Both plakoglobin and β-catenin can be divided into three different subdomains (Fig. ). The central domain is composed of 12 repetitions of 42 amino acids each, known as armadillo repeats, after the β-catenin ortholog in Drosophila melanogaster
. This region, very similar in both proteins (with a 76% identity) (7
), has a basic pI, and its structure has been determined; it forms a super helix composed of 36 small α-helices (3 per each armadillo repeat) (19
). Tyr654 lies on the last repeat of this domain, and Tyr142 lies on the limit of this domain and the N-terminal tail. Contrarily to the armadillo repeat domain, the N- and C-terminal tails are mainly acidic (in both the pI is 4.4) and the degree of conservation between β-catenin and plakoglobin is very low (29 and 41%, respectively, for the N- and C-terminal tails). As a consequence of the homologies in these domains, Tyr residues equivalent to β-catenin tyrosines 142 and 654 can be located in plakoglobin: they correspond to tyrosines 133 and 643, respectively. The similarity among the sequences surrounding equivalent Tyr residues in β-catenin and plakoglobin is shown in Fig. . On the other hand, β-catenin Tyr86, placed in the N-terminal tail, does not have a correspondent residue in plakoglobin; the equivalent amino acid is Ser74.
FIG. 1. Diagram of β-catenin and plakoglobin. The three different domains that form these two proteins are shown. The amino acid sequences near the three indicated Tyr residues in β-catenin are shown, as are the equivalent sequences in plakoglobin. (more ...)
We determined whether protein kinases that specifically phosphorylate these three Tyr residues in β-catenin also do it in plakoglobin. As shown in Fig. , recombinant EGFR or purified erbB2 phosphorylates β-catenin. The phosphorylation is not observed when a β-catenin Tyr654→Phe mutant was used as the substrate but was not affected by similar mutations in Tyr86 or Tyr142. These data indicate that EGFR or erbB2 exclusively phosphorylates Tyr654 in β-catenin.
FIG. 2. EGFR presents different substrate specificities of phosphorylation on β-catenin and plakoglobin. (A) GST-β-catenin fusion proteins (6.7 pmol) were phosphorylated with 0.5 U of recombinant EGFR kinase or RWP1 cell extracts transfected with (more ...)
Similar in vitro assays were performed with recombinant plakoglobin or several fragments as substrates. Plakoglobin was efficiently phosphorylated by EGFR, but different from β-catenin, EGFR did not phosphorylate the plakoglobin armadillo domain, where Tyr643 is located, but modified the C-terminal tail (Fig. ). Data from other authors have shown that three Tyr residues in this domain (Tyr693, Tyr724, and Tyr729) are phosphorylated after EGFR stimulation (12
). To verify that Tyr643 was not being modified, the same in vitro assays were performed with a Tyr643→Phe mutant. This plakoglobin form incorporated phosphate identically to the wild-type form (Fig. ), indicating that Tyr643 is not a substrate of EGFR.
The relevance of plakoglobin phosphorylation by EGFR was also determined. Interaction with desmoplakin was significantly decreased after phosphorylation (Fig. ), in agreement with previously published data (12
). No changes were observed after phosphorylation in the interaction with desmoglein, E-cadherin, α-catenin, or other cofactors related to the transcriptional activity of this protein (TBP and Tcf-4) (Fig. ).
Together, these results indicate that EGFR modifies β-catenin and plakoglobin differently and exerts distinct effects on the interaction of these proteins with some shared partners, such as E-cadherin. These observations prompted us to extend the study to other Tyr kinases, like Src, Fer, or Fyn, which phosphorylate β-catenin.
Src kinase phosphorylates β-catenin mainly on Tyr86, although Tyr654 is also modified with a lower efficiency (39
). In plakoglobin, phosphate was incorporated mostly in a fragment comprising armadillo repeats 6 to 12, and to a lesser extent in the C-terminal tail (Fig. ). The plakoglobin Tyr643→Phe mutation greatly diminished the amount of PTyr present after Src treatment (Fig. ), suggesting that this residue, present in the 12th armadillo repeat, was the main target of this kinase.
FIG. 3. Mapping of residues involved in plakoglobin phosphorylation by Src, Fer, and Fyn kinases. Five picomoles of GST-plakoglobin deletion mutants (A) or the indicated point mutants (B) was phosphorylated with either 0.5 U of recombinant Src (upper panel), (more ...)
The effect of Fer kinase was also quite different in β-catenin and plakoglobin. Fer and Fyn specifically phosphorylate β-catenin Tyr142, blocking the interaction with α-catenin (35
). Fer phosphorylated plakoglobin, but results with armadillo fragments indicated that this kinase was modifying armadillo repeats 7 to 12, comprising aa 381 to 673, but not armadillo repeats 1 to 6, the fragment that contains Tyr133, the equivalent residue to β-catenin Tyr142 (Fig. ). Fer was unable to modify this residue, as could be concluded from the experiments performed with plakoglobin Tyr→Phe mutants. Replacement of either Tyr643 or Tyr133 by Phe did not modify the phosphorylation of plakoglobin (Fig. ). Thus, in plakoglobin, Fer phosphorylates a residue different from Tyr133 and Tyr643. We investigated the involvement of Tyr549, the only residue located within plakoglobin armadillo repeats 7 to 12 that is not present in β-catenin (Phe561 in β-catenin). Mutation of Tyr549 to Phe confirmed that this Tyr was the target of Fer action: phosphorylation of plakoglobin Tyr549→Phe by this kinase was completely abolished (Fig. ).
Similar results were obtained with Fyn kinase. However, although the plakoglobin armadillo fragment 7-12 was again the best substrate for this kinase, the armadillo repeat fragment 1-6 was also modified to a lesser extent. None of the terminal tails were phosphorylated (Fig. ). Experiments with Tyr→Phe mutants gave evidence that Fyn mainly phosphorylated Tyr549 and that it phosphorylated Tyr133 with a much lower activity (Fig. ).
We investigated the effect of these kinase activities on the interaction of plakoglobin with several partners. Pull-down assays revealed that phosphorylation by Src decreased the binding of plakoglobin with two components of the adherens junctions, α-catenin and E-cadherin, whereas no changes with desmoglein, TBP, or Tcf-4 were detected (Fig. ). On the other hand, the affinity of phosphorylated plakoglobin for desmoplakin is higher than that of the unmodified protein (Fig. ). This change in affinities was further analyzed by using recombinant proteins: phosphorylation of plakoglobin by Src caused a 2.3-fold decrease in the affinity for E-cadherin (Fig. ) and a 4.1-fold rise in the binding to desmoplakin (Fig. ). However, neither Src phosphorylation nor the plakoglobin mutant Y643E, designed to mimic the phosphorylation at this residue, had a direct effect on the interaction with α-catenin (Fig. ). The apparent contradiction between this result and the decrease in α-catenin binding observed in the pull-down assays can be reconciled if plakoglobin-α-catenin binding is dependent on the presence of E-cadherin, similar to the coordinated interaction of E-cadherin and α-catenin with β-catenin previously reported (10
). Indeed, we found that the association of α-catenin and E-cadherin with plakoglobin is interdependent. Previous binding of E-cadherin to plakoglobin significantly facilitated the association of α-catenin (Fig. ), although preassociation of α-catenin with plakoglobin did not modify E-cadherin binding to the complex (Fig. ). Therefore, the decrease observed in α-catenin binding upon plakoglobin phosphorylation by Src (Fig. ) is likely the consequence of reduced E-cadherin binding. No changes were observed in the desmoplakin-plakoglobin binding upon addition of E-cadherin or α-catenin, indicating that, as expected, effects on the affinity for desmoplakin are not dependent on adherens junction proteins (Fig. ).
FIG. 4. Phosphorylation of plakoglobin by Src decreases its interaction with α-catenin and E-cadherin and increases plakoglobin-desmoplakin association. (A) Five picomoles of GST or GST-plakoglobin fusion proteins was phosphorylated with 0.5 U of pp60 (more ...)
The relevance of Fer phosphorylation was also investigated. Fer affected plakoglobin interaction with desmoplakin and α-catenin. Contrary to what was previously observed for α-catenin-β-catenin interaction, phosphorylation by Fer raised the affinity of plakoglobin for α-catenin (Fig. ); using recombinant proteins, this increase was estimated to be 4.4-fold (Fig. ). On the other hand, Fer-phosphorylated plakoglobin showed a sevenfold decrease in the affinity for desmoplakin (Fig. ). No effect was detected on the interaction of plakoglobin with E-cadherin, desmoglein, TBP, or Tcf-4.
FIG. 5. Phosphorylation of plakoglobin by Fer and Fyn kinases decreases plakoglobin-desmoplakin interaction and increases plakoglobin-α-catenin association. (A and D) GST or GST-plakoglobin fusion proteins (5 pmol) were phosphorylated with Fer purified (more ...)
The effects of Fyn phosphorylation on the association of plakoglobin with α-catenin and desmoplakin were comparable to those obtained with Fer; i.e., an increased binding to α-catenin and a disruption of the interaction with desmoplakin (Fig. ). These changes were further confirmed in binding assays with different amounts of recombinant proteins (Fig. and G). Moreover, phosphorylation of plakoglobin by Fyn, and not by Fer, also modified the interaction with the transcriptional factor Tcf-4 (Fig. ). We consider that effects on this binding may likely be mediated by the phosphorylation of plakoglobin Tyr133, a residue that is modified by Fyn and not by Fer, albeit with a low efficiency. Even so, Fyn phosphorylation reduced the affinity of plakoglobin for Tcf-4 more than twofold (Fig. ). No changes were observed in the interaction with other factors.
All these results indicate that phosphorylation of plakoglobin residues Tyr133, Tyr549, and Tyr643 affects its ability to interact with α-catenin, E-cadherin, desmoplakin, and Tcf-4. To confirm our conclusions, each of these three residues was individually replaced with Glu to mimic the phosphorylation charge. The results obtained with these mutants were consistent with those obtained with the kinases. The plakoglobin Tyr643→Glu mutant showed reduced binding to α-catenin and E-cadherin and a rise in the affinity for desmoplakin. On the contrary, the Tyr549→Glu mutant presented the opposite alterations: an increase in binding to α-catenin and a decreased affinity for desmoplakin without modifications in the association with other factors (Fig. ). Finally, the Tyr133→Glu mutant showed a complete disruption in the interaction with α-catenin and a parallel increase in desmoplakin binding (Fig. ). Thus, this mutant has an behavior opposite to what was previously observed upon Fyn phosphorylation of wild-type plakoglobin (Fig. ), where the predominant effect is caused by the more efficient modification of Tyr549 (Fig. ). A decrease in Tcf-4 binding was also observed in the Tyr133→Glu mutant (Fig. ).
FIG. 6. Effect of Tyr-to-Glu point mutants in the association of plakoglobin to its cellular cofactors. Pull-down assays were performed by incubating 8 pmol of GST or GST-plakoglobin fusion proteins with 200 μg of whole-cell extracts from SW480. Protein (more ...)
The results shown so far indicate that the effects of phosphorylation of plakoglobin are quite complex, and modification of a specific Tyr residue affects the interaction of several partners. In addition, the action of a specific tyrosine kinase can simultaneously promote the disassembly of the interactions with components of the adherens junctions and the establishment of associations with components of desmosomes. This is what was expected after Src phosphorylation which, upon Tyr643 phosphorylation, reduces binding to α-catenin and E-cadherin and increases the association with desmoplakin. In turn, Fer stimulation, through modification of Tyr549, causes diminished binding of plakoglobin to components of desmosomes (desmoplakin) and increased interaction with adherens junction proteins (α-catenin).
To verify these conclusions, epithelial RWP1 cells were transiently cotransfected with the different Tyr kinases and wild-type plakoglobin (labeled by a poly-His tag). Plakoglobin complexes were purified by Ni2+-agarose, and its association with the different components of adherens junction and desmosome complexes was examined. Overexpression of Src promoted a significant change in the interactions established by plakoglobin; as predicted, higher levels of desmoplakin and lower levels of E-cadherin and α-catenin were purified with this protein (Fig. ). On the other hand, transfection of Fer exerted the opposite effect: strengthening the association between plakoglobin and α-catenin while disrupting the interaction with desmoplakin (Fig. ). Therefore, our results indicate that these Tyr kinases modulate the interaction of plakoglobin with adherens junctions and desmosome components. Thus, as a consequence of Src phosphorylation, plakoglobin would be mainly associated with desmosomes while Fer activation would promote the preferential linking of plakoglobin to adherens junctions.
FIG. 7. Src and Fyn modify the association of plakoglobin to its cellular partners. RWP1 cells were cotransfected with 5 μg of pcDNA3.1His-plakoglobin and pCMV-Src (A) or pcDNA3.1His-Fer (B), with empty vectors as controls. After 48 h, cell extracts were (more ...)
We also analyzed what happens in conditions in which cell-to-cell contacts are disrupted. Transfection of ras oncogenes to epithelial cells have been shown to disrupt intercellular adhesion (22
). In IEC intestinal cells, K-ras transfection stimulates the activity of Fer, Fyn, Src, and EGFR tyrosine kinases and induced the phosphorylation of β-catenin in residues Tyr142 and Tyr654, which was correlated with a downregulated binding to α-catenin and E-cadherin (35
). Accordingly, a change in the subcellular distribution of β-catenin was detected after K-ras transfection. Differently than control IEC cells, in which β-catenin was restricted to the cell periphery, IEC K-ras cells presented β-catenin reactivity more dislocated from these regions (Fig. ). Similar results have been obtained by other groups (22
). On the contrary, in IEC cells, plakoglobin showed a more diffuse localization; the protein was observed in the intercellular contacts but also in the cytosol. In IEC K-ras cells, the distribution of this protein seemed to be more localized to the loose contacts established by these cells (Fig. ). Plakoglobin also showed an increased tyrosine phosphorylation after K-ras transfection (Fig. ). Residues Tyr549 and Tyr643 are modified in IEC K-ras cells, since mutation of both amino acids to Phe reduced the phosphorylation of this protein (Fig. ). Probably as a consequence of Tyr549 phosphorylation, a higher association of plakoglobin with α-catenin was observed in coimmunoprecipitation experiments, either immunoprecipitation with antiplakoglobin and blotting with anti-α-catenin MAbs or vice versa (Fig. and C). Accordingly, a Tyr549→Phe plakoglobin mutant that cannot be phosphorylated in this residue showed a lower association with α-catenin in IEC K-ras cells than wild-type plakoglobin. These results indicate that plakoglobin Tyr549 phosphorylation and increased binding to α-catenin occur even under conditions with low levels of cell-to-cell interactions.
FIG. 8. Localization of β-catenin and plakoglobin in IEC and IEC K-ras cells. Cells were grown on glass coverslips as indicated in Materials and Methods and fixed before they reached confluence. The distribution of β-catenin and plakoglobin was (more ...)
FIG. 9. K-ras transfection of IEC epithelial cells induces plakoglobin phosphorylation and upregulates binding to α-catenin. (A and C) Three hundred micrograms of whole-cell extracts from control and K-ras IEC18 cells were immunoprecipitated (IP) with (more ...)
In addition to its role in the formation of adherens junctions and desmosomes, plakoglobin has also been involved in the regulation of gene transcription (see the introduction). As observed in Fig. , plakoglobin overexpression stimulated the transcriptional activity of β-catenin-Tcf-4 complexes, determined by using the TOP plasmid (24
). This effect may be explained by the competition between β-catenin and plakoglobin for binding to several cofactors that has been detected in several systems (45
). We checked whether plakoglobin mutants in the phosphorylation sites with altered interactions with E-cadherin and α-catenin behaved differently than the wild-type form on Tcf-4-mediated transcription. IEC K-ras cells were transiently transfected with a Tyr549→Phe mutant which shows decreased binding to α-catenin with respect to the control (Fig. ). As shown in Fig. , the stimulatory activity on the TOP activity of this mutant was significantly lower than that of the control. Similar assays were performed with MDCK and RWP-1 cells which show well-formed contacts. Whereas wild-type plakoglobin stimulated TOP activity, plakoglobin mutants with lower affinities for α-catenin (Tyr133→Glu) or E-cadherin and α-catenin (Tyr643→Glu) could not stimulate (and even inhibited) Tcf-4-β-catenin transcriptional activity (Fig. ). Binding assays rendered compatible results. Transfection of wild-type plakoglobin to MDCK cells stimulated the amount of β-catenin bound to Tcf-4; on the contrary, the Tyr643→Glu mutant did not modify this association (Fig. ). These results indicate that the enhanced transcriptional activity induced by plakoglobin was dependent on its phosphorylation status and suggest that displacement of β-catenin from its interaction with other components of adherens junctions is relevant for the plakoglobin transcriptional activity.
FIG. 10. Tyrosine phosphorylation of plakoglobin affects β-catenin-dependent transcription. (A) The indicated cells were cotransfected with plakoglobin plasmid mutants inserted into pcDNA3.1His (150 ng), TOP-FLASH (20 ng), and pTK-Renilla (20 ng) luciferase (more ...)