RESUMO
In Wnt signaling, beta-catenin and plakoglobin transduce signals to the nucleus through interactions with TCF-type transcription factors. However, when plakoglobin is artificially engineered to restrict it to the cytoplasm by fusion with the transmembrane domain of connexin (cnxPg), it efficiently induces a Wnt-like axis duplication phenotype in Xenopus. In Xenopus embryos, maternal XTCF3 normally represses ventral expression of the dorsalizing gene Siamois. Two models have been proposed to explain the Wnt-like activity of cnxPg: 1) that cnxPg inhibits the machinery involved in the turnover of cytosolic beta-catenin, which then accumulates and inhibits maternal XTCF3, and 2) that cnxPg directly acts to inhibit XTCF3 activity. To distinguish between these models, we created a series of N-terminal deletion mutations of cnxPg and examined their ability to induce an ectopic axis in Xenopus, activate a TCF-responsive reporter (OT), stabilize beta-catenin, and colocalize with components of the Wnt signaling pathway. cnxPg does not colocalize with the Wnt pathway component Dishevelled, but it does lead to the redistribution of APC and Axin, two proteins involved in the regulation of beta-catenin turnover. Expression of cnxPg increases levels of cytosolic beta-catenin; however, this effect does not completely explain its signaling activity. Although cnxPg and Wnt-1 stabilize beta-catenin to similar extents, cnxPg activates OT to 10- to 20-fold higher levels than Wnt-1. Moreover, although LEF1 and TCF4 synergize with beta-catenin and plakoglobin to activate OT, both suppress the signaling activity of cnxPg. In contrast, XTCF3 suppresses the signaling activity of both beta-catenin and cnxPg. Both exogenous XLEF1 and XTCF3 are sequestered in the cytoplasm of Xenopus cells by cnxPg. Based on these data, we conclude that, in addition to its effects on beta-catenin, cnxPg interacts with other components of the Wnt pathway, perhaps TCFs, and that these interactions contribute to its signaling activity.
Assuntos
Proteínas do Citoesqueleto/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras , Transdução de Sinais , Transativadores , Proteínas de Peixe-Zebra , Animais , Proteína Axina , Caderinas/metabolismo , Linhagem Celular , Conexinas/genética , Proteínas do Citoesqueleto/metabolismo , Desmoplaquinas , Imunofluorescência , Genes Reporter , Proteínas de Homeodomínio/genética , Humanos , Membranas Intracelulares/metabolismo , Modelos Moleculares , Plasmídeos , Proteínas/metabolismo , Deleção de Sequência , Fatores de Transcrição/genética , Proteínas Wnt , Proteína Wnt1 , Xenopus , Proteínas de Xenopus , beta Catenina , gama CateninaRESUMO
Vertebrates have two Armadillo-like proteins, beta-catenin and plakoglobin. Mutant forms of beta-catenin with oncogenic activity are found in many human tumors, but plakoglobin mutations are not commonly found. In fact, plakoglobin has been proposed to suppress tumorigenesis. To assess differences between beta-catenin and plakoglobin, we compared several of their biochemical properties. After transient transfection of 293T cells with an expression vector encoding either of the two proteins, soluble wild type beta-catenin does not significantly accumulate, whereas soluble wild type plakoglobin is readily detected. As anticipated, beta-catenin is stabilized by the oncogenic mutation S37A; however, the analogous mutation in plakoglobin (S28A) does not alter its half-life. S37A-beta-catenin activates a TCF/LEF-dependent reporter 20-fold more potently than wild type beta-catenin, and approximately 5-fold more potently than wild type or S28A plakoglobin. These differences may be attributable to an enhanced affinity of S37A beta-catenin for LEF1 and TCF4, as observed here by immunoprecipitation assays. We show that the carboxyl-terminal domain is largely responsible for the difference in signaling and that the Armadillo repeats account for the remainder of the difference. The relatively weak signaling by plakoglobin and the failure of the S28A mutation to enhance its stability, may explain why plakoglobin mutations are infrequent in malignancies.
Assuntos
Proteínas do Citoesqueleto/fisiologia , Transdução de Sinais/fisiologia , Transativadores , Proteínas de Peixe-Zebra , Linhagem Celular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desmoplaquinas , Humanos , Fator 1 de Ligação ao Facilitador Linfoide , Mutação Puntual , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição TCF , Proteína 2 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Transfecção , Proteínas Wnt , beta Catenina , gama CateninaRESUMO
Using a functional screen in Xenopus embryos, we identified a novel function for the HMG box protein XSox17 beta. Ectopic expression of XSox17 beta ventralizes embryos by inhibiting the Wnt pathway downstream of beta-catenin but upstream of the Wnt-responsive gene Siamois. XSox17 beta also represses transactivation of a TCF/LEF-dependent reporter construct by Wnt and beta-catenin. In animal cap experiments, it both activates transcription of endodermal genes and represses beta-catenin-stimulated expression of dorsal genes. The inhibition activity of XSox17 beta maps to a region C-terminal to the HMG box; this region of XSox17 beta physically interacts with the Armadillo repeats of beta-catenin. Two additional Sox proteins, XSox17 alpha and XSox3, likewise bind to beta-catenin and inhibit its TCF-mediated signaling activity. These results reveal an unexpected mechanism by which Sox proteins can modulate Wnt signaling pathways.