ABSTRACT
A role for Rac1 GTPase in canonical Wnt signaling has recently been demonstrated, showing that it is required for ß-catenin translocation to the nucleus. In this study, we investigated the mechanism of Rac1 stimulation by Wnt. Upregulation of Rac1 activity by Wnt3a temporally correlated with enhanced p120-catenin binding to Rac1 and Vav2. Vav2 and Rac1 association with p120-catenin was modulated by phosphorylation of this protein, which was stimulated upon serine/threonine phosphorylation by CK1 and inhibited by tyrosine phosphorylation by Src or Fyn. Acting on these two post-translational modifications, Wnt3a induced the release of p120-catenin from E-cadherin, enabled the interaction of p120-catenin with Vav2 and Rac1, and facilitated Rac1 activation by Vav2. Given that p120-catenin depletion disrupts gastrulation in Xenopus, we analyzed p120-catenin mutants for their ability to rescue this phenotype. In contrast to the wild-type protein or other controls, p120-catenin point mutants that were deficient in the release from E-cadherin or in Vav2 or Rac1 binding failed to rescue p120-catenin depletion. Collectively, these results indicate that binding of p120-catenin to Vav2 and Rac1 is required for the activation of this GTPase upon Wnt signaling.
Subject(s)
Catenins/metabolism , Proto-Oncogene Proteins c-vav/metabolism , Wnt3A Protein/pharmacology , rac1 GTP-Binding Protein/metabolism , Animals , Cadherins/metabolism , Cell Line , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cytosol/drug effects , Cytosol/metabolism , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/metabolism , Enzyme Activation/drug effects , Gastrulation/drug effects , Humans , Models, Biological , Mutant Proteins/metabolism , Phosphorylation/drug effects , Phosphoserine/metabolism , Phosphotyrosine/metabolism , Protein Binding/drug effects , Protein Transport/drug effects , Signal Transduction/drug effects , Xenopus/embryology , Xenopus/metabolism , beta Catenin/metabolism , Delta CateninABSTRACT
p120-catenin is an E-cadherin-associated protein that modulates E-cadherin function and stability. In response to Wnt3a, p120-catenin is phosphorylated at Ser268 and Ser269, disrupting its interaction with E-cadherin. Here, we describe that Wnt-induced p120-catenin phosphorylation at Ser268 and Ser269 also enhances its binding to the transcriptional factor Kaiso, preventing Kaiso-mediated inhibition of the ß-catenin-Tcf-4 transcriptional complex. Kaiso-mediated repression of this complex is due to its association not only with Tcf-4 but also with ß-catenin. Disruption of Tcf-4-Kaiso and ß-catenin-Kaiso interactions by p120-catenin not only releases Tcf-4 and ß-catenin enabling its mutual association and the formation of the transcriptional complex but also permits Kaiso binding to methylated CpG islands, an interaction that is weakly inhibited by p120-catenin. Consequently, Wnt stimulates Kaiso association to the CDKN2A promoter, which contains CpG sequences, in cells where these sequences are extensively methylated, such as HT-29 M6, an effect accompanied by decreased expression of its gene product. These results indicate that, when released from E-cadherin by Wnt3a-stimulated phosphorylation, p120-catenin controls the activity of the Kaiso transcriptional factor, enhancing its binding to repressed promoters and relieving its inhibition of the ß-catenin-Tcf-4 transcriptional complex.
Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Catenins/metabolism , Transcription Factors/metabolism , Transcriptional Activation , Wnt3A Protein/metabolism , Cadherins/metabolism , Catenins/genetics , CpG Islands , Genes, p16 , Humans , Methylation , Phosphorylation , Promoter Regions, Genetic , Protein Binding , Signal Transduction/genetics , Transcription Factor 4 , beta Catenin/metabolism , Delta CateninABSTRACT
p120-catenin is an E-cadherin-associated protein that modulates E-cadherin function and stability. We describe here that p120-catenin is required for Wnt pathway signaling. p120-catenin binds and is phosphorylated by CK1ε in response to Wnt3a. p120-catenin also associates to the Wnt co-receptor LRP5/6, an interaction mediated by E-cadherin, showing an unexpected physical link between adherens junctions and a Wnt receptor. Depletion of p120-catenin abolishes CK1ε binding to LRP5/6 and prevents CK1ε activation upon Wnt3a stimulation. Elimination of p120-catenin also inhibits early responses to Wnt, such as LRP5/6 and Dvl-2 phosphorylation and axin recruitment to the signalosome, as well as later effects, such as ß-catenin stabilization. Moreover, since CK1ε is also required for E-cadherin phosphorylation, a modification that decreases the affinity for ß-catenin, p120-catenin depletion prevents the increase in ß-catenin transcriptional activity even in the absence of ß-catenin degradation. Therefore, these results demonstrate a novel and crucial function of p120-catenin in Wnt signaling and unveil additional points of regulation by this factor of ß-catenin transcriptional activity different of ß-catenin stability.
Subject(s)
Casein Kinase 1 epsilon/metabolism , Catenins/metabolism , Wnt Proteins/pharmacology , Adaptor Proteins, Signal Transducing/metabolism , Cadherins/genetics , Cadherins/metabolism , Casein Kinase 1 epsilon/genetics , Catenins/genetics , Cell Line, Tumor , Dishevelled Proteins , Humans , Immunoprecipitation , LDL-Receptor Related Proteins/metabolism , Low Density Lipoprotein Receptor-Related Protein-5 , Low Density Lipoprotein Receptor-Related Protein-6 , Mass Spectrometry , Phosphoproteins/metabolism , Phosphorylation/drug effects , Protein Binding/drug effects , Protein Binding/genetics , Signal Transduction/drug effects , Signal Transduction/genetics , Delta CateninABSTRACT
The transcription factor SNAIL1 is a master regulator of epithelial to mesenchymal transition. SNAIL1 is a very unstable protein, and its levels are regulated by the E3 ubiquitin ligase beta-TrCP1 that interacts with SNAIL1 upon its phosphorylation by GSK-3beta. Here we show that SNAIL1 polyubiquitylation and degradation may occur in conditions precluding SNAIL1 phosphorylation by GSK-3beta, suggesting that additional E3 ligases participate in the control of SNAIL1 protein stability. In particular, we demonstrate that the F-box E3 ubiquitin ligase FBXl14 interacts with SNAIL1 and promotes its ubiquitylation and proteasome degradation independently of phosphorylation by GSK-3beta. In vivo, inhibition of FBXl14 using short hairpin RNA stabilizes both ectopically expressed and endogenous SNAIL1. Moreover, the expression of FBXl14 is potently down-regulated during hypoxia, a condition that increases the levels of SNAIL1 protein but not SNAIL1 mRNA. FBXL14 mRNA is decreased in tumors with a high expression of two proteins up-regulated in hypoxia, carbonic anhydrase 9 and TWIST1. In addition, Twist1 small interfering RNA prevents hypoxia-induced Fbxl14 down-regulation and SNAIL1 stabilization in NMuMG cells. Altogether, these results demonstrate the existence of an alternative mechanism controlling SNAIL1 protein levels relevant for the induction of SNAIL1 during hypoxia.
Subject(s)
F-Box Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Binding Sites , Blotting, Western , Cell Hypoxia , Cell Line , Cell Line, Tumor , Down-Regulation , F-Box Proteins/genetics , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Immunoprecipitation , Mice , Mutation , NIH 3T3 Cells , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphorylation , Protein Binding , RNA Interference , Snail Family Transcription Factors , Transcription Factors/genetics , Transfection , Twist-Related Protein 1/genetics , Twist-Related Protein 1/metabolism , Ubiquitin-Protein Ligases/genetics , UbiquitinationABSTRACT
UNLABELLED: Ergometric exercise stress tests (EST) give important information about the cardiovascular (CV) response to increased demands. The expected EST-related changes in variables like blood pressure and heart rate are known, but those in the arterial biomechanics are controversial and incompletely characterized. AIMS: a) to characterize the regional and local arterial biomechanical behavior in response to EST, and its temporal profile in the post-EST recovery phase and (b) to compare different arteries biomechanical response to EST. METHODS: In 16 non-trained healthy young subjects the carotid-femoral pulse wave velocity and the carotid, femoral and brachial arterial distensibility were non-invasively evaluated before (Rest) and after EST. Post exercise recordings were obtained 0-1, 4-5, and 9-10 minutes after exercise. RESULTS: The EST resulted in an early increase in the arterial stiffness, evidenced by regional and local parameters. There were quali-quantitative differences among the arterial local stiffness response to EST, when analyzing conjunctly the different postEST recovery stages. The biomechanical changes could not be explained only by blood pressure variations.
Subject(s)
Arteries/physiology , Muscles/pathology , Adult , Arteries/pathology , Biomechanical Phenomena , Biomedical Engineering/methods , Blood Pressure , Cardiovascular Diseases/diagnosis , Carotid Arteries/pathology , Elasticity , Electrocardiography , Exercise Test , Female , Femoral Artery/pathology , Humans , MaleABSTRACT
UNLABELLED: Current methods used to evaluate the endothelial function have limitations. The analysis of the pulse wave velocity (PWV) response to transient ischaemia could be an alternative to evaluate the endothelial dynamics. AIMS: To analyze (a) the carotid-radial PWV temporal profile during flow mediated dilatation test, and (b) the PWV changes considering its main vascular geometrical (diameter) and intrinsic (elastic modulus) determinants. METHODS: Sixteen healthy young adults were included. The carotid-radial PWV (strain gauge mechano-transducers), wall thickness and brachial diameter (B-Mode ultrasound) were measured before (basal state), during a forearm cuff inflation (5 minutes) and after its deflation (10 minutes). The PWV, brachial diameter and elastic modulus changes and temporal profile were analyzed (basal state, 15, 30, 45, and 60 seconds after cuff deflation). RESULTS: Transient ischaemia was associated with arterial stiffness changes, evidenced by carotid-radial PWV variations. The PWV and diastolic diameter changes, and temporal profiles differed. The arterial stiffness changes could not be explained only by geometrical (diameter) changes. CONCLUSION: The carotid-radial PWV analysis, evaluated using robust and simple available techniques, could be used in the clinical practice to study the vascular response to transient ischaemia and the endothelial function.