The organization of adherens junctions and desmosomes at the cardiac intercalated disc is independent of gap junctions.
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Adherens junctions and desmosomes are responsible for mechanically coupling myocytes in the heart and are found closely apposed to gap junction plaques at the intercalated discs of cardiomyocytes. It is not known whether loss of cardiac gap junctions, such as described in cardiac disease states, may influence the expression patterns of other intercalated disc-associated proteins. We investigated whether the major cardiac gap junction protein connexin43 (Cx43) may be responsible for regulating adherens junctions, desmosomes and their associated catenins, in terms of abundance and localization at the intercalated discs of cardiomyocytes. In order to study the effect of loss of cardiac gap junctions on the intercalated disc-associated proteins, we used a combination of immunoblotting, immunofluorescence with confocal microscopy and electron microscopy to evaluate heart tissue from mice with cardiac-specific conditional knockout of Cx43. We found that the cardiac adherens junctions, desmosomes and their associated catenins, as well as vinculin and ZO-1, maintain their normal abundance, structural appearance and localization in the absence of Cx43. We conclude from these data that Cx43 is not required for the organization of the cell adhesion junctions and their associated catenins at the intercalated disc in the adult cardiac myocyte. (+info)
VE-cadherin-induced Cdc42 signaling regulates formation of membrane protrusions in endothelial cells.
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The cytoplasmic domain of cadherins and the associated catenins link the cytoskeleton with signal transduction pathways. To study the signaling function of non-junctional VE-cadherin, which can form during the loss VE-cadherin homotypic adhesion, wild type VE-cadherin or VE-cadherin cytoplasmic domain (DeltaEXD) was expressed in sub-confluent endothelial cells. We observed that Cdc42 was activated in transfected cells and that these cells also developed Cdc42-dependent >70-microm-long plasma membrane protrusions. The formation of these structures required actin polymerization, and they developed specifically in endothelial cells as compared with epithelial cells. Expression of the VE-cadherin cytoplasmic domain lacking the beta-catenin binding site also induced Cdc42 activation; thus, its activation cannot be ascribed to beta-catenin binding. However, these cells were not able to form the protrusions. These results suggest that the cytoplasmic domain of non-junctional VE-cadherin can serve as a scaffold involved in Cdc42 activation at the endothelial plasma membrane. beta-Catenin and the associated alpha-catenin may serve as support sites for actin polymerization, leading to formation of long plasma membrane protrusions. Thus, non-junctional VE-cadherin actively participates in inside-out signaling at the plasma membrane, leading to the development of endothelial membrane protrusions. (+info)
N-cadherin-catenin complexes form prior to cleavage of the proregion and transport to the plasma membrane.
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Cadherins are calcium-dependent glycoproteins that function as cell-cell adhesion molecules and are linked to the actin cytoskeleton via catenins. Newly synthesized cadherins contain a prosequence that must be proteolytically removed to generate a functional adhesion molecule. The goal of this study was to examine the proteolytic processing of N-cadherin and the assembly of the cadherin-catenin complex in cells that express endogenous N-cadherin. A monoclonal antibody specific for the proregion of human N-cadherin was generated and used to examine N-cadherin processing. Our data show that newly synthesized proN-cadherin is phosphorylated and proteolytically processed prior to transport to the plasma membrane. In addition, we show that beta-catenin and plakoglobin associate only with phosphorylated proN-cadherin, whereas p120(ctn) can associate with both phosphorylated and non-phosphorylated proN-cadherin. Immunoprecipitations using anti-proN-cadherin showed that cadherin-catenin complexes are assembled prior to localization at the plasma membrane. These data suggest that a core N-cadherin-catenin complex assembles in the endoplasmic reticulum or Golgi compartment and is transported to the plasma membrane where linkage to the actin cytoskeleton can be established. (+info)
p120 Catenin-associated Fer and Fyn tyrosine kinases regulate beta-catenin Tyr-142 phosphorylation and beta-catenin-alpha-catenin Interaction.
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beta-Catenin has a key role in the formation of adherens junction through its interactions with E-cadherin and alpha-catenin. We show here that interaction of beta-catenin with alpha-catenin is regulated by the phosphorylation of beta-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 alpha-catenin-beta-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 beta-catenin-alpha-catenin interaction. (+info)
Minimal mutation of the cytoplasmic tail inhibits the ability of E-cadherin to activate Rac but not phosphatidylinositol 3-kinase: direct evidence of a role for cadherin-activated Rac signaling in adhesion and contact formation.
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Classic cadherins are adhesion-activated cell signaling receptors. In particular, homophilic cadherin ligation can directly activate Rho family GTPases and phosphatidylinositol 3-kinase (PI3-kinase), signaling molecules with the capacity to support the morphogenetic effects of these adhesion molecules during development and disease. However, the molecular basis for cadherin signaling has not been elucidated, nor is its precise contribution to cadherin function yet understood. One attractive hypothesis is that cadherin-activated signaling participates in stabilizing adhesive contacts (Yap, A. S., and Kovacs, E. M. (2003) J. Cell Biol. 160, 11-16). We now report that minimal mutation of the cadherin cytoplasmic tail to uncouple binding of p120-ctn ablated the ability of E-cadherin to activate Rac. This was accompanied by profound defects in the capacity of cells to establish stable adhesive contacts, defects that were rescued by sustained Rac signaling. These data provide direct evidence for a role of cadherin-activated Rac signaling in contact formation and adhesive stabilization. In contrast, cadherin-activated PI3-kinase signaling was not affected by loss of p120-ctn binding. The molecular requirements for E-cadherin to activate Rac signaling thus appear distinct from those that stimulate PI3-kinase, and we postulate that p120-ctn may play a central role in the E-cadherin-Rac signaling pathway. (+info)
Characterization of cadherin-24, a novel alternatively spliced type II cadherin.
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Cadherins comprise a superfamily of calcium-dependent cell-cell adhesion molecules. Within the superfamily are six subfamilies including type I and type II cadherins. Both type I and type II cadherins are composed of five extracellular repeat domains with conserved calcium-binding motifs, a single pass transmembrane domain, and a highly conserved cytoplasmic domain that interacts with beta-catenin and p120 catenin. In this study, we describe a novel cadherin, cadherin-24. It is a type II cadherin with a 781-codon open reading frame, which encodes a type II cadherin protein complete with five extracellular repeats containing calcium-binding motifs, a transmembrane domain, and a conserved cytoplasmic domain. Cadherin-24 has the unusual feature of being alternatively spliced in extracellular repeat 4. This alternative exon encodes 38 in-frame amino acids, resulting in an 819-amino-acid protein. Sequence analysis suggests the presence of beta-catenin and p120 catenin-binding sequences, and immunoprecipitation experiments confirm the ability of both forms of the novel cadherin to associate with alpha-catenin, beta-catenin, and p120 catenin. In addition, aggregation assays show that both forms of cadherin-24 mediate strong cell-cell adhesion. (+info)
Protein kinase C modifications of VE-cadherin, p120, and beta-catenin contribute to endothelial barrier dysregulation induced by thrombin.
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The adherens junction is a multiprotein complex consisting of the transmembrane vascular endothelial cadherin (VEC) and cytoplasmic catenins (p120, beta-catenin, plakoglobin, alpha-catenin) responsible for the maintenance of endothelial barrier function. Junctional disassembly and modifications in cadherin/catenin complex lead to increased paracellular permeability of the endothelial barrier. However, the mechanisms of junctional disassembly remain unclear. In this study, we used the proinflammatory mediator thrombin to compromise the barrier function and test the hypothesis that phosphorylation-induced alterations of VEC, beta-catenin, and p120 regulate junction disassembly and mediate the increased endothelial permeability response. The study showed that thrombin induced dephosphorylation of VEC, which is coupled to disassembly of cell-cell contacts, but VEC remained in aggregates at the plasma membrane. The cytoplasmic catenins dissociated from the VEC cytoplasmic domain in thin membrane projections formed in interendothelial gaps. We also showed that thrombin induced dephosphorylation of beta-catenin and phosphorylation of p120. Thrombin-induced interendothelial gap formation and increased endothelial permeability were blocked by protein kinase C inhibition using chelerythrine and Go-6976 but not by LY-379196. Chelerythrine also prevented thrombin-induced phosphorylation changes of the cadherin/catenin complex. Thus the present study links posttranslational modifications of VEC, beta-catenin, and p120 to the mechanism of thrombin-induced increase in endothelial permeability. (+info)
p120 Catenin is required for growth factor-dependent cell motility and scattering in epithelial cells.
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Cadherin-mediated cell-cell adhesion is dynamically modulated during epithelial-mesenchymal transition triggered by activation of receptor tyrosine kinases (RTK) in epithelial cells. Several cadherin-binding proteins have been identified that control cell-cell adhesion. However, the mechanisms by which intercellular adhesion and cell motility are coregulated are still unknown. Here, we delineate a hitherto uncharted cooperation between RTKs, RhoA GTPase, and p120 catenin in instructing a motile behavior to epithelial cells. We found that expression of an N-terminus-deleted p120 catenin in a variety of epithelial cell types, including primary keratinocytes, effectively competes for endogenous p120 at cadherin binding sites and abrogates EGF-stimulated cell motility as well as HGF-induced cell scattering. The deleted mutant also inhibits the PI3K-dependent RhoA activation ensuing receptor activation. Conversely, we also show that the ectopic expression of full-length p120 in epithelial cells promotes cytoskeletal changes, stimulates cell motility, and activates RhoA. Both motogenic response to p120 and RhoA activation require coactivation of signaling downstream of RTKs as they are suppressed by ablation of the Ras/PI3K pathway. These studies demonstrate that p120 catenin is a necessary target of RTKs in regulating cell motility and help define a novel pathway leading to RhoA activation, which may contribute to the early steps of metastatic invasion. (+info)