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

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.

Beta-catenin plays a dual role in cell function. 1 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 (1). Second, β-catenin acts as inducer of gene expression that control an array of embryonic and adult processes (2,3). Beta-catenin is a key effector of Wnt signaling, a pathway first identified in Drosophila (4) and soon found to be highly conserved throughout the animal kingdom and to affect cell growth and differentiation in many cell types (5,6). In this role, β-catenin functions as an activator of T cell-factor (TCF) / lymphoid enhancer factor (LEF) transcription factors to stimulate transcription of a variety of growth-related genes, including c-Myc (7) and cyclin D1 (8). Mutations of β-catenin or of its regulatory proteins are frequently found in various types of cancers, and they result in accumulation of β-catenin and in activation of TCF/TCFdependent gene transcription (9,10).
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 (APC) and axin, and protein kinases, casein-kinase 1 alpha (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 (11), leading to its ubiquitination and proteasomal degradation (12,13). 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 (14). In addition to phosphorylation by CK1 (at S 45 site) and GSK3 at the amino terminus,  β-catenin can be also phosphorylated at the C-terminus on Y 654 by Src tyrosine kinases, and this phosphorylation affects the association of β-catenin with cadherin resulting in a loss of cell-cell adhesion (15).
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 show that (i) βcatenin 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).
The cDNA for human β-catenin was kindly provided by Dr. Stephen W. Byers, Georgetown University, Washington, DC). The βcatenin 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 PKA inhibitor protein (PKI) gene under control of CMV promoter was described previously (16). The cDNA for Myc-tagged dominant negative PKA mutant was described previously (17). The cDNA for PKA catalytic subunit (cPKA) was from Stratagene.
Non-radioactive in vitro assay for PKA activity in cell lysates. Following stimulation with desired agonists, the cells (grown in 12-well plates) are lysed in 0.1 ml/well lysis buffer containing 25 mM HEPES (pH 7.5), 0.5 % NP-40, protease inhibitors (1 mg/ml leupeptin, 1 mg/ml aprotinin, 1 mM PMSF) and phosphatase inhibitors (1 mM NaF, 200 mM Naorthovanadate). The lysates are cleared from insoluble material by centrifugation at 20,000 g for 10 minutes, and 5 µl cleared lysates are subjected to a kinase reaction with the fluorescence-labeled PKA substrate, Kemptide (Promega) following the manufacturer's protocol. The reaction is stopped by boiling the samples for 10 min. The phosphorylated Kemptide is separated by 0.8% agarose electrophoresis and the fluorescence of the phosphorylated kemptide is detected and quantified by Luminescent Image Analyzer LAS-3000 (Fujifilm).
Immunoprecipitation. Generally, the lysates from 2x10 6 transfected cells were used for one immunoprecipitation. The cells were washed twice with the ice-cold PBS and lysed in the extraction buffer (300 µl per 10 6 cells) containing 150 mM NaCl, 25 mM HEPES (pH 7.4), 0.5% NP-40, 2 mM EDTA, 2 mM EGTA, 100 µM orthovanadate, and protease inhibitors (200 µM PMSF, 2 µg /ml leupeptin and 2 µg / ml aprotinin). The lysates were cleared by centrifugation at 20,000 x g for 5 min. The immunoprecipitation of endogenous β-catenin was performed by overnight incubation of cleared lysates with agarose-conjugated goat anti-βcatenin antibodies (Santa Cruz Biotechnology, sc-1496), followed by three washes with 1 ml lysis buffer. Immunoprecipitation of FLAG-tagged βcatenin proteins was performed using agaroseconjugated mouse anti-FLAG antibodies (Sigma-Aldrich, 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 the Laemmli buffer and subjected to Western blotting with desired antibodies. In vitro 32 P-labeling of β-catenin. FLAG-tagged-WT β-catenin or its PKAphosphorylation mutants were expressed in COS7 cells and purified using anti-FLAG-agarose beads. The beads were washed once with PKA assay buffer (20mM Tris-HCl (pH 7.4), 100 mM NaCl, 10 mM MgCl 2 , 1mM ATP) then incubated with 45 µl of PKA assay buffer supplemented with 0.1 mM DTT, 5 units of purified protein kinase A catalytic subunit (Promega, V5340) and 5 µCi of [γ-32 P]ATP at 30 °C for 30 min. The reaction was stopped by addition of 15 µl of 4X Laemmli buffer and heating for 5min at 95ºC. Proteins were subjected to SDS-PAGE, followed by autoradiography Western blotting with desired antibodies. 32 P-labeling of β-catenin in intact cells. COS7 cells were transfected with cDNAs for Nterminally transacted (∆N100), FLAG-tagged WTβ-catenin or its PKA-phosphorylation mutants and serum starved for 24 hours. Cells were incubated with 0.25 mCi / ml 32 P-orthophosphate in a phosphate-free and serum-free medium, followed by three washes and stimulation with 10 µM FSK for 10 minutes. Cell 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 thymidine kinase promoter (tk-RL), and with the other plasmids of interest. The total amount of DNA was equalized in all samples by using the corresponding empty vector. Following stimulation with desired agonists, the cells were lysed, the luciferase activity was measured and normalized to the corresponding Renilla activity, using the duallucifease assay kit (Promega). The normalized FOPflash values were subtracted from the corresponding TOPflash values and expressed as mean +/-SD.
Indirect immunofluorescence microscopy. Immunofluorescence was performed as described previously (18). 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, permeabilized in 0.2% Triton-X100 in PBS for 5 min, followed by incubation with 1% BSA in PBS for 1 hr. The cells were then incubated with monoclonal anti-FLAG antibodies (5 µg/ml in PBS/BSA as above) overnight at 4˚C for 1 hr, washed 4 times with PBS, followed by incubation with fluorescein-conjugated goat antimouse IgG (10 µg/ml in PBS/BSA as above) for 1 hr at room temperature. The slides were additionally washed 4 times with PBS, and the cover slips 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 (pvalue cut-offs are indicated in the figure legends).

RESULTS.
Phosphorylation of β-catenin by PKA. We first tested the possibility that the endogenous βcatenin can be phosphorylated by PKA, using PKAS antibodies directed against the phosphorylated PKA substrates (RXXpS/pT, Cell Signaling Technology). Figure 2A shows stimulation of PKA by a cAMP-rising agent, forskolin (FSK), and inhibition by adenovirus (Ad)-mediated transduction of PKA inhibitor, PKI (AdPKI). The specificity of AdPKI was previously confirmed by its inability to affect MAP kinase activation induced by various agonists (16). 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 lystates followed by Western blotting with PKAS antibodies, a single band that co-migrated with β-catenin was detected, and 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.
We identified two potential PKAphosphorylation sites on β-catenin (serine-552, serine-675; Fig. 1) and generated alanine substitutions to examine the function. We first examined whether PKA can phosphorylate βcatenin and the above mutants in vitro, and found that PKA readily phosphorylated the wild type (WT) β-catenin (Fig. 3). The S 552 A or S 675 A mutation partially attenuated this effect, whereas the double-mutation (S 552 A,/S 675 A) completely abolished phosphorylation of β-catenin by PKA (Fig. 3). We then examined whether FSK can simulate PKA-dependent phosphorylation of βcatenin in intact cells. To dissect the CK1-and GSK3-dependent phosphorylation, we generated the FLAG-tagged truncated versions of β-catenin and of its PKA-phosphorylation mutants by deleting the N-terminal (∆N100) 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 32 P-labelled cells, followed by autoradiography. As shown in figure  4, FSK stimulated phosphorylation of ∆N100-WTβ-catenin; this phosphorylation was partially attenuated by the S 552 A or S 675 A mutations, and was abolished by mutation of both sites. The smear (above the β-catenin band, Fig. 4A), nonspecifically precipitated by FLAG antibodies, serves as an internal control for the presence of 32 P-labeling in all the original lysates. Together, these data convincingly demonstrate that β-catenin can be phosphorylated by PKA at S 552 and S 675 sites both in vitro and in intact cells.
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 figure 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 (17).
It has been suggested that PKA can phosphorylate and inactivate GSK3 resulting in decreased phosphorylation and increased stability of β-catenin (19)(20)(21). 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 phospho-specific 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 βcatenin (Fig. 6D), or with phospho-(pS 33 /pS 37 /pT 41 )-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.
We then examined how the PKAdependent phosphorylation of ectopically expressed β-catenin affects its transcriptional activity. Overexpression of FLAG-tagged WT-βcatenin 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 S 552 A did not change the TCF/LEF-reporter activity, but the S 675 A or the S 552 A/ S 675 A 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 a result of an increased transcription by the CMV promoter (driving transcription of βcatenin and its mutants). Likewise, FSK treatment also increased the expression of CMV-driven βgalactosidase 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) while it still stimulated the TCF/LEF activity in non-transfected cells (Fig. 5A)). Importantly, the expression of both WT-and of 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 S 675 A or S 552 A/ S 675 A mutants. This suggests that the S 675 A mutation attenuates the ability of FSK to promote the β-cateninmediated TCF/LEF activity.
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 human embryonic kidney HEK293 cells. As shown in figure 8A, cPKA overexpression in HEK293 cells promoted the signaling of WT-β-catenin or its S 552 A mutant to a much greater extent than that of S 675 A or S 552 A/ S 675 A 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 S 675 site, and (ii) this effect of PKA does not seem to involve stabilization of β-catenin.
To further address the latter notion, we examined the rate of β-catenin degradation following the inhibition of protein synthesis by cycloheximide (CHX). As shown in figure 9A, a three-hour treatment of cells with CHX 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 PKAphosphorylation mutants in the degradation rate. We also examined how PKA affects the intracellular localization of β-catenin. As shown on figure 9B, overexpressed β-catenin localized mainly to the nucleus as expected. However, forskolin stimulation for 15 min (which results in maximal phosphorylation at both the S 552 and S 675 sites) did not affect the localization of WT-βcatenin (Fig. 9B). Furthermore, the alanine substitution of either S 552 or S 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 βcatenin signaling without affecting its stability or intracellular localization.
Given that the S 675 phosphorylation site (responsible for the promoting effect of PKA) is located within the transactivation domain of βcatenin, we then hypothesized that phosphorylation by PKA promotes β-catenin signaling at the level of its transcriptional activity, through modulation of its interaction with coactivators, such as CREB Binding Protein (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 (22)(23)(24). Therefore, we examined whether phosphorylation of β-catenin by PKA is important for β-catenin-CBP interaction by co-immunoprecipitation. As shown on figure 10, WT-β-catenin and the S 552 A mutant bound specifically to CBP, and their binding was significantly increased by 50% after FSK treatment. In contrast, the S 675 A 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 S 675 site under basal conditions (as the S 675 A mutation abolishes this basal phosphorylation, Fig. 4C), which may explain an appreciable binding of β-catenin to CBP under control conditions (no FSK, Fig. 10). Together, these data suggest that phosphorylation of β-catenin by PKA at the S 675 site is important for its interaction with CBP, which may explain how PKA promotes transcriptional activity of βcatenin.

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 (S 552 and S 675 ) distinct from those phosphorylated by CK1, GSK3 or Src kinases; (ii) phosphorylation of βcatenin by PKA at the S 675 site promotes its transcriptional activity by a non-canonical mechanism that does not involve stabilization of β-catenin; and (iii) phosphorylation of β-catenin at the S 675 site facilitates the interaction between βcatenin and CBP, which may provide a molecular mechanism by which PKA promotes the transcriptional activity of β-catenin.
The role of cAMP/PKA pathway in modulation of cell growth is highly complex and cell-specific: it stimulates cell growth in many cell types while inhibiting cell growth in others (27). 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 (16). The present study suggests that in COS7 and HEK293 cells, PKA facilitates the signaling of β-catenin, which is consistent the recent report demonstrating that prostaglandin E2, through PKA activation, promotes β-catenin signaling in colon cancer cells (28). However, in the latter report, the promoting effect of PKA was attributed to phosphorylation and inactivation of GSK3, whereas our results point to the role of a direct phosphorylation of βcatenin by PKA at the S 675 site.
Finally, we sought to examine whether the phosphorylation of β-catenin by PKA affects its interaction with cadherin, for the following reasons: (i) the S 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 S 552 phosphorylation site is located within the 10 th armadillo repeat of βcatenin that may be important for interaction with cadherin (25), and (iii) the β-catenin / cadherin interaction was reported to be regulated by PKA, albeit through phosphorylation of cadherin (26). As shown in figure 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, figure 11C also shows that GSK3dependent phosphorylation (at S 33 /S 37 /T 41 sites) of ectopic β-catenin is not affected by PKAdependent phosphorylation of β-catenin (at S 552 or S 675 sites), which is consistent with the results on the endogenous β-catenin phosphorylation by GSK3 (Fig. 6C).
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 S 33 /S 37 /T 41 sites (Figs. 6C, 11C), (ii) does not protect βcatenin from degradation (Fig. 9A), (iii) does not affect the amount of endogenous β-catenin (Fig.  6D), and 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 propose one potential mechanism, wherein phosphorylation of β-catenin by PKA at the S 675 site promotes the interaction between β-catenin and its co-activator, CBP, whereas S 675 A mutation nearly abolishes this interaction.
While this work was in progress, an independent study by Hino et al was published showing that PKA can phosphorylate β-catenin at the S 675 site (32). Consistent with our results, these authors showed that phosphorylation of β-catenin at this site promotes TCF-dependent gene transcription, whereas S 675 A mutation attenuates this effect. However, Hino et al suggest that phosphorylation of β-catenin at S 675 site leads to stabilization of β-catenin, which is different from what our data suggest showing no stabilization and no protection of β-catenin from degradation. While acknowledging that such discrepancy could be a 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 S 552 as a site of phosphorylation by PKA, which was not reported previously (Figs. 3, 4). Even though its mutation has a lesser effect on 32 P incorporation into βcatenin as does the S 675 A mutation (Figs. 3A, 4A), the S 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 don't know what the functional significance of βcatenin phosphorylation at the S 552 site is. Few possibilities exist: (i) it has no significance -this can be argued by a notion that every modification of the protein is meaningful; (ii) we have missed the effect of the S 552 A mutation due to an overwhelming overexpression of the mutant -this is unlikely because under the same transfection conditions, the effect of the S 675 A mutation was readily detectable (Figs. 7, 8, 10); and (iii) phosphorylation of β-catenin at the S 552 site may be important for an other, previously unrecognized function of β-catenin. These unanswered questions are currently being addressed in our laboratory.

34.
Orford   Purified β-catenin and the corresponding mutants were subjected to a kinase reaction with 32 P-γ-ATP and with or without the purified catalytic subunit of PKA (cPKA) as described in "Experimental Procedures". Whole kinase reactions were then subjected to electrophoresis, followed by autoradiography (A) or Western blotting with β-catenin antibodies (B). C, 32 P-densitometry (panel A) was normalized to the corresponding levels of β-catenin (panel B) and expressed as mean ±SD 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 *. Figure 4. Phosphorylation of ∆N100-β-catenin and its mutants in intact cells. COS7 cells were transfected with empty vector or with cDNAs for FLAG-tagged ∆N100-β-catenin and the corresponding mutants, labeled with 32 P-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, 32 P-densitometry (panel A) was normalized to the corresponding levels of ∆N100-β-catenin (panel B) and expressed as mean ±SD 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 *.   COS7 cells were transfected with cDNAs for TCF-luciferase reporters (TOPflash or FOPflash) along with a control renilla plasmid (tk-RL), and with the cDNAs for FLAG-tagged WT-β-catenin or its PKAphosphorylation mutants, as indicated. Following stimulation of cells with 10 µM FSK for 12 hours, the TCF-reporter activity was measured as described in "Experimental Procedures" (A), and cell lysates were analyzed by Western blotting with FLAG antibodies (B). Data represent mean ±SD from a representative (out 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 *. Figure 8. The S 675 A mutation attenuates β-catenin signaling induced by cPKA overexpression in HEK293 cells. HEK293 cells were transfected with TOPflash, FOPflash and tk-RL reporter plasmids, with cDNAs for the FLAG-tagged WT-β-catenin or its PKA-phosphorylation mutants (as in figure 7), 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 in "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 ±SD from a representative (out 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 *.  The co-immunoprecipitation of β-catenin with CBP was assessed as described previously (33). COS7 cells were transfected with cDNA for CBP together with cDNAs for a stabilized (S37A mutant (34)) FLAG-tagged β-catenin or its PKA-phosphorylation mutants, as indicated. Cells were stimulated with 10 µM 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; WB: FLAG) were digitally quantified and expressed as % of WT control (mean ±SD from three independent experiments). Statistical significance was analyzed using Student's t-test. Statistically significant differences (p < 0.05) are indicated by *.