RESUMEN
Axin is a negative regulator of canonical Wnt signaling, which promotes the degradation of beta-catenin, the major effector in this signaling cascade. While many protein-binding domains of Axin have been identified, their significance has not been evaluated in vivo. Here, we report the generation and analysis of mice carrying modified Axin alleles in which either the RGS domain or the six C-terminal amino acids (C6 motif) were deleted. The RGS domain is required for APC-binding, while the C6 motif has been implicated in the activation of c-Jun N-terminal kinase, but is not required for the effects of Axin on the Wnt/beta-catenin pathway, in vitro. Both mutant Axin alleles caused recessive embryonic lethality at E9.5-E10.5, with defects indistinguishable from those caused by a null allele. As Axin-DeltaRGS protein was produced at normal levels, its inability to support embryogenesis confirms the importance of interactions between Axin and APC. In contrast, Axin-DeltaC6 protein was expressed at only 25-30% of the normal level, which may account for the recessive lethality of this allele. Furthermore, many Axin(DeltaC6/DeltaC6) embryos that were heterozygous for a beta-catenin null mutation survived to term, demonstrating that early lethality was due to failure to negatively regulate beta-catenin.
Asunto(s)
Desarrollo Embrionario/genética , Proteínas Represoras/química , Proteínas Represoras/fisiología , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/fisiología , Animales , Animales Modificados Genéticamente , Proteína Axina , Secuencia de Bases , Células Cultivadas , Embrión de Mamíferos , Embrión no Mamífero , Viabilidad Fetal/genética , Eliminación de Gen , Genes Letales , Ratones , Datos de Secuencia Molecular , Proteínas Mutantes/fisiología , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , Proteínas RGS/química , Proteínas RGS/genética , Proteínas Represoras/genética , Proteínas Wnt/fisiología , Xenopus/embriología , Proteínas de XenopusRESUMEN
Axin is a scaffold protein for the beta-catenin destruction complex, and a negative regulator of canonical Wnt signaling. Previous studies implicated the six C-terminal amino acids (C6 motif) in the ability of Axin to activate c-Jun N-terminal kinase, and identified them as a SUMOylation target. Deletion of the C6 motif of mouse Axin in vivo reduced the steady-state protein level, which caused embryonic lethality. Here, we report that this deletion (Axin-DeltaC6) causes a reduced half-life in mouse embryonic fibroblasts and an increased susceptibility to ubiquitination in HEK 293T cells. We confirmed the C6 motif as a SUMOylation target in vitro, and found that mutating the C-terminal SUMOylation target residues increased the susceptibility of Axin to polyubiquitination and reduced its steady-state level. Heterologous SUMOylation target sites could replace C6 in providing this protective effect. These findings suggest that SUMOylation of the C6 motif may prevent polyubiquitination, thus increasing the stability of Axin. Although C6 deletion also caused increased association of Axin with Dvl-1, this interaction was not altered by mutating the lysine residues in C6, nor could heterologous SUMOylation motifs replace the C6 motif in this assay. Therefore, some other specific property of the C6 motif seems to reduce the interaction of Axin with Dvl-1.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Fosfoproteínas/metabolismo , Proteínas Represoras/metabolismo , Ubiquitinación/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos , Animales , Proteína Axina , Línea Celular , Proteínas Dishevelled , Humanos , Ratones , Fosfoproteínas/genética , Proteínas Represoras/genética , Eliminación de Secuencia , Transducción de Señal/fisiología , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
Axin is a central component of the canonical Wnt signal transduction machinery, serving as a scaffold for the beta-catenin destruction complex. The related protein Axin2/Conductin, although less extensively studied, is thought to perform similar functions. Loss of Axin causes early embryonic lethality, while Axin2-null mice are viable but have craniofacial defects. Mutations in either gene contribute to cancer in humans. The lack of redundancy between Axin and Axin2 could be due to their different modes of expression: while Axin is expressed ubiquitously, Axin2 is expressed in tissue- and developmental-stage-specific patterns, and its transcription is induced by canonical Wnt signaling. Alternatively, the two proteins might have partially different functions, a hypothesis supported by the observation that they differ in their subcellular localizations in colon epithelial cells. To test the functional equivalence of Axin and Axin2 in vivo, we generated knockin mice in which the Axin gene was replaced with Myc-tagged Axin or Axin2 cDNA. Mice homozygous for the resulting alleles, Axin(Ax) or Axin(Ax2), express no endogenous Axin but express either Myc-Axin or Myc-Axin2 under the control of the Axin locus. Both Axin(Ax/Ax) and Axin(Ax2/Ax2) homozygotes are apparently normal and fertile, demonstrating that the Axin and Axin2 proteins are functionally equivalent.