Regulation of beta -Catenin Structure and Activity by Tyrosine Phosphorylation

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 276, No. 23, Issue of June 8, pp. 20436 –20443, 2001 Printed in U.S.A. Regulation of b-Catenin Structure and Activity by Tyrosine Phosphorylation* Received for publication, January 9, 2001, and in revised form, March 8, 2001 Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M100194200 José Piedra‡§, Daniel Martı́nez‡, Julio Castaño‡, Susana Miravet‡§, Mireia Duñach‡i, and Antonio Garcı́a de Herreros¶i From the ¶Unitat de Biologia Cel.lular i Molecular, Institut Municipal d’Investigació Mèdica, Universitat Pompeu Fabra, c/Dr. Aiguader 80, 08003 Barcelona, Spain and ‡Unitat de Biofı́sica, Departament de Bioquı́mica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain cación y Ciencia and CIRIT (Generalitat de Catalunya), respectively. i To whom correspondence should be addressed. Tel.: 34-93-581-1870; Fax: 34-93-581-1907; E-mail: mireia.dunach@uab.es (for M. Duñach) or Tel.: 34-93-221-1009; Fax: 34-93-221-3237; E-mail: agarcia@imim.es (for A. Garcia de Herreros) 1 The abbreviations used are: TBP, TATA-binding protein; GST, glutathione S-transferase; cytoE-cadh, cytosolic domain of E-cadherin; mAb, monoclonal antibody; Tyr(P), phosphotyrosine. 20436 This paper is available on line at http://www.jbc.org Downloaded from http://www.jbc.org/ by guest on May 21, 2020 b-Catenin plays a dual role as a key effector in the sequence in the first armadillo repeat of b-catenin (3). It has regulation of adherens junctions and as a transcrip- been proposed that the interactions of b-catenin with these two tional coactivator. Phosphorylation of Tyr-654, a residue proteins are regulated by tyrosine phosphorylation (4, 5). In the placed in the last armadillo repeat of b-catenin, de- case of E-cadherin, we have recently demonstrated that phoscreases its binding to E-cadherin. We show here that phorylation of tyrosine residue 654 diminishes the association phosphorylation of Tyr-654 also stimulates the associa- of b-catenin to this protein by a factor of 10 (6). This residue is tion of b-catenin to the basal transcription factor TATA- modified in vivo by effectors that concomitantly decrease binding protein. The structural bases of these different b-catenin-E-cadherin binding (6). On the other hand, there is affinities were investigated. Our results indicate that no direct evidence so far that modification of any Tyr residue on the b-catenin C-terminal tail interacts with the arma- b-catenin inhibits its interaction with a-catenin. dillo repeat domain, hindering the association of the In addition to its structural role in cellular junctions, b-catearmadillo region to the TATA-binding protein or to Enin is a critical component of the wnt-signaling pathway that cadherin. Phosphorylation of b-catenin Tyr-654 degoverns cell fate in early embryogenesis (7, 8). Activation of creases armadillo-C-terminal tail association, uncoverpathway induces the stabilization free b-catenin, its This article been withdrawn by this the authors. Errors were identified inof several ing the last armadillo repeats. In ahas C-terminal-depleted translocation to the nucleus, and its binding to members of the b-catenin, the presencefigures. of a negative charge Evaluation byat theTyr-654 Journal with image analysis software determined that does not affect the interaction of the TATA-binding pro- LEF-1/TCF family of transcription factors (7, 8). Interaction of images were reused represent conditions inthem the -catenin b-cateninexperimental with these factors converts to transcriptional tein to the armadillo domain. However, in to the case of different (9) and expression of several genes E-cadherin, the establishment of ion in pairs its activators immunoblot Fig.dominates 2, the -catenin immunoblots in stimulates Fig. 5, the the E-cadherin association with b-catenin, and its binding is greatly containing Tcf-4-responsive sequences in their promoter (10 – immunoblot in Fig. 6A, the Tcf-4 immunoblot in Fig. 6B, and the -catenin dependent on the absence of a negative charge at Tyr- 14). In the absence of wnt stimulus, cytosolic b-catenin is deimmunoblot in Fig.blocks 7B. The raw are no longer available to validateitsthe graded through a mechanism requiring binding to the tumor 654. Thus, phosphorylation of Tyr-654 the E-data suppressor gene product polyposis evenThe though thehave steric cadherin-b-catenin interaction, information. authors expressed the opinion that adenomatous none of these errors coli (8). The hindrance of the C-tailaffect is no the longer These exact role adenomatous polyposis coli in the regulation of finalpresent. conclusions of rethis article that,ofaccording to them, have been sults explain how phosphorylation of b-catenin in Tyr- b-catenin levels has not been perfectly explained, although it is extensively during these years.to facilitate the formation of a complex between b-cate654 modifies the tertiary structurevalidated of this protein and 15 thought the interaction with its different partners. nin and axin/axil, glycogen synthase 3-B, and b-TCRP/slimb (8). The domains of b-catenin involved in transcriptional activab-Catenin was initially described as a protein involved in the tion have been localized in the N- and C-terminal parts of this regulation of E-cadherin function, since it binds to the cytoplas- molecule (15, 16). The C-terminal tail of b-catenin, when fused mic domain of this protein and is necessary for linkage of to LEF-1, has been shown to be sufficient to promote transacE-cadherin to the actin cytoskeleton (1). Sequences involved in tivation (15). Although the mechanism underlying this activaE-cadherin and a-catenin binding have been identified in tion is not totally known, the N- and C-terminal transactivab-catenin; association of E-cadherin requires armadillo repeats tion domains of b-catenin interact with a growing list of nuclear 4 –12 situated in the central part of b-catenin (2). On the other factors that include the TATA-binding protein (TBP)1 (16), hand, a-catenin binding is limited to a short 31-amino acid Pontin (17), Teashirt (18), Sox17 and 13 (19), histone deacetylase (20), SMAD4 (21), the retinoic acid receptor (22), and the CREB binding protein and related proteins (23–26). One of the * This work was supported by La Marató de TV3 Grant 983110 (to essential roles of b-catenin-Tcf-4 complex consists in recruiting A. G. H.), Ministerio de Ciencia y Tecnologı́a Grant PM99-0064 (to the basal transcriptional machinery to the promoters of wntM. D.), FEDER-Fondo Nacional I1D Fund Grants 2FD97-1491-C02-01 and sensitive genes. A key component of this transcriptional com2FD97-1491-C02-02 (to A. G. H. and M. D., respectively), and Direcció General de Recerca Grants 1999SGR00245 and 1999SGR00102. The plex is TBP, which interacts with two different domains of costs of publication of this article were defrayed in part by the payment b-catenin necessary for transactivation (16). of page charges. This article must therefore be hereby marked “adverAs mentioned above, phosphorylation of b-catenin Tyr-654 tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate severs b-catenin-E-cadherin binding (6). Since this residue is this fact. located in a domain involved in TBP binding (16), we have § Recipients of predoctoral fellowships awarded by Ministerio de Edu- Regulation of b-Catenin Structure by Phosphorylation investigated the possible role of this phosphorylation in the interaction between b-catenin and TBP. EXPERIMENTAL PROCEDURES incubated in a final volume of 0.3 ml with 20 ml of a 50% (w/v) suspension of nickel nitrilotriacetic acid-agarose (Qiagen, Hilden, Germany) for 30 min at 4 °C. Beads were washed with radioimmune precipitation buffer, and bound proteins were eluted with electrophoresis sample buffer. Samples were separated by 10% SDS-polyacrylamide gel electrophoresis and analyzed by Western blot. To reprove the membranes, blots were stripped as described (28). The absence of signal after stripping was always checked by incubating with the correspondent secondary antibody and ECL reagent. Analysis of b-Catenin-mediated Transcriptional Activity—b-Catenin-mediated transcription was performed transfecting NIH-3T3 fibroblasts, SW-480 cells, or E-cadherin-deficient MiaPaca-2 pancreas cells with a plasmid containing three copies of the Tcf-4 binding site upstream a firefly luciferase reporter gene (plasmid TOP-FLASH) as described (29). The activity of the product of the Renilla luciferase gene under the control of a constitutive thymidine kinase promoter (Promega) was used as control. Assays were always performed in triplicate; the average of the results of 3– 4 independent transfections 6 S.D. is given. Protease Sensitivity of b-Catenin—1 mg of the different forms of b-catenin, phosphorylated or not by pp60c-src, were incubated in the presence of trypsin (60 ng) at 24 °C in a final volume of 100 ml in a buffer containing 90 mM Tris-HCl, pH 8.5, 2 mM CaCl2, and 4 mM dithiothreitol. Reactions were stopped at different digestion times from 1 to 90 min with electrophoresis loading buffer and boiled for 4 min. The extent of the digestion was determined analyzing the samples by SDS-polyacrylamide gel electrophoresis and Western blot with a mAb anti-bcatenin C terminus, which recognizes an epitope situated between amino acids 696 and 781 of this protein. A quantitation of the reaction was performed scanning the autoradiograms and representing the amount of full-length b-catenin at the different times of incubation relative to the initial time. RESULTS b-Catenin is a good substrate of pp60c-src tyrosine kinase in vitro; this kinase modifies specifically Tyr-86 and Tyr-654, located in the N-terminal domain and in the last armadillo repeat of b-catenin, respectively (6) (see Fig. 1). Although Tyr-86 is phosphorylated with a higher stoichiometry, only modification of Tyr-654 alters the interaction of b-catenin with E-cadherin. Since Tyr-654 is located in the domain of interaction with TBP, we examined whether tyrosine phosphorylation of b-catenin influences the association with this factor. As shown in Fig. 2, phosphorylation of b-catenin by pp60c-src greatly increased its interaction with TBP in pull-down assays (by 6-fold). To analyze the specific influence of Tyr-654 phosphorylation, b-catenin mutants were used in which Tyr-86 and Tyr-654 were replaced by Phe. The same amounts of pulled down TBP were obtained when phosphorylated wild-type b-catenin or phosphorylated Tyr-86 3 Phe mutant were used as bait (Fig. 2). In this case the amount of Tyr(P) incorporated to the b-catenin form was greatly reduced, since only Tyr-654 was phosphorylated (Fig. 2). On the other hand, binding of TBP to the Tyr-654 3 Phe mutant was not increase after phosphorylation, demonstrating that phosphorylation of this residue is involved in the augmented interaction of TBP and b-catenin (Fig. 2). To confirm these results, binding of TBP to b-catenin mutants Tyr-86 3 Glu and Tyr-654 3 Glu was determined. These forms were generated to mimic the effect of phosphorylation in these two residues. b-Catenin Tyr-654 3 Glu interacted much better with TBP than the wild-type form; the amount of pulled down TBP was eight times greater (Fig. 2). Therefore, it could be demonstrated that the introduction of a negative charge in Tyr-654 enhances b-catenin binding to TBP. We also noticed that phosphorylation of Tyr-86 exerted an opposite effect on b-catenin association to TBP. b-Catenin Tyr-86 3 Glu consistently pulled down a lower amount of TBP than wild-type b-catenin (approximately a 30% less); phosphorylation of Tyr-86 in b-catenin Tyr-654 3 Phe exerted a similar action (Fig. 2). Although consistently detected, this negative Downloaded from http://www.jbc.org/ by guest on May 21, 2020 Expression of Recombinant Proteins—Expression and purification of full-length b-catenin, fragments 1–106 and 696-end, and b-catenin point mutants Tyr-86 3 Glu, Tyr-86 3 Phe, Tyr-654 3 Glu and Tyr-654 3 Phe have been previously described (6). A DNA fragment corresponding to the complete 12 armadillo repeats (amino acids 138 – 683) was amplified from entire b-catenin cDNA by polymerase chain reaction using oligonucleotides corresponding to nucleotide sequences 358 –372 and 2035–2047. The 1.7-kilobase amplification fragment was inserted in the BamHI-SmaI sites of a pGEX-6P-1 plasmid and expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Armadillo fragments comprising repeats 7–12 (amino acids 422– 683) and 10 –12 (amino acids 575– 696) were generated cutting the entire armadillo domain cDNA with EcoRI-EcoRV or EcoICRI-EcoRV and inserting in pGEX 6P-2 digested with EcoRI-SmaI or pGEX 6P-3 digested with SmaI. The b-catenin deletion mutants used in this study are presented in Fig. 1, indicating which part of the molecule they comprise. The 1– 80-amino acid fragment of Tcf-4 was generated from pcDNA3-hTcf-4 cutting with BamHI and SmaI and inserting in pGEX6P-1 plasmid. Phosphorylation of b-catenin mutant forms by recombinant pp60c-src protein kinase (from Upstate Biotechnology, Inc.) was performed as described (6). To avoid a possible interference of this kinase in the binding assay, once phosphorylated the GST-b-catenin protein was purified by chromatography on glutathione-Sepharose 4B as indicated below. b-Catenin Binding Assays—The indicated amounts of b-catenin proteins or the 12 armadillo repeats were incubated with different concentrations of N- and C-terminal-GST-b-catenin tails (or GST as a control) at ratios from 1:1 to 5:1 (GST protein versus b-catenin) for 30 min at 20 °C. Incubations were performed in binding buffer: 50 mM Tris-HCl, pH 7.3, 150 mM NaCl, 3 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and 0.1% (w/v) Triton X-100 in a final volume of 200 ml. In some experiments binding to the cytosolic domain of E-cadherin (cytoE-cadh) or to the Tcf-4 b-catenin binding domain (Tcf-4-(1– 80)) was performed in these same conditions. Protein complexes were isolated by incubation with 40 ml of a 50% (w/v) suspension of glutathione-Sepharose 4B for 30 min at 20 °C. Beads were collected by spinning in a microcentrifuge and washed three times with binding buffer. Samples were separated by SDS-polyacrylamide gel electrophoresis, and the presence of bound proteins in the complex was analyzed by Western blot with specific monoclonal antibodies (mAbs) against b-catenin C terminus (Transduction Laboratories, Lexington, KY), b-catenin armadillo core (Alexis Biochemicals, San Diego, CA), E-cadherin cytosolic domain (Transduction Labs), or Tcf-4 N terminus (Santa Cruz Biotechnology). Lysate pull-down assays were performed incubating 12 pmol of GST or GSTb-catenin with 50 mg of SW-480 total cell extract in the conditions mentioned above. Samples were purified by glutathione-Sepharose chromatography and the presence of TBP or Tcf-4 in the complex was determined by Western blot with specific mAbs from Transduction Laboratories and Santa Cruz Biotechnology, respectively. Immunoblots were developed with peroxidase-conjugated secondary antibody followed by enhanced chemiluminiscence detection system (ECL, Pierce). The autoradiograms were scanned, and the values obtained were either compared with known amounts of recombinant proteins included as reference (b-catenin binding assays) or with the value obtained for wild-type full-length b-catenin (pull-down assays). Transient Transfections and Analysis of Transfectants—Assays were performed in SW-480 cell line, which although it contains high levels of b-catenin (like most intestinal epithelial cells), it is deficient in Ecadherin (27). Absence of E-cadherin precludes that the observed differences in TBP binding by the different b-catenin mutants could be attributed to impaired transport to the nucleus due to a distinct association to E-cadherin. Cells were grown in Dulbecco’s modified Eagle’s medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Life Technologies). When 80% confluent, cells were transfected with the indicated plasmids using LipofectAMINE (Life Technologies) according to the instructions of the manufacturer. After transfection, cells were incubated for 48 h in Dulbecco’s modified Eagle’s medium plus 10% fetal calf serum. Cell extracts were prepared in radioimmune precipitation buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 mM EDTA) supplemented with 10 mg/ml aprotinin, 20 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 0.25 mM Na3VaO4. Lysates were centrifuged at 13,000 rpm in a microcentrifuge for 5 min at 4 °C. 250 mg of extract were 20437 20438 Regulation of b-Catenin Structure by Phosphorylation FIG. 1. Diagram of b-catenin. The three different domains that form this protein are shown. The 12 armadillo repeats of b-catenin are represented with numbered boxes, and the two tyrosine residues phosphorylated by pp60c-src are also indicated. The deletion mutants used in this article are depicted, indicating which parts of the molecule they comprise. wt, wild type. Downloaded from http://www.jbc.org/ by guest on May 21, 2020 FIG. 2. Phosphorylation of Tyr-654 enhances binding of b-catenin to TBP but not to Tcf-4. 11 pmol of GST or GST-b-catenin fusion proteins were phosphorylated by pp60c-src in the conditions indicated under “Experimental Procedures.” Pull-down assays were performed incubating the GST proteins with 50 mg of total cell extracts prepared from SW-480 cells. Protein complexes were pelleted down by affinity on glutathione-Sepharose beads, and proteins bound to the complex were analyzed by SDS- polyacrylamide gel electrophoresis and Western blot with anti-TBP mAb. Membranes were stripped and re-analyzed with mAb against Tyr(P), b-catenin, or Tcf-4. wt, wild-type b-catenin; Y86F, Y654F, Y86E, and Y654E correspond to b-catenin mutants Tyr-86 3 Phe, Tyr-654 3 Phe, Tyr-86 3 Glu, and Tyr-654 3 Glu, respectively. The estimated molecular weights of the bands detected with each antibody are shown. The autoradiograms were scanned in a densitometer, and the results obtained (numbers below the lanes) are presented relative to the value obtained for wild-type b-catenin (or phosphorylated wild-type b-catenin in the case of the analysis with Tyr(P) mAb). Only the upper band in the analysis of b-catenin was employed for this analysis; the lower band corresponds to a degradation product of this protein occasionally observed in our preparations that does not interfere in the assay. effect of Tyr-86 phosphorylation on TBP binding was clearly less important than the positive effect observed after Tyr-654 phosphorylation. Probably for this reason, no significant differences were observed in the interaction of TBP to the Tyr-654 3 Glu mutant or to the double mutant Tyr-86 3 Glu/Tyr-654 3 Glu (data not shown). The effects of b-catenin phosphorylation on its association to a well known co-factor, Tcf-4, were determined. No differences in the amount of this protein pulled down by GST-b-catenin were observed after phosphorylation of this molecule or when b-catenin Tyr-86 3 Glu and Tyr-654 3 Glu mutants were analyzed (Fig. 2). The same results were obtained when in vitro binding of recombinant b-catenin and Tcf-4 was determined (not shown). The in vivo association between b-catenin and TBP was also investigated. SW-480 cells were chosen for these assays because they contain very little E-cadherin, and most of the b-catenin is not retained in the membrane by this molecule. Cells were transfected with wild type or Tyr-654 3 Glu b-catenin labeled with polyhistidine and the X-Press® tag to facilitate their purification and identification. Transfected forms were purified by Ni21-agarose, and the amount of associated Regulation of b-Catenin Structure by Phosphorylation 20439 TBP was determined. As shown in Fig. 3A, TBP associated in vivo better with b-catenin mutant Tyr-654 3 Glu than with the wild-type form (2.5-fold better). This higher association correlated with a greater stimulation of b-catenin-Tcf-4-mediated transcription. Overexpression of wild-type b-catenin in SW-480 cells induced a significant increase (60% stimulation) in the activity of a reporter gene placed under the control of a b-catenin- and Tcf-4-sensitive promoter (TOP plasmid) (30). Expression of Tyr-654 3 Glu b-catenin mutant raised the activity of this promoter to a higher extent (194% stimulation) (Fig. 3B). Similar stimulations of TOP activity were obtained in other cell lines (Fig. 3B). Our results indicate that phosphorylation of b-catenin Tyr654 regulates not only the interaction with E-cadherin but with TBP as well. The structural basis of these differences was investigated. Three different regions can be distinguished in b-catenin with distinct charge distributions: the N- and Cterminal tails, with pIs close to 4.5, and the armadillo repeat domain, which presents a basic pI of 8.3 (31). It has been proposed that b-catenin C-terminal region directly interacts with the armadillo domain (32–34). The association between the complete armadillo domain (amino acids 138 – 683) and the N- and C-terminal regions of b-catenin was studied using binding assays with recombinant proteins. Both N-tail (amino acids 1–106) and C-tail (amino acids 696-end) interacted with the armadillo domain. Binding of the C terminus to the armadillo domain requires sequences upstream of the last six armadillo repeats, since a recombinant protein comprising only repeats 7–12 (amino acids 422– 683) associated to the C-terminal tail much worse than the complete armadillo domain (Fig. 4B). A possible effect of phosphorylation of b-catenin Tyr-654, located in the last armadillo repeat, on armadillo-C-tail association was studied. Phosphorylation of this residue decreased armadillo-C-tail interaction (Fig. 4A) but did not modify the binding of the armadillo domain with the N-tail (Fig. 4C). Consequently, b-catenin C-terminal tail also interacted better with FIG. 4. Tyrosine phosphorylation of b-catenin residue 654 inhibits association of the C-terminal tail with the armadillo repeat domain. Panels A and B, 9 pmol of GST fusion proteins containing the indicated forms of b-catenin were incubated with 30 pmol of b-catenin C terminus (696-end) tail in a final volume of 200 ml as described under “Experimental Procedures.” Panel C, interaction of armadillo domain with N-tail was determined by incubating 8 pmol of GST-b-catenin N terminus (1–106) with 2 pmol of b-catenin (138 – 683) (armadillo domain). Protein complexes were purified with glutathioneSepharose and analyzed by SDS-polyacrylamide gel electrophoresis and Western blot (WB) with anti-b-catenin mAbs that recognize the C terminus or the armadillo domain. Phosphorylation of wild-type (WT) arm b-catenin (138 – 683) were carried out with recombinant pp60c-src for 4 h at 23 °C. The numbers below the lanes indicate the amount of bound protein. These values were calculated comparing the result of the scanning of the corresponding lanes with known amounts of b-catenin (0.5 pmol) included as internal references (St) in the same blots. the wild-type armadillo domain than with an armadillo form containing the Tyr-654 3 Glu mutation (Fig. 4A). These results suggest that, in its native conformation, b-catenin is folded with its C-tail interacting with the armadillo repeats. Phosphorylation of Tyr-654 disrupts this interaction and releases the C-terminal tail. To prove this model, experiments of limited trypsin proteolysis of b-catenin were performed, and the extent of unfolding of the C-tail was followed by measuring the rate of disappearance of b-catenin reactivity using an antibody that recognizes only the intact C-tail. As shown in Fig. 5, the b-catenin mutant Tyr-654 3 Glu presented a higher susceptibility to proteolysis than the wild- Downloaded from http://www.jbc.org/ by guest on May 21, 2020 FIG. 3. b-Catenin mutant Tyr-654 3 Glu associates better with TBP and increases Tcf-4-mediated transcription to a higher extent than wild-type (wt) b-catenin. Panel A, in vivo association between b-catenin and TBP. SW-480 cells were transfected with 5 mg of pcDNA3-His-b-catenin (wild type or Tyr-654 mutant forms) or empty vector as control. After 48 h, cell extracts were prepared, His-tagged b-catenin was purified by chromatography on nickel-agarose, and associated TBP was analyzed by Western blot (WB) with TBP mAb. To verify that the extent of ectopic expression was similar in the different cases, blots were reanalyzed with an anti-XpressTM antibody corresponding to a tag that labels the transgen. The estimated molecular masses of the bands detected with each antibody are indicated. The numbers below the lanes indicate the results of the scanning of the two autoradiograms. Panel B, stimulation of Tcf-4-mediated transcription by wild-type b-catenin or Tyr-654 3 Glu mutant (Y654E). NIH-3T3 fibroblasts, SW-480, and MiaPaca-2 cells were cotransfected with b-catenin plasmids (150 ng), TOP-FLASH (20 ng), and pTK-Renilla (20 ng) luciferase plasmids. Relative luciferase activity was determined with a dual luciferase reporter assay system 48 h after transfection and normalized using the Renilla luciferase activity for each sample. Fold activation was calculated by comparing levels of luciferase activity to the pcDNA.3 plasmid alone. 20440 Regulation of b-Catenin Structure by Phosphorylation type form. A faster degradation of the wild-type protein was also observed when it was phosphorylated by pp60c-src. In this case, differences in sensitivity to trypsin proteolysis were less evident, probably due to the incomplete phosphorylation of Tyr-654 in our conditions (6). Phosphorylation of either wild type or Tyr-86 3 Phe mutant produced the same patterns of trypsin digestion (not shown), discarding possible effects due to phosphorylation of Tyr-86 in our assay. We have also analyzed whether binding of the armadillo repeat domain to the C-tail affected the interaction of b-catenin FIG. 6. b-Catenin C-tail, but not Ntail, restricts interaction of the armadillo domain to cytoE-cadh. 0.7 pmol of GST fusion proteins containing b-catenin or the armadillo domain (138 – 683) were incubated with 3 pmol of cytoE-cadh (panel A) or Tcf-4-(1– 80) (panel B). When indicated, binding assays were supplemented with b-catenin C-tail (696-end) (26 and 52 pmol) or N-tail (1–106) (22 and 44 pmol). The amount of associated cytoEcadh or Tcf-4-(1– 80) was determined as above using mAbs specific for these two proteins. The numbers below the lanes indicate the amount of bound protein calculated as in Fig. 4 using known amounts of cytoE-cadh or Tcf-4-(1– 80) as internal standards (St). WT, wild type. WB, Western blot. Downloaded from http://www.jbc.org/ by guest on May 21, 2020 FIG. 5. Phosphorylation of b-catenin tyrosine residue 654 modifies its sensitivity to proteolysis. 1 mg of either wild-type (wt) b-catenin, Tyr-654 3 Glu mutant (Y654E) or phosphorylated b-catenin by pp60c-src were incubated with 60 ng of trypsin at 24 °C. Trypsin digestion was stopped with electrophoresis sample buffer at the indicated times, and samples were analyzed by SDS-polyacrylamide gel electrophoresis and Western blot with a mAb against b-catenin C-tail. The arrowheads indicate the migration of GST-b-catenin (120 kDa); the lower bands represent degradation fragments generated by trypsin. The numbers below the lanes correspond to the percentage of full-length b-catenin remaining after trypsin treatment. Numbers were calculated scanning the autoradiograms. with E-cadherin or Tcf-4. Interaction of the armadillo repeats with the cytosolic domain of E-cadherin (cytoE-cadh) was disrupted by the addition of the C-tail (696-end), indicating that both protein domains interact within the same region of the armadillo domain (Fig. 6A). On the contrary, the addition of b-catenin N-tail (1–106) did not modify armadillo-cytoE-cadh association (Fig. 6A). It is remarkable that the armadillo domain bound cytoE-cadh significantly better than full-length b-catenin (Fig. 6A), supporting the conclusion that removal of the C-tail facilitates the interaction with cytoE-cadh. On the other hand, binding of the armadillo domain to a Tcf-4 fragment containing the b-catenin-binding site was not modified by the addition of both b-catenin terminal tails (Fig. 6B). The binding site for TBP to the b-catenin C-terminal domain has been ascribed to amino acids 630 –729, with residues 630 – 675 contributing critically to this association (16). In our hands, TBP binds uniquely to the armadillo domain (amino acids 138 to 683) and not to the C-tail (amino acids 696-end) (Fig. 7A). As in the case of cytoE-cadh, b-catenin armadillo domain also bound TBP significantly better than full-length b-catenin (Fig. 7A), indicating that the C-tail restricted the interaction with TBP. The association of TBP to the 12 armadillo repeats was also competed by preincubation with b-catenin C-tail (Fig. 7A) but not with Tcf-4 (data not shown). At this point, we also considered the possibility that phosphorylation of Tyr-654 might be inducing alterations in TBP binding independent of the presence of the C-tail. As shown in Fig. 7A, this is not the case; either the wild-type armadillo domain as well as the phosphorylated form of this protein or the Tyr-654 3 Glu mutant pulled down similar amounts of TBP. Thus, these results indicate that changes in TBP binding upon phosphorylation of Tyr-654 are basically due to the release of b-catenin C-terminal tail from the armadillo domain, allowing a better interaction of the last armadillo repeats with TBP. Our results on the binding of the armadillo domain to TBP and to E-cadherin differ in their sensitivity to tyrosine phosphorylation. As shown in Fig. 6 and 7A, whereas phosphorylation of Tyr-654 decreases binding of E-cadherin, it does not modify the interaction of TBP to the armadillo domain. This result suggests that both proteins interact with this domain in a different way. One possibility is that TBP is not binding through an interaction based in the establishment of ion pairs, Regulation of b-Catenin Structure by Phosphorylation 20441 as it has been proposed for E-cadherin. Another possibility is that both proteins interact with different surfaces of the armadillo domain. To explore these possibilities, binding of TBP to full-length b-catenin or to the armadillo domain was performed in the presence of an excess of cytoE-cadh. As shown in Fig. 7B, the addition of a 10-fold molar excess of cytoE-cadh did not modify the amount of TBP bound to the armadillo domain, whereas it increased the amount of TBP pulled down by fulllength b-catenin. This result suggests that, although TBP and E-cadherin interact with overlapping armadillo repeats, both proteins bind to different faces of b-catenin. DISCUSSION b-Catenin has been shown to act both as a regulator of E-cadherin-dependent cell-to-cell adhesion and as an essential mediator in the wnt-signaling pathway (8, 35). Experimental data indicate that the presence of b-catenin in the cellular junctions is controlled by tyrosine phosphorylation (5, 36 – 40). We have previously demonstrated that phosphorylation of Tyr654, a residue located in the 12th and last armadillo repeat of b-catenin, modifies the association of this protein to E-cadherin (6). The armadillo repeat domain has been shown to be essential for the binding of b-catenin to its many binding partners, as E-cadherin and the transcription factor Tcf-4. However, binding of both proteins does not show the same requirements; whereas Tcf-4 associates mainly to repeats 3– 8 (41), E-cadherin requires the last 8 repeats (2, 9, and 42). Therefore, it makes sense that, as we show in this article (Fig. 2), modification of a residue placed at the 12th armadillo repeat does not affect Tcf-4 binding. Armadillo repeat 12 has also been characterized as part of the C terminus-transactivating element required for activation of gene expression (16). Our results indicate that phosphorylation of b-catenin Tyr-654 increases binding of this protein to TBP both in vitro and in vivo, and this greater association correlates with a higher stimulation of Tcf-4-b-catenin transcriptional activity. This higher stimulation of Tcf-4 transcriptional activity observed in vivo by b-catenin Tyr-654 3 Glu mutant is not a consequence of its impaired association to E-cadherin, since it is observed in cells that present very low levels of E-cadherin. In any case, our data suggest that phosphorylation of Tyr-654 is relevant not only for disruption of b-catenin-E-cadherin binding but for stimulation of the interaction of b-catenin to the basal transcriptional machinery as well. These results are consistent with the fact that the nonjunctional pool of b-catenin is preferentially phosphorylated on tyrosine (37). According to our results, phosphorylation of Tyr-654 affects binding of b-catenin to TBP by releasing the restriction created by the C-tail. This restriction is evidenced by the fact that the armadillo domain interacts better with TBP than the complete b-catenin and also by the inhibitory effect of the C-tail on the binding of TBP to the armadillo domain. These data have suggested a working model, presented in Fig. 8, which proposes that, when not phosphorylated and not bound to any ligand, b-catenin would adopt a folded conformation in which the Cterminal tail and the N-tail interact with the armadillo repeat domain. This conformation would prevent the binding to armadillo repeats of low affinity ligands and would select those (such as E-cadherin) presenting high association constants. Phosphorylation of tyrosine residue 654 would remove the C-tail and allow a better access of TBP to the last armadillo repeats. As Downloaded from http://www.jbc.org/ by guest on May 21, 2020 FIG. 7. b-Catenin C-tail restricts interaction of the armadillo domain to TBP. 10 pmol of GST fusion proteins containing the indicated domains of b-catenin, either wild type (WT) or with the Tyr-654 3 Glu mutation, were phosphorylated by pp60c-src or incubated with 100 pmol of b-catenin C-tail (696-end) (panel A) or 100 pmol of cytoE-cadh (panel B) when specified. Fusion proteins and bound proteins were purified by glutathione-Sepharose chromatography and incubated with 50 mg of total cell extract from SW-480 cells. Pulled down TBP was analyzed as in Fig. 1. The same samples were reblotted with mAbs against b-catenin armadillo domain (138 – 683), b-catenin C-tail (696-end), or cytoE-cadh. The estimated molecular masses of GST-b-catenin (120 kDa), GST-arm (86 kDa), GST-C-tail (36 kDa), and C-tail (10 kDa) are shown. The numbers below the lanes correspond to the values obtained scanning the autoradiograms, presented relative to the value obtained for wild-type full-length b -catenin. WB, Western blot. 20442 Regulation of b-Catenin Structure by Phosphorylation depicted in Fig. 8. The interference of the C-tail on the binding of these two proteins indicates that the C-tail can interact with, and possibly hide, both binding surfaces. Accordingly, interaction of E-cadherin with b-catenin displaces the C-tail from its binding to the armadillo domain and facilitates the interaction of factors as TBP that associates to the other side of this domain (see Fig. 8). Although a simultaneous interaction of E-cadherin and TBP with b-catenin is evidently not physiological (E-cadherin and TBP are localized in different cellular compartments), it is possible that a similar role to E-cadherin might be played by other factors interacting with the same binding surface. Nevertheless, a definitive validation of our model of b-catenin regulation by tyrosine phosphorylation would require the determination of the complete structure of this molecule and the characterization of the effect of the two terminal tails in the interaction of b-catenin with its numerous protein partners (35). Acknowledgments—We thank Santiago Roura, Josep Baulida, and Esteve Padrós for their help at different stages of the study and Drs. E. Batlle, R. Kemler, and H. Clevers for reagents. REFERENCES indicated, association of Tcf-4, which takes place mainly through armadillo repeats 3– 8, would not be affected by the C-tail. Thus, the presence or absence of a negative charge at Tyr654 would act as a key for opening or closing b-catenin and would affect the association of this protein to factors binding to the last armadillo repeats and, possibly, to the C-tail as well. In some cases, as for TBP (Fig. 7), after removal of the C-tail the presence or not of a phosphate in Tyr-654 does not modify the interaction of proteins with the armadillo domain. However, in other cases the introduction of a negative charge at this position might hamper the binding of b-catenin with factors like E-cadherin that interact mainly by charge complementarity (31) (see Fig. 6). Thus, for these proteins the negative effect caused on the establishment of ion pairs would predominate over the removing of the steric interference caused by the C-tail. We have also demonstrated that, although E-cadherin and TBP associate to the same armadillo repeats, they do not appear to compete directly for binding to the armadillo domain. As mentioned before, these results suggest that E-cadherin and TBP are interacting with different surfaces of this domain, as Downloaded from http://www.jbc.org/ by guest on May 21, 2020 FIG. 8. Proposed model of regulation of b-catenin binding to TBP and E-cadherin by Tyr-654 phosphorylation. This model illustrates the association to b-catenin of TBP (symbolized by the blue disc), the cytosolic domain of E-cadherin (blue belt), and the N-terminal domain of Tcf-4 (pink cylinder). In b-catenin, the cylinders represent the a-helices that constitute the armadillo domain, and the red dot stands for the phosphate that modifies Tyr-654. In the absence of phosphorylation (left) b-catenin adopts a closed conformation with the C-tail interacting with the armadillo repeats (middle left). E-cadherin binds strongly to these repeats through the establishment of ion pairs between its acidic residues and the positively charged groove in the armadillo domain and can displace the C-tail (bottom left). On the other hand, TBP is not able to overcome this restriction while interacting with the last armadillo repeats (upper left). Phosphorylation of Tyr-654 (symbolized by the introduction of a red dot in the armadillo domain, right side) hampers binding of the C-tail, opening b-catenin and making the last armadillo repeats more accessible (middle right). Therefore, separation of the C-tail makes the binding of TBP much easier (upper right). However, although the armadillo domain is much more accessible, E-cadherin does not bind to phosphorylated b-catenin since the presence of a negative charge makes it difficult to establish the correct ion pairs required for the interaction between these two proteins (lower right). Binding of Tcf-4 (upper) does not require the last armadillo repeats of b-catenin and, thus, is not affected by Tyr-654 phosphorylation. 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Cell Biol. 11, 567–572 Downloaded from http://www.jbc.org/ by guest on May 21, 2020 Regulation of β-Catenin Structure and Activity by Tyrosine Phosphorylation José Piedra, Daniel Marti?nez, Julio Castaño, Susana Miravet, Mireia Duñach and Antonio Garci?a de Herreros J. Biol. Chem. 2001, 276:20436-20443. doi: 10.1074/jbc.M100194200 originally published online March 13, 2001 Access the most updated version of this article at doi: 10.1074/jbc.M100194200 Alerts: • When this article is cited • When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts Downloaded from http://www.jbc.org/ by guest on May 21, 2020 This article cites 42 references, 30 of which can be accessed free at http://www.jbc.org/content/276/23/20436.full.html#ref-list-1