RESUMO
In common with other p120-catenin subfamily members, Xenopus ARVCF (xARVCF) binds cadherin cytoplasmic domains to enhance cadherin metabolic stability or, when dissociated, modulates Rho-family GTPases. We report here that xARVCF binds and is stabilized by Xenopus KazrinA (xKazrinA), a widely expressed conserved protein that bears little homology to established protein families, and which is known to influence keratinocyte proliferation and differentiation and cytoskeletal activity. Although we found that xKazrinA binds directly to xARVCF, we did not resolve xKazrinA within a larger ternary complex with cadherin, nor did it co-precipitate with core desmosomal components. Instead, screening revealed that xKazrinA binds spectrin, suggesting a potential means by which xKazrinA localizes to cell-cell borders. This was supported by the resolution of a ternary biochemical complex of xARVCF-xKazrinA-xß2-spectrin and, in vivo, by the finding that ectodermal shedding followed depletion of xKazrin in Xenopus embryos, a phenotype partially rescued with exogenous xARVCF. Cell shedding appeared to be the consequence of RhoA activation, and thereby altered actin organization and cadherin function. Indeed, we also revealed that xKazrinA binds p190B RhoGAP, which was likewise capable of rescuing Kazrin depletion. Finally, xKazrinA was found to associate with δ-catenins and p0071-catenins but not with p120-catenin, suggesting that Kazrin interacts selectively with additional members of the p120-catenin subfamily. Taken together, our study supports the essential role of Kazrin in development, and reveals the biochemical and functional association of KazrinA with ARVCF-catenin, spectrin and p190B RhoGAP.
Assuntos
Proteínas do Domínio Armadillo/metabolismo , Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Epiteliais/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Espectrina/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Proteínas do Domínio Armadillo/química , Proteínas do Domínio Armadillo/genética , Caderinas/genética , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Linhagem Celular , Células Epiteliais/química , Células Epiteliais/enzimologia , Proteínas Ativadoras de GTPase/genética , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Espectrina/genética , Técnicas do Sistema de Duplo-Híbrido , Xenopus/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Using an animal model system and depletion-rescue strategies, we have addressed the requirement and functions of armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF) and p120 catenins in early vertebrate embryogenesis. We find that xARVCF and Xp120 are essential to development given that depletion of either results in disrupted gastrulation and axial elongation, which are specific phenotypes based on self-rescue analysis and further criteria. Exogenous xARVCF or Xp120 cross-rescued depletion of the other, and each depletion was additionally rescued with (carefully titrated) dominant-negative RhoA or dominant-active Rac. Although xARVCF or Xp120 depletion did not appear to reduce the adhesive function of C-cadherin in standard cell reaggregation and additional assays, C-cadherin levels were somewhat reduced after xARVCF or Xp120 depletion, and rescue analysis using partial or full-length C-cadherin constructs suggested contributory effects on altered adhesion and signaling functions. This work indicates the required functions of both p120 and ARVCF in vertebrate embryogenesis and their shared functional interplay with RhoA, Rac, and cadherin in a developmental context.