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J. Biol. Chem., Vol. 275, Issue 45, 35170-35175, November 10, 2000

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Regulation of Lef-mediated Transcription and p53-dependent Pathway by Associating -Catenin with CBP/p300^*

Makoto Miyagishi^§, Ryouji Fujii^§, Mitsutoki Hatta^§, Eisaku Yoshida^§, Natsumi Araya^§, Akira Nagafuchi^¶, Satoru Ishihara^¶, Toshihiro Nakajima^§, and Akiyoshi Fukamizu^§

From the  Center for Tsukuba Advanced Research Alliance, ^§ Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8577, and the ^¶ Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan

Received for publication, April 17, 2000, and in revised form, July 7, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CBP and its homologue p300 play significant roles in cell differentiation, cell cycle, and anti-oncogenesis. We demonstrated that -catenin, recently known as a potent oncogene, and CBP/p300 are associated through its CH3 region, which is a primary target of adenoviral oncoprotein E1A and various nuclear proteins, such as p53, cyclin E, and AP-1, and both are colocalized in the nuclear bodies. CBP/p300 potentiated Lef-mediated transactivation of -catenin, and E1A, a potent inhibitor of CBP/p300, repressed its transactivation. Furthermore, overexpression of stable -catenin mutant competitively suppressed the p53-dependent pathway. These may be a key mechanism of -catenin involved in oncogenic events underlying disruption of tumor suppressor function through CBP/p300.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The coactivator CBP/p300 family is a central regulator of gene expression (1-5) and important in cell differentiation, cell cycle, and anti-oncogenesis (6-9). The importance of CBP/p300 in such cellular functions is supported by their ability to potentiate the activity of a large number of nuclear factors such as adenoviral oncoprotein E1A, p53, cyclin E, and AP-1 (10-14). In addition, CBP/p300 has been proposed as a bridging protein, bringing together proteins of the RNA polymerase II-dependent basal transcription complex with either specific transcription factors or other cofactors.

Drosophila Armadillo and its vertebrate homologue -catenin are scaffold proteins in cadherin-mediated cell-cell adhesion, and they function as signal transducers to the nucleus in Wnt/Wingless pathway (15-17). -Catenin translocated to the nucleus regulates expression of various downstream genes by acting as a coactivator of Lef/TCF family. Furthermore, recent studies suggested that stable mutants of -catenin leading to accumulation in the nucleus are implicated in the initiation of some forms of colon cancers and melanomas (18-21).

The involvement of -catenin in its nuclear functions, including transcriptional activation and oncogenic events, addresses the existence of bridging nuclear factors for recruitment of RNA polymerase II. To test this hypothesis, in the present study, we explored the possible association of -catenin with CBP/p300 and attempted to define the biological function of CBP/p300 and -catenin interaction.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture and Transfections and Reporter Gene Assays-- HeLa cells, L cells, and HEK293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. HCT116 cells were maintained in McCoy 5A supplemented with 10% fetal bovine serum. Transfections were performed by the calcium phosphate method. Thirty ng of a RSV-Renilla luciferase control plasmid were included in each transfection experiment to control for the efficiency of transfection. To ensure equal DNA amounts, empty plasmids were added in each transfection. The luciferase activity was measured with AutoLumat (Berthold). The values were normalized to Renilla luciferase activity as an internal control.

Antibodies-- Anti--catenin (8A5) and anti-CBP (5614) antibodies were obtained according to the procedures described previously (22, 23).

Plasmids-- pTOPFlash and pFOPFlash were provided by H. Clevers (19). A series of CBP deletion mutants, pRc/RSV-mCBP-HA and pG5b-Luc, have been described previously (24). p53 expression plasmid (pCIcchp53) was obtained from RIKEN Gene Bank (25). pGAL4 control vector was generated by inserting the GAL4 DNA binding domain (aa 1-147) of pGBT9 (CLONTECH) into pcDNA3 (Invitrogen). pGAL4-p53(1-83) was generated by PCR¹-based subcloning into pGAL4.

pcEF9-mCBP was generated by inserting full-length mouse CBP into pcEF9. -Catenin deletion mutants were made by digesting a full-length -catenin with appropriate enzymes or PCR-based subcloning into pcDNA3-HA. GST-CBP-(1679-1891) and biotinylated tag fused CBP-(1679-1891) were generated by inserting EcoRI-SmaI fragment (aa 1679-1891) into pGEX5X-1 (Amersham Pharmacia Biotech) and PinPoint Xa1 (Promega), respectively. GST--catenin or GST--catenin-(550-781) was made by inserting a full-length -catenin or a fragment (aa 550-781) generated by PCR into pGEX5X-1. HA-PML expression and Lef-1 expression plasmids were made by reverse transcription-PCR-based cloning into pcDNA3-HA. HA-tagged stable -catenin mutant (pEFBCHA) was generated by inserting C-terminal HA-tagged full-length -catenin mutant (S33A, S37A, T41A, S45A) downstream of EF-1 promoter. HdNB was made by inserting N-terminal truncated -catenin (N89) in frame to N-terminal HA-tag. FLAG-tagged stable -catenin mutant (pEFFlagMMB) was generated by inserting N-terminal FLAG-tagged full-length -catenin mutant (S33A, S37A, T41A, S45A) downstream of EF-1 promoter. p300 and p300CH3 expression plasmids and wild type and mutants E1A were described previously (23).

Immunofluorescence and TUNEL Assay-- HeLa cells were plated onto glass coverslips and transfected using Fugene-6 reagents (Roche Molecular Biochemicals) at 60% confluence. Thirty-six h after transfection, cells were treated with 200 ng/ml doxorubicin (Sigma) and incubated additional 12-16 h. Cells were then fixed with 4% paraformaldehyde in PBS for 20 min and permeabilized by treatment with 0.1% Triton X-100 in PBS for 30 min. After blocking with 1% bovine serum albumin, 0.1% Tween 20 in PBS for 30 min, cells were incubated with rat anti-HA monoclonal antibody (1:200 dilution; 3F10, Roche Molecular Biochemicals), anti-p53 mouse monoclonal antibody (1:500 dilution; pAb421 oncogene science), followed by staining with Cy5-conjugated anti-rat and Cy3-conjugated anti-mouse secondary antibodies (1:1000 dilution each; Amersham Pharmacia Biotech). TUNEL staining was performed by an in situ apoptosis detection kit (Takara). Immunofluorescence was analyzed under a confocal microscopy.

Immunoprecipitation and Western Blot Analysis-- HEK293T cells were transfected using lipofectAMINE reagents (Life Technologies, Inc.), and nuclear extracts were prepared as described previously (24). Approximately 200 µg of nuclear extracts were incubated with anti-HA antibody (12CA5) coupled with protein A/G-agarose in buffer A (20 mM HEPES (pH 7.9), 150 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 0.05% Tween 20, 0.1 mM Na₃VO₄, 20 mM NaF, and protease inhibitors) at 4 °C overnight, and washed five times with the same buffer. For coimmunoprecipitation of endogenous -catenin with CBP, cells were lysed in Co-IP buffer (10 mM HEPES (pH 7.5), 150 mM KCl, 0.1% Nonidet P-40, and protease inhibitors) and subjected to immunoprecipitation with anti-CBP antibody (5614). Western blot analysis of the immunoprecipitates was done as described previously (24).

In Vitro Binding Assay and in Vitro Competitive Binding Assay-- In vitro binding assay was performed as described previously (24). For in vitro competitive binding assay, in vitro translated ³⁵S-labeled p53 was incubated with 2 µg of biotinylated tag-fused CBP (1679-1860) or control biotinylated tag bound to streptavidin beads in buffer A containing 250 µg/ml of GST, GST--catenin-(550-781), or GST--catenin-(661-781) for 3 h. After washing five times with buffer A, the pull-down complexes were fractionated by SDS-polyacrylamide gel electrophoresis and were analyzed by an image analyzer (BAS2000; Fujix).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Interaction of -Catenin with Coactivator CBP/p300-- We first performed coimmunoprecipitation experiments using an HA-tagged stable -catenin mutant. HEK293T cells were transiently transfected with the HA-tagged -catenin expression plasmid or a mock plasmid. Western blotting of HA--catenin complexes immunoprecipitated with anti-HA antibody revealed that endogenous CBP/p300 was coimmunoprecipitated with HA--catenin (Fig. 1A). Bacterially expressed GST--catenin also captured in vitro translated CBP and p300 with comparable binding to in vitro translated Lef-1 (Fig. 1B). To evaluate the interaction between -catenin and CBP/p300 under more physiological conditions, we analyzed endogenous -catenin and CBP/p300 in colon carcinoma cell line, HCT116 cells, in which -catenin is mutated and thereby stabilized. As shown in Fig. 1D, endogenous -catenin could be efficiently coimmunoprecipitated with endogenous CBP/p300. Further analysis using in vitro translated CBP fragments (Fig. 1A) revealed that -catenin specifically interacted with the CH3 region of CBP (Fig. 1E), which is known as a binding site of various nuclear proteins concerning cell cycle regulation and apoptosis, such as E1A, cyclin E/CDK2, p53, and AP-1. To define the CBP interaction domain for -catenin binding, we constructed a series of -catenin deletion mutants (Fig. 1A). GST-pull-down assay using GST-fused CBP CH3 fragment (aa 1679-1891) indicated that CBP interacted with both of the N- and C-terminal armadillo (Arm) repeat domains, but not with the N- and C-terminal hydrophilic activation domains (20, 26) (Fig. 1F). These results indicated that the CH3 region of CBP/p300 interacts with the -catenin Arm repeat domain in vitro.

View larger version (38K): [in this window] [in a new window]   Fig. 1.   In vivo and in vitro binding of -catenin with CBP. A, coimmunoprecipitation of CBP using HA-tagged stable -catenin mutant. Nuclear extracts from HEK293T cells transfected with 1 µg of HA-tagged stable -catenin mutant expression plasmids or an empty control plasmid were immunoprecipitated with anti-HA antibody (12CA10, Roche Molecular Biochemicals) and were then subjected to immunoblotting with anti-CBP (5614), which is cross-reactive with p300 (top) or anti-HA (12CA10) antibody (center). Immunoblotting of nuclear extracts with anti-CBP antibody shows a quantitation of the experiment (bottom). B, in vitro binding assay using in vitro translated 35S-labeled CBP, p300, or Lef-1, with GST or GST--catenin. C, schematic representations of CBP, -catenin, and their deletion mutants used in this study. CH, cysteine/histidine-rich region; KIX, KIX domain; BR, bromodomain; HAT, histone acetyltransferase domain; p/CIP, p300/CBP-interacting protein. D, coimmunoprecipitation of endogenous -catenin using anti-CBP antibody. Cell extracts from HCT116 cells were immunoprecipitated with anti-CBP antibody (5614) or normal mouse IgG as a control. E, in vitro binding assay using in vitro translated 35S-labeled CBP deletion mutants and GST or GST--catenin. The minimal binding domain of CBP is aa 1805-1891. F, in vitro binding assay using in vitro translated 35S-labeled -catenin deletion mutants and GST or GST-CBP-(1679-1891).

Colocalization of -Catenin and CBP/p300-- To further confirm the interaction between CBP/p300 and -catenin, we examined its cellular localization using mouse fibroblast L cells. Since this cell line does not express E-cadherin (27), we predicted that -catenin is translocated to the nucleus. Overexpression of -catenin in L cells results in nuclear translocation and formation of speckle-like aggregates (Fig. 2A, panel a), in which endogenous CBP was also translocated to the same speckles (Fig. 2A, panels b and c). A similar speckled pattern was observed in HeLa cells, when both CBP and -catenin were transfected together (Fig. 2B, panels d-f). In contrast, transfection of either CBP or -catenin alone showed uniform distribution in the nucleus (Fig. 2B, panels g and h). These results suggested physical association of CBP/p300 with -catenin in the nucleus. Recently, a similar speckled-like structure called PML nuclear bodies (NBs) or PODs (PML oncogenic domains) has been found in the nucleus (18). Its function is not yet fully understood, but it has been shown that a variety of nuclear proteins (PML, Sp100, SUMO-1, etc.) are present. Therefore, we examined whether nuclear aggregates formed by CBP and -catenin are identical to this nuclear structure. As shown in Fig. 2C (panels i-k), HA-PML was colocalized in the CBP--catenin aggregates, indicating the nuclear aggregates formed by CBP and -catenin are NBs.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Colocalization of CBP and -catenin in the nuclear structure. A, a stable -catenin mutant is colocalized with endogenous CBP in L cells. L cells were transfected with stable -catenin mutant expression vector (HdNB) (panels a and b) or an empty vector (panel c), and were stained with anti-CBP (panels b and c) (Santa Cruz Biotechnology; sc-583) or anti--catenin (8A5) (panel a) antibodies. The anti--catenin (8A5) antibody is able to preferentially detect -catenin expressed in L cells. B, coexpression of CBP and stable -catenin mutant causes the formation of nuclear aggregates in HeLa cells. HeLa cells were transfected with CBP expression plasmid (panels d, e, f, and g) and/or HA-tagged stable -catenin mutant expression plasmid (pEFMMBCHA) (panels d, e, f, and h) and stained with anti-CBP (5614) (panels d and g) and anti-HA (3F10) (panels e and h) antibodies. C, HA-PML coexists in the nuclear aggregates. HeLa cells were transfected together with FLAG-tagged stable -catenin mutant (pEFFlagMMB), CBP and HA-PML-expression plasmids (panels i, j, and k) and incubated with anti-FLAG (M2, Eastman Kodak Co.) (panel i), anti-CBP (5614) (panel j), and anti-HA (3F10, Roche Molecular Biochemicals) (panel k) antibodies, followed by staining with Cy2-conjugated goat anti-mouse, Cy3-conjugated goat anti-rabbit, and Cy5-conjugated goat anti-rat secondary antibodies, respectively.

Function of CBP/p300 as a Coactivator for Lef/TCF-mediated Transcription-- To investigate the functional significance of the interaction between CBP/p300 and -catenin, we examined the effect of p300 on -catenin-mediated transcription using a luciferase reporter plasmid containing Lef binding sites (pTOPflash) or mutant Lef binding sites (pFOPflash) (19). Cotransfection of stable -catenin mutant and Lef-1 in L cells results in significant enhancement of luciferase activity (Fig. 3A). p300 enhanced this -catenin-Lef-mediated transactivation in a dose-dependent manner, while a p300CH3, which has the 33-amino acid deletion in the CH3 region, had no effect (Fig. 3B). CBP also had the same synergistic effect as p300 (data not shown). In HCT116 cells, the transcription activity of pTOPFlash reporter was two to three times higher than that of pFOPFlash as a result of stabilization of -catenin mutant (Fig. 3C). p300, but not p300CH3, could enhance this -catenin-mediated transactivation in a dose-dependent manner (Fig. 3C). Furthermore, E1A, which binds to CBP/p300 and inhibits CBP/p300-dependent transactivation, repressed the -catenin-dependent transactivation in HCT116 (Fig. 3D). E1AmCR2 mutant that lacks the ability to interact with retinoblastoma protein but does bind to CBP also inhibited the -catenin-dependent transactivation. In contrast, E1AN, which is defective for CBP/p300 binding, and E1ACR1, which is defective for both CBP/p300 and retinoblastoma binding, were impaired in repression (Fig. 3D). To further verify the CBP/p300 dependence on the -catenin-mediated transactivation, we utilized GAL4 fusion system using -catenin-GAL4 DNA binding domain fusion (GAL4--catenin) expression plasmid and G5b-Luc reporter containing five GAL4 binding sites and E1b promoter. p300 potentiated GAL4--catenin transactivation, whereas p300CH3 had no effect (Fig. 3E). These results indicated that Lef-directed transactivation by -catenin is mediated by CBP/p300.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Involvement of CBP/p300 in -catenin-mediated Lef transactivation. A, -catenin potentiates Lef-mediated transactivation in L cells. L cells were transiently transfected with 100 ng of pTOPflash and 10 ng of stable -catenin mutant expression plasmid (pEFMMBCHA) and/or 10 ng of Lef-1 expression plasmid or empty control plasmid. B, p300 potentiates Lef-mediated transactivation by -catenin in L cells. L cells were transfected with 100 ng of pTOPflash, 10 ng of stable -catenin mutant expression plasmid or empty control plasmid, and the indicated amount of p300 or p300CH3 expression plasmid. C, p300 enhances -catenin-Lef-mediated transactivation in HCT116 cells. HCT116 cells were transfected with 100 ng of pTOPFlash or pFOPFlash and the indicated amounts of p300 or p300CH3 expression plasmid. D, E1A suppresses Lef-mediated transactivation by -catenin. HCT116 cells were transfected with 100 ng of pTOPFlash or pFOPFlash, and 1 ng of wild-type E1A, E1AN, E1ACR1, or E1AmCR2 mutants. E, p300, but not p300CH3, enhances transactivation activity of GAL4--catenin. L cells were transfected with 100 ng of G5b-Luc reporter plasmid, 1 ng of GAL4 or GAL4--catenin, and 500 ng of p300 or p300CH3 expression plasmid.

Inhibition of CBP/p300-mediated p53-dependent Pathway by -Catenin-- The accumulation of stable -catenin mutant in the nucleus is known to provide the possibility leading to oncogenesis (18). We next considered that the interaction between -catenin and CBP might contribute to a certain oncogenic event, such as suppression of apoptosis. In p53-dependent apoptosis, p53 needs for interaction with CBP/p300 through the CH3 region (11), which is the same binding region as -catenin. Furthermore, different transcription factors that associate with CBP/p300 as a cofactor were known to antagonize one another by competing limiting amounts of CBP/p300, as reported for AP-1 and nuclear receptor (14), Stat2 and NF-B (29), and p53 and NF-B (30). These data prompted us to examine whether -catenin acts to inhibit p53-dependent pathway by inhibiting the interaction of p53 with CBP.

We first examined the competitive effect of -catenin expression in p53-dependent transactivation using a Gal4-fused p53 activation domain (aa 1-83) expression plasmid and luciferase reporter plasmid containing Gal4 binding sites (G5b-Luc), because it has been reported that CBP/p300 potentiates p53 transactivation through the N-terminal transactivation domain of p53 (11). As shown in Fig. 4A, stable -catenin mutant inhibited transactivation of p53 activation domain at comparable levels of a dominant-negative CBP CH3 fragment (11, 31). We next performed in vitro competitive binding assay to address the possibility of direct competition between -catenin and p53 for association with CBP. As shown in Fig. 4B, GST--catenin (550-781 aa) competed directly with p53 for association with the CH3 fragment of CBP in a dose-dependent manner, whereas a control GST protein and -catenin mutant, both of which defect the binding of CBP/p300, did not.

View larger version (39K): [in this window] [in a new window]   Fig. 4.   Suppression of p53-dependent pathway by stable -catenin mutant. A, stable -catenin mutant suppresses transactivation activity of GAL4-p53-(1-83). HeLa cells were transfected with 0.01 ng of pGAL-p53-(1-83) and stable -catenin mutant expression plasmid or pEF-HA-CBP-(1679-1891) or an empty control plasmid. B, in vitro competitive binding assay using in vitro translated 35S-labeled p53, biotinylated tag-fused CBP-(1679-1860) and GST, GST--catenin-(550-781), and GST--catenin-(661-781). GST--catenin-(550-781) inhibits the binding p53 with the CBP CH3 region in a dose-dependent manner, whereas GST and GST--catenin-(661-781) have no effect. C, HeLa cells were transfected with 0.5 µg of p53 expression plasmid and 0.5 µg of HA-tagged stable -catenin expression plasmid or an empty control plasmid. TUNEL assay and immunostaining were performed as described under "Experimental Procedures." Arrowheads indicate the cells expressing p53 but not -catenin.

Finally, we tested whether -catenin is able to suppress p53-dependnt apoptosis. HeLa cells were transiently transfected with a p53 expression plasmid and were subjected to following treatment with 200 ng/ml doxorubicin, which is a DNA damage-inducing agent and known to enhance p53-dependent apoptosis (11). Approximately 90% of p53-transfected cells were positive for TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP end labeling) staining, and microscopic analysis revealed several particular apoptotic features, such as cell shrinkage and highly nuclear condensation (Fig. 4C, panels c-e). Coexpression of stable HA--catenin mutant significantly suppressed p53-dependent apoptosis (Fig. 4C, panels f-h, Table I), and immunocytochemical staining with anti-HA antibody revealed that a few cells, which undergo apoptosis, express low or undetectable levels of -catenin. In contrast, almost all cells expressing high levels of -catenin appeared to escape apoptosis (Fig. 4C, panels f-i). Collectively, these results suggested that stable -catenin mutant suppresses p53-dependent pathway by competitively binding to CBP/p300.

View this table: [in this window] [in a new window]   Table I Effect of -catenin on p53-dependent apoptosis in HeLa cells Apoptotic index represents the percentage of TUNEL positive cells in p53-transfected HeLa cells. Values were determined by counting more than 200 cells from randomly chosen fields.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A recent study has described that Drosophila CBP represses an inappropriate activation of Lef-mediated signal at the low levels of -catenin (32). Our data from the reporter gene assay revealed that CBP enhanced Lef-mediated transactivation, suggesting the differential modes of action of -catenin expression between the normal and transformed cells. In the normal cells, as the nuclear levels of wild-type -catenin are thought to be maintained at a low level by GSK-3-dependent ubiquitination pathway, CBP/p300 would be preferentially recruited to Lef/TCF, acting to repress the Wingless pathway. On the other hand, in the transformed cells, when -catenin is mutated, stabilized, and present at high levels in the nucleus, CBP/p300 might be dominantly recruited to -catenin by the following two mechanisms. (i) Most population of CBP is occupied by -catenin because of a much higher levels of -catenin than those of Lef/TCF. (ii) High levels of -catenin inhibit the binding between Lef and CBP by the association of -catenin with Lef and/or CBP and cause the conversion from a Lef-CBP suppression complex to a Lef--catenin-CBP activation complex.

Although the function of PML bodies is not fully understood, several recent studies provide clues to understand PML bodies. For instance, Doucas et al. (33) have shown that the overexpression of PML results in the recruitment of CBP to PML bodies, accompanied by a synergistic enhancement of retinoic acid receptor transactivation. Torii et al. (34) demonstrated that the recruitment of Daxx to PML bodies affects Fas-induced apoptosis. Furthermore, PML has been reported to be essential for irradiation-induced apoptosis (35, 36). Thus, it is possible that the recruitment of -catenin and CBP/p300 to PML bodies plays a significant role in the regulation of transcription and apoptosis.

A p53-dependent pathway was recently shown to involve apoptosis through CBP/p300, considering the dominant-negative effect of the CH3 fragment (11). Since the cellular level of CBP/p300 is limiting, different transcription factors associated with CBP/p300 as a cofactor are reported to antagonize one another. For example, the repression of AP-1 activity by nuclear receptors was reported in the earlier study (14), and competitive inhibition between Stat2 and NF-(29) and p53 and NF-(30) has been described more recently. In view of these results, it is possible that molecules that tightly bind to the CH3 region of CBP/p300 prevent the p53-dependent pathway. However, suppression of the p53-dependent pathway by the CH3-binding molecules would not always occur, because in many cases cells appear to be transformed by a combination of various oncogenic events. In the case of -catenin, in addition to its ability to inhibit tumor suppresser functions of p53, a certain combinatorial action of other effectors, such as the transcriptional activation of downstream oncogenes (c-Myc or cyclin D), might be required for subsequent oncogenic transformation (28, 37). Therefore, the up-regulation of downstream oncogenes by CBP, -catenin, and Lef and the concomitant suppression of p53-dependent pathway by the competitive mechanism may be key events for oncogenesis by -catenin.

	ACKNOWLEDGEMENTS

We thank Dr. H. Clevers (pTOPFlash and pFOPFlash), Drs. Y. Katsura and S. Fujimoto (LEF-1 expression plasmid), and Dr. H. Hamada (pCIcchp53) for their kind gift of plasmids. We acknowledge the Fukamizu laboratory members for their helpful discussion and encouragement.

	FOOTNOTES

^* This work was supported by grants from the "Research for the Future" Program (The Japan Society for the Promotion of Science: JSPS-RFTF 97L00804); the Ministry of Education, Science, Sports, and Culture of Japan; The Asahi Glass Foundation; and The Nissan Science 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: Center for Tsukuba Advanced Research Alliance, Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan. Tel.:/Fax: 81-298-53-6070; E-mail: akif@tara.tsukuba.ac.jp.

Published, JBC Papers in Press, July 20, 2000, DOI 10.1074/jbc.C000258200

	ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; GST, glutathione S-transferase; HA, hemagglutinin; PBS, phosphate-buffered saline; aa, amino acid(s); NBs, nuclear bodies.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				W. N. de Vries, A. V. Evsikov, B. E. Haac, K. S. Fancher, A. E. Holbrook, R. Kemler, D. Solter, and B. B. Knowles Maternal {beta}-catenin and E-cadherin in mouse development Development, September 15, 2004; 131(18): 4435 - 4445. [Abstract] [Full Text] [PDF]