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Originally published In Press as doi:10.1074/jbc.M508153200 on November 23, 2005

J. Biol. Chem., Vol. 281, Issue 3, 1401-1411, January 20, 2006
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Presenilin-1 Interacts with Plakoglobin and Enhances Plakoglobin-Tcf-4 Association

IMPLICATIONS FOR THE REGULATION OF beta-CATENIN/Tcf-4-DEPENDENT TRANSCRIPTION*

Imma Raurell{ddagger}1, Julio Castaño{ddagger}2, Clara Francí§, Antonio García de Herreros§3, and Mireia Duñach{ddagger}4

From the {ddagger}Unitat de Biofísica, Departament de Bioquímica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain and §Unitat de Biologia Cellular i Molecular, Institut Municipal d'InvestigacióMèdica, Universitat Pompeu Fabra, E-08003 Barcelona, Spain

Received for publication, July 26, 2005 , and in revised form, November 23, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Alzheimer disease-linked Presenilin-1 (PS1) is a negative modulator of beta-catenin/Tcf-4 activity. However, the mechanism underlying this effect is not well understood. We show here that the effects of PS1 on the activity of this complex in epithelial cells are independent of its {gamma}-secretase activity and its interaction with beta-catenin. As presented in this report PS1 also binds plakoglobin with similar affinity as beta-catenin, although this interaction does not involve equivalent residues in the two catenins. Moreover, PS1 association with plakoglobin enhances the interaction of this molecule with Tcf-4 and prevents its binding to DNA. These effects were observed with the unprocessed form of PS1, which has higher affinity for plakoglobin and beta-catenin than processed PS1. These results provide a new explanation for the effects of PS1 on gene transcription mediated by beta-catenin in epithelial cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
beta-catenin and plakoglobin ({gamma}-catenin) are two related proteins that play a crucial role in cell-cell adhesion. In adherens junctions, beta-catenin and plakoglobin independently link the cytosolic domain of cadherins to {alpha}-catenin, which in turn directly or indirectly interacts with actin filaments (1–3). In addition, plakoglobin is a component of desmosomes, where it mediates the association of the desmosomal cadherins and the intermediate filament cytoskeleton (3–5). beta-catenin and plakoglobin are members of the armadillo family of proteins and share a common central structural motif containing twelve armadillo repeats (consisting of 42 amino acids each) with 76% identity (6). In contrast, the amino and carboxyl termini of beta-catenin and plakoglobin are acidic and share only 29 and 41% identity, respectively. The interaction of both beta-catenin and plakoglobin with other members of adherens junctions and desmosomes is controlled by phosphorylation of specific Tyr residues in both molecules (2, 7–9).

In addition to its function in cell adhesion, beta-catenin participates in the Wnt pathway (10–12). When released from the junction complex, beta-catenin migrates to the nucleus where, through interaction with the Tcf5 family of transcriptional factors, it triggers a wide variety of genes that in turn activate genes involved in embryonic development and tumorigenesis (11, 12). The translocation of beta-catenin to the nucleus is tightly controlled by the activity of a complex involved in beta-catenin degradation. This complex includes the product of the tumor suppressor adenomatous polyposis gene, axin, and the Thr/Ser protein kinases, CKI{alpha} and glycogen synthase kinase 3beta (11, 13). As a result of the activity of this complex, beta-catenin is phosphorylated initially by CKI{alpha} on Ser-45 and sequentially by glycogen synthase kinase 3beta on Thr-41 and Ser-37 and -33. Upon phosphorylation, beta-catenin is polyubiquitinated by beta-TrCP ubiquitin ligase and degraded by the proteasome. The activity of the complex is controlled by a signaling pathway triggered by Wnt factors that stabilize cytosolic beta-catenin (10, 11). Recently, an alternative mechanism of beta-catenin degradation, depending on the activity of Siah-1 protein, was described (14, 15).

Plakoglobin is also involved in the Wnt pathway. In some systems, plakoglobin activates Tcf-mediated transcription indirectly, interfering with the degradation of beta-catenin by the proteasome and increasing the levels of beta-catenin in the cytosol and nucleus (16). However, the direct interaction of plakoglobin with Tcf-4, in a subdomain other than beta-catenin, inhibits binding of Tcf-4 to DNA and causes plakoglobin to work as a negative regulator of the beta-catenin-Tcf-4 complexes (17, 18). Accordingly, the effects of transgenic overexpression of both proteins in mouse epidermis are opposed: whereas beta-catenin induces hyperproliferation and hair follicle differentiation, plakoglobin suppresses both cell fate decisions (19, 20). Recent results from our laboratory show that the armadillo domain of both plakoglobin and beta-catenin interacts with the two binding sites at Tcf-4, placed between amino acids 1–53 and 53–80. However, binding of the armadillo domain of plakoglobin to Tcf-4-(1–53) is blocked by the presence of the two terminal tails of this molecule. Similarly, interaction of full-length beta-catenin with Tcf-4-(51–80) is repressed, unless the two tails of this molecule are deleted (21).

Presenilin-1 (PS1) is the main gene responsible for familial forms of Alzheimer disease (22). PS1 gene product is an 8-transmembrane protein with a molecular mass of 50 kDa that is normally itself processed and generates 2 amino- and carboxyl-terminal polypeptides of 30 and 20 kDa, respectively, which remain associated (23). PS1 forms part of the proteolytic {gamma}-secretase complex, a protease involved in the processing of beta-amyloid precursor proteins and other type-I transmembrane proteins, including several Notch family members (23, 24). Increasing evidence indicates that PS1 is an important negative regulator of beta-catenin/Tcf-4-mediated transcription. Drosophila presenilin was identified as a negative modifier of wingless/Wnt in a genetic screening (25). In addition, mutants deficient in Drosophila presenilin accumulate armadillo/beta-catenin in cytoplasm (26). In transgenic mice, loss of PS1 in keratinocytes causes high beta-catenin/Tcf-mediated signaling, epidermal hyperplasia, and tumors (27). Finally, PS1 deficiency in primary fibroblasts leads to increased activity of Tcf-4-dependent transcription of cyclin D1 gene (28).


Figure 1
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  		FIGURE 1.
PS1 affects beta-catenin/Tcf-4-mediated transcription. A, the indicated cells were cotransfected with PS1 inserted into pcDNA3.1His plasmid (+, 150 ng; ++, 300 ng), TOP-FLASH (50 ng), or pGL3-Lef1 human promoter (50 ng) and pTK-Renilla (50 ng) luciferase plasmids. Relative luciferase activity was determined with a dual luciferase reporter assay system 48 h after transfection, and results were normalized by using the Renilla luciferase activity for each sample. Percentage activity was calculated by comparing levels of luciferase activity and levels obtained after transfection of the pcDNA3.1His plasmid alone. B, RWP1 and SW-480 cells were transfected with VP16-Tcf-4 (150 ng), TOP-FLASH (50 ng), and pTK-Renilla (50 ng) luciferase plasmids in the presence or absence of pcDNA3.1His-PS1 (150 ng). The results show the average ± S.D. of three-four experiments performed in triplicate. C, SW-480 cells were transfected with 5 µg of pcDNA3.1His-PS1 or empty vector as a control. After 48 h, cell extracts were prepared as described under "Experimental Procedures" and 20 µg analyzed by Western blotting with antibodies anti-c-Myc, anti-PS1, or anti-{alpha}-catenin used to verify that equal amounts of protein were loaded. +, present; –, absent.

 
However, the molecular basis of this inhibitory effect of PS1 onbeta-catenin-dependent transcription is not clearly understood. PS1 interacts with beta-catenin (29, 30) and E-cadherin (31) in epithelial cells. According to some authors, PS1 might down-regulate beta-catenin-mediated transcription by enhancing the stability of the E-cadherin-beta-catenin complex (32). In parallel, loss or mutation of PS1 increases the stability of beta-catenin (28, 33), whereas other authors have not detected this effect (34–36). Moreover, PS1 interacts with glycogen synthase kinase 3beta (33–34), although the regulation of beta-catenin-mediated transcription seems to be independent of the activity of this kinase (36–37). In this study, we analyzed the regulation of beta-catenin signaling by PS1 in epithelial cell lines and report that the effect on beta-catenin-mediated transcription is not dependent on the down-regulation of this protein. We show that PS1 interacts in these cell lines with plakoglobin and facilitates its interaction with Tcf-4, decreasing the capability to bind DNA of this essential element in beta-catenin signaling.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Expression of Recombinant Proteins and Protein Binding Assays—The preparation of all the plasmids encoding beta-catenin, Tcf-4, and plakoglobin deletion and point mutants has been described elsewhere (7, 9, 38), except pGEX-beta-catenin arm repeats 1–6. This plasmid was prepared inserting the BamH1/EcoRI fragment of beta-catenin in the same sites of pGEX-6P (Amersham Biosciences). cDNAs inserted in pGEX-6P plasmid were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins and purified by affinity chromatography on glutathione-Sepharose (7). When required, GST was removed by cleaving with Pre-Scission protease (Amersham Biosciences). Pulldown assays were performed using purified recombinant proteins fused to a GST tag and extracts from RWP1 cells as described (9). Glutathione-Sepharose-bound proteins were analyzed by Western blot with specific monoclonal antibodies (mAbs) against beta-catenin, plakoglobin, {alpha}-catenin, E-cadherin, c-Myc, Rho (all from BD Biosciences) or Tcf-4 (clone 6H5–3; Upstate%20Biotechnology">Upstate Biotechnology, Lake Placid, NY). PS1 was analyzed with a polyclonal antibody from Calbiochem raised against the carboxyl-terminal fragment. As shown, this antibody mostly recognizes a band of 50 kDa after PS1 transfection to cell lines, although other bands of smaller molecular mass were eventually detected. These bands correspond to partially proteolyzed forms and were also detected with another polyclonal antibody from Santa Cruz Biotechnology. In any case, the data considered in this work only refer to the 50-kDa molecular mass band. Other polyclonal antibodies used were phosphorylated Ser-33, Ser-37, and Thr-41 in beta-catenin (Cell Signaling, Beverly, MA), Tcf-4 (amino-terminal; Santa Cruz Biotechnology), and GST (Amersham Biosciences). The blots were re-analyzed with a different antibody after the membranes were stripped as described (39).


Figure 2
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  		FIGURE 2.
PS1 does not down-regulate beta-catenin in SW-480 cells. A, SW-480 cells were transfected with 5 µg of either pcDNA3.1His-PS1, pcDNA3.1His-APC, or empty vector as a control. After 48 h, cell extracts were prepared as described under "Experimental Procedures." 20 µg of untransfected or transfected SW-480 total cell extracts were analyzed by SDS-PAGE and Western blot with antibodies against beta-catenin, PS1, and E-cadherin as a control. Cells transfected as above were treated with the proteasome inhibitor MG132 (10 µM) for 6 h before preparing the extracts. Cell extracts were analyzed by Western blot with the mentioned antibodies and with a monoclonal antibody specific for phosphorylated Ser-33 in beta-catenin. B, SW-480 cells were cotransfected with 7 µg of beta-catenin, 6 µg of Rho, and 10 µg of PS1, APC, or empty vector, all of them inserted into pcDNA3.1His. After 48 h, cell extracts were prepared, His-tagged beta-catenin was purified by chromatography on nickel-agarose, and the levels of ectopicbeta-catenin were analyzed by Western blotting with anti-beta-catenin and anti-PS1 mAbs. Membrane was re-analyzed against anti-Rho as a control to check that similar levels of purification were obtained in all cases.

 
Transient Transfections—The cell lines RWP1, MiaPaca-2, SW-480, and SW-620 were used in this study. SW-480 and SW-620 were established from a primary colon adenocarcinoma and a metastasis from the same patient and express a mutant form of APC (40). RWP1 and MiaPaca-2 were established from pancreas tumors; RWP1 presents also a mutation in APC,6 whereas the status of this gene in MiaPaca-2 cells is unknown. Cells were routinely grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Transient expression of ectopic proteins was achieved in 50% confluent cells with the indicated cDNAs inserted in pcDNA3-His plasmid (Invitrogen) and using Lipofectamine (Invitrogen) according to the instructions of the manufacturer. Cells were analyzed 48 h after transfection.


Figure 3
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  		FIGURE 3.
PS1 associates with beta-catenin and plakoglobin in SW-480 cells. SW-480 and RWP1 cells were transfected with 15 µg of pcDNA3.1His-PS1 or with empty vector as a control. After 48 h, cell extracts were prepared, and 300 µg of each cell extract were immunoprecipitated with anti-beta-catenin or with anti-plakoglobin, and the associated proteins were analyzed with specific mAbs against presenilin-1, beta-catenin, and plakoglobin. WB, Western blotting; +, present; –, absent.

 
Reporter Gene Assays—Cells were transfected as above with the two promoters sensitive to the activity of beta-catenin/Tcf-4 complex: Top-Flash, a synthetic promoter that contains three copies of the Tcf-4 binding site upstream of a firefly luciferase reporter gene (42), and a fragment of human Lef-1 promoter (–1874/+58) inserted in pGL3 plasmid (43). PS1 or the indicated plasmids and controls were cotransfected when indicated. Activity of the product of the Renilla luciferase gene under the control of a constitutive thymidine kinase promoter (Promega) was used as a control. Assays were always performed in triplicate; the average of the results of three-four independent transfections ± S.D. is given.

Immunoprecipitation—Cell extracts were prepared from cultured cells resuspended in lysis buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% digitonin, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) on ice for 30 min. Lysates were cleared at 12,000 x g for 15 min at 4 °C. 300 µg of cell extracts were incubated with 4 µg/ml of antibody for 5 h at 4°C. Precipitated material was removed by centrifugation at 12,000 x g, and the resulting supernatant was incubated for 90 min with 30 µl of protein A-agarose (Sigma). Immunoprecipitates were washed three times with lysis buffer; bound proteins were directly eluted with electrophoresis sample buffer and either analyzed by Western blot or used for kinase assays. Alternatively, ectopically expressed proteins were purified with nickel-nitrilotriacetic acid-agarose (Qiagen). 250 µg of cell extracts were incubated in a final volume of 300 µl with 20 µl of a 50% (w/v) suspension of nickel-agarose for 1 h at 4°C. Proteins present in the complex were analyzed by Western blot using specific mAbs.

Immunofluorescence—Cells were transfected with pcDNA3-PS1, pcDNA3-Tcf-4, and pcDNA3-plakoglobin when indicated. After transfection, cells were plated on glass coverslips, fixed with 4% paraformaldehyde for 30 min, and permeabilized by incubation with 1% SDS for 10 min. Blocking was performed for 1 h with phosphate buffered-saline containing 0.1% saponin and 1% bovine seroalbumin. Mouse mAb anti-Tcf4 or rabbit polyclonal antisera anti-PS1 were used to analyze the distribution of these proteins. After washing, binding of primary antibodies was detected by anti-mouse and anti-rabbit antibodies conjugated to fluorescein isothiocyanate (Dako) and Alexa 488 (Molecular Probes), respectively. Finally, fluorescence was viewed through a TCS-SP2 Leica confocal microscope.


Figure 4
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  		FIGURE 4.
Mapping of PS1 binding sites on beta-catenin and plakoglobin. A, diagram of beta-catenin and plakoglobin. The three different domains that form these two proteins are shown. B–F, pulldown assays were performed by incubating 10 pmol GST or GST fusion proteins containing beta-catenin wt (B), GST-beta-catenin deletion mutants (C), or the indicated point mutants (D), GST-plakoglobin wt (B), GST-plakoglobin deletion mutants (E), or the indicated point mutants (F). Fusion proteins were incubated with 500µg of whole-cell extracts from RWP1 transfected with PS1; protein complexes were affinity purified with glutathione-Sepharose and analyzed by SDS-PAGE and Western blotting (WB) with anti-PS1. Blots were reanalyzed with anti-GST, anti-beta-catenin (D), or anti-plakoglobin (F) to ensure that similar levels of fusion protein forms were present in all cases. In the Input lane, a sample of 5% of the total cell extract used for the assay was loaded. Autoradiograms were scanned; the values obtained for PS1 were referred to the corresponding fusion protein and presented below each panel, considering as control the binding to the wild-type protein. These values did not differ more than 10% in the three experiments performed.

 
Tcf-4-DNA Binding Assays—Tcf-4-DNA binding assays were performed by incubation of 200 ng of two oligonucleotides corresponding to the Tcf-4 binding site TBE2 (5'-CTCTTGATCAAAGC GCGG-3') within the myc promoter and labeled with biotin, with 200 µg of transfected total cell extract for 45 min at 4 °C (18). Incubations were performed in the presence of 20 mM HEPES, pH 7.6, 150 mM KCl, 3 mM MgCl2, 10% glycerol, and 0.3 mg/ml bovine seroalbumin in a final volume of 200 µl. 20 µg of poly(dI-dC) were used as a nonspecific competitor. Protein complexes were isolated by incubation with 40 µl of a 50% (w/v) suspension of streptavidin-agarose (Sigma) for 30 min at 4 °C. Beads were collected by spinning in a microcentrifuge and washed three times with binding buffer. Samples were separated by SDS-PAGE, and the presence of bound proteins in the complex was analyzed by Western blot with specific mAbs against beta-catenin, plakoglobin, or Tcf-4 (amino terminus).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
As mentioned above (see Introduction), PS1 inhibits the Wnt/beta-catenin signaling pathway. We checked whether this effect occurred in epithelial cell lines. As shown in Fig. 1A, PS1 overexpression in colon SW-480 cells down-regulated the activity of two promoters sensitive to beta-catenin/Tcf-4, the widely used synthetic TOP promoter and a fragment of human Lef-1 promoter (–1874/+58) containing the elements responsive to Tcf-4 (43). The activity of both promoter fragments was also affected by co-expression of a mutant form of Tcf-4 lacking the beta-catenin binding domain that acts as a constitutive repressor of beta-catenin target genes (not shown). Inhibition of beta-catenin/Tcf-4-dependent transcription by PS1 was observed in cell lines such as pancreas MiaPaca-2 and colon SW-620 (Fig. 1A). Other cell lines, such as pancreas RWP1, seem more resistant to effects of PS1, although the endogenous activity of TOP and Lef-1 promoters is similar to those in SW-480 or RWP1 cells (Fig. 1A). As expected, because c-Myc protein is a target of beta-catenin/Tcf-4 (44), its levels were lower in cells transfected with PS1 (Fig. 1C).


Figure 5
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  		FIGURE 5.
Repression of beta-catenin-dependent transcription by PS1 is not dependent on {gamma}-secretase activity or interaction with beta-catenin. A, SW-480 cells were transfected with pcDNA3.1Myc-His-PS1 (300 ng), TOP-FLASH (50 ng), and pTK-Renilla (50 ng) luciferase plasmids. 24 h after transfection, 5 µM {gamma}-secretase inhibitor LS-685,458 (Calbiochem) was added to the medium for 24 h. Relative luciferase activity was detected 48 h after transfection. B, RWP1 cells were cotransfected with beta-catenin mutants inserted into pcDNA3.1His (150 ng), TOP-FLASH (50 ng), and pTK-Renilla (50 ng) luciferase plasmids in the presence or absence of pcDNA3.1His-PS1 (150 ng). Relative luciferase activity was determined with a dual luciferase reporter assay system 48 h after transfection, and the result was normalized using the Renilla luciferase activity for each sample. Percentage activity was calculated by comparing levels of luciferase activity to those obtained after transfection of the pcDNA3.1His plasmid alone. Values are the average ± S.D. of three experiments performed in triplicate.

 
To study the element affected by PS1, cells were transfected with a plasmid encoding a VP16-Tcf-4 fusion protein. This protein stimulates the activity of promoters containing functional Tcf-4 binding elements independently of beta-catenin, because it contains the transactivation element of VP16. As shown in Fig. 1B, transfection of this plasmid greatly increased the activity of TOP reporter in both SW-480 cells and in RWP1. Addition of PS1 did not prevent the up-regulation caused by VP16-Tcf-4 in SW-480, but stimulated it a little.

PS1 promotes beta-catenin degradation through axin-independent stimulation of the activity of glycogen synthase kinase 3beta kinase on beta-catenin Ser-33, Ser-37, and Thr-41 (33). We examined whether PS1 inhibition of TOP signaling in SW-480 was due to destabilization of beta-catenin after its phosphorylation. This does not seem to be the case, because no increases in phosphorylation of these three residues of beta-catenin were observed after PS1 transfection in SW-480 cells although the proteasome inhibitor MG321 was added to the cells (Fig. 2A). More conclusively, PS1 did not promote the down-regulation of beta-catenin (Fig. 2, A and B). As observed, PS-1 caused no change in the levels of beta-catenin, whether endogenously or ectopically expressed. As a control, transfection of APC induced a significant down-regulation of this catenin, accompanied by increased phosphorylation in Ser-33 (Fig. 2).

The possibility that inhibition was related to the interaction of beta-catenin with PS1 was also investigated. Both proteins were co-immunoprecipitated either in SW-480 cells or in RWP1 cells (Fig. 3). We detected that PS1 also interacted with the beta-catenin homologue plakoglobin in both cell lines.

Plakoglobin and beta-catenin consist of a central armadillo repeat domain, very similar in both proteins, and two more divergent tails (Fig. 4A). The association of PS1 and beta-catenin was also detected by pulldown assays, using GST-beta-catenin as bait (Fig. 4B). A similar amount of PS1 was also bound by GST-plakoglobin (Fig. 4B), indicating that the affinities of both proteins for PS1 are similar. The interaction sites of PS1 in both proteins were determined. PS1 had a slightly greater affinity for the armadillo repeat domain than for the entire beta-catenin, similar to what has been described for other beta-catenin-binding proteins (39). PS1 associated with armadillo repeats 7–12 to the same extent as the complete central domain and with repeats 10–12 only slightly less, indicating that these repeats are the main binding site for PS1 (Fig. 4C). On the other hand, armadillo repeats 1–6 barely interact with PS1. Deletion of the beta-catenin amino-tail, which strengthens the interaction of the carboxyl-tail with the armadillo domain and reduces the interaction of this element with other beta-catenin-binding proteins (41), decreases the affinity for PS1 (Fig. 4C). The effects of beta-catenin point mutants that modify the interaction with other proteins were also studied. Tyr-654 -> Glu (Y654E), a mutant that mimics the phosphorylation of this residue by epidermal growth factor receptor or other tyrosine kinases, decreases 4-fold the association between beta-catenin and PS1, but Tyr-142 -> Glu did not modify this interaction (Fig. 4D). In this regard, it is worth mentioning that Tyr-654 is placed in armadillo repeat 12 (Fig. 4A).

Despite its close similarity to beta-catenin, plakoglobin interacted differently with PS1. The plakoglobin armadillo domain showed slightly less affinity for PS1 than the full-length protein (Fig. 4E). The effect of removing the carboxyl- or the amino-tails was also small. The armadillo repeats involved in PS1 binding were identified as 1–6, suggesting that the interaction of PS1 with plakoglobin requires a more amino-terminal part of this domain than in the case of beta-catenin. This conclusion was reinforced by the fact that mutation of the plakoglobin equivalent residue to beta-catenin Tyr-654, Tyr-643, did not down-regulate PS1 binding but, to the contrary, increased it (Fig. 4F).


Figure 6
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  		FIGURE 6.
Plakoglobin mediates PS1 interaction with Tcf-4. A, RWP1 and SW-480 cells were transfected with 7.5 µg of pcDNA3.1His-Tcf-4, in the absence or presence of 8.5 µg of pcDNA3.1Myc-His-PS1 or empty vector. B, GST-Tcf-4-(1–53) or GST-Tcf-4-(51–110) (30 pmol) were preincubated with 60 pmol beta-catenin or plakoglobin, when indicated. Afterward, pulldown assays were performed by incubating the GST proteins with 500 µg of total cell extracts from RWP1 transfected with pcDNA3.1Myc-His-PS1. C, SW-480 cells were transfected with 7.5 µg of pcDNA3.1His-Tcf-4-(1–80), either wild-type or the S60E mutant, and the presence or absence of 8.5 µg of pcDNA3.1His-PS1 or empty vector. After 48 h, cell extracts were prepared, 500 (A) or 350 (C) µg of whole-cell extracts were immunoprecipitated with anti-Tcf-4 antibody, and the complexes were analyzed by immunoblotting with the indicated antibodies. D, GST-plakoglobin fusion proteins containing the full-length protein or the armadillo repeats domain (11 pmol) were preincubated with 23 pmol Tcf-4-(1–80) and pulldown assays performed as above with 500 mg of RWP1 transfected with pcDNA3.1Myc-His-PS1. In the Input lane, a sample corresponding to 5% of the total cell extract used for the assay was loaded. +, present; –, absent.

 


Figure 7
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  		FIGURE 7.
PS1 prevents binding of Tcf-4 to DNA and stabilizes Tcf-4 outside the nucleus. A, SW-480 cells were transfected with 7 µg of pcDNA3.1His-Tcf-4 in the absence or presence of 9 µg of pcDNA3-PS1. 200 ng of the biotinylated oligo containing the binding sequence for Tcf-4 in the c-myc promoter (Oligo PrMyc) or synthetic TOP promoter (Oligo PrTop) was incubated with 300 µg of cell lysates prepared from transfected SW-480 cells. The biotinylated oligo was purified by chromatography on streptavidin-agarose, and associated Tcf-4 was analyzed by Western blot with polyclonal antibody against Tcf-4. Membranes were stripped and reanalyzed with antibodies against beta-catenin, plakoglobin, and PS1. Input, a sample corresponding to 10% of the total extract used for the assay; Cont, no specific oligo added. B, SW-480 cells were transfected with Tcf-4 and PS1 cDNAs inserted in pcDNA3 and plakoglobin when indicated. Subcellular localization of Tcf-4 and PS1 was determined by immunofluorescence as described, using a mAb anti-Tcf-4 and a rabbit polyclonal anti-PS1. The same immunoreactivity as obtained for Tcf-4 in the absence of plakoglobin was obtained in the absence of PS1. Controls performed without primary antibodies showed no signal.

 
These results indicate that PS1 interacts with both beta-catenin and plakoglobin, although they did not use binding sites placed in equivalent residues. In addition, phosphorylation of Tyr-654 in beta-catenin affected its interaction with PS1.

It has been suggested that PS1 might prevent beta-catenin-mediated transcription through its direct interaction with beta-catenin or by increasing the binding of this protein to the cytosolic domain of E-cadherin, generated by the PS1-dependent {gamma}-secretase activity. Both possibilities were ruled out in our cell lines. First, blocking of {gamma}-secretase activity with LS-685,458, a widely used specific inhibitor (45, 46), did not prevent the effect of PS1 on beta-catenin-dependent transcription in epithelial cell lines (Fig. 5A). Moreover, PS1 repression of beta-catenin-mediated transcription in RWP1 cells was increased by ectopic transfection of beta-catenin. As Fig. 5B shows, beta-catenin forms containing Y654E mutant were more active than wild-type form in stimulating the activity of the TOP promoter. Although this mutant has less interaction with PS-1 and with E-cadherin, the activity induced by Y654E and Y654E,Y142E beta-catenin forms was sensitive to inhibition by PS1 (Fig. 5B), indicating that PS1 affects beta-catenin-mediated transcription by a mechanism independent of its direct interaction with beta-catenin.

As mentioned in the Introduction, beta-catenin and plakoglobin bind to different subdomains in Tcf-4 and promote opposite effects. Whereas beta-catenin increases the recruitment of positive transcriptional cofactors necessary for gene activation, plakoglobin inhibits DNA binding of Tcf-4. We checked whether PS1 binding interfered with the interaction of beta-catenin or plakoglobin with Tcf-4 or whether, on the contrary, PS1 and Tcf-4 were present in the same complex. As shown in Fig. 6A, PS1 and Tcf-4 co-immunoprecipitated in both SW-480 and RWP1 cells. Transfection of PS1 consistently increased the amount of plakoglobin present in Tcf-4 immunocomplexes in SW-480 and only very slightly in RWP1 cells. beta-catenin was also immunoprecipitated with Tcf-4; PS1 also increases the association between these two proteins although slightly less than in the case of plakoglobin, and only in SW-480 cells.

Pulldown assays using fragments of Tcf-4 fused to GST indicated that PS1 interacted through the same regions used by beta-catenin and plakoglobin, amino acids 1–50 and 51–80, respectively (Fig. 6B). Preincubation with either beta-catenin or plakoglobin increased the amount of PS1 retained by glutathione-Sepharose, suggesting that these proteins mediate the interaction of PS1 and Tcf-4. However, the effect was much stronger when plakoglobin was used (Fig. 6B), suggesting that binding of Tcf-4 to PS1 is mainly mediated by this protein. Moreover, plakoglobin also associated better with Tcf-4 when PS1 was present, reinforcing the conclusion of the experiment shown in Fig. 6A.

We also analyzed the effects of PS1 binding on the association of beta-catenin and plakoglobin with wild-type Tcf-4 and with a mutant in which Ser-60 was replaced by a Glu. It has been reported that this mutation selectively decreases binding of Tcf-4 to plakoglobin but does not decrease it to beta-catenin (18). Transfection of PS1 increased the amount of beta-catenin that could be precipitated with wild-type Tcf-4, but the increase was only very modest with Tcf-4 S60E (Fig. 6C). Better PS1 stimulation was observed when binding of Tcf-4 to plakoglobin was analyzed. Surprisingly, PS1 also up-regulated binding of plakoglobin to Tcf-4 S60E, indicating that PS1 modifies plakoglobin-Tcf-4 association and becomes insensitive to Ser-60 phosphorylation.

The mechanism involved in stimulation of plakoglobin-Tcf-4 association by PS1 was further investigated. As shown in Fig. 6D, the armadillo repeat domain binds to Tcf-4 with a higher affinity than the full-length protein, suggesting that the two tails prevent binding of this element. Similar effects of the tails have been reported for the association of other cofactors with the armadillo repeats of beta-catenin or plakoglobin (21, 38, 39). PS1 increased 3.5-fold (as determined after densitometry of Fig. 6D) the amount of Tcf-4 bound to plakoglobin but did not affect the interaction of this factor with the armadillo repeat domain, suggesting that the effect of PS1 consisted in the elimination of the restriction imposed by the tails. Binding of PS1 to plakoglobin was only slightly affected by elimination of the tails, as reported above (Fig. 4E).

We examined whether PS1-Tcf-4 binding was accompanied by a lower association of Tcf-4 with DNA. SW-480 cell extracts from control or PS1-transfected cells were incubated with an oligonucleotide containing the Tcf-4 binding site present in the c-myc promoter. As shown in Fig. 7A, PS1 prevented Tcf-4 interacting with DNA. As expected, beta-catenin was detected in the DNA-bound complexes, but plakoglobin was not.

The inhibition of Tcf-4 binding to DNA by PS1 may be due to an intrinsic inhibitory effect of PS1 or plakoglobin in the complex that precludes interaction to the DNA or to an altered subcellular distribution of Tcf-4. Therefore, the localization of Tcf-4 and PS1 was studied by immunofluorescence. As observed in Fig. 7B, PS1 immunoreactivity was detected in the cytosol, with a mainly perinuclear distribution, and it was completely excluded from the nucleus. On the other hand, Tcf-4 was present in both cellular compartments. Tcf-4 reactivity was not substantially altered by ectopic expression of PS1 unless plakoglobin was also simultaneously transfected; in these cells a reproducible decrease in Tcf-4 nuclear immunoreactivity was observed (Fig. 7B). These results suggest that PS1 interacts with Tcf-4 in an extranuclear compartment only in the presence of plakoglobin, and this interaction retains Tcf-4 outside the nucleus and precludes its binding to target promoters.

These results indicate that PS1, through its binding to plakoglobin, prevents Tcf-4 binding to DNA. Therefore, Tcf-4 is the limiting factor in beta-catenin-dependent transcription in PS1-transfected cells. Promoter reporter assays confirmed this conclusion because, in SW-480 cells transfected with Tcf-4, TOP activity was barely affected by expression of PS1 (Fig. 8). Moreover, cotransfection of plakoglobin returned sensitivity to PS1. In this case, the repression of PS1 was much higher than in the control cells, an effect that was probably due to the stimulation of TOP activity observed after addition of plakoglobin.

As observed in our assays, PS1 migrated in our SDS gels mainly as a band of ~50 kDa, suggesting that it was not being processed in our cell lines. To determine whether this modification might alter PS1 binding to Tcf-4 and plakoglobin, we analyzed cellular extracts from cell lines in which PS1 was predominantly present in the processed form. As shown in Fig. 9, when binding of processed and unprocessed forms of PS1 to GST fusion proteins was analyzed, the PS1 carboxyl-terminal fragment bound with lower affinity to beta-catenin (~5-fold lower, Fig. 9B) and plakoglobin (~3-fold) than the 50-kDa form. Binding to Tcf-4 was barely detectable for the processed PS1 (Fig. 9A). Similar results were obtained when a mixed extract containing both forms was used as source of PS1. Although the most abundant band in this extract corresponded to the 20-kDa band, the 50-kDa form was much more represented in the fraction bound to GST-beta-catenin or GST-plakoglobin (Fig. 9A). In these conditions the 50 kDa associated 6- and 5-fold better, approximately, to beta-catenin and plakoglobin, respectively. Therefore, these results suggest that interaction of Tcf-4 is restricted to the unprocessed form of PS1 because of its greater affinity for plakoglobin.


Figure 8
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  		FIGURE 8.
Ectopic Tcf-4 expression prevents PS1 effects on beta-catenin-dependent transcription. SW-480 cells were transfected with pcDNA3-Tcf-4 (+, 150 ng; ++, 300 ng) (grey columns) pcDNA3-PS1 (150–300 ng) (black columns) or empty vector (white columns) TOP-FLASH (50 ng), and pTK-Renilla luciferase (50 ng) plasmids. In panel B pcDNA3-Plakoglobin was transfected when indicated. Relative luciferase activity was determined with a dual luciferase reporter assay system 48 h after transfection, and the result was normalized by using the Renilla luciferase activity for each sample. Percentage activity was calculated by comparing levels of luciferase activity with those obtained after transfection of the pcDNA3.1His plasmid alone. Values are the average ± S.D. of four experiments performed in triplicate.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Because PS1 was identified as a negative modifier of Wnt/wingless pathway in epithelial cells (27), several alternative mechanisms to explain this result have been put forward. Initial studies ruled out the possibility of this inhibitory effect being associated with its function as a component of the {gamma}-secretase complex (28, 37). However, because PS1 interacts with beta-catenin and E-cadherin (31, 32) it might enhance the stability of this complex, precluding the transit of beta-catenin to the nucleus, a step required for its transcriptional activity. Moreover, PS1 works under certain conditions as a scaffolding protein, facilitating the phosphorylation of beta-catenin by protein kinase A and subsequently by glycogen synthase kinase 3beta. The mechanism of this paired phosphorylation is independent of the presence of axin, a protein normally required for coupling protein kinase CK1 to the complex (33). Protein kinase A, in the case of PS1, and CK1, for axin, are responsible for the initial priming that allows the subsequent phosphorylation by glycogen synthase kinase 3beta that leads to protein destabilization. However, although we detected decreases in beta-catenin-mediated transcription in our epithelial cell lines, these changes were not accompanied by down-regulation of beta-catenin. Moreover, use of beta-catenin mutants deficient in PS1 binding reinforced our conclusion that effects of PS1 on beta-catenin transcriptional activity were independent of beta-catenin binding. The reasons for this discrepancy are unknown, although several explanations are possible. First, protein kinase A activity in our cell lines may be low and unable to phosphorylate beta-catenin Ser-45, even in the presence of PS1. The second reason is due to the lack of functional APC gene product in our cell lines (40). Although the requirement of this protein for PS1-induced beta-catenin down-regulation has not been established, it is likely that, as in the case of axin, PS1 permits beta-catenin degradation only when it is in a complex with APC. Finally, the effects of PS1 on beta-catenin degradation might depend on the processing of PS1 and might only be detected when the protein has been proteolyzed in the two amino- and carboxyl-terminal fragments. We considered this last possibility unlikely, because unprocessed PS1 has a greater affinity for beta-catenin than the processed protein, but it is still possible that only the processed protein has the capability to anchor protein kinase A to beta-catenin.


Figure 9
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  		FIGURE 9.
Processed PS1 has lower affinity for plakoglobin, beta-catenin, and Tcf-4 than the 50-kDa form. A, pulldown assays were performed by incubation of 10 pmol GST, GST-beta-catenin, GST-plakoglobin, or 30 pmol GST-Tcf-4-(1–110) with 1.5 mg of total cell extracts from murine embryonic fibroblasts (MEFs), 300 µg of extracts from RWP1 transfected with PS1, or a mixture composed of 200 µg of RWP1-PS1 and 1 mg of MEFs extracts. When indicated, GST-Tcf-4-(1–110) was preincubated with 60 pmol beta-catenin or plakoglobin. Protein complexes were affinity purified with glutathione-Sepharose and analyzed by SDS-PAGE and Western blotting (WB) with anti-PS1, anti-GST, anti-beta-catenin, or anti-plakoglobin. In the Input lane, a sample corresponding to 10% MEFs extract, 3% RWP1 extract, or 4% mixed extract was loaded. B, autoradiograms were scanned, and the values obtained for binding of the two PS1 forms to beta-catenin (gray bars) and plakoglobin (black bars) were represented with respect to the corresponding inputs. The results show the average ± range of two experiments.

 
In any case, our results have identified plakoglobin as a protein with high affinity for PS1. As mentioned in the Introduction, plakoglobin, also called {gamma}-catenin, is a protein very similar to beta-catenin, especially in the central armadillo repeat domain, which is the binding site for PS1. However, the two proteins do not associate with PS1 through equivalent residues in their molecules; whereas beta-catenin uses the last six armadillo repeats, PS1 binds to the first six repeats in plakoglobin. Moreover, the dependence of this binding on phosphorylation in specific tyrosine residues is also different. PS1-beta-catenin interaction is reduced by modification of Tyr-654, a residue placed in the last armadillo repeat of beta-catenin that also controls the interaction with E-cadherin (7). However, phosphorylation of the equivalent residue in plakoglobin, Tyr-643, does not repress PS1 binding. Therefore, growth factors that modulate phosphorylation of these tyrosine residues in the two catenins may modulate PS1 binding to one or the other protein. Preliminary results indicate that addition of epidermal growth factor to RWP1 cells transfected with PS1 increases its interaction with plakoglobin7 and evidence that association of PS1 to catenins can be modulated by extracellular signals. This dependence on tyrosine phosphorylation of PS1-catenin binding also helps explain why effects of PS1 on beta-catenin-dependent transcription might be different in distinct cell lines.

Association of PS1 with plakoglobin and beta-catenin also presents several interesting features. First, it enhances the interaction of plakoglobin and Tcf-4. Plakoglobin binds to this transcriptional factor through amino acids 53–80 and is repressed by phosphorylation of Tcf-4 Ser-60 (18). Our results indicate that plakoglobin interacts better with Tcf-4 when PS1 is overexpressed and the effect of phosphorylation is low, suggesting that in the presence of PS1 the characteristics of the interaction Tcf-4-plakoglobin are modified. Tcf-4 associates with the armadillo domain of plakoglobin and beta-catenin, and this binding is affected by the two tails of the catenins. As shown in Fig. 6D, and reported also for bindings of other cofactors to plakoglobin and beta-catenin (8, 21), the armadillo domain interacts much better with Tcf-4 than the entire protein. The stimulation by PS1 on Tcf-4 binding is not observed on the plakoglobin armadillo domain, indicating that PS1 relieves the inhibition caused by the tails. We have previously reported that binding of factors to the armadillo domain of plakoglobin and beta-catenin is sometimes cooperative because the interaction of a first factor (for instance, interaction of plakoglobin to E-cadherin) potentiates further association of a second factor ({alpha}-catenin) (9). This effect is due to the unfolding of the molecule by the first cofactor that separates the tails from the armadillo domain and renders the binding site for the second factor more accessible. We propose that a similar action is carried out by PS1 through its interaction with repeats 1–6 of plakoglobin, potentiating the subsequent binding of Tcf-4 to the same repeats, probably through a different surface.

Association of plakoglobin to Tcf-4 blocks the interaction of Tcf-4 and DNA. Two complementary explanations account for this. First, in the presence of PS1, a substantial part of Tcf-4 is present in an extranuclear compartment; it cannot locate in the nucleus and is therefore inactive. Second, plakoglobin interaction with Tcf-4 prevents binding of the complex to DNA, which might facilitate Tcf-4 nuclear export.

All these data fit previous reports indicating that plakoglobin is indeed a repressor of epithelial cell proliferation and its effects are not merely due to the stimulation of cell-cell adhesion (47). It is also likely that some of the positive effects reported so far for plakoglobin on beta-catenin-dependent signaling, such as those shown in Fig. 8, are due to the displacement of beta-catenin from adherens junctional complexes, thus facilitating its transport to the nucleus and increasing the activity of the beta-catenin-Tcf-4 complex (48). Therefore, the role of plakoglobin as positive or negative effector of beta-catenin-dependent signaling depends on its binding to Tcf-4, an interaction modulated by PS1 and possibly by post-translational modifications of plakoglobin.

Finally, it must be remembered that these results have been obtained using epithelial cell lines. Moreover, as mentioned above, the interaction of PS1 and beta-catenin and plakoglobin depends on the post-translational modification of these two proteins and on the processing of PS1, two parameters that may vary in different cell lines and circumstances. Therefore, the relative contribution of plakoglobin to the modulation of beta-catenin-mediated transcription may differ in cell lines from distinct origins and would not be the same in neurons, fibroblasts, or epithelial cells. In any case, the data given here provide evidence for a new mechanism controlling the activity of this transcriptional complex and help explain the function of plakoglobin as a repressor of this pathway.


   FOOTNOTES
 
* This work was supported in part by Fundació La Caixa Grant 01/045-00 (to A. G. H.) and by Spanish Ministry of Science and Technology Grants BMC2003-00410 (to M. D.) and SAF2003-0234 (to A. G. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Recipient of a postgraduate fellowship from the Instituto de Salud Carlos III. Back

2 Recipient of a postgraduate fellowship from the Ministry of Education. Back

3 To whom correspondence may be addressed. Tel.: 34-93-221-1009; Fax: 34-93-221-3237; E-mail: agarcia{at}imim.es. 4 To whom correspondence may be addressed. Tel.: 34-93-581-1870; Fax: 34-93-581-1907; E-mail: mireia.dunach{at}uab.es.

5 The abbreviations used are: Tcf, family of T-cell transcription factors; APC, adenomatous polyposis coli; GST, glutathione S-transferase; mAb, monoclonal antibody; PS1, presenilin-1. Back

6 G. Capellà and F. X. Real, personal communication. Back

7 I. Raurell, A. Garcia de Herreros, and M. Duñach, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Drs. R. Killick and J. Baulida for providing reagents. We also thank Dr. C. Saura for helpful comments on this work. The technical assistance of Neus Ontiveros is appreciated.



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