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Genes to Cells (2008) 13, 67-77. doi:10.1111/j.1365-2443.2007.01149.x
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Defining the function of β-catenin tyrosine phosphorylation in cadherin-mediated cell–cell adhesion

 Junji Tominaga, Yoshitaka Fukunaga, Edgardo Abelardo and Akira Nagafuchi*

Division of Cellular Interactions, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
β-catenin is a key protein in cadherin–catenin cell adhesion complex and its tyrosine phosphorylation is believed to cause destruction of junctional apparatus. The broad spectrum of substrates for kinases and phosphatases, however, does not rule out tyrosine phosphorylation of other junctional proteins as the main culprit in reduction of cell adhesion activity. Further, the endogenous β-catenin perturbs detailed functional analysis of phosphorylated mutant β-catenin in living cells. To directly evaluate the effect of β-catenin tyrosine phosphorylation in cell adhesion, we utilized F9 cells in which expression of endogenous β-catenin and its closely related protein plakoglobin were completely shut down. We also used {alpha}-catenin-deficient ({alpha}D) cells to evaluate the role of {alpha}-catenin on β-catenin tyrosine phosphorylation. We show that β-catenin with phosphorylation mutation at 654th tyrosine forms functional cadherin–catenin complex to mediate strong cadherin-mediated cell adhesion. Moreover, we show that 64th and 86th tyrosines are mainly phosphorylated in F9 cells, especially in the absence of {alpha}-catenin. Phosphorylation of these tyrosine residues, however, does not affect cadherin-mediated cell adhesion activity. Our data identified a novel site phosphorylated by endogenous tyrosine kinases in β-catenin. We also demonstrate that tyrosine phosphorylation of β-catenin might regulate cadherin-mediated cell adhesion in a more complicated way than previously expected.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
β-catenin is a dual function molecule which plays pivotal roles both in cadherin-mediated cell adhesion and Wnt-signaling cascade (see Harris & Peifer 2005 for review). In the former, β-catenin directly binds both E-cadherin and {alpha}-catenin to maintain functional cadherin–catenin cell adhesion complex (Aberle et al. 1994). In the latter, β-catenin directly binds with LEF/TCF to form transcriptional complex and functions in the nucleus (Behrens et al. 1996; Huber et al. 1996; Molenaar et al. 1996). The main function of β-catenin in cadherin–catenin complex is the molecular linkage between E-cadherin and {alpha}-catenin. In fact, when {alpha}-catenin is covalently linked to E-cadherin lacking β-catenin-binding site, this E-cadherin-{alpha}-catenin fusion protein functions as a cell adhesion molecule. However, adhesive mode of the fusion is a little different from that of wild-type cadherin–catenin complex (Nagafuchi et al. 1994). Thus, it was suggested that β-catenin has a regulatory function in this complex, although the molecular mechanisms involved are unclear.

It is believed that endogenous tyrosine kinases are involved in phosphorylation of junctional proteins. Phosphotyrosine-modified proteins are highly concentrated at adherens junction (AJ) in various tissues (Takata & Singer 1988). It was also reported that the specific proto-oncogenic tyrosine kinases, c-src and c-yes are enriched in AJ (Tsukita et al. 1991). Furthermore, incubation of cells with pervanadate, a potent inhibitor of tyrosine phosphatase, induces tyrosine phosphorylation of junctional proteins and destruction of AJ (Volberg et al. 1992). In these conditions, it was also demonstrated that β-catenin itself was phosphorylated (Ozawa & Kemler 1998).

Studies on over-expression of active tyrosine kinases and in vitro phosphorylation analysis demonstrated tyrosine phosphorylation of β-catenin as a possible regulatory mechanism for cadherin-mediated cell adhesion. Originally, it was reported that in v-src tyrosine kinase transfectants and in RSV-transformed cultured chick lens cells, exogenous tyrosine kinases suppress cadherin-based cell adhesion with concomitant destruction or structural modification of AJs (Warren & Nelson 1987; Kellie 1988; Volberg et al. 1991). Moreover, it was demonstrated that in v-src transfected cells, β-catenin was preferentially tyrosine phosphorylated in the cadherin–catenin complex and increased phosphorylation of β-catenin appeared to be associated with dysfunction of cadherin (Matsuyoshi et al. 1992; Behrens et al. 1993; Hamaguchi et al. 1993).

Recently, several tyrosine residues were identified as major phosphorylation sites which affect cadherin–catenin complex formation. The 142nd tyrosine (Y142) in β-catenin, which is present at the {alpha}-catenin binding domain, is phosphorylated by Fyn and Fer kinase, and its phosphorylation prevents interaction with {alpha}-catenin (Piedra et al. 2003). The 654th tyrosine (Y654) is phosphorylated by EGFR and its phosphorylation results in release of β-catenin from E-cadherin (Roura et al. 1999). Src was also reported to phosphorylate 86th tyrosine (Y86) in addition to Y654 (Roura et al. 1999). The effect of phosphorylation of Y86, however, is not clear. The point mutations of tyrosine to glutamic acid (phosphorylation mutation) at Y142 and Y654 cause the release of {alpha}-catenin and E-cadherin, respectively, from β-catenin. The mutation to phenylalanine (unphosphorylated mutant) does not affect β-catenin function (Roura et al. 1999; Piedra et al. 2003). These findings strongly suggest that tyrosine kinases induce the dysfunction of cadherin through tyrosine phosphorylation of β-catenin.

However, there are several controversial issues in this hypothesis. The most important point is that the treatment with pervanadate or exogenous expression of tyrosine kinases increases phosphorylation level of various proteins besides β-catenin. We previously reported that these hyperphosphorylations affect cadherin-mediated cell adhesion independent of β-catenin (Takeda et al. 1995). It is also not confirmed whether endogenous tyrosine kinases actually phosphorylate tyrosine residues mentioned above. Moreover, the function of phosphorylation mutant β-catenin could not be strictly verified in living cells, as the cells used in the experiments express wild-type endogenous β-catenin.

Recently, we succeeded in isolating β-catenin/plakoglobin-double null (BPD) cells from mouse teratocarcinoma F9 cells using gene targeting technology (Fukunaga et al. 2005). F9 cells express all cadherin–catenin complex components and show strong cell adhesion activity. The strong cadherin-mediated cell adhesion activity was abolished in BPD cells. Re-expression of wild-type β-catenin in BPD cells restored the adhesion activity. Thus, BPD would be an ideal model to examine the function of β-catenin in living cells.

To define the specific role of β-catenin tyrosine phosphorylation, we used BPD cells expressing various mutant β-catenins. {alpha}-catenin-deficient F9 ({alpha}D) and {alpha}-catenin-deficient BPD (BPD-{alpha}D) cells were also used to elucidate the role of {alpha}-catenin on β-catenin tyrosine phosphorylation. We found out that phosphorylated mutant Y654E forms functional cadherin–catenin complex to mediate strong cell adhesion. Phosphorylation mutation at Y142, on the other hand, affects {alpha}-catenin interaction, as previously reported. Interestingly, mutant β-catenin failing to interact with {alpha}-catenin was highly phosphorylated. Deletion mutant and point mutant β-catenins demonstrated that not Y142 and Y654, but Y64 and Y86 are mainly phosphorylated in {alpha}D cells. Cell dissociation assay revealed that the phosphorylation mutation at Y64 and Y86 does not affect cadherin-mediated cell adhesion activity. Finally, luciferase reporter assay showed that any mutation at 64th and/or 86th tyrosine residues does not abolish the potential of β-catenin signaling function in LEF/TCF-dependent transcription.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Glutamic acid mutation of Y142, but not Y654, of β-catenin affected cadherin-mediated cell adhesion

We first addressed whether phosphorylation of 142nd and 654th tyrosine residues affected β-catenin function in the absence of endogenous β-catenin using BPD cells. For this purpose, we prepared constructs consisting of phosphorylated and unphosphorylated mutant β-catenin where tyrosine residues were converted to glutamic acid and phenylalanine, respectively. (For the sake of simplicity, mutant β-catenin was represented using one letter form of amino acid and the number of the mutated residue. For example, phosphorylated mutant at 142nd tyrosine was represented as Y142E.)

In an effort to examine the effect of β-catenin phosphorylation at Y142 and Y654, we isolated BPD transfectant cells stably expressing wild-type β-catenin, Y142E, Y142F and Y654E mutants. Western blot analysis confirmed that each molecule was expressed in each transfectant. The expression levels of mutant β-catenin were similar to that of wild-type β-catenin. Using these transfectants, we examined cadherin-dependent cell adhesion activity. Cell morphology under monolayer culture condition reflects cell adhesion activity. Although adhesive parental F9 cells form tightly packed colonies, BPD cells, which lack strong cell adhesion activity, sparsely grew on culture dish (Fig. 1A). Re-expression of wild-type β-catenin restored colony formation activity on BPD transfectants. Consistent with previous reports, Y142E mutant did not restore colony formation activity on BPD transfectants, although Y142F did. Interestingly, Y654E expressing BPD transfectant cells formed closely packed colonies similar to BPD cell transfectants re-expressing wild-type β-catenin.


Figure 1
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  		Figure 1  Phosphorylated and unphosphorylated mutants at 142nd and 654th tyrosine. (A) Cell morphology. F9, parental F9 cells; BPD, β-catenin/plakoglobin-double null F9 cells; BPD-β (wt), BPD transfectants re-expressing wild-type β-catenin; Y142F, Y142E, Y654E, BPD transfectants expressing indicated mutant β-catenin. Note that BPD-β, Y142F and Y654E adhere to neighboring cells and form compact colonies, likely F9 cells. On the other hand, BPD and Y142E cannot form compact colonies. Bar; 50 µm. (B) Immunoprecipitation of wild-type β-catenin (wt) and its mutant Y142F, Y142E and Y654E. Western blots of immunoprecipitates were probed with anti-E-cadherin (E-cad), anti-β-catenin (β-cat), anti-{alpha}-catenin ({alpha}-cat) or anti-phosphotyrosine (PY20) antibodies. {alpha}-catenin is barely detected in immunoprecipitate derived from Y142E expressing cells and tyrosine of β-catenin was more efficiently phosphorylated in these cells. In immunoprecipitate derived from Y654E expressing cells, E-cadherin was clearly detected, although the amount seemed relatively lower than other samples. *Position of β-catenin. The positions of molecular weight markers (in thousands) are indicated on the left. (C) Cell adhesion activity of BPD cells and their transfectants expressing wild-type β-catenin (wt) and its mutant Y142E and Y654E. The confluent cell sheet of each cell line was treated with 0.01% trypsin in the presence (TC) or absence (TE) of Ca2+, and then dissociated by pipetting 10 times. Cell adhesion activities are represented by the cell dissociation index (NTC/NTE; see Experimental procedures). The lower the value of NTC/NTE is, the higher the activity of cell adhesion is. Note that Y654E mutant restored strong cell adhesion activity.

 
Then, we examined the effect of mutation at 142nd and 654th tyrosine residues on cadherin–catenin complex formation. β-catenin or its mutant expressed in BPD transfectants was immunoprecipitated with anti-β-catenin antibodies. Immunoprecipitates were subjected to SDS-PAGE and Western blot analysis was performed with anti-E-cadherin, anti-β-catenin and anti-{alpha}-catenin antibodies. The results demonstrated that {alpha}-catenin was barely co-precipitated with Y142E mutant (Fig. 1B). On the other hand, E-cadherin was co-precipitated with all mutants, although the amount of E-cadherin associated with Y654E mutant was relatively lower than that of β-catenin and other mutants. These data strongly suggest that Y654E mutant forms functional complex with E-cadherin in the absence of endogenous β-catenin. To verify this, we performed dissociation assay using BPD cells and their transfectants expressing wild-type β-catenin, Y142E and Y654E mutant. As shown in Fig. 1C, Y142E did not restore strong cell adhesion activity on BPD cells. Y654E expressing cells, however, showed strong cell adhesion activity similar to wild-type β-catenin-expressing cells. These data confirm that phosphorylation mutation at 142nd tyrosine prevents the interaction of β-catenin with {alpha}-catenin and abolishes β-catenin function in cadherin-mediated strong cell adhesion. On the other hand, it shows that Y654E mutant can support strong cell adhesion, although its affinity to E-cadherin might be lower than that of wild-type β-catenin.

High-level tyrosine phosphorylation of β-catenin in the absence of {alpha}-catenin

To examine whether 142nd and 654th tyrosines are physiological phosphorylation sites in F9 cells, immunoprecipitates from BPD transfectants re-expressing β-catenin and its mutants were subjected to Western blot analysis using anti-phosphotyrosine antibodies. To eliminate the effect of endogenous tyrosine phosphatase(s), cells were treated with pervanadate, a known phosphatase inhibitor, before lysate preparation. Anti-phosphotyrosine antibodies recognized wild-type β-catenin, Y142F and Y654E in similar fashion (Fig. 1B). These results indicate that 142nd and 654th tyrosines are not main phosphorylation sites in F9 cells and other tyrosine residue(s) besides these two are phosphorylated by endogenous tyrosine kinase(s).

Interestingly, the tyrosine phosphorylation of Y142E mutant was clearly detected in the immunoprecipitates (Fig. 1B). This suggests that {alpha}-catenin prevents tyrosine phosphorylation of β-catenin. To validate this observation, we compared the tyrosine phosphorylation of β-catenin in F9 cells and {alpha}D cells. As reported previously, the expression levels of E-cadherin and β-catenin in {alpha}D cells are similar to that of parental F9 cells (Maeno et al. 1999). Immunoprecipitation analysis showed similar amount of E-cadherin and β-catenin were precipitated with anti-β-catenin antibodies from F9 cells and {alpha}D cells (Fig. 2A). Western blot analysis with anti-phosphotyrosine antibodies, however, clearly demonstrated that not only β-catenin but also E-cadherin was effectively phosphorylated in {alpha}D cells.


Figure 2
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  		Figure 2  Tyrosine phosphorylation of β-catenin in {alpha}-catenin-deficient cells. (A) Immunoprecipitation of β-catenin from parental F9 cells (F9) and {alpha}-catenin-deficient F9 cells ({alpha}D). Western blots of immunoprecipitates were probed with anti-E-cadherin (E-cad), anti-β-catenin (β-cat), anti-{alpha}-catenin ({alpha}-cat) and anti-phosphotyrosine (PY20) antibodies. Note that both β-catenin and E-cadherin were highly phosphorylated at tyrosine residues in {alpha}-catenin-deficient cells. *Position of β-catenin. (B) Morphology of {alpha}-catenin-deficient BPD (BPD-{alpha}D) cells and their transfectants re-expressing β-catenin (BPD-{alpha}D-β). In the absence of {alpha}-catenin, β-catenin cannot support compact colony formation. Bar, 50 µm. (C) Expression of cadherin–catenin complex components in BPD, BPD-{alpha}D and BPD-{alpha}D-β cells. Western blots of total cell lysate were probed with anti-E-cadherin (E-cad), anti-β-catenin (β-cat) or anti-{alpha}-catenin ({alpha}-cat) antibodies. Both {alpha}-catenin and β-catenin do not express in BPD-{alpha}D cells. (D) Immunoprecipitation of β-catenin expressed in BPD transfectants (BPD-β) or BPD-{alpha}D transfectants (BPD-{alpha}D-β). Western blots of immunoprecipitates were probed with anti-E-cadherin (E-cad), anti-β-catenin (β-cat), anti-{alpha}-catenin ({alpha}-cat) or anti-phosphotyrosine (PY20) antibodies. Note that β-catenin in BPD-{alpha}D-β cells was more efficiently phosphorylated than that in BPD-β cells. *Position of β-catenin. The positions of molecular weight markers (in thousands) are indicated on the left.

 
To confirm whether β-catenin is efficiently phosphorylated in the absence of {alpha}-catenin, we isolated BPD-{alpha}D cells (Fig. 2B). In these cells, neither β-catenin nor {alpha}-catenin was detected (Fig. 2C). Expression of plakoglobin, a closely-related protein of β-catenin, was also not detected (data not shown). Then, we isolated BPD-{alpha}D transfectants which were stably expressing wild-type β-catenin and compared the level of tyrosine phosphorylation of expressed β-catenin in BPD-{alpha}D transfectants with that of BPD transfectants. Immunoprecipitation analysis showed that similar amount of E-cadherin and β-catenin was recovered with anti-β-catenin antibodies (Fig. 2D). Consistent with the result obtained from {alpha}D cells, β-catenin expressed in BPD-{alpha}D transfectants was more efficiently phosphorylated than that in BPD transfectants (Fig. 2D). Furthermore, E-cadherin was likewise highly phosphorylated in β-catenin-positive BPD-{alpha}D transfectants, although its function is not clear. These data confirmed that β-catenin is highly phosphorylated in the absence of {alpha}-catenin.

Tyr64 and Tyr86 of β-catenin were highly phosphorylated by endogenous kinase(s) in the absence of {alpha}-catenin

In β-catenin, {alpha}-catenin binding site is located at the boundary between N-terminal domain and armadillo repeat domain and includes 142nd tyrosine (Fig. 3). As β-catenin was highly phosphorylated in the absence of {alpha}-catenin, it is likely that the phosphorylation site(s) is/are located around the region of N-terminal domain of β-catenin. To determine the tyrosine phosphorylation domain of β-catenin, we constructed an expression vector for mutant β-catenin where the N-terminal domain was replaced with GFP polypeptide (Fig. 4A). We isolated BPD-{alpha}D cells stably expressing this N-terminal deletion mutant β-catenin ({Delta}N) and compared the phosphorylation level of expressed {Delta}N with that of wild-type β-catenin. Immunoprecipitation analysis showed that similar amount of E-cadherin and β-catenin or its mutant was recovered with anti-β-catenin antibodies (Fig. 4B). As shown above, wild-type β-catenin in BPD-{alpha}D is highly phosphorylated judging from the Western blot analysis with anti-phosphotyrosine antibodies. In contrast, tyrosine phosphorylation of {Delta}N mutant β-catenin was barely detected. These results strongly suggest that the tyrosine residues in the N-terminal domain were phosphorylated in F9 cells. Sequence analysis showed that there are four tyrosine resides, including 142nd tyrosine, in the N-terminal domain of β-catenin (Fig. 3). In an effort to confirm the tyrosine phosphorylation in this domain and to identify the specific tyrosine residues phosphorylated, we constructed three expression vectors for unphosphorylated mutant β-catenin, Y30F, Y64F and Y86F, and isolated BPD-{alpha}D transfectants stably expressing these mutants. Then we examined tyrosine phosphorylation of these mutants, Y142F mutant and wild-type β-catenin in BPD-{alpha}D transfectants (Fig. 4C). Y30F and Y142F were well phosphorylated similar to wild-type β-catenin. In contrast, phosphorylation of Y64F and Y86F mutant was significantly reduced. These data suggest that 64th and 86th tyrosines are phosphorylated in F9 cells by endogenous enzymes. To confirm this, we constructed Y64/86F mutant where both 64th and 86th tyrosines were mutated to phenylalanine. We isolated BPD-{alpha}D transfectants expressing this mutant and analyzed its phosphorylation, and found that Y64/86F was barely phosphorylated. These results indicate that 64th and 86th tyrosine residues were the main phosphorylation sites of β-catenin in F9 derivative cells.


Figure 3
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  		Figure 3  N-terminal domain sequence of mouse β-catenin, plakoglobin and Drosophila armadillo. The 185 amino acids of mouse β-catenin N-terminal domain and corresponding sequence of mouse plakoglobin and Drosophila armadillo were aligned using the program "Multiple Alignment (Pep)" with default parameters (gap penalty is –12 for GAP Insert and –4 for GAP extend) in GENETYX-Mac Ver. 12.2.0 (Genetyx Corp., Japan). All tyrosine residues are bolded. Tyrosine residues in β-catenin are also numbered. Asterisks and dots show amino acids identical among all and two of three polypeptides, respectively. Underline in β-catenin sequence shows {alpha}-catenin binding region.

 

Figure 4
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  		Figure 4  Identification of tyrosine residues phosphorylated in β-catenin. (A) Schematic drawing of β-catenin and N-terminal deletion mutant β-catenin ({Delta}N). In {Delta}N, GFP replaces N-terminus region of β-catenin, which include N-terminal domain and first armadillo repeat. Molecular weight of {Delta}N is nearly equal to that of β-catenin. Boxes show armadillo repeats. (B) Immunoprecipitation of β-catenin or {Delta}N mutant expressed in BPD-{alpha}D transfectants. Western blots of immunoprecipitates were probed with anti-E-cadherin (E-cad), anti-β-catenin (β-cat) or anti-phosphotyrosine (PY20) antibodies. Note that tyrosine phosphorylation of {Delta}N is barely detected. E-cadherin is phosphorylated in both immunoprecipitates. *Position of β-catenin or {Delta}N mutant. The positions of molecular weight markers (in thousands) are indicated on the left. (C) Immunoprecipitation with anti-E-cadherin mAb from BPD-{alpha}D transfectants expressing mutant β-catenin with unphosphorylated point mutation(s). Western blots of immunoprecipitates were probed with anti-E-cadherin (E-cad), anti-β-catenin (β-cat) or anti-phosphotyrosine (PY20) antibodies. Note that tyrosine phosphorylation of Y64F and Y86F is reduced and that of Y64/86F is barely detected.

 
Mutations of 64th and/or 86th tyrosine of β-catenin did not affect cadherin-mediated cell adhesion

The phosphorylation level of 64th and 86th tyrosine is regulated by {alpha}-catenin in F9 cell derivatives. We, then, addressed the effect of these tyrosine residues on cadherin–catenin complex formation and cadherin-mediated cell adhesion. For this purpose, we constructed expression vectors for phosphorylated mutant β-catenin, Y64E, Y86E and Y64/86E, and isolated BPD transfectants expressing these mutants. In addition to these newly-isolated transfectants, we also used BPD transfectants expressing wild-type and unphosphorylated mutant β-catenin in the subsequent experiments. Immunoprecipitation analysis showed all mutants form complex with E-cadherin and {alpha}-catenin in similar manner with wild-type β-catenin (Fig. 5A). Cell dissociation assay revealed all mutants restored strong cell adhesion activity on BPD cells like wild-type β-catenin (Fig. 5B). Immunofluorescent analysis also showed that all mutants localized at cell–cell contact sites with E-cadherin just like how wild-type β-catenin behaved (data not shown). Thus, 64th and 86th tyrosines do not affect the basic cell adhesion activity mediated by E-cadherin.


Figure 5
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  		Figure 5  Phosphorylated and unphosphorylated mutant β-catenin at 64th and 86th tyrosines. (A) Effect on cadherin–catenin complex formation. Immunoprecipitation with anti E-cadherin mAb from BPD transfectants expressing phosphorylated (Y64E, Y86E and Y64/86E) and unphosphorylated (Y64F, Y86F and Y64/86F) mutant β-catenin. Western blots of immunoprecipitates were probed with anti-E-cadherin (E-cad), anti-β-catenin (β-cat) or anti-{alpha}-catenin ({alpha}-cat) antibodies. Note that all mutants form complex with E-cadherin and {alpha}-catenin. (B) Effect on cell–cell adhesion. The confluent cell sheet of each cell line was treated with 0.01% trypsin in the presence (TC) or absence (TE) of Ca2+, and then dissociated by pipetting 10 times. Cell adhesion activities are represented by the cell dissociation index (NTC/NTE; see Experimental procedures). The lower the value of NTC/NTE is, the higher the activity of cell adhesion is. Note that all mutant β-catenin restored strong cell adhesion activity.

 
β-catenin with mutations at 64th and/or 86th tyrosine activated LEF/TCF-dependent transcription

In an effort to clarify the involvement of 64th and 86th tyrosine in β-catenin signaling function, we examined the transcriptional activity of non-phosphorylated and phosphorylated mutant β-catenin at 64th and/or 86th tyrosine residues. When the expression vectors for mutant β-catenins were transiently transfected into BPD cells, polypeptides with molecular weights similar to wild-type β-catenin were expressed (Fig. 6A). Luciferase reporter assay showed that LEF/TCF-dependent transcriptional activity was highly induced in BPD cells expressing mutant β-catenins (Fig. 6A).


Figure 6
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  		Figure 6  LEF/TCF-dependent transcriptional activity in BPD cells expressing β-catenin mutants. (A) The effect of mutant β-catenins on TOPflash luciferase reporter activity. The expression vector for each β-catenin mutant was transiently introduced into BPD cells with pTOPflash and pCMV-renilla luciferase reporters. As negative and positive controls, expression vectors for GFP and β-catenin, respectively, were used. The value of BPD cells transfected with β-catenin was set at 100. Note that luciferase activity of all mutants, except phosphorylated mutant at 64th tyrosine, showed double or triple (Y64/86F) increase comparing to wild-type β-catenin. The expression level of each mutant protein (β-cat) was similar (upper panel). Eukaryotic elongation factor 2 (EF2) levels were used as an internal control (lower panel) (B) Effect of Wnt-3a on TOPflash luciferase reporter activity in BPD cells stably expressing mutant β-catenins. Cells were transiently transfected with pTOPflash and pCMV-renilla luciferase reporters, then treated with conditioned media from L cells expressing Wnt-3a (black bars) or with that from parental L cells (white bars) for 15 h. The value of BPD cells expressing wild-type β-catenin treated withWnt-3a was set at 100. Note that any mutation at 64th and/or 86th tyrosine residues did not abolish the potential of β-catenin signaling function in LEF/TCF-dependent transcription.

 
F9 cells are known to be sensitive to Wnt-3a signal. We then examined the responsiveness of each mutant β-catenin on Wnt-3a. Among isolated BPD transfectants, we selected one for each mutant β-catenin, which shows highest increase of LEF/TCF-dependent transcriptional activity responding to Wnt-3a. Using pTOPflash and pCMV-renilla as reporters, we examined the response of these cells to a Wnt-3a stimulus (Fig. 6B). In the absence of Wnt-3a, all clones showed only trace levels of transcriptional activity. In the presence of Wnt-3a, significant increase of nuclear activities was observed in all transfectants. The transcriptional activity of Y86 mutant β-catenin was similar to that of wild-type β-catenin. Mutation at 64th tyrosine residue seemed to reduce the transcriptional activity. The expression level of mutant β-catenins, however, could not be strictly adjusted both in transient and stable expressions. Thus, it is difficult to judge whether the different inductions by mutant β-catenins is influenced by qualitative difference or quantitative difference of each mutant. It, however, was clearly showed that any mutation at 64th and/or 86th tyrosine residues do not abolish the potential of β-catenin signaling function in LEF/TCF-dependent transcription.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
We examined tyrosine phosphorylation of β-catenin by endogenous enzyme(s) using BPD cells expressing various mutant β-catenin. {alpha}D cells and BPD-{alpha}D cells were also used to define the role of {alpha}-catenin on β-catenin phosphorylation. Using the merit of BPD cells, the role of tyrosine residues endogenously to be phosphorylated in β-catenin was examined from the view of cell adhesion. Here, we will discuss regulatory mechanisms of β-catenin tyrosine phosphorylation in F9 cells. Furthermore, we will also discuss the physiological function of β-catenin tyrosine phosphorylation in cadherin-mediated cell adhesion.

Regulation of β-catenin tyrosine phosphorylation

We used pervanadate, a common phosphatase inhibitor, in this study. This means that the phosphorylation of β-catenin observed here was performed by endogenous tyrosine kinase(s). It is not clear whether constitutive phosphorylation, which is usually cancelled by phosphatase activity, occurred or inhibition of phosphatase activated endogenous kinases. In either case, it is noticed that phosphatase, but not kinase, could regulate phosphorylation level of β-catenin in steady state F9 cells. So far, involvement of tyrosine kinases and phosphatase in β-catenin function has been reported (Matsuyoshi et al. 1992; Behrens et al. 1993; Hamaguchi et al. 1993; Wadham et al. 2003; van Buul et al. 2005; Yan et al. 2006). In most reports, however, the target of kinase and phosphates is not necessarily specified to β-catenin. The use of β-catenin/plakoglobin-null cells like BPD cells and phosphorylated (or unphosphorylated) mutant β-catenins will be required to examine the roles of tyrosine phosphorylation in β-catenin function.

Phosphorylation of Y142 and Y654 is well studied using various experimental systems (Roura et al. 1999; Piedra et al. 2003). In F9 cell derivatives, however, these residues were barely phosphorylated and Y64 and Y86 were primarily phosphorylated. It has been reported that Y86 and Y654 were phosphorylated by Src kinase (Roura et al. 1999), and Y142 was by Fer and Fyn kinases (Piedra et al. 2003). The kinase, which preferentially phosphorylates Y64, has not yet been identified. Interestingly, among these tyrosine residues, Y64 and Y86 are not conserved in Armadillo, a Drosophila homologue of β-catenin (Fig. 3). Thus, these tyrosines are phosphorylated under mammalian specific manner by unidentified kinase. On the other hand, it has been reported that pervanadate treatment causes dissociation of {alpha}-catenin from β-catenin in leukemia cell transfectants expressing exogenous E-cadherin (Ozawa & Kemler 1998). This strongly suggests that phosphorylation of Y142 (see below) occurred in these cells. In the presence of pervanadate, various tyrosine residues would be phosphorylated depending on the cellular context.

We showed that high level tyrosine phosphorylation of β-catenin was observed in cells lacking {alpha}-catenin expression. Y142E β-catenin mutant, which has lower affinity to {alpha}-catenin, was also highly phosphorylated even in the presence of endogenous {alpha}-catenin. These results indicate that the {alpha}-catenin binding to β-catenin, but not the cytoplasmic {alpha}-catenin, prevents the phosphorylation of β-catenin. In the absence of {alpha}-catenin, 64th and 86th tyrosine was preferentially phosphorylated. As {alpha}-catenin-binding domain of β-catenin is identified at amino acids 118–146 near these tyrosine residues (Aberle et al. 1996), it is likely that {alpha}-catenin sterically prevents the access of tyrosine kinase(s) which phosphorylate these tyrosine residues of β-catenin.

Effects of tyrosine phosphorylation in cadherin–catenin complex formation

It was reported that phosphorylation of Y142 and Y654 affect the interaction of β-catenin with {alpha}-catenin and E-cadherin, respectively (Roura et al. 1999; Piedra et al. 2003). Consistent with previous reports, phosphorylation mutation at Y142 clearly prevented {alpha}-catenin interaction with β-catenin in F9 cell derivatives. Y654E phosphorylated mutant, however, formed complex with E-cadherin and supported cadherin-mediated cell adhesion in similar fashion as wild-type β-catenin. Closer examination of the data of these previous reports revealed that Y654E mutant merely showed lower affinity to E-cadherin and not failure to interact with this molecule (Roura et al. 1999; Piedra et al. 2001; Xu et al. 2004). In the complete absence of endogenous β-catenin and plakoglobin, Y654E phosphorylated mutant forms functional cadherin–catenin complex. This is consistent with another report that claims activation of Src kinase, which preferentially phosphorylates Y654, did not affect cadherin–catenin complex formation (Papkoff 1997). It is, however, possible that Y654E mutant does not fully mimic the functions of the phosphorylated form. For the complete loss of binding function of β-catenin with E-cadherin, real phosphorylation of 654th tyrosine might be required.

In the absence of {alpha}-catenin, Y64 and Y86 of β-catenin were preferentially phosphorylated in F9 cell derivatives. Mutation at these tyrosine sites, either phosphorylated or unphosphorylated forms, did not affect β-catenin function in cadherin-mediated cell adhesion. All mutants form complex with E-cadherin and {alpha}-catenin, and restored cell adhesion activity in BPD cells. On the other hand, any mutation at these tyrosine residues did not abolish the signaling function of β-catenin. It is possible that the glutamate replacement might partially mimic phosphorylation of these residues. To the minimum, 64th and 86th tyrosines are dispensable for basic function of β-catenin.

In this paper, we have shown that phosphorylation of 654th tyrosine might regulate cadherin-mediated cell adhesion in more complicated way than previously expected. It was also revealed that 64th and 86th tyrosine residues are phosphorylated by endogenous kinase(s), when phosphatases were inactivated. These tyrosine residues are preferentially phosphorylated in the absence of {alpha}-catenin. However, modification of both 64th and 86th tyrosines does not affect the basic cadherin-mediated cell adhesion activity and LEF/TCF-dependent transcription activity. We must now start looking for roles of β-catenin phosphorylation on subtle regulation in cell–cell adhesion and/or Wnt signal.


   Experimental procedures
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 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cell culture and phase contrast images

Mouse F9 teratocarcinoma cells (Sherman & Miller 1978) and their derivatives were cultured in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum (BIOSOURCE, Bethesda, MD). Isolation of {alpha}-catenin-deficient ({alpha}D) and β-catenin/plakoglobin-double null (BPD) F9 cells were previously reported (Maeno et al. 1999; Fukunaga et al. 2005). Culture dishes or cover slips were pre-coated with 0.2% gelatin for 15 min. Phase contrast microscopic images were acquired using a microscope (ECLIPSE TS100, Nikon, Tokyo, Japan) equipped with a digital camera (Coolpix 990, Nikon).

{alpha}-Catenin-deficient BPD cells (BPD-{alpha}D cells) were generated by gene targeting. The targeting vector for the {alpha}-catenin gene and the method of gene targeting has been described in our previous report (Maeno et al. 1999). Upon electroporation and selection with G418, one clone, Clone51, contained the correct recombination events among 72 clones analyzed. Clone51 was subjected to antibiotic selection using higher concentration of G418. Then, we isolated four clones among 36 clones analyzed, in which expression of {alpha}-catenin polypeptide was not detected and both alleles of the {alpha}-catenin gene were disrupted. Among these four {alpha}-catenin-deficient clones, Clone21 was selected for the subsequent analysis. To create a G418-sensitive {alpha}D clone, the drug-resistance genes in disrupted alleles were removed using the Cre-pac method (Taniguchi et al. 1998). Several G418-sensitive clones were isolated and Clone5 was used as BPD-{alpha}D cells in subsequent experiments.

Expression vector and isolation of stable transfectants

pCAG-βABCD, expression vector for a full-length β-catenin was described elsewhere (Shimizu et al., 2008). pCAG-NGFP, expression vector for GFP fusion, was described previously (Matsuda et al. 2004). For an N-terminal-deleted β-catenin fused to GFP ({Delta}N) we constructed pCAG-β-G-BCD expression vector. In this expression vector, XbaI-SalI fragment of pCAG-βABCD, which encodes 183–781 amino acids of β-catenin, was inserted into XhoI-EcoRI sites of pCAG-NGFP. An in-frame linker was inserted at the connection with GFP. As results, N-terminal domain (amino acids 1–182) of β-catenin, which includes {alpha}-catenin-binding site, was replaced with GFP polypeptide in {Delta}N. To construct expression vectors for phosphorylated and unphosphorylated β-catenin mutants, we used pCAG-βABCD as a basic expression vector. In this vector, we generated mutation at a codon for tyrosine residue in β-catenin N-terminal region using Quick Change XL Site-Directed mutagenesis kit (Stratagene, La Jolla, CA) and a complementary primer set. For phosphorylated and unphosphorylated mutants, codon for tyrosine (TAC or TAT) was changed to that of glutamic acid (GAA or GAG) and that of phenylalanine (TTC or TTT), respectively.

For the isolation of stable transfectants, expression vectors were transfected into BPD cells or BPD-{alpha}D cells using LipofectamineTM 2000 system (Invitrogen, Carlsbad, CA). Cells were subjected to G418 selection at 400 µg/mL for 2 weeks. At least three clones were isolated from each transfectant and one, which showed a common phenotype, was chosen as the representative clone.

Antibodies

The following primary antibodies were used for immunoprecipitation and immunoblot analyses: rat anti-mouse E-cadherin mAb, ECCD-2 (Shirayoshi et al. 1986), rat anti-mouse {alpha}-catenin mAb, {alpha}18 (Nagafuchi & Tsukita 1994), mouse anti-β-catenin mAb (BD Bioscience, Rockville, MD), mouse anti-phosphotyrosine mAb (PY20, BD Biosciences).

Immunoprecipitation analysis

To protect tyrosine phosphorylated β-catenin from tyrosine phosphatases, cells were treated with pervanadate (1 mM sodium orthovanadate, 0.01% H2O2), an inhibitor of phosphotyrosine phosphatase, for 20 min. Cells harvested from a confluent culture in 9-cm dishes were lysed using 1 mL of extraction buffer (1% NP-40, 1 mM CaCl2, 1 mM NaF, 10 mM Na4P2O7, 2 mM Pefabloc SC Plus (Roche, Basel, Switzerland), 10 µg/mL leupeptin and 10 µg/mL aprotinin in HEPES-buffered Mg2+-free saline) and centrifuged at 20 400 g for 20 min. The cell extract was pre-adsorbed 3 times with 50 µL beads, then preincubated with anti-E-cadherin or anti-β-catenin mAb for 30 min, and finally adsorbed with 20 µL beads. The beads were extensively washed with the extraction buffer and then suspended in SDS lysis buffer. Anti-rat IgG agarose beads (American Qualex, San Clemente, CA) and protein-G sepharose beads (GE Healthcare, Little Chalfont, UK) were used for anti-E-cadherin and anti-β-catenin antibodies, respectively.

Western blot analysis

SDS-PAGE and immunoblotting were performed as previously described (Imamura et al. 1999). Samples solubilized in SDS sample buffer were separated by SDS-PAGE. For immunoblotting, proteins were electrophoretically transferred onto nitrocellulose sheets. Nitrocellulose membranes were then incubated with primary antibody. Antibody detection was performed using GE Healthcare biotin–streptavidin kit according to the manufacturer's instructions.

Trypsin treatment and dissociation of cells

Cells were trypsinized using two different methods for the differential removal of E-cadherin, as previously described (Takeichi 1977). Cells were treated with 0.01% trypsin with 1 mM CaCl2 (TC) or 1 mM EDTA (TE) at 37 ºC for 20 min. Generally, cadherins remain intact following TC treatment, but are digested following TE treatment. For the cell dissociation assay, cells treated with TC or TE were dissociated by pipetting 10 times and counted for the total particle number. The extent of cell dissociation was represented by the ratio of the total particle number following TC treatment (NTC) to TE treatment (NTE).

Transient transfection and luciferase assay

BPD cells (24-well plate) were transfected with 1.0 µg plasmid by LipofectamineTM 2000 system for 5 h, then cultured in normal medium for another 15 h. The cells were lysed for luciferase assay and Western blot assay. Luciferase activity was measured using a Dual Luciferase Assay System (Promega, Madison, WI) and a lumiphotometer. For Wnt-3a assays, cells were transfected with 0.5 µg of pTOPflash and 2 ng pCMV-renilla reporter vectors, then cultured in a conditioned medium from L cells or L cells secreting Wnt-3a. To examine the effect of β-catenin and their mutants, 0.5 µg of the indicated expression vectors were mixed with 0.5 µg pTOPflash and 2 ng pCMV-renilla reporter vectors. Total DNA was adjusted to 1.0 µg by pCAG-NGFP, if necessary.


   Acknowledgements
 
We are grateful to all laboratory members (Division of Cellular Interactions of the Institute of Molecular Embryology and Genetics at Kumamoto University) for their participation in helpful discussions. We would also like to thank M. Matsuda for pCAG-NGFP, H. Niwa for pCAGGSneodelEcoRI, M. van de Wetering and H. Clevers for pTOPflash, E. Morikawa and T. Magarikaji for excellent technical assistance.

Part of this work at the Department of Cellular Interactions of the Institute of Molecular Embryology and Genetics of Kumamoto University was supported by a grant to A.N. from Grants-in-Aid for Scientific Research and Cancer Research and a Grant-in-Aid for 21st Century COE Research "Cell Fate Regulation Research and Education Unit" from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Shigeo Hayashi

* Correspondence: E-mail: naga-san{at}kumamoto-u.ac.jp


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 Discussion
 Experimental procedures
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Received: 10 September 2007
Accepted: 16 October 2007





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