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Phosphorylation of β-Catenin by Cyclic AMP-dependent Protein Kinase*

  • Sebastien Taurin
    Affiliations
    Department of Medicine, University of Chicago, Chicago, Illinois 60637
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  • Nathan Sandbo
    Affiliations
    Department of Medicine, University of Chicago, Chicago, Illinois 60637
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  • Yimin Qin
    Affiliations
    Department of Medicine, University of Chicago, Chicago, Illinois 60637
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  • Darren Browning
    Affiliations
    Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912
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  • Nickolai O. Dulin
    Correspondence
    To whom correspondence should be addressed: Section of Pulmonary and Critical Care Medicine, Dept. of Medicine, University of Chicago, 5841 S. Maryland Ave., MC 6076, Chicago, IL 60637. Tel.: 773-702-5198;
    Affiliations
    Department of Medicine, University of Chicago, Chicago, Illinois 60637
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  • Author Footnotes
    * This study was supported by National Institutes of Health Grant HL071755 (to N. O. D.), American Heart Association Grant AHA0235405Z (to N. O. D.), and American Heart Association post-doctoral fellowship awards (to S. T. and N. S.). 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.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental data.
Open AccessPublished:February 13, 2006DOI:https://doi.org/10.1074/jbc.M508778200
      β-Catenin is a signaling molecule that promotes cell proliferation by the induction of gene transcription through the activation of T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factors. The canonical mechanism of the regulation of β-catenin involves its phosphorylation by casein kinase 1 at the Ser-45 site and by glycogen synthase kinase 3 (GSK3) at the Thr-41, Ser-37, and Ser-33 sites. This phosphorylation targets β-catenin to ubiquitination and degradation by the proteasome system. Mitogenic factors promote β-catenin signaling through the inhibition of GSK3, resulting in reduced β-catenin phosphorylation, its stabilization, and subsequent accumulation in the nucleus, where it stimulates TCF/LEF-dependent gene transcription. In the present study, we have shown that (i) β-catenin can be phosphorylated by protein kinase A (PKA) in vitro and in intact cells at two novel sites, Ser-552 and Ser-675; (ii) phosphorylation by PKA promotes the transcriptional activity (TCF/LEF transactivation) ofβ-catenin; (iii) mutation of Ser-675 attenuates the promoting effect of PKA; (iv) phosphorylation by PKA does not affect the GSK3-dependent phosphorylation ofβ-catenin, its stability, or intracellular localization; and (v) phosphorylation at the Ser-675 site promotes the binding of β-catenin to its transcriptional coactivator, CREB-binding protein. In conclusion, this study identifies a novel, noncanonical mechanism of modulation of β-catenin signaling through direct phosphorylation of β-catenin by PKA, promoting its interaction with CREB-binding protein.
      β-Catenin plays a dual role in cell function. First, being a major component of the cell-cell adherens junctions, β-catenin links members of the cadherin family of transmembrane cell-cell adhesion receptors to the actin cytoskeleton (
      • Ozawa M.
      • Baribault H.
      • Kemler R.
      ). Second, β-catenin acts as an inducer of gene expression that controls an array of embryonic and adult processes (
      • Huelsken J.
      • Behrens J.
      ,
      • Miller J.R.
      • Hocking A.M.
      • Brown J.D.
      • Moon R.T.
      ). β-Catenin is a key effector of Wnt signaling, a pathway first identified in Drosophila (
      • Nusslein-Volhard C.
      • Wieschaus E.
      ) and soon found to be highly conserved throughout the animal kingdom and to affect cell growth and differentiation in many cell types (
      • Willert K.
      • Nusse R.
      ,
      • Polakis P.
      ). In this role, β-catenin functions as an activator of T-cell factor (TCF)
      The abbreviations used are: TCF, T-cell factor; CK1, casein kinase 1; CBP, CREB-binding protein; CREB, cAMP-response element-binding protein; FSK, forskolin; GSK3, glycogen synthase kinase 3; LEF, lymphoid enhancer factor; PKA, protein kinase A; PKAS, PKA substrate; PKI, PKA inhibitor protein; WT, wild-type; HEK, human embryonic kidney; PBS, phosphate-buffered saline; Ad, adenovirus.
      2The abbreviations used are: TCF, T-cell factor; CK1, casein kinase 1; CBP, CREB-binding protein; CREB, cAMP-response element-binding protein; FSK, forskolin; GSK3, glycogen synthase kinase 3; LEF, lymphoid enhancer factor; PKA, protein kinase A; PKAS, PKA substrate; PKI, PKA inhibitor protein; WT, wild-type; HEK, human embryonic kidney; PBS, phosphate-buffered saline; Ad, adenovirus.
      /lymphoid enhancer factor (LEF) transcription factors to stimulate transcription of a variety of growth-related genes, including c-myc (
      • He T.C.
      • Sparks A.B.
      • Rago C.
      • Hermeking H.
      • Zawel L.
      • da Costa L.T.
      • Morin P.J.
      • Vogelstein B.
      • Kinzler K.W.
      ) and cyclin D1 (
      • Tetsu O.
      • McCormick F.
      ). Mutations of β-catenin or of its regulatory proteins are frequently found in various types of cancers, and they result in the accumulation of β-catenin and in the activation of TCF/TCF-dependent gene transcription (
      • Morin P.J.
      • Sparks A.B.
      • Korinek V.
      • Barker N.
      • Clevers H.
      • Vogelstein B.
      • Kinzler K.W.
      ,
      • Bienz M.
      • Clevers H.
      ).
      In quiescent cells, β-catenin is maintained in the cytoplasm at low levels. This is provided by its interaction with the scaffolding proteins, adenomatous polyposis coli and axin, and protein kinases, casein-kinase 1α (CK1α) and glycogen synthase kinase 3 (GSK3), which phosphorylate β-catenin on Ser-45 and then on Ser-33/Ser-37/Thr-41 sites, respectively (
      • Rubinfeld B.
      • Albert I.
      • Porfiri E.
      • Fiol C.
      • Munemitsu S.
      • Polakis P.
      ), leading to its ubiquitination and proteasomal degradation (
      • Aberle H.
      • Bauer A.
      • Stappert J.
      • Kispert A.
      • Kemler R.
      ,
      • Liu C.
      • Li Y.
      • Semenov M.
      • Han C.
      • Baeg G.
      • Tan Y.
      • Zhang Z.
      • Lin X.
      • He X.
      ). The Wnt and other growth stimuli lead to inhibition of GSK3, resulting in a decreased phosphorylation of β-catenin at Ser-33/Ser-37/Thr-41 sites, its stabilization, accumulation and subsequent translocation to the nucleus (
      • van Noort M.
      • Meeldijk J.
      • van der Zee R.
      • Destree O.
      • Clevers H.
      ). In addition to phosphorylation by CK1 and GSK3 at the N terminus, β-catenin can also be phosphorylated at the C terminus on Tyr-654 by Src tyrosine kinases, and this phosphorylation affects the association of β-catenin with cadherin, resulting in a loss of cell-cell adhesion (
      • Roura S.
      • Miravet S.
      • Piedra J.
      • de Herreros A.G.
      • Dunach M.
      ).
      While analyzing the primary structure of β-catenin, we found two putative sites of phosphorylation by PKA that are distinct from those phosphorylated by CK1, GSK3, or Src kinases (Fig. 1). In this study, we have shown that (i) β-catenin can be phosphorylated by PKA at these sites and (ii) phosphorylation at one of these sites promotes the transcriptional activity of β-catenin by facilitating its interaction with a transcriptional co-activator, CREB-binding protein (CBP).
      Figure thumbnail gr1
      FIGURE 1Schematic diagram of the known phosphorylation sites and the putative PKA phosphorylation sites of β-catenin.

      EXPERIMENTAL PROCEDURES

      Cell Culture, DNA Reagents, and Antibodies—COS7 cells and human embryonic kidney (HEK)293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mml-glutamine, 100 units/ml streptomycin, 250 ng/ml amphotericin B, and 100 units/ml penicillin. Twenty-four hours prior to stimulation, the cells were serum-deprived using Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin and 2 mm l-glutamine. Transient transfections were performed using Lipofectamine-PLUS reagent (Invitrogen) following the manufacturer's recommendations.
      The cDNA for human β-catenin was kindly provided by Dr. Stephen W. Byers, Georgetown University, Washington, D. C.). The β-catenin mutants were generated by site-directed mutagenesis and confirmed by sequencing. The TCF/LEF luciferase reporter constructs (TOPflash and FOPflash) were from Upstate Biotechnology. The replication-deficient adenovirus (Ad) encoding a complete sequence for the PKA inhibitor protein (PKI) gene under the control of the cytomegalovirus promoter was described previously (
      • Hogarth D.K.
      • Sandbo N.
      • Taurin S.
      • Kolenko V.
      • Miano J.M.
      • Dulin N.O.
      ). The cDNA for Myc-tagged dominant negative PKA mutant was described previously (
      • Davis A.
      • Hogarth K.
      • Fernandes D.
      • Solway J.
      • Niu J.
      • Kolenko V.
      • Browning D.
      • Miano J.M.
      • Orlov S.N.
      • Dulin N.O.
      ). The cDNA for the PKA catalytic subunit (cPKA) was from Stratagene.
      The antibodies were from the following sources: anti-PKA substrate (PKAS, catalog number 9624), anti-phospho-pS33/pS37/pT41-β-catenin (catalog number 9561), and anti-phospho-GSK-3α/β (catalog number 9331) were from Cell Signaling Technology; anti-β-catenin (catalog number sc-7939), anti-cPKA (catalog number sc-903), anti-CREB-binding protein (CBP, catalog number sc-369), and anti-N-cadherin (catalog number sc-7939) were from Santa Cruz Biotechnology; anti-GSK-3α/β (catalog number 44-610) was from BIOSOURCE International; anti-FLAG M2 (catalog number F1804) and anti-β-actin (catalog number A5441) were from Sigma-Aldrich.
      Nonradioactive in Vitro Assay for PKA Activity in Cell Lysates—Following stimulation with the desired agonists, the cells (grown in 12-well plates) were lysed in 0.1 ml/well lysis buffer containing 25 mm HEPES (pH 7.5), 0.5% Nonidet P-40, protease inhibitors (1 mg/ml leupeptin, 1 mg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride), and phosphatase inhibitors (1 mm NaF, 200 mm Na-orthovanadate). The lysates were cleared from insoluble material by centrifugation at 20,000 × g for 10 min, and 5 μl of cleared lysates were subjected to a kinase reaction with the fluorescence-labeled PKA substrate, Kemptide (Promega) following the manufacturer's protocol. The reaction was stopped by boiling the samples for 10 min. The phosphorylated Kemptide was separated by 0.8% agarose electrophoresis, and the fluorescence of the phosphorylated Kemptide was detected and quantified by a Luminescent Image Analyzer LAS-3000 (Fujifilm).
      Western Blotting—Unless indicated, cells were lysed in radioimmune precipitation assay buffer (containing 25 mm HEPES (pH 7.5), 150 mm NaCl, 1% Triton X-100, 0.1% SDS, 2 mm EDTA, 2 mm EGTA, 10% glycerol, 1 mm NaF, 200 μm sodium-orthovanadate, and protease inhibitors (1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride). The lysates were cleared from insoluble material by centrifugation at 20,000 × g for 10 min, boiled in Laemmli buffer, subjected to polyacrylamide gel electrophoresis, and analyzed by Western blotting with the desired primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies (Calbiochem), and developed by enhanced chemiluminescence reaction (Pierce). The digital chemiluminescence images were taken by a Luminescent Image Analyzer LAS-3000 (Fujifilm).
      Immunoprecipitation—Generally, the lysates from 2 × 106 transfected cells were used for one immunoprecipitation. The cells were washed twice with ice-cold PBS and lysed in the extraction buffer (300 μl/106 cells) containing 150 mm NaCl, 25 mm HEPES (pH 7.4), 0.5% Nonidet P-40, 2 mm EDTA, 2 mm EGTA, 100 μm orthovanadate, and protease inhibitors (200 μm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, and 2 μg/ml aprotinin). The lysates were cleared by centrifugation at 20,000 × g for 5 min. The immunoprecipitation of endogenous β-catenin was performed by overnight incubation of the cleared lysates with agarose-conjugated goat anti-β-catenin antibodies (Santa Cruz Biotechnology, catalog number sc-1496), followed by three washes with 1 ml of lysis buffer. Immunoprecipitation of FLAG-tagged β-catenin proteins was performed using agarose-conjugated mouse anti-FLAG antibodies (Sigma-Aldrich, catalog number A2220). Immunoprecipitation of CBP was performed using anti-CBP antibodies followed by incubation with protein A/protein G-conjugated agarose beads (The immune complexes were boiled in Laemmli buffer and subjected to Western blotting with the desired antibodies.
      In Vitro 32P Labeling of β-Catenin—FLAG-tagged wild-type (WT) β-catenin or its PKA phosphorylation mutants were expressed in COS7 cells and purified using anti-FLAG-agarose beads. The beads were washed once with PKA assay buffer (20 mm Tris-HCl (pH 7.4), 100 mm NaCl, 10 mm MgCl2, 1 mm ATP) and then incubated with 45 μl of PKA assay buffer supplemented with 0.1 mm dithiothreitol, 5 units of purified protein kinase A catalytic subunit (Promega, V5340), and 5 μCi of [γ-32P]ATP at 30 °C for 30 min. The reaction was stopped by the addition of 15 μl of 4× Laemmli buffer and heating for 5 min at 95 °C. Proteins were subjected to SDS-PAGE, followed by autoradiography Western blotting with the desired antibodies.
      32P Labeling of β-Catenin in Intact Cells—COS7 cells were transfected with cDNAs for N-terminally truncated (ΔN100) FLAG-tagged WT-β-catenin or its PKA phosphorylation mutants and serum-starved for 24 h. The cells were incubated with 0.25 mCi/ml [32P]orthophosphate in a phosphate-free and serum-free medium, followed by three washes and stimulation with 10 μm forskolin (FSK) for 10 min. The cells were then lysed, and the cell lysates were subjected to immunoprecipitation with FLAG antibodies as described above, followed by electrophoresis, autoradiography, or Western blotting with FLAG antibodies.
      TCF/LEF-Luciferase Reporter Assay—Cells grown on 24-well plates were transfected in triplicates or quadruplicates with cDNAs (20 ng/well) for TCF-luciferase reporter (TOPflash) or its mutated control reporter (FOPflash) along with the Renilla reporter driven by the thymidine kinase promoter (tk-RL) and with the other plasmids of interest. The total amount of DNA was equalized in all of the samples by using the corresponding empty vector. Following stimulation with the desired agonists, the cells were lysed and the luciferase activity was measured and normalized to the corresponding Renilla activity using the dual-luciferase assay kit (Promega). The normalized FOPflash values were subtracted from the corresponding TOPflash values and expressed as mean ± S.D.
      Indirect Immunofluorescence Microscopy—Immunofluorescence was performed as described previously (
      • Dulin N.O.
      • Pratt P.
      • Tiruppathi C.
      • Niu J.
      • Voyno-Yasenetskaya T.
      • Dunn M.J.
      ). Cells grown on glass chamber slides and transfected with FLAG-tagged β-catenin cDNA were stimulated with or without 10 μm forskolin for 15 min, washed twice with ice-cold PBS, fixed in 4% paraformaldehyde in PBS for 15 min at room temperature, washed again with PBS, and permeabilized in 0.2% Triton-X100 in PBS for 5 min, followed by incubation with 1% bovine serum albumin in PBS for 1 h. The cells were then incubated with monoclonal anti-FLAG antibodies (5 μg/ml in PBS/bovine serum albumin, as above) overnight at 4 °C for 1 h and washed four times with PBS, followed by incubation with fluorescein-conjugated goat anti-mouse IgG (10 μg/ml in PBS/bovine serum albumin, as above) for 1 h at room temperature. The slides were additionally washed four times with PBS, and the coverslips were mounted using Gel/Mount aqueous mounting medium (Fisher, Pittsburgh, PA). The confocal images (sections near the bottom of the cell) were taken under a confocal fluorescent microscope.
      Statistics—The Bonferroni t test for multiple comparisons was utilized to test for statistically significant differences. The appropriate level of significance was determined for each analysis, according to the number of simultaneous comparisons tested against the reference group (p value cutoffs are indicated in the figure legends).

      RESULTS

      Phosphorylation of β-Catenin by PKA—We first tested the possibility that the endogenous β-catenin can be phosphorylated by PKA using PKAS antibodies directed against the phosphorylated PKA substrates ((RXX(pS/pT), Cell Signaling Technology). Fig. 2A shows stimulation of PKA by a cAMP-rising agent, FSK, and inhibition by adenovirus (Ad)-mediated transduction of the PKA inhibitor PKI (AdPKI). The specificity of AdPKI was previously confirmed by its inability to affect mitogen-activated protein kinase activation induced by various agonists (
      • Hogarth D.K.
      • Sandbo N.
      • Taurin S.
      • Kolenko V.
      • Miano J.M.
      • Dulin N.O.
      ). Western blotting of cell lysates with PKAS antibodies revealed a PKA-dependent phosphorylation of a number of proteins, and this PKAS recognition was attenuated by AdPKI transduction (Fig. 2B). When β-catenin was immunoprecipitated from cell lysates followed by Western blotting with PKAS antibodies, a single band that co-migrated with β-catenin was detected only after FSK stimulation (Fig. 2C). Overexpression of PKI prevented the recognition of immunoprecipitated β-catenin by PKAS antibodies. The equal amount of β-catenin in the immunoprecipitation samples was confirmed by probing with β-catenin antibodies (Fig. 2D). These data suggest that endogenous β-catenin can be phosphorylated in a PKA-dependent manner in response to FSK.
      Figure thumbnail gr2
      FIGURE 2Endogenous β-catenin is recognized by antibodies against phosphorylated PKA substrates (PKAS). COS7 cells were stimulated with 10 μm forskolin (FSK) for 10 min and lysed. A, a nonradioactive in vitro PKA assay of cell lysates was performed as described previously (
      • Hogarth D.K.
      • Sandbo N.
      • Taurin S.
      • Kolenko V.
      • Miano J.M.
      • Dulin N.O.
      ). B, total lysates were analyzed by Western blotting (IB) with PKAS antibodies. C and D, endogenous β-catenin was immunoprecipitated (IP) from cell lysates, and the immune complexes were analyzed by Western blotting with antibodies against PKAS (C) or β-catenin (D).
      We identified two potential PKA phosphorylation sites on β-catenin (serine 552, serine 675; Fig. 1) and generated alanine substitutions to examine the function. We first examined whether PKA could phosphorylate β-catenin and the above mutants in vitro and found that PKA readily phosphorylated the WT β-catenin (Fig. 3). The S552A or S675A mutation partially attenuated this effect, whereas the double mutation (S552A/S675A) completely abolished the phosphorylation of β-catenin by PKA (Fig. 3). We then examined whether FSK could stimulate PKA-dependent phosphorylation of β-catenin in intact cells. To dissect the CK1- and GSK3-dependent phosphorylations, we generated the FLAG-tagged truncated versions of β-catenin and of its PKA phosphorylation mutants by deleting the N-terminal (ΔN100) region containing the phosphorylation sites for CK1 or GSK3. We then overexpressed these truncated β-catenin proteins in COS7 cells and examined their phosphorylation in response to FSK by immunoprecipitation from 32P-labeled cells, followed by autoradiography. As shown in Fig. 4, FSK stimulated phosphorylation of ΔN100-WT-β-catenin; this phosphorylation was partially attenuated by the S552A or S675A mutations and was abolished by mutation of both sites. The smear (Fig. 4A, above the β-catenin band), nonspecifically precipitated by FLAG antibodies, served as an internal control for the presence of 32P labeling in all of the original lysates. Together, these data convincingly demonstrate that β-catenin can be phosphorylated by PKA at Ser-552 and Ser-675 sites both in vitro and in intact cells.
      Figure thumbnail gr3
      FIGURE 3In vitro phosphorylation of β-catenin and its mutants by PKA. Purified β-catenin and the corresponding mutants were subjected to a kinase reaction with [γ-32P]ATP with (+) or without (–) the purified catalytic subunit of PKA (cPKA) as described under “Experimental Procedures.” Whole kinase reactions were then subjected to electrophoresis followed by autoradiography (A) or Western blotting with β-catenin antibodies (B). C, 32P densitometry (panel A) was normalized to the corresponding levels of β-catenin (B) and expressed as mean ± S.D. from three independent experiments. Statistical significance was analyzed using the Bonferroni t test for three comparisons. Statistically significant differences (p < 0.017) are indicated by an asterisk.
      Figure thumbnail gr4
      FIGURE 4Phosphorylation of ΔN100-β-catenin and its mutants in intact cells. COS7 cells were transfected with empty vector or with cDNAs for FLAG-tagged ΔN100-β-catenin and the corresponding mutants labeled with [32P]orthophosphate and stimulated with 10 μm FSK for 10 min. The proteins were immunoprecipitated with FLAG antibodies followed by electrophoresis and autoradiography (A) or Western blotting with FLAG antibodies (B). C, 32P densitometry (A) was normalized to the corresponding levels of ΔN100-β-catenin (B) and expressed as mean ± S.D. from three independent experiments. Statistical significance was analyzed using the Bonferroni t test for three comparisons. Statistically significant differences (p < 0.017) are indicated by an asterisk.
      Phosphorylation of β-Catenin Promotes Its Transcriptional Activity—To understand the functional significance of β-catenin phosphorylation by PKA, we first examined how the stimulation of cells with FSK affects the signaling of endogenous β-catenin, using TCF/LEF-luciferase reporter (TOPflash/FOPflash). As shown in Fig. 5A, FSK stimulated a significant induction of TCF/LEF reporter (TOPflash) without affecting the control (mutant) reporter activity (FOPflash). Overexpression of dominant negative PKA mutant (dnPKA) abolished FSK-induced TCF/LEF activity, indicating the role of PKA for this effect (Fig. 5B). The efficiency and specificity of dnPKA in inhibiting PKA activity in intact cells was previously confirmed (
      • Davis A.
      • Hogarth K.
      • Fernandes D.
      • Solway J.
      • Niu J.
      • Kolenko V.
      • Browning D.
      • Miano J.M.
      • Orlov S.N.
      • Dulin N.O.
      ).
      Figure thumbnail gr5
      FIGURE 5PKA activation promotes TCF/LEF-dependent gene transcription. COS7 cells were transfected with cDNAs for TCF-luciferase reporters (TOPflash or FOPflash) along with a Renilla reporter driven by thymidine kinase promoter tk-RL and with an empty vector or cDNA for the Myc-tagged PKA dominant negative mutant (Myc-dnPKA). Following stimulation of cells with 10 μm FSK for 12 h, TCF-reporter activity was measured as described under “Experimental Procedures” (A), and cell lysates were analyzed by Western blotting with Myc antibodies (B). Data represent mean ± S.D. from a representative (of three) experiment performed in triplicates. RLU, relative light units.
      It has been suggested that PKA can phosphorylate and inactivate GSK3 resulting in decreased phosphorylation and increased stability of β-catenin (
      • Li M.
      • Wang X.
      • Meintzer M.K.
      • Laessig T.
      • Birnbaum M.J.
      • Heidenreich K.A.
      ,
      • Khaled M.
      • Larribere L.
      • Bille K.
      • Aberdam E.
      • Ortonne J.P.
      • Ballotti R.
      • Bertolotto C.
      ,
      • Tanji C.
      • Yamamoto H.
      • Yorioka N.
      • Kohno N.
      • Kikuchi K.
      • Kikuchi A.
      ). We examined whether this could explain the effect of FSK on TCF/LEF induction in our experiments. In COS7 cells, FSK stimulated a rapid and prolonged phosphorylation of GSK3α (a major GSK3 isoform in these cells), as assessed by phosphospecific antibodies (Fig. 6A). Nevertheless, the total levels of β-catenin, as well as its GSK3-dependent phosphorylation, were not significantly affected by FSK, as assessed by Western blotting with antibodies against total β-catenin (Fig. 6D), or with phospho-(pS33/pS37/pT41)-specific β-catenin antibodies (Fig. 6C). Together, these data suggest that, in COS7 cells, PKA promotes the signaling of endogenous β-catenin without affecting the levels of its expression.
      Figure thumbnail gr6
      FIGURE 6PKA activation does not affect GSK3-dependent phosphorylation of β-catenin or its expression levels. COS7 cells were stimulated with 10 μm FSK for the indicated times, followed by Western blotting of cell lysates with antibodies against phospho-GSK3α/β (A), total GSK3α/β (B), phospho-(pS33/pS37/pT41)-β-catenin (C), or total β-catenin (D).
      We then examined how the PKA-dependent phosphorylation of ectopically expressed β-catenin affects its transcriptional activity. Overexpression of FLAG-tagged WT-β-catenin on its own in COS7 cells resulted in a 5–7-fold activation of TCF/LEF-reporter, as expected (not shown), but this effect was further potentiated by FSK treatment (Fig. 7A). Mutation of β-catenin at S552A did not change the TCF/LEF-reporter activity, but the S675A or the S552A/S675A mutations decreased the effect of FSK by half. Western blotting of cell lysates with FLAG antibodies revealed that FSK facilitated the expression of ectopic β-catenin (Fig. 7B), which was probably the result of an increased transcription by the cytomegalovirus promoter (driving transcription of β-catenin and its mutants). Likewise, FSK treatment also increased the expression of cytomegalovirus-driven β-galactosidase or green fluorescent protein (data not shown). This may partially, but not entirely, contribute to a FSK-induced TCF/LEF activity in β-catenin-transfected cells (note that FSK did not affect the expression of endogenous β-catenin (Fig. 6D); whereas it still stimulated the TCF/LEF activity in nontransfected cells (Fig. 5A)). Importantly, the expression of both WT and all three PKA phosphorylation mutants of β-catenin was equally increased upon FSK stimulation, whereas the FSK-induced TCF/LEF reporter activity was significantly reduced in cells transfected with S675A or S552A/S675A mutants. This suggests that the S675A mutation attenuates the ability of FSK to promote the β-catenin-mediated TCF/LEF activity.
      Figure thumbnail gr7
      FIGURE 7The S675A mutation attenuates β-catenin signaling induced by forskolin in COS7 cells. COS7 cells were transfected with cDNAs for TCF-luciferase reporters (TOP-flash or FOPflash) along with a control Renilla plasmid (tk-RL) and with the cDNAs for FLAG-tagged WT-β-catenin or its PKA phosphorylation mutants, as indicated. Following stimulation of cells with 10 μm FSK for 12 h, the TCF-reporter activity was measured as described under “Experimental Procedures” (A), and cell lysates were analyzed by Western blotting with FLAG antibodies (B). Data represent mean ± S.D. from a representative (of at least three) experiment performed in triplicates. Statistical significance was analyzed using the Bonferroni t test for two comparisons. Statistically significant differences (p < 0.025) are indicated by an asterisk.
      To test whether the results obtained in FSK-stimulated COS7 cells could be reproduced in other cells and by other means of PKA stimulation, we overexpressed the PKA-catalytic subunit (cPKA) in HEK293 cells. As shown in Fig. 8A, cPKA overexpression in HEK293 cells promoted the signaling of WT-β-catenin or its S552A mutant to a much greater extent than that of S675A or S552A/S675A mutants. Similar to our observations using FSK-stimulated COS7 cells (Fig. 7), cPKA overexpression in HEK293 cells resulted in increased levels of β-catenin and its mutants but all to the same extent (Fig. 8C). Thus, the very similar results obtained by two different approaches strongly suggest that (i) PKA promotes the TCF/LEF activity induced by β-catenin, at least in part, through phosphorylation of β-catenin at the Ser-675 site and (ii) this effect of PKA does not seem to involve stabilization of β-catenin.
      Figure thumbnail gr8
      FIGURE 8The S675A mutation attenuates β-catenin signaling induced by cPKA overexpression in HEK293 cells. HEK293 cells were transfected with TOPflash, FOP-flash, and tk-RL reporter plasmids with cDNAs for the FLAG-tagged WT-β-catenin or its PKA phosphorylation mutants (as in ) together with empty vector or with cDNA for the PKA catalytic subunit (cPKA), as indicated. Twenty-four hours post-transfection, the TCF-reporter activity was measured as described under “Experimental Procedures” (A), and cell lysates were analyzed by Western blotting with antibodies against PKA (B) or against the FLAG epitope (C). Data represent mean ± S.D. from a representative (of three) experiment performed in triplicates. Statistical significance was analyzed using the Bonferroni t test for two comparisons. Statistically significant differences (p < 0.025) are indicated by an asterisk.
      To further address the latter notion, we examined the rate of β-catenin degradation following the inhibition of protein synthesis by cycloheximide. As shown in Fig. 9A, a 3-h treatment of cells with cycloheximide resulted in a 60% decrease of exogenous β-catenin but not of β-actin. Importantly, (i) treatment of cells with FSK did not protect β-catenin from degradation, and (ii) there was no significant difference between WT-β-catenin and its PKA phosphorylation mutants in the degradation rate. We also examined how PKA affects the intracellular localization of β-catenin. As shown on Fig. 9B, overexpressed β-catenin localized mainly to the nucleus as expected. However, forskolin stimualtion for 15 min (which results in maximal phosphorylation at both the Ser-552 and Ser-675 sites) did not affect the localization of WT-β-catenin (Fig. 9B). Furthermore, the alanine substitution of either the Ser-552 or Ser-675 sites also had no obvious effect on β-catenin localization under basal or forskolin-stimulated conditions; it still remained largely in the nucleus (data not shown). Together, these data suggest that PKA promotes β-catenin signaling without affecting its stability or intracellular localization.
      Figure thumbnail gr9
      FIGURE 9Phosphorylation by PKA does not affect the stability of β-catenin or its nuclear localization. A, COS7 cells were transfected with cDNAs for FLAG-tagged WT-β-catenin or its PKA phosphorylation mutants, as indicated. The cells were then pretreated with 10 μg/ml cycloheximide (CHX) for 30 min followed by the addition of 10 μm FSK for 3 h. The cells were then lysed, and the lysates were analyzed by Western blotting with antibodies against FLAG or against β-actin, as indicated. B, COS7 cells were transfected with cDNA for FLAG-tagged WT-β-catenin, stimulated with 10 μm FSK for 15 min and subjected to immunofluorescent microscopy with FLAG antibodies. No significant change was observed in the localization of WT-β-catenin upon FSK treatment (B) or in the localization of the PKA phosphorylation mutants of β-catenin (data not shown). Scale bar, 20 μm.
      Given that the Ser-675 phosphorylation site (responsible for the promoting effect of PKA) is located within the transactivation domain of β-catenin, we then hypothesized that phosphorylation by PKA promotes β-catenin signaling at the level of its transcriptional activity, through modulation of its interaction with co-activators, such as CBP. CBP is an acetyltransferase that promotes the activity of several transcription factors (i.e. CREB) by their acetylation. CBP acetylates β-catenin and increases its transcriptional activity (
      • Hecht A.
      • Vleminckx K.
      • Stemmler M.P.
      • van Roy F.
      • Kemler R.
      ,
      • Wolf D.
      • Rodova M.
      • Miska E.A.
      • Calvet J.P.
      • Kouzarides T.
      ,
      • Levy L.
      • Wei Y.
      • Labalette C.
      • Wu Y.
      • Renard C.-A.
      • Buendia M.A.
      • Neuveut C.
      ). Therefore, we examined whether phosphorylation of β-catenin by PKA is important for β-catenin-CBP interaction by co-immunoprecipitation. As shown on Fig. 10, WT-β-catenin and the S552A mutant bound specifically to CBP, and their binding was significantly increased by 50% after FSK treatment. In contrast, the S675A mutation resulted in a dramatic reduction of β-catenin binding to CBP, and, importantly, FSK no longer promoted this binding. Note that β-catenin is partially phosphorylated at the Ser-675 site under basal conditions (as the S675A mutation abolishes this basal phosphorylation) (Fig. 4C), which may explain an appreciable binding of β-catenin to CBP under control conditions (Fig. 10, no FSK (–)). Together, these data suggest that phosphorylation of β-catenin by PKA at the Ser-675 site is important for its interaction with CBP, which may explain how PKA promotes transcriptional activity of β-catenin.
      Figure thumbnail gr10
      FIGURE 10Phosphorylation of β-catenin by PKA at the Ser-675 site promotes its association with CBP. The co-immunoprecipitation of β-catenin with CBP was assessed as described previously (
      • Takemaru K.-I.
      • Moon R.T.
      ). COS7 cells were transfected with cDNA for CBP together with cDNAs for a stabilized (S37A mutant (
      • Orford K.
      • Crockett C.
      • Jensen J.P.
      • Weissman A.M.
      • Byers S.W.
      )) FLAG-tagged β-catenin or its PKA phosphorylation mutants, as indicated. Cells were stimulated with 10μm FSK for 15 min, lysed, followed by immunoprecipitation (IP) of cell lysates with normal IgG or with anti-CBP antibodies as indicated (A). The immune complexes (A) or total cell lysates (B) were immunoblotted with antibodies against CBP or against the FLAG epitope as indicated. C, the amounts of CBP-bound β-catenin or its mutants (IP: CBP; FLAG) were digitally quantified and expressed as percentage of WT control (mean ± S.D. from three independent experiments). Statistical significance was analyzed using Student's t test. Statistically significant differences (p < 0.05) are indicated by an asterisk.
      Finally, we sought to examine whether the phosphorylation of β-catenin by PKA affects its interaction with cadherin, for the following reasons: (i) the Ser-552 phosphorylation site does not seem important for modulation of β-catenin transcriptional activity, suggesting that phosphorylation at this site may modulate the other function of β-catenin at cell-cell adherens junctions, (ii) the Ser-552 phosphorylation site is located within the 10th armadillo repeat of β-catenin that may be important for interaction with cadherin (
      • Huber A.H.
      • Weis W.I.
      ), and (iii) the β-catenin/cadherin interaction was reported to be regulated by PKA, albeit through phosphorylation of cadherin (
      • Boucher M.J.
      • Laprise P.
      • Rivard N.
      ). As shown in Fig. 11A, endogenous N-cadherin readily and specifically co-immunoprecipitated with FLAG-WT-β-catenin. However, stimulation of cells with FSK had no effect on the interaction of N-cadherin with either WT-β-catenin, or with its PKA phosphorylation mutants. Thus, at least as assessed by co-immunoprecipitation approach, phosphorylation of β-catenin by PKA does not seem to affect its interaction with N-cadherin. In addition, Fig. 11C also shows that GSK3-dependent phosphorylation (at Ser-33/Ser-37/Thr-41 sites) of ectopic β-catenin is not affected by PKA-dependent phosphorylation of β-catenin (at Ser-552 or Ser-675 sites), which is consistent with the results on the endogenous β-catenin phosphorylation by GSK3 (Fig. 6C).
      Figure thumbnail gr11
      FIGURE 11Phosphorylation by PKA does not affect the interaction of β-catenin with N-cadherin or its phosphorylation by GSK3. COS7 cells were transfected with empty vector or with cDNAs for FLAG-tagged WT-β-catenin or its PKA phosphorylation mutants, as indicated. The β-catenin proteins were then immunoprecipitated with FLAG antibodies, and the immune complexes were analyzed by Western blotting with N-cadherin antibodies (A), FLAG antibodies (B), or phospho-(pS33/pS37/pT41)-specific β-catenin antibodies (C).

      DISCUSSION

      The present study describes three major findings. (i) β-Catenin can be phosphorylated by PKA in vitro and in intact cells at novel sites (Ser-552 and Ser-675) distinct from those phosphorylated by CK1, GSK3, or Src kinases. (ii) Phosphorylation of β-catenin by PKA at the Ser-675 site promotes its transcriptional activity by a noncanonical mechanism that does not involve the stabilization of β-catenin. (iii) Phosphorylation of β-catenin at the Ser-675 site facilitates the interaction between β-catenin and CBP, which may provide a molecular mechanism by which PKA promotes the transcriptional activity of β-catenin.
      The role of the cAMP/PKA pathway in the modulation of cell growth is highly complex and cell-specific; it stimulates cell growth in many cell types while inhibiting cell growth in others (
      • Stork P.J.S.
      • Schmitt J.M.
      ). Moreover, even in the same cell type (vascular smooth muscle cells), PKA can promote or inhibit cell proliferation dependent on the agonist that stimulates its activity (
      • Hogarth D.K.
      • Sandbo N.
      • Taurin S.
      • Kolenko V.
      • Miano J.M.
      • Dulin N.O.
      ). The present study suggests that, in COS7 and HEK293 cells, PKA facilitates the signaling of β-catenin, which is consistent with the recent report demonstrating that prostaglandin E2, through PKA activation, promotes β-catenin signaling in colon cancer cells (
      • Shao J.
      • Jung C.
      • Liu C.
      • Sheng H.
      ). However, in the latter report, the promoting effect of PKA is attributed to phosphorylation and inactivation of GSK3, whereas our results point to the role of a direct phosphorylation of β-catenin by PKA at the Ser-675 site.
      Regarding the mechanism by which phosphorylation of β-catenin by PKA promotes its transcriptional activity, we first showed that phosphorylation of β-catenin by PKA: (i) does not affect the GSK3-dependent phosphorylation of endogenous or ectopic β-catenin at the Ser-33/Ser-37/Thr-41 sites (Figs. 6C and 11C), (ii) does not protect β-catenin from degradation (Fig. 9A), (iii) does not affect the amount of endogenous β-catenin (Fig. 6D), and (iv) does not affect the nuclear localization of ectopic β-catenin (Fig. 9B). Thus, our data suggest that PKA promotes β-catenin signaling by a noncanonical mechanism. In this study, we have proposed one potential mechanism wherein phosphorylation of β-catenin by PKA at the Ser-675 site promotes the interaction between β-catenin and its co-activator, CBP, whereas S675A mutation nearly abolishes this interaction.
      It has been recently shown that the phosphoserine-binding protein 14-3-3ζ interacts with β-catenin and potentiates its transcriptional activity by an unknown mechanism (
      • Tian Q.
      • Feetham M.C.
      • Tao W.A.
      • He X.C.
      • Li L.
      • Aebersold R.
      • Hood L.
      ). Given that both PKA phosphorylation sites on β-catenin (RRTS552MGGT and KRLS675VELT) (Fig. 1) match the type-2 consensus 14-3-3 binding motif (RX1–2(pS/pT)X2–3(pS/pT)) (
      • Fu H.
      • Subramanian R.R.
      • Masters S.C.
      ), we explored whether 14-3-3 interacts with β-catenin through these sites. However, we have failed to coimmunoprecipitate these two proteins despite multiple efforts using different conditions (data not shown). In contrast, a positive control interaction between the regulator of G protein signaling (RGS3) and 14-3-3 (experiments performed in parallel) was readily detectable, as we have shown previously (
      • Niu J.
      • Scheschonka A.
      • Druey K.M.
      • Davis A.
      • Reed E.
      • Kolenko V.
      • Bodnar R.
      • Voyno-Yasenetskaya T.
      • Du X.
      • Kehrl J.
      • Dulin N.O.
      ).
      While this work was in progress, an independent study by Hino et al. (
      • Hino S.-i.
      • Tanji C.
      • Nakayama K.I.
      • Kikuchi A.
      ) has been published showing that PKA can phosphorylate β-catenin at the Ser-675 site. Consistent with our results, these authors show that phosphorylation of β-catenin at this site promotes TCF-dependent gene transcription, whereas the S675A mutation attenuates this effect. However, Hino et al. (
      • Hino S.-i.
      • Tanji C.
      • Nakayama K.I.
      • Kikuchi A.
      ) suggest that phosphorylation of β-catenin at the Ser-675 site leads to stabilization of β-catenin, which is different from what our data, showing no stabilization and no protection of β-catenin from degradation, suggest. Although acknowledging that such a discrepancy could be the result of multiple experimental factors, including cell-type specificity, cell growth conditions, density, etc., we propose here an additional possible mechanism by which PKA promotes the transcriptional activity of β-catenin, namely by facilitating its interaction with CBP (Fig. 10).
      Finally, we also identified Ser-552 as a site of phosphorylation by PKA, which was not reported previously (Figs. 3 and 4). Even though its mutation has a lesser effect on 32P incorporation into β-catenin, as does the S675A mutation (Figs. 3A and 4A), the Ser-552 site is surely phosphorylated by PKA, because its mutation abolishes the recognition of β-catenin by antibodies against phosphorylated PKA substrates (data not shown). At this time, we do not know what the functional significance is of β-catenin phosphorylation at the Ser-552 site. Few possibilities exist. (i) It has no significance; this can be argued by the notion that every modification of the protein is meaningful. (ii) We have missed the effect of the S552A mutation due to an overwhelming overexpression of the mutant; this is unlikely, because under the same transfection conditions, the effect of the S675A mutation was readily detectable (Figs. 7, 8, and 10). (iii) Phosphorylation of β-catenin at the Ser-552 site may be important for another previously unrecognized function of β-catenin. These unanswered questions are currently being addressed in our laboratory.

      Acknowledgments

      We thank Dr. Stephen W. Byers for providing the cDNA for β-catenin.

      Supplementary Material

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