mg/ml bovine serum albumin for 4 h. The cells were washed and incubated for 1 h with the anti-plakoglobin or anti-beta -catenin antibody. After washing with PBS, they were further incubated for 1 h with AlexaTM 594 goat anti-mouse IgG (Molecular Probes, Eugene, OR). Coverslips were washed with PBS, mounted on glass slides, and viewed with a confocal laser-scanning microscope (TCS-NT®, Leica-laser-technik GmbH, Heidelberg, Germany).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Formation of Complex between Axin and Plakoglobin-- Plakoglobin shares 66% overall identity with beta -catenin. Both proteins have armadillo repeats in the central region, and the identity of their armadillo repeats is 77%. We have shown that Axin directly binds to the armadillo repeats of beta -catenin (45, 51). Therefore, we first examined whether Axin forms a complex with plakoglobin in intact cells. Myc-rAxin and HA-plakoglobin were expressed in COS cells (Fig. 1A). When the lysates of cells expressing Myc-rAxin and HA-plakoglobin were immunoprecipitated with the anti-Myc antibody, HA-plakoglobin was coprecipitated with Myc-rAxin (Fig. 1A). When the lysates expressing HA-plakoglobin alone were immunoprecipitated with the anti-Myc antibody, HA-plakoglobin was not observed in the immune complex (Fig. 1A). We next examined whether Axin forms a complex with endogenous plakoglobin in SW480 cells, because this cell line contains increased cytoplasmic beta -catenin and plakoglobin because of the truncation of APC (57). Plakoglobin in SW480 cells was observed as two bands (Fig. 1B). Although we do not know the reason for this, other reports also showed similar results with the antibody that we used (38, 58). When the lysates of SW480 cells stably expressing Myc-rAxin were immunoprecipitated with the anti-Myc antibody, endogenous plakoglobin was detected in the Myc-rAxin immune complex (Fig. 1B). To determine which region of rAxin is required for the complex formation with plakoglobin, various deletion mutants of Myc-rAxin were expressed with HA-plakoglobin in COS cells. HA-plakoglobin was coprecipitated with Myc-rAxin (full length) and Myc-rAxin-(298-506) but not with Myc-rAxin-(1-353), Myc-rAxin-(508-832), or Myc-rAxin-(713-832) (Fig. 1C). These results indicate that the region containing residues 298-506 of rAxin is necessary and sufficient for its complex formation with plakoglobin in intact cells.


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  		Fig. 1.   Formation of complex between Axin and plakoglobin in intact cells. A, interaction of plakoglobin with rAxin in COS cells. The lysates (20 µg of protein) of cells expressing HA-plakoglobin alone (lane 2) or both Myc-rAxin and HA-plakoglobin (lane 3) were probed with the anti-Myc and anti-HA antibodies. A lysate of COS cells transfected with vectors only was used as a control (lane 1). The same lysates (200 µg of protein) were immunoprecipitated with the anti-Myc antibody (lanes 4 and 5). The immunoprecipitates were probed with the anti-Myc and anti-HA antibodies. B, interaction of rAxin with endogenous plakoglobin in SW480 cells. Lysates (60 µg of protein) of wild type SW480 cells (lane 1) or SW480 cells stably expressing Myc-rAxin (full length) (lane 2) were probed with the anti-Myc and anti-plakoglobin antibodies. The same lysates (800 µg of protein) were immunoprecipitated with the anti-Myc antibody, and the immunoprecipitates were probed with the anti-Myc and anti-plakoglobin antibodies (lanes 3 and 4). C, region of rAxin that interacts with plakoglobin. The lysates (250 µg of protein) expressing HA-plakoglobin with Myc-rAxin (full length) (lane 1), Myc-rAxin-(1-353) (lane 2), Myc-rAxin-(298-506) (lane 3), Myc-rAxin-(508-832) (lane 4), or Myc-rAxin-(713-832) (lane 5) were immunoprecipitated with the anti-Myc antibody, and the immunoprecipitates were probed with the anti-HA antibody. IP, immunoprecipitation; PG, plakoglobin; Ig, immunoglobulin; Ab, antibody. The arrows, large arrowhead, and small arrowheads indicate the positions of the Myc-rAxin, endogenous plakoglobin, and HA-plakoglobin, respectively. The results shown are representative of three independent experiments.

Direct Interaction of Axin with Plakoglobin-- To examine whether Axin binds directly to plakoglobin, GST-plakoglobin (full length) and MBP-rAxin (full length) were purified (Fig. 2B). GST-plakoglobin bound to MBP-rAxin in a dose-dependent manner, and the Kd value of the binding was approximately 270 nM (Fig. 2A). These results indicate that plakoglobin binds to Axin directly. To determine the regions of plakoglobin and Axin that are necessary for their interaction, various deletion mutants of plakoglobin and rAxin were purified as GST and MBP fusion proteins, respectively (Fig. 2B). MBP-rAxin was precipitated with GST-plakoglobin (full length) and GST-plakoglobin-(185-399) but not with GST-plakoglobin-(1-121) or GST-plakoglobin-(399-744) (Fig. 2C). Plakoglobin-(185-399) contains armadillo repeats 2-7. Consistent with the observations in intact cells, GST-plakoglobin was precipitated with MBP-rAxin (full length), MBP-rAxin-(298-832), and MBP-rAxin-(298-506) but not with MBP-rAxin-(1-229) or MBP-rAxin-(713-832) (Fig. 2C). These results indicate that the region containing amino acid residues 298-506 of rAxin binds directly to armadillo repeats 2-7 of plakoglobin. Because rAxin-(298-506) also binds directly to armadillo repeats 2-7 of beta -catenin (45), plakoglobin and beta -catenin may share the same binding site on rAxin. To examine this possibility, GST-plakoglobin was incubated with MBP-rAxin in the presence of GST-beta -catenin. GST-beta -catenin inhibited the interaction of GST-plakoglobin with MBP-rAxin in a dose-dependent manner (Fig. 3). These results indicate that plakoglobin and beta -catenin interact with overlapping and perhaps identical sites on Axin.


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  		Fig. 2.   Direct interaction of Axin with plakoglobin. A, kinetics of the binding of plakoglobin to rAxin. MBP-rAxin (5 pmol) immobilized on amylose resin was incubated with the indicated concentrations of GST-plakoglobin. MBP-rAxin was precipitated by centrifugation, and the amounts of interacted GST-plakoglobin were quantified by densitometric tracing. B, purification of GST-plakoglobin and MBP-rAxin. GST-plakoglobin (full length) (lane 1), GST-plakoglobin-(1-121) (lane 2), GST-plakoglobin-(185-399) (lane 3), GST-plakoglobin-(399-744) (lane 4), MBP-rAxin (full length) (lane 5), MBP-rAxin-(1-229) (lane 6), MBP-rAxin-(298-832) (lane 7), MBP-rAxin-(298-506) (lane 8), or MBP-rAxin-(713-832) (lane 9) (500 ng of each protein) was visualized with Coomassie Brilliant Blue staining. C, regions of plakoglobin and rAxin that are necessary for their interaction. After 250 nM GST-plakoglobin (full length) (lane 1), GST-plakoglobin-(1-121) (lane 2), GST-plakoglobin-(185-399) (lane 3), or GST-plakoglobin-(399-744) (lane 4) was incubated with 500 nM MBP-rAxin (full length) for 1 h, GST-plakoglobin and its deletion mutants were precipitated with glutathione-Sepharose 4B. The precipitates were probed with the anti-MBP antibody. After MBP-rAxin (full length) (lane 5), MBP-rAxin-(1-229) (lane 6), MBP-rAxin-(298-832) (lane 7), MBP-rAxin-(298-506) (lane 8), or MBP-rAxin-(713-832) (lane 9) (7.5 pmol of each) immobilized on amylose resin was incubated with 500 nM GST-plakoglobin (full length) for 1 h, MBP-rAxin and its deletion mutants were precipitated by centrifugation. The precipitates were probed with the anti-plakoglobin antibody. PG, plakoglobin. The arrows and arrowheads indicate the positions of MBP-rAxin, GST-plakoglobin, and their deletion mutants. The results shown are representative of three independent experiments.


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  		Fig. 3.   Effect of beta -catenin on the interaction of plakoglobin with Axin. MBP-rAxin (full length) (3 pmol) immobilized on amylose resin was incubated with 500 nM GST-plakoglobin (full length) () and the indicated concentrations of GST-beta -catenin (full length) (open circle ). After MBP-rAxin was precipitated by centrifugation, the precipitates were probed with the anti-beta -catenin and anti-plakoglobin antibodies (upper and middle panels). The amounts of interacted GST-plakoglobin or GST-beta -catenin with MBP-rAxin were quantified by densitometric tracing (lower panel). PG, plakoglobin. The arrow and arrowhead indicate the positions of GST-beta -catenin and GST-plakoglobin, respectively. The results shown are representative of three independent experiments.

Phosphorylation of Plakoglobin by GSK-3beta in the Presence of Axin-- Plakoglobin has a consensus sequence of the phosphorylation site for GSK-3beta , and deletion of the N-terminal region containing this site stabilizes plakoglobin (59). Therefore, it is conceivable that GSK-3beta phosphorylates plakoglobin. However, no direct evidence for this has been reported. Because Axin forms a complex with GSK-3beta and plakoglobin, we examined whether GSK-3beta phosphorylates plakoglobin in the conditions under which these proteins form a complex with Axin. In the absence of MBP-rAxin, no phosphorylation of GST-plakoglobin by GST-GSK-3beta was observed, whereas in its presence the phosphorylation was greatly increased (Fig. 4A). Axin was also phosphorylated by GSK-3beta directly (45). MBP-rAxin-(298-506) contains a minimal region that interacts with both GSK-3beta and plakoglobin and the phosphorylation sites for GSK-3beta (Ref. 45 and Fig. 2). MBP-rAxin-(298-506) also enhanced GSK-3beta -dependent phosphorylation of plakoglobin (Fig. 4A). These results indicate that rAxin-(298-506) is sufficient for GSK-3beta -dependent phosphorylation of plakoglobin. GST-GSK-3beta phosphorylated GST-plakoglobin in time- and dose-dependent manners in the presence of MBP-rAxin-(298-506) (Fig. 4, B and C). MBP-rAxin-(298-506) enhanced the phosphorylation of GST-plakoglobin by GST-GSK-3beta in a dose-dependent manner (Fig. 4D). Thus, rAxin promotes GSK-3beta -dependent phosphorylation of plakoglobin.


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  		Fig. 4.   Phosphorylation of plakoglobin by GSK-3beta in the presence of Axin. A, GST-plakoglobin (full length) (1.2 µM) was incubated with 180 nM GST-GSK-3beta in the absence (lane 1) or presence of 100 nM MBP-rAxin (full length) (lane 2) or MBP-rAxin-(298-506) (lane 3) for 30 min. The arrows, large arrowhead, and small arrowhead indicate the positions of MBP-rAxin, GST-plakoglobin, and GST-GSK-3beta , respectively. B, time course. GST-plakoglobin (1.2 µM) was incubated with 180 nM GST-GSK-3beta in the presence of 200 nM MBP-rAxin-(298-506) for the indicated times. Upper panel, autoradiography; lower panel, the radioactivities incorporated into GST-plakoglobin. C, dependence on dose of GSK-3beta . GST-plakoglobin (1.2 µM) was incubated with the indicated concentrations of GST-GSK-3beta in the presence of 100 nM MBP-rAxin-(298-506) for 10 min. D, dependence on dose of rAxin. GST-plakoglobin (1.2 µM) was incubated with 180 nM GST-GSK-3beta in the presence of the indicated concentrations of MBP-rAxin-(298-506) for 10 min. PG, plakoglobin. The results shown are representative of three independent experiments.

Down-regulation of Plakoglobin by Axin-- Finally we examined whether Axin regulates the stability of plakoglobin. Although expression of Axin (full length) in SW480 cells induces the degradation of beta -catenin (49, 52), the level of plakoglobin was not changed by expression of rAxin (full length) (Fig. 1B). It has been shown that deletion of the regulators of G protein signaling domain from Axin enhances its ability to down-regulate beta -catenin (49). Therefore, we expressed EGFP-rAxin-(298-832), in which the regulators of G protein signaling domain is deleted, in SW480 cells and detected cytoplasmic plakoglobin by immunofluorescence staining. EGFP-rAxin-(298-832) formed irregular particles as shown in the previous reports (Fig. 5, A and C) (52, 54). EGFP-hEpsin that was used as a control showed diffuse expression (Fig. 5, E and G). beta -Catenin was stained homogeneously in cytoplasm (Fig. 5, B and F). Plakoglobin had a diffuse cytosolic pattern of expression with some areas of particular or vesicular staining (Fig. 5, D and H). Consistent with the previous observations (49, 52), more than 90% of rAxin-(298-832)-expressing cells exhibited a marked diminution of beta -catenin immunofluorescence staining (n = 300) (Fig. 5B). rAxin-(298-832) also down-regulated plakoglobin (Fig. 5D), but its efficiency was less potent (23% of transfected cells) compared with that for beta -catenin (n = 300). EGFP-hEpsin did not affect the level of either beta -catenin or plakoglobin significantly (Fig. 5, F and H). These results suggest that the stability of plakoglobin is regulated by Axin as well as that of beta -catenin, although the efficiency of the degradation of plakoglobin by Axin might be different from that of beta -catenin.


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  		Fig. 5.   Degradation of plakoglobin by Axin in SW480 cells. SW480 cells grown on coverslips were transfected with pEGFP-c3/rAxin-(298-832) (A-D) or pEGFP-c1/hEpsin (E-H), which was used as a control. At 48 h after the transfection, the cells were fixed and permeabilized. The cells were stained with the anti-beta -catenin (B and F) or anti-plakoglobin (D and H) antibody in red. Transfected cells were visualized with spontaneous fluorescence of EGFP in green (A, C, E, and G). The arrows indicate transfected cells. Scale bar, 10 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Regulation of the expression of beta -catenin and plakoglobin is important in morphogenetic events during embryonic development (34, 35, 58, 60) and in the process of tumorigenesis (61-64). Although the molecular mechanism by which the stability of beta -catenin is regulated has become clear, that for regulation of plakoglobin is not fully understood. In this study, we have addressed the regulation of the cellular plakoglobin level. We have demonstrated for the first time that Axin directly binds to plakoglobin, that it enhances GSK-3beta -dependent phosphorylation of plakoglobin, and that it down-regulates plakoglobin. Taken together with the observations that Axin enhances GSK-3beta -dependent phosphorylation of beta -catenin, resulting in the degradation of beta -catenin (45, 49, 51), these findings suggest that Axin regulates the stability of both beta -catenin and plakoglobin.

The phosphorylation of serine/threonine residues 29-45 of beta -catenin is necessary for its degradation, and deletion of this region results in the stabilization of beta -catenin (23, 36, 37). In Xenopus embryos, plakoglobin behaves like beta -catenin in that deletion of the 17 amino acids including the possible phosphorylation site for GSK-3beta from the N terminus of plakoglobin leads to an increase in the stability of the protein (59). GSK-3beta and beta -catenin directly bind to separate, adjacent sites on Axin. Because beta -catenin inhibits the binding of plakoglobin to Axin, beta -catenin and plakoglobin may share the same binding site on Axin. Therefore, it is possible that Axin enhances GSK-3beta -dependent phosphorylation of plakoglobin by simultaneously binding to GSK-3beta and plakoglobin. It has been shown that the phosphorylation of beta -catenin by GSK-3beta in the presence of Axin is required for the complex formation with beta TrCP/FWD, resulting in beta -catenin being ubiquitinated and degraded by the proteasome pathway (50). Ubiquitination of plakoglobin is also observed in A431 cells treated with a proteasome inhibitor (38). Taken together, these results imply that the phosphorylation of plakoglobin by GSK-3beta in the Axin complex could result in its degradation by the ubiquitin-proteasome pathway.

We have found that the armadillo repeats 2-7 of plakoglobin bind to Axin. This region of plakoglobin is also necessary to interact with the cytoplasmic tails of desmocollin, desmoglein, and cadherin (11, 17, 18). Expression of either desmoglein or desmocollin in mouse L cells decreases the rate of plakoglobin degradation (7). Expression of E-cadherin induces the accumulation of beta -catenin (7, 65), and the binding region of beta -catenin to Axin overlaps with that to E-cadherin (17, 45). These results indicate that the bindings of plakoglobin and beta -catenin to desmoglein and desmocollin, and E-cadherin, respectively, rescue plakoglobin and beta -catenin from the degradation pathway. It has been shown that plakoglobin interacts with desmoglein in the insoluble fraction of Madin-Darby canine kidney cells and that plakoglobin is more heavily phosphorylated in the soluble fraction than in the insoluble fraction (66). Therefore, plakoglobin complexed with desmoglein does not bind to Axin and may not be efficiently phosphorylated by GSK-3beta and thereby be stabilized.

Although plakoglobin and beta -catenin are structurally homologous, whether their functions are similar is not clear. Plakoglobin, like beta -catenin, directly binds to cadherin (18), alpha -catenin (16), APC (17), and Tcf/Lef (67, 68). Expression of Lef-1 in Madin-Darby canine kidney cells induces nuclear translocation of beta -catenin but not of plakoglobin, suggesting that these proteins may differ in their specificities for transcriptional factors (68). The ability of plakoglobin to activate Lef-1 is much lower than that of beta -catenin (68). In the Drosophila female germ line, mammalian beta -catenin and plakoglobin can complement an armadillo mutation (58). However, in embryonic signaling assays, plakoglobin has no detectable activity, whereas beta -catenin has weak activity (58). Therefore, to definitively determine whether plakoglobin can induce transactivation, it is necessary to use beta -catenin-null cells. Expression of plakoglobin in Xenopus embryos induces axis duplication (35) that was similar to that observed with beta -catenin expression (34). These results suggest that plakoglobin and beta -catenin have similar activities in at least axis formation in Xenopus embryos. However, it has been demonstrated that plakoglobin induces Wnt signaling in Xenopus embryos, even when it is tethered to the plasma membranes (60), suggesting that plakoglobin could stimulate the Wnt signaling by relieving the negative action on transcriptional activation of Wnt-responsive genes or by competition and displacement of endogenous beta -catenin from its association with the degradation system, followed by stabilization and nuclear translocation of beta -catenin. Our present results suggest that Axin degrades both beta -catenin and plakoglobin in similar manners but that plakoglobin is less sensitive to this proteolysis. Overexpression of plakoglobin may lead to the accumulation of beta -catenin by blocking the proteasome degradation system.

Because mutations in the phosphorylation site of beta -catenin for GSK-3beta have been discovered in several human tumors, including colorectal cancers and melanoma (62-64), it is thought that beta -catenin acts as an oncogene. However, plakoglobin suppresses the tumorigenesis of cells, and its expression level is often lost in tumor cells (19, 61). Although the exact roles of plakoglobin in tumorigenesis are not known, possible mechanisms whereby plakoglobin affects the functions of beta -catenin have been suggested. For example, the plakoglobin/Tcf activates genes that are tumor suppressive or the plakoglobin/Tcf forms an inactive complex and thus inhibits transactivation by beta -catenin. In addition to these possibilities, one more possibility is conceivable based on our results. Plakoglobin competes with beta -catenin for the binding to cadherin and alpha -catenin. beta -Catenin released from the adherens complex may bind to Axin, leading to beta -catenin degradation. Indeed, it has been shown that overexpression of plakoglobin results in a decrease in the level of beta -catenin (69). Further studies are necessary to understand the cellular functions regulated by beta -catenin and plakoglobin, of which the stability is controlled by Axin.

    ACKNOWLEDGEMENTS

We are grateful to Drs. W. W. Franke, E. Tahara, Q. Hu, and M. Nakata for the gifts of plasmids, cell lines, and antibodies, respectively. We thank the Research Center for Molecular Medicine and Research Facilities for Laboratory Animal Sciences, Hiroshima University School of Medicine, for the use of its facilities.

    FOOTNOTES

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

To whom correspondence should be addressed. Tel.: 81-82-257-5130; Fax: 81-82-257-5134; E-mail: akikuchi@mcai.med.hiroshima-u. ac.jp.

    ABBREVIATIONS

The abbreviations used are: GSK-3beta , glycogen synthase kinase-3beta ; Tcf, T-cell-specific factor; Lef, lymphoid enhancer binding factor; APC, adenomatous polyposis coli; HA, hemagglutinin; MBP, maltose-binding protein; GST, glutathione S-transferase; PBS, phosphate-buffered saline; EGFP, enhanced green fluorescent protein; rAxin, rat Axin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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