RESUMEN
The central regulator of the Wnt/ß-catenin pathway is the Axin/APC/GSK3ß destruction complex (DC), which, under unstimulated conditions, targets cytoplasmic ß-catenin for degradation. How Wnt activation inhibits the DC to permit ß-catenin-dependent signaling remains controversial, in part because the DC and its regulation have never been observed in vivo Using bimolecular fluorescence complementation (BiFC) methods, we have now analyzed the activity of the DC under near-physiological conditions in Drosophila By focusing on well-established patterns of Wnt/Wg signaling in the developing Drosophila wing, we have defined the sequence of events by which activated Wnt receptors induce a conformational change within the DC, resulting in modified Axin-GSK3ß interactions that prevent ß-catenin degradation. Surprisingly, the nucleus is surrounded by active DCs, which principally control the degradation of ß-catenin and thereby nuclear access. These DCs are inactivated and removed upon Wnt signal transduction. These results suggest a novel mechanistic model for dynamic Wnt signal transduction in vivo.
Asunto(s)
Proteína Axina/metabolismo , Complejo de Señalización de la Axina/fisiología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Vía de Señalización Wnt/fisiología , beta Catenina/fisiología , Animales , Animales Modificados Genéticamente , Proteína Axina/química , Complejo de Señalización de la Axina/química , Complejo de Señalización de la Axina/metabolismo , Tipificación del Cuerpo/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Embrión no Mamífero , Prueba de Complementación Genética , Glucógeno Sintasa Quinasa 3 beta/química , Imagen Óptica , Fosforilación/genética , Unión Proteica/genética , Conformación Proteica , Pliegue de Proteína , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismo , Proteínas Wnt/metabolismo , Proteínas Wnt/fisiología , Vía de Señalización Wnt/genética , beta Catenina/metabolismoRESUMEN
Wnt signaling provides a paradigm for cell-cell signals that regulate embryonic development and stem cell homeostasis and are inappropriately activated in cancers. The tumor suppressors APC and Axin form the core of the multiprotein destruction complex, which targets the Wnt-effector beta-catenin for phosphorylation, ubiquitination and destruction. Based on earlier work, we hypothesize that the destruction complex is a supramolecular entity that self-assembles by Axin and APC polymerization, and that regulating assembly and stability of the destruction complex underlie its function. We tested this hypothesis in Drosophila embryos, a premier model of Wnt signaling. Combining biochemistry, genetic tools to manipulate Axin and APC2 levels, advanced imaging and molecule counting, we defined destruction complex assembly, stoichiometry, and localization in vivo, and its downregulation in response to Wnt signaling. Our findings challenge and revise current models of destruction complex function. Endogenous Axin and APC2 proteins and their antagonist Dishevelled accumulate at roughly similar levels, suggesting competition for binding may be critical. By expressing Axin:GFP at near endogenous levels we found that in the absence of Wnt signals, Axin and APC2 co-assemble into large cytoplasmic complexes containing tens to hundreds of Axin proteins. Wnt signals trigger recruitment of these to the membrane, while cytoplasmic Axin levels increase, suggesting altered assembly/disassembly. Glycogen synthase kinase3 regulates destruction complex recruitment to the membrane and release of Armadillo/beta-catenin from the destruction complex. Manipulating Axin or APC2 levels had no effect on destruction complex activity when Wnt signals were absent, but, surprisingly, had opposite effects on the destruction complex when Wnt signals were present. Elevating Axin made the complex more resistant to inactivation, while elevating APC2 levels enhanced inactivation. Our data suggest both absolute levels and the ratio of these two core components affect destruction complex function, supporting models in which competition among Axin partners determines destruction complex activity.
Asunto(s)
Proteínas del Dominio Armadillo/metabolismo , Complejo de Señalización de la Axina/metabolismo , Proteínas de Drosophila/metabolismo , Factores de Transcripción/metabolismo , Vía de Señalización Wnt , Animales , Animales Modificados Genéticamente , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/química , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/genética , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas del Dominio Armadillo/química , Proteínas del Dominio Armadillo/genética , Proteína Axina/química , Proteína Axina/genética , Proteína Axina/metabolismo , Complejo de Señalización de la Axina/química , Complejo de Señalización de la Axina/genética , Línea Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteolisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción Genética , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMEN
The Wnt/beta-catenin pathway has many key roles in the development of animals, including a conserved and central role in the specification of the primary (antero-posterior) body axis. The posterior expression of Wnt ligands and the anterior expression of secreted Wnt inhibitors are known to be conserved during the larval metamorphosis of tapeworms. However, their downstream signaling components for Wnt/beta-catenin signaling have not been characterized. In this work, we have studied the core components of the beta-catenin destruction complex of the human pathogen Echinococcus multilocularis, the causative agent of alveolar echinococcosis. We focused on two Axin paralogs that are conserved in tapeworms and other flatworm parasites. Despite their divergent sequences, both Axins could robustly interact with one E. multilocularis beta-catenin paralog and limited its accumulation in a heterologous mammalian expression system. Similarly to what has been described in planarians (free-living flatworms), other beta-catenin paralogs showed limited or no interaction with either Axin and are unlikely to function as effectors in Wnt signaling. Additionally, both Axins interacted with three divergent GSK-3 paralogs that are conserved in free-living and parasitic flatworms. Axin paralogs have highly segregated expression patterns along the antero-posterior axis in the tapeworms E. multilocularis and Hymenolepis microstoma, indicating that different beta-catenin destruction complexes may operate in different regions during their larval metamorphosis.
Asunto(s)
Proteína Axina/genética , Complejo de Señalización de la Axina/genética , Echinococcus multilocularis/genética , Glucógeno Sintasa Quinasa 3/genética , Proteínas del Helminto/genética , Hymenolepis/genética , beta Catenina/genética , Secuencia de Aminoácidos , Animales , Proteína Axina/química , Proteína Axina/metabolismo , Complejo de Señalización de la Axina/química , Echinococcus multilocularis/crecimiento & desarrollo , Echinococcus multilocularis/metabolismo , Perfilación de la Expresión Génica , Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas del Helminto/química , Humanos , Hymenolepis/crecimiento & desarrollo , Hymenolepis/metabolismo , Larva/metabolismo , Filogenia , Alineación de Secuencia , beta Catenina/metabolismoRESUMEN
Wnt/ß-catenin signaling plays important roles in both ontogenesis and development. In the absence of a Wnt stimulus, ß-catenin is degraded by a multiprotein "destruction complex" that includes Axin, APC, GSK3B, and FBXW11. Although the key molecules required for transducing Wnt signals have been identified, a quantitative understanding of this pathway has been lacking. Here, we calculated the absolute number of ß-catenin destruction complexes by absolute protein quantification using LC-MS/MS. Similar amounts of destruction complex-constituting proteins and ß-catenin interacted, and the number of destruction complexes was calculated to be about 1468 molecules/cell. We demonstrated that the calculated number of destruction complexes was valid for control of the ß-catenin destruction rate under steady-state conditions. Interestingly, APC had the minimum expression level among the destruction complex components at about 2233 molecules/cell, and this number approximately corresponded to the calculated number of destruction complexes. Decreased APC expression by siRNA transfection decreased the number of destruction complexes, resulting in ß-catenin accumulation and stimulation of the transcriptional activity of T-cell factor. Taken together, our results suggest that the amount of APC expression is the rate-limiting factor for the constitution of ß-catenin destruction complexes.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/genética , Complejo de Señalización de la Axina/genética , Vía de Señalización Wnt/genética , beta Catenina/genética , Proteína Axina/genética , Complejo de Señalización de la Axina/química , Complejo de Señalización de la Axina/metabolismo , Regulación de la Expresión Génica/genética , Glucógeno Sintasa Quinasa 3 beta/genética , Células HCT116 , Humanos , Fosforilación , ARN Interferente Pequeño/genética , Ubiquitina-Proteína Ligasas/genética , beta Catenina/aislamiento & purificación , Proteínas con Repetición de beta-Transducina/genéticaRESUMEN
Upon binding of a Wnt ligand to the frizzled (FZD)-low density lipoprotein receptor related protein 5/6 (LRP5/6) receptor complex, the ß-catenin destruction complex, composed of Axin1, adenomatous polyposis coli (APC), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1), is immediately inactivated, which causes ß-catenin stabilization. However, the molecular mechanism of signal transduction from the receptor complex to the ß-catenin destruction complex is controversial. Here we show that Wnt3a treatment promotes the dissociation of the Axin1-APC complex in glioblastoma cells cultured in serum-free medium. Experiments with the GSK3 inhibitor BIO suggest that Axin1-APC dissociation was controlled by phosphorylation. Introduction of a phosphomimetic mutation into Thr160 of Axin1, located in the APC-binding region RGS, abrogated the interaction of Axin1 with APC. Consistent with these observations, the Axin1 phosphomimetic mutant lost the ability to reduce ß-catenin stability and to repress ß-catenin/TCF-dependent transcription. Taken together, our results suggest a novel mechanism of Wnt signaling through the dissociation of the ß-catenin destruction complex by Axin1 Thr160 modification.
Asunto(s)
Proteína Axina/química , Proteína Axina/metabolismo , Complejo de Señalización de la Axina/química , Complejo de Señalización de la Axina/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/química , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Proteína Axina/genética , Sitios de Unión , Materiales Biomiméticos/química , Materiales Biomiméticos/metabolismo , Quinasa de la Caseína I/química , Quinasa de la Caseína I/metabolismo , Línea Celular Tumoral , Glioblastoma/metabolismo , Glucógeno Sintasa Quinasa 3/química , Glucógeno Sintasa Quinasa 3/metabolismo , Células HEK293 , Humanos , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/antagonistas & inhibidores , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/antagonistas & inhibidores , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Mutagénesis Sitio-Dirigida , Fosforilación , Estabilidad Proteica , ARN Interferente Pequeño/genética , Treonina/química , Vía de Señalización Wnt , beta Catenina/química , beta Catenina/metabolismoRESUMEN
The axis inhibition (Axin) scaffold protein colocalizes ß-catenin, casein kinase Iα, and glycogen synthetase kinase 3ß by their binding to Axin's long intrinsically disordered region, thereby yielding structured domains with flexible linkers. This complex leads to the phosphorylation of ß-catenin, marking it for destruction. Fusing proteins with flexible linkers vastly accelerates chemical interactions between them by their colocalization. Here we propose that the complex works by random movements of a "stochastic machine," not by coordinated conformational changes. This non-covalent, modular assembly process allows the various molecular machine components to be used in multiple processes.
Asunto(s)
Complejo de Señalización de la Axina/química , Modelos Moleculares , Procesamiento Proteico-Postraduccional , Regulación Alostérica , Complejo de Señalización de la Axina/fisiología , Quinasa de la Caseína I/química , Humanos , Fosforilación , Estructura Cuaternaria de Proteína , Transporte de Proteínas , Proteolisis , Procesos Estocásticos , Vía de Señalización Wnt , beta Catenina/químicaRESUMEN
Most cases of colorectal cancer are linked to mutational inactivation of the Adenomatous polyposis coli (APC) tumour suppressor. APC downregulates Wnt signalling by enabling Axin to promote the degradation of the Wnt signalling effector ß-catenin (Armadillo in flies). This depends on Axin's DIX domain whose polymerization allows it to form dynamic protein assemblies ('degradasomes'). Axin is inactivated upon Wnt signalling, by heteropolymerization with the DIX domain of Dishevelled, which recruits it into membrane-associated 'signalosomes'. How APC promotes Axin's function is unclear, especially as it has been reported that APC's function can be bypassed by overexpression of Axin. Examining apc null mutant Drosophila tissues, we discovered that APC is required for Axin degradasome assembly, itself essential for Armadillo downregulation. Degradasome assembly is also attenuated in APC mutant cancer cells. Notably, Axin becomes prone to Dishevelled-dependent plasma membrane recruitment in the absence of APC, indicating a crucial role of APC in opposing the interaction of Axin with Dishevelled. Indeed, co-expression experiments reveal that APC displaces Dishevelled from Axin assemblies, promoting degradasome over signalosome formation in the absence of Wnts. APC thus empowers Axin to function in two ways-by enabling its DIX-dependent self-assembly, and by opposing its DIX-dependent copolymerization with Dishevelled and consequent inactivation.