Alteration of cadherin-mediated cell-cell adhesion is frequently associated to tyrosine phosphorylation of p120-and ␤-catenins. We have examined the role of this modification in these proteins in the control of ␤-catenin/E-cadherin binding using in vitro assays with recombinant proteins. Recombinant pp60 c-src efficiently phosphorylated both catenins in vitro, with stoichiometries of 1.5 and 2.0 mol of phosphate/mol of protein for ␤-catenin and p120-catenin, respectively. pp60 c-src phosphorylation had opposing effects on the affinities of ␤-catenin and p120 for the cytosolic domain of E-cadherin; it decreased (in the case of ␤-catenin) or increased (for p120) catenin/E-cadherin binding. However, a role for p120-catenin in the modulation of ␤-catenin/E-cadherin binding was not observed, since addition of phosphorylated p120-catenin did not modify the affinity of phosphorylated (or unphosphorylated) ␤-catenin for E-cadherin. The phosphorylated Tyr residues were identified as Tyr-86 and Tyr-654. Experiments using point mutants in these two residues indicated that, although Tyr-86 was a better substrate for pp60 c-src , only modification of Tyr-654 was relevant for the interaction with E-cadherin. Transient transfections of different mutants demonstrated that Tyr-654 is phosphorylated in conditions in which adherens junctions are disrupted and evidenced that binding of ␤-catenin to E-cadherin in vivo is controlled by phosphorylation of ␤-catenin Tyr-654.

␤-Catenin has a key role in the formation of adherens junction through its interactions with E-cadherin and ␣-catenin. We show here that interaction of ␤-catenin with ␣-catenin is regulated by the phosphorylation of ␤-catenin Tyr-142. This residue can be phosphorylated in vitro by Fer or Fyn tyrosine kinases. Transfection of these kinases to epithelial cells disrupted the association between both catenins. We have also examined whether these kinases are involved in the regulation of this interaction by K-ras. Stable transfectants of the K-ras oncogene in intestinal epithelial IEC18 cells were generated which show little ␣-catenin-␤-catenin association with respect to control clones; this effect is accompanied by increased Tyr-142 phosphorylation and activation of Fer and Fyn kinases. As reported for Fer, Fyn kinase is constitutively bound to p120 catenin; expression of K-ras induces the phosphorylation of p120 catenin on tyrosine residues increasing its affinity for E-cadherin and, consequently, promotes the association of Fyn with the adherens junction complex. Yes tyrosine kinase also binds to p120 catenin but only upon activation, and stimulates Fer and Fyn tyrosine kinases. These results indicate that p120 catenin acts as a docking protein facilitating the activation of Fer/Fyn tyrosine kinases by Yes and demonstrate the role of these p120 catenin-associated kinases in the regulation of ␤-catenin-␣-catenin interaction.

␤-Catenin plays a dual role as a key effector in the regulation of adherens junctions and as a transcriptional coactivator. Phosphorylation of Tyr-654, a residue placed in the last armadillo repeat of ␤-catenin, decreases its binding to E-cadherin. We show here that phosphorylation of Tyr-654 also stimulates the association of ␤-catenin to the basal transcription factor TATAbinding protein. The structural bases of these different affinities were investigated. Our results indicate that the ␤-catenin C-terminal tail interacts with the armadillo repeat domain, hindering the association of the armadillo region to the TATA-binding protein or to Ecadherin. Phosphorylation of ␤-catenin Tyr-654 decreases armadillo-C-terminal tail association, uncovering the last armadillo repeats. In a C-terminal-depleted ␤-catenin, the presence of a negative charge at Tyr-654 does not affect the interaction of the TATA-binding protein to the armadillo domain. However, in the case of E-cadherin, the establishment of ion pairs dominates its association with ␤-catenin, and its binding is greatly dependent on the absence of a negative charge at Tyr-654. Thus, phosphorylation of Tyr-654 blocks the Ecadherin-␤-catenin interaction, even though the steric hindrance of the C-tail is no longer present. These results explain how phosphorylation of ␤-catenin in Tyr-654 modifies the tertiary structure of this protein and the interaction with its different partners.␤-Catenin was initially described as a protein involved in the regulation of E-cadherin function, since it binds to the cytoplasmic domain of this protein and is necessary for linkage of E-cadherin to the actin cytoskeleton (1). Sequences involved in E-cadherin and ␣-catenin binding have been identified in ␤-catenin; association of E-cadherin requires armadillo repeats 4 -12 situated in the central part of ␤-catenin (2). On the other hand, ␣-catenin binding is limited to a short 31-amino acid sequence in the first armadillo repeat of ␤-catenin (3). It has been proposed that the interactions of ␤-catenin with these two proteins are regulated by tyrosine phosphorylation (4, 5). In the case of E-cadherin, we have recently demonstrated that phosphorylation of tyrosine residue 654 diminishes the association of ␤-catenin to this protein by a factor of 10 (6). This residue is modified in vivo by effectors that concomitantly decrease ␤-catenin-E-cadherin binding (6). On the other hand, there is no direct evidence so far that modification of any Tyr residue on ␤-catenin inhibits its interaction with ␣-catenin.In addition to its structural role in cellular junctions, ␤-catenin is a critical component of the wnt-signaling pathway that governs cell fate in early embryogenesis (7,8). Activation of this pathway induces the stabilization of free ␤-catenin, its translocation to the nucleus, and its binding to members of the LEF-1/TCF family of transcription factors (7,8). Interaction of ␤-catenin with these factors converts them to transcriptional activators (9) and stimulates the expression of several g...

Plakoglobin is a protein closely related to ␤-catenin that links desmosomal cadherins to intermediate filaments. Plakoglobin can also substitute for ␤-catenin in adherens junctions, providing a connection between E-cadherin and ␣-catenin. Association of ␤-catenin with E-cadherin and ␣-catenin is regulated by phosphorylation of specific tyrosine residues; modification of ␤-catenin Tyr654 and Tyr142 decreases binding to E-cadherin and ␣-catenin, respectively. We show here that plakoglobin can also be phosphorylated on tyrosine residues, but unlike ␤-catenin, this modification is not always associated with disrupted association with junctional components. Protein tyrosine kinases present distinct specificities on ␤-catenin and plakoglobin, and phosphorylation of ␤-catenin-equivalent Tyr residues of plakoglobin affects its interaction with components of desmosomes or adherens junctions differently. For instance, Src, which mainly phosphorylates Tyr86 in ␤-catenin, modifies Tyr643 in plakoglobin, decreasing the interaction with E-cadherin and ␣-catenin and increasing the interaction with the ␣-catenin-equivalent protein in desmosomes, desmoplakin. The tyrosine kinase Fer, which modifies ␤-catenin Tyr142, lessening its association with ␣-catenin, phosphorylates plakoglobin Tyr549 and exerts the contrary effect: it raises the binding of plakoglobin to ␣-catenin. These results suggest that tyrosine kinases like Src or Fer modulate desmosomes and adherens junctions differently. Our results also indicate that phosphorylation of Tyr549 and the increased binding of plakoglobin to components of adherens junctions can contribute to the upregulation of the transcriptional activity of the ␤-catenin-Tcf-4 complex observed in many epithelial tumor cells.␤-Catenin and plakoglobin (also known as ␥-catenin) are two closely related proteins essential for the establishment and maintenance of cell-cell contacts among epithelial cells. In adherens junctions, ␤-catenin links the cytosolic domain of the transmembrane protein E-cadherin to ␣-catenin, which in turn directly or indirectly associates with the actin cytoskeleton (2, 33, 38). Plakoglobin can substitute for ␤-catenin in adherens junctions. In addition, plakoglobin is a component of the desmosomes, where it mediates the association of desmosomal cadherins, desmocollin and desmoglein, to desmoplakin and the intermediate filament cytoskeleton (15,21). This role of plakoglobin is specific and cannot be exerted by ␤-catenin, even though both proteins are structurally similar. Besides this role, ␤-catenin also has a signaling activity as a member of the Wnt pathway. When released from E-cadherin and ␣-catenin, ␤-catenin can migrate to the nucleus where, through its interaction with the Tcf family of transcription factors, it can activate the transcription of a rapidly increasing number of genes involved in embryonic development and tumorigenesis (5, 37).Probably as a consequence of the importance of this pathway, the translocation of ␤-catenin to the nucleus is tightly controlled thro...