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[This corrects the article DOI: 10.1371/journal.pone.0181724.].
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Members of the Dickkopf (Dkk) family of Wnt antagonists interrupt Wnt-induced receptor assembly and participate in axial patterning and cell fate determination. One family member, DKK3, does not block Wnt receptor activation. Loss of Dkk3 expression in cancer is associated with hyperproliferation and dysregulated ß-catenin signaling, and ectopic expression of Dkk3 halts cancer growth. The molecular events mediating the DKK3-dependent arrest of ß-catenin-driven cell proliferation in cancer cells are unknown. Here we report the identification of a new intracellular gene product originating from the Dkk3 locus. This Dkk3b transcript originates from a second transcriptional start site located in intron 2 of the Dkk3 gene. It is essential for early mouse development and is a newly recognized regulator of ß-catenin signaling and cell proliferation. Dkk3b interrupts nuclear translocation ß-catenin by capturing cytoplasmic, unphosphorylated ß-catenin in an extra-nuclear complex with ß-TrCP. These data reveal a new regulator of one of the most studied signal transduction pathways in metazoans and provides a novel, completely untapped therapeutic target for silencing the aberrant ß-catenin signaling that drives hyperproliferation in many cancers.
Asunto(s)
Proliferación Celular/genética , Proliferación Celular/fisiología , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiología , Animales , Citoplasma/genética , Citoplasma/metabolismo , Citoplasma/fisiología , Femenino , Ratones Endogámicos C57BL , Neoplasias/genética , Neoplasias/metabolismo , Embarazo , Ratas , Ratas Sprague-Dawley , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Proteínas con Repetición de beta-Transducina/genética , Proteínas con Repetición de beta-Transducina/metabolismoRESUMEN
Recent work has led to the identification of novel endocytic compartments with functional roles in both protein trafficking and growth factor signal transduction. The phosphatidylinositol 3-phosphate binding, FYVE domain-containing protein WDFY2 is localized to a distinct subset of early endosomes, which are localized close to the plasma membrane. Here, we find that the serine/threonine kinase Akt interacts with these endosomes in an isoform-specific manner. Using quantitative fluorescence microscopy we demonstrate specific co-localization of WDFY2 with endogenous Akt2, but not Akt1. Moreover, depletion of WDFY2 leads to impaired phosphorylation of Akt in response to insulin due to isoform specific reduction of Akt2, but not Akt1, protein levels, and to a marked reduction in the insulin-stimulated phosphorylation of numerous Akt substrates. This is accompanied by an impairment in insulin-stimulated glucose transport and, after prolonged silencing, a reduction in the level of expression of adipogenic genes. We propose that WDFY2-enriched endosomes serve as a scaffold that enables specificity of insulin signaling through Akt2.
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Proteínas Portadoras/fisiología , Endosomas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Células 3T3-L1 , Animales , Transporte Biológico , Western Blotting , Glucosa/metabolismo , Hipoglucemiantes/farmacología , Insulina/farmacología , Ratones , Microscopía Fluorescente , Fosforilación , Isoformas de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas del Sistema de Dos HíbridosRESUMEN
Type I deiodinase is the best characterized member of a small family of selenoenzymes catalyzing the bioactivation and disposal of thyroid hormone. This enzyme is an integral membrane protein composed of two 27-kDa subunits that assemble into a functional enzyme after translation using a highly conserved sequence of 16 amino acids in the C-terminal half of the polypeptide, (148)DFLXXYIXEAHXXDGW(163). In this study, we used alanine scanning mutagenesis to identify the key residues in this domain required for holoenzyme assembly. Overexpression of sequential alanine-substituted mutants of a dimerization domain-green fluorescent protein fusion showed that sequence (152)IYI(154) was required for type I enzyme assembly and that a catalytically active monomer was generated by a single I152A substitution. Overexpression of the sequential alanine-substituted dimerization domain mutants in type II selenodeiodinase-expressing cells showed that five residues ((153)FLIVY(157)) at the beginning and three residues ((164)SDG(166)) at the end of this region were required for the assembly of the type II enzyme. In vitro binding analysis revealed a free energy of association of -60 +/- 5 kJ/mol for the noncovalent interaction between dimerization domain monomers. These data identify and characterize the essential residues in the dimerization domain that are responsible for the post-translational assembly of selenodeiodinases.
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Yoduro Peroxidasa/química , Selenio/química , Alanina/química , Secuencia de Aminoácidos , Animales , Unión Competitiva , Dimerización , Proteínas Fluorescentes Verdes/química , Datos de Secuencia Molecular , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/química , TermodinámicaRESUMEN
Thyroid hormone metabolism is catalyzed by a small family of selenoenzymes. Type I deiodinase (D1) is the best characterized family member and is an integral membrane protein composed of two 27-kDa subunits that assemble to a functional holoenzyme after translation. To characterize the protein domain(s) responsible for this post-translational assembly event, we used deletion/truncation analysis coupled with immune depletion assays to map the dimerization domain of D1. The results of our studies show that a highly conserved sequence of 16 amino acids in the C-terminal half of the D1 subunit, -D148FL-YI-EAH-DGW163-, serves as the dimerization domain. Based on the high conservation of this domain, we synthesized a novel bait peptide-green fluorescent protein fusion probe (DDD(GFP)) to examine holoenzyme assembly of other family members. Overexpression of either the DDD(GFP) or an inert D1 subunit (M4) into SeD2 (accession number U53505)-expressing C6 cells specifically led to the loss of >90% of the catalytic activity. Catalytically inactive D2 heterodimers composed of SeD2: DDD(GFP) subunits were rescued by specific immune precipitation with anti-SeD2 IgG, suggesting that SeD2 requires two functional subunits to assemble a catalytically active holoenzyme. These findings identify and characterize the essential dimerization domain responsible for post-translational assembly of selenodeiodinases and show that family members can intermingle through this highly conserved protein domain.