Similitudes y diferencias en la estructura y función de [Beta]-catenina y plakoglobina

Consultable des del TDXTítol obtingut de la portada digitalitzadaLa b-catenina y plakoglobina (también llamada g-catenina) son dos proteínas homólogas esenciales para la formación y el mantenimiento de los contactos célula-célula entre células epiteliales. En las uniones adherentes, b-catenina y plakoglobina interaccionan de forma independiente con el dominio citoplasmático de los receptores de adhesión celular de la familia de las cadherinas, uniéndolos al citoesqueleto de actina mediante la asociación con a-catenina. Plakoglobina también es un componente de la capa submembranal de los desmosomas. Además de su papel estructural en las uniones adherentes, b-catenina interviene en señalización celular actuando como miembro de la vía Wnt. Está menos claro si su homóloga plakoglobina también presenta esta papel en señalización. El objetivo principal de este trabajo ha consistido en caracterizar las analogías y diferencias de b-catenina y plakoglobina en su interacción con el factor de transcripción Tcf-4 y con componentes de las uniones adherentes y los desmosomas. Hemos determinado que Tcf-4 es fosforilado in vitro por la proteína quinasa CK2 en los amino ácidos Ser-58-Ser-59-Ser-60. Hemos comprobado que la fosforilación de estos residuos no modifica la interacción del Tcf-4 con b-catenina pero si reduce su asociación con plakoglobina. Se han comparado los sitios de unión de estas dos proteínas con el Tcf-4; mientras que b-catenina requiere los primeros 50 amino ácidos del Tcf-4, plakoglobina interacciona principalmente con los residuos 51-80. Se han detectado in vitro complejos ternarios formados por b-catenina/Tcf-4/plakoglobina, indicando que es posible la unión simultánea de las dos proteínas armadillo al Tcf-4. Se han llevado a cabo experimentos utilizando una forma mutada del Tcf-4 cuya interacción con plakoglobina se encuentra disminuída y se ha comprobado que la unión de plakoglobina afecta negativamente a la actividad transcripcional del Tcf-4. Estos resultados indican que el Tcf-4 contiene dos sitios de unión diferentes para b-catenina y plakoglobina y que la interacción de esta última regula la actividad transcripcional del complejo. Había evidencias de que la fosforilación en residuos tirosina de los componentes de los complejos de adhesión regulaba la estabilidad de estos complejos. Hemos caracterizado que la fosforilación de b-catenina en los residuos Tyr-142 y Tyr-654 disminuye la interacción de esta proteína con a-catenina y E-cadherina, respectivamente. La identificación de las tirosina quinasas que catalizan estas modificaciones indica que la Tyr-142 de b-catenina es un buen sustrato de las quinasas Fer y Fyn, mientras que la Tyr-654 es modificada por el receptor de EGF o proteínas relacionadas. Una regulación similar se ha propuesto para plakoglobina en los desmosomas y uniones adherentes. Nuestros resultados indican que, aunque b-catenina y plakoglobina son dos proteínas homólogas, las mismas tirosina quinasas fosforilan diferentes residuos en las dos proteínas. Además, la fosforilación de residuos equivalentes causa efectos totalmente diferentes en la interacción de plakoglobina y b-catenina con sus cofactores celulares. Por ejemplo, Src que fosforila principalmente la Tyr-86 en b-catenina, modifica la Tyr-643 en plakoglobina disminuyendo su interacción con E-cadherina y a-catenina y aumentando su asociación con la proteína equivalente a a-catenina en los desmosomas, la desmoplakina. Por otro lado, la tirosina quinasa Fer, que modifica la Tyr-142 de b-catenina disminuyendo su asociación con a-catenina, fosforila la Tyr-549 de plakoglobina y ejerce el efecto contrario: aumenta la unión plakoglobina-a-catenina. Estos resultados sugieren que tirosina quinasas como Src o Fer modulan de forma diferente los desmosomas y las uniones adherentes y promueven el paso de plakoglobina de un tipo de complejo de adhesión a otro. Nuestros resultados también sugieren que la interacción de plakoglobina con los miembros de las uniones adherentes previene la translocación de plakoglobina al núcleo y su inhibición de la actividad del Tcf-4.The b-catenin and plakoglobin (also known as g-catenin) are two closely related proteins essential for the establishment and maintenance of cell-cell contacts among epithelial cells. In adherens junctions, b-catenin and plakoglobin independently bind to the cytoplasmic domain of cell-cell adhesion receptors of the cadherin family, linking them to the actin cytoskeleton by an association with a-catenin. Plakoglobin is also a component of the submembranal plaque of desmosomes. In addition to its structural role in adherens junctions, b-catenin has a signaling activity as a member of the Wnt pathway. It is still unclear whether its close homologue, plakoglobin, also has a signaling role. In this work we have studied the similarities and differences on the interaction of b-catenin and plakoglobin with the transcription factor Tcf-4 and members of the adherens junctions and desmosomes. We show that Tcf-4 can be phosphorylated in vitro by protein kinase CK2 in amino acids Ser-58-Ser-59-Ser-60. Phosphorylation of these residues does not modify the interaction of Tcf-4 with b-catenin but reduces its association to plakoglobin. The binding sites of Tcf-4 for these two proteins were compared; whereas b-catenin requires the N-terminal first 50 amino acids, plakoglobin interacts mainly with residues 51-80. Ternary complexes composed by b-catenin/Tcf-4/plakoglobin could be detected in vitro, demonstrating that simultaneous binding of the two armadillo proteins to Tcf-4 is posible. Experiments performed using a Tcf-4 mutant with decreased interaction to plakoglobin demonstrated that binding to this protein negatively affected the transcriptional activity of Tcf-4. These results indicates that Tcf-4 contains two different sites for binding b-catenin and plakoglobin, and the interaction of the latter hinders the transcriptional activity of the complex. Phosphorylation in tyrosine residues of components of adhesion complexes regulates the stability of these complexes. We have described that phosphorylation of b-catenin residues Tyr-654 and Tyr-142 specifically decreases the interaction of this protein with E-cadherin and a-catenin, respectively. The identification of the Tyr kinases that catalyze these modifications indicates that b-catenin Tyr-142 is a good substrate of Fer and Fyn kinases, whereas Tyr-654 is modified by EGF receptor or related proteins. A similar modulation has been proposed for plakoglobin action on desmosomes and adherens junctions. Our results indicate that, although b-catenin and plakoglobin are two closely related proteins, the same protein kinase phosphorylate different residues in the two proteins. Moreover phosphorylation of equivalent residues cause totally different effects on the interaction of plakoglobin and b-catenin with their cellular partners. For instance, Src that phosphorylates mainly Tyr-86 in b-catenin, modifies Tyr-643 in plakoglobin decreasing the interaction with E-cadherin and a-catenin and increasing the interaction with the a-catenin-equivalent protein in desmosomes, desmoplakin. On the other hand, the tyrosine kinase Fer, that modifies b-catenin Tyr-142 lessening its association to a-catenin, phosphorylates plakoglobin Tyr-549 and exerts the contrary effect: augmentates plakoglobin-a-catenin binding. These results suggest that tyrosine kinases as Src or Fer modulate desmosomes and adherens junctions differently and promote the switch of plakoglobin from one type of adhesion complex to the other. Our results also suggest that interaction of plakoglobin to members of the adherens junctions prevents the translocation of plakoglobin to the nucleus and its inhibition of Tcf-4 action

Interplay of Molecular Hydrogelators and SDS Affords Responsive Soft Matter Systems with Tunable Properties

The gelation efficiency of low molecular weight bolaamphiphilic hydrogelators 1 and 2 is influenced by the presence of SDS micelles. Similarly, the critical micellar concentration value of SDS is reduced in the presence of the studied molecular hydrogelators. Rheological measurements indicate that the strength of the hydrogels can be modulated with SDS, the gels becoming weaker in the presence of micelles This behavior has been rationalized with the help of NMR studies using diffusion measurements and NOE correlations. The results obtained clearly point to the formation of mixed micelles composed of SDS and the hydrogelators. In the case of 1, the gelator:SDS ratio in the mixed micelles has been estimated from solubility studies to be ca. 1:2.5. Electron microscopy reveals that when SDS is present, the morphology of the xerogels is modified in its appearance at the micrometer scale but fibers with diameter in the nanometer range are observe in all the cases. The interplay between the surfactant and the gelators provides with new possibilities for the modulation of both gel and micelle formation. Examples are shown to highlight the potential usefulness of this type of interconnected system. In one case the release of a gel entrapped dye is modulated by the presence of SDS and sodium chloride. In another example, an intricate system that responds to a temperature excursion by irreversible micelle disassembly is shown.We thank Spanish Ministry of Science and Innovation (Grants CTQ2009-13961 and CTQ2012-37735) and Universitat Jaume I (Grant P1.1B2012-25) for financial support

Freezing Capture of Polymorphic Aggregates of Bolaamphiphilic L-Valine-Based Molecular Hydrogelators

Nanostructured xerogels have been prepared by the freeze-drying of hydrogels and aggregates formed by bolaamphiphilic l-valine derivatives after aging under different environmental conditions. A wide variety of shapes and sizes has been achieved by a simple methodology. These nanostructures have been studied by SEM and WAXD and a dramatic influence of structural flexibility on the kinetics of aggregation has been observed. Such flexibility and a modulation of the hydrophobic effect have shown a profound influence in the packing of these compounds and revealed a high degree of polymorphism

Molecular Hydrogels from Bolaform Amino Acid Derivatives: A Structure–Properties Study Based on the Thermodynamics of Gel Solubilization

Insight is provided into the aggregation thermodynamics associated to hydrogel formation by molecular gelators derived from L-valine and L-isoleucine. Solubility data from NMR measurements are used to extract thermodynamic parameters for the aggregation in water. It is concluded that at room temperature and up to 55 °C, these systems form self-assembled fibrillar networks in water with quite low or zero enthalpic component, whereas the entropy of the aggregation is favorable. These results are explained by considering that the hydrophobic effect is dominant in the self-assembly. However, studies by NMR and IR spectroscopy reveal that intermolecular hydrogen bonding is also a key issue in the aggregation process of these molecules in water. The low enthalpy values measured for the self-assembly process are ascribed to the result of a compensation of the favorable intermolecular hydrogen-bond formation and the unfavorable enthalpy component of the hydrophobic effect. Additionally, it is shown that by using the hydrophobic character as a design parameter, enthalpy-controlled hydrogel formation, as opposed to entropy-controlled hydrogel formation, can be achieved in water if the gelator is polar enough. It is noteworthy that these two types of hydrogels, enthalpy-versus entropy-driven hydrogels, present quite different response to temperature changes in properties such as the minimum gelator concentration (mgc) or the rheological moduli. Finally, the presence of a polymorphic transition in a hydrogel upon heating above 70 °C is reported and ascribed to the weakening of the hydrophobic effect upon heating. The new soft polymorphic materials present dramatically different solubility and rheological properties. Altogether these results are aimed to contribute to the rational design of molecular hydrogelators, which could be used for the tailored preparation of this type of soft materials. The reported results could also provide ground for the rationale of different self-assembly processes in aqueous media

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

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

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

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