RESUMO
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.
Assuntos
Cateninas/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Proteína Wnt3A/farmacologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Caderinas/metabolismo , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Ativação Enzimática/efeitos dos fármacos , Gastrulação/efeitos dos fármacos , Humanos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Fosfotirosina/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Xenopus/embriologia , Xenopus/metabolismo , beta Catenina/metabolismo , delta CateninaRESUMO
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.
Assuntos
Caseína Quinase 1 épsilon/metabolismo , Cateninas/metabolismo , Proteínas Wnt/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Caderinas/genética , Caderinas/metabolismo , Caseína Quinase 1 épsilon/genética , Cateninas/genética , Linhagem Celular Tumoral , Proteínas Desgrenhadas , Humanos , Imunoprecipitação , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Espectrometria de Massas , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , delta CateninaRESUMO
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.