Susana Miravet

Regulation of E-cadherin/Catenin association by tyrosine phosphorylation

Alteration of cadherin-mediated cell-cell adhesion is frequently associated to tyrosine phosphorylation of p120- and β-catenins. We have examined the role of this modification in these proteins in the control of β-catenin/E-cadherin binding usingin vitro assays with recombinant proteins. Recombinant pp60c-src efficiently phosphorylated both catenins in vitro, with stoichiometries of 1.5 and 2.0 mol of phosphate/mol of protein for β-catenin and p120-catenin, respectively. pp60c-src phosphorylation had opposing effects on the affinities of β-catenin and p120 for the cytosolic domain of E-cadherin; it decreased (in the case of β-catenin) or increased (for p120) catenin/E-cadherin binding. However, a role for p120-catenin in the modulation of β-catenin/E-cadherin binding was not observed, since addition of phosphorylated p120-catenin did not modify the affinity of phosphorylated (or unphosphorylated) β-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 pp60c-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 β-catenin to E-cadherin in vivo is controlled by phosphorylation of β-catenin Tyr-654.

p120 Catenin-Associated Fer and Fyn Tyrosine Kinases Regulate β-Catenin Tyr-142 Phosphorylation and β-Catenin-α-Catenin Interaction

ABSTRACT β-Catenin has a key role in the formation of adherens junction through its interactions with E-cadherin and α-catenin. We show here that interaction of β-catenin with α-catenin is regulated by the phosphorylation of β-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 α-catenin-β-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 β-catenin-α-catenin interaction.

Regulation of β-catenin structure and activity by tyrosine phosphorylation.

β-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 β-catenin, decreases its binding to E-cadherin. We show here that phosphorylation of Tyr-654 also stimulates the association of β-catenin to the basal transcription factor TATA-binding protein. The structural bases of these different affinities were investigated. Our results indicate that the β-catenin C-terminal tail interacts with the armadillo repeat domain, hindering the association of the armadillo region to the TATA-binding protein or to E-cadherin. Phosphorylation of β-catenin Tyr-654 decreases armadillo-C-terminal tail association, uncovering the last armadillo repeats. In a C-terminal-depleted β-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 β-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-β-catenin interaction, even though the steric hindrance of the C-tail is no longer present. These results explain how phosphorylation of β-catenin in Tyr-654 modifies the tertiary structure of this protein and the interaction with its different partners.

Tyrosine Phosphorylation of Plakoglobin Causes Contrary Effects on Its Association with Desmosomes and Adherens Junction Components and Modulates β-Catenin-Mediated Transcription

ABSTRACT Plakoglobin is a protein closely related to β-catenin that links desmosomal cadherins to intermediate filaments. Plakoglobin can also substitute for β-catenin in adherens junctions, providing a connection between E-cadherin and α-catenin. Association of β-catenin with E-cadherin and α-catenin is regulated by phosphorylation of specific tyrosine residues; modification of β-catenin Tyr654 and Tyr142 decreases binding to E-cadherin and α-catenin, respectively. We show here that plakoglobin can also be phosphorylated on tyrosine residues, but unlike β-catenin, this modification is not always associated with disrupted association with junctional components. Protein tyrosine kinases present distinct specificities on β-catenin and plakoglobin, and phosphorylation of β-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 β-catenin, modifies Tyr643 in plakoglobin, decreasing the interaction with E-cadherin and α-catenin and increasing the interaction with the α-catenin-equivalent protein in desmosomes, desmoplakin. The tyrosine kinase Fer, which modifies β-catenin Tyr142, lessening its association with α-catenin, phosphorylates plakoglobin Tyr549 and exerts the contrary effect: it raises the binding of plakoglobin to α-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 β-catenin-Tcf-4 complex observed in many epithelial tumor cells.

β-Catenin N- and C-terminal tails modulate the coordinated binding of adherens junction proteins to β-catenin

β-Catenin plays a central role in the establishment and regulation of adherens junctions because it interacts with E-cadherin and, through α-catenin, with the actin cytoskeleton. β-Catenin is composed of three domains: a central armadillo repeat domain and two N- and C-terminal tails. The C-tail interacts with the armadillo domain and limits its ability to bind E-cadherin and other cofactors. The two β-catenin tails are mutually inter-regulated because the C-tail is also necessary for binding of the N-tail to the armadillo domain. Moreover, the N-tail restricts the interaction of the C-tail with the central domain. Depletion of either of the two tails has consequences for the binding of factors at the other end: deletion of the C-tail increases α-catenin binding, whereas deletion of the N-tail blocks E-cadherin interaction to the armadillo repeats. As an effect of the interconnection of the tails, the association of α-catenin and E-cadherin to β-catenin is interdependent. Thus, binding of α-catenin to the N-tail, through conformational changes that affect the C-tail, facilitates the association of E-cadherin. These results indicate that different cofactors of β-catenin bind coordinately to this protein and indicate how the two terminal ends of β-catenin exquisitely modulate intermolecular binding within junctional complexes.

β-Catenin and Plakoglobin N- and C-tails Determine Ligand Specificity

β-Catenin and plakoglobin are related proteins involved in the regulation of adherens junctions and desmosomes. Moreover, by binding to Tcf-4, they can act as transcriptional modulators of genes involved in embryonic development and tumorigenesis. However, they associate to distinct Tcf-4 subdomains causing opposing effects on Tcf-4 binding to DNA: whereas β-catenin does not affect this binding, plakoglobin prevents it. Both proteins are composed by two N- and C-tails and a central armadillo repeat domain. Interaction of Tcf-4, as well as other desmosomal or adherens junction components, with β-catenin or plakoglobin takes place through the central armadillo domain. Here we show that, as reported for β-catenin, plakoglobin terminal tails also interact with the central domain and regulate the ability of this region to bind to different cofactors. Moreover the specificity of the interaction of β-catenin and plakoglobin with different subdomains in Tcf-4 or with other junctional components resides within the terminal tails and not in the armadillo domain. For instance, a chimeric protein in which the central domain of β-catenin was replaced by that of plakoglobin presented the same specificity as wild-type β-catenin. Therefore, the terminal tails of these proteins are responsible for discerning among binding of factors to the armadillo domain. These results contribute to the understanding of the molecular basis of the interactions established by these key regulators of epithelial tumorigenesis.

Canine adenovirus downstream processing protocol.

Adenovirus vectors are efficient gene delivery tools. A major caveat with vectors derived from common human adenovirus serotypes is that most adults are likely to have been exposed to the wild-type virus and exhibit active immunity against the vectors. This preexisting immunity limits their clinical success. Strategies to circumvent this problem include the use of nonhuman adenovirus vectors. Vectors derived from canine adenovirus type 2 (CAV-2) are among the best-studied representatives. CAV-2 vectors are particularly attractive for the treatment of neurodegenerative disorders. In addition, CAV-2 vectors have shown great promise as oncolytic agents in virotherapy approaches and as vectors for recombinant vaccines. The rising interest in CAV-2 vectors calls for the development of scalable GMP compliant production and purification strategies. A detailed protocol describing a complete scalable downstream processing strategy for CAV-2 vectors is reported here. Clarification of CAV-2 particles is achieved by microfiltration. CAV-2 particles are subsequently concentrated and partially purified by ultrafiltration-diafiltration. A Benzonase(®) digestion step is carried out between ultrafiltration and diafiltration operations to eliminate contaminating nucleic acids. Chromatography purification is accomplished in two consecutive steps. CAV-2 particles are first captured and concentrated on a propyl hydrophobic interaction chromatography column followed by a polishing step using DEAE anion exchange monoliths. Using this protocol, high-quality CAV-2 vector preparations containing low levels of contamination with empty viral capsids and other inactive vector forms are typically obtained. The complete process yield was estimated to be 38-45 %.

Construction, Production, and Purification of Recombinant Adenovirus Vectors

Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. In this chapter, a standard procedure for their generation and small-scale production is described. Homologous recombination in E. coli between shuttle plasmids and full-length adenovirus backbones (E1-deleted) is used for the generation of recombinant adenoviral vectors genomes. The adenovirus genomes are then analyzed to confirm their identity and integrity, and further linearized and transfected to generate a recombinant adenoviral vector in permissive human cells. These vectors are then purified by two sequential CsCl gradient centrifugations and subjected to a chromatography step in order to eliminate the CsCl and exchange buffers. Finally, the viral stock is characterized through the quantification of its viral particle content and its infectivity.