The canonical Wnt signaling pathway controls normal embryonic development, cellular proliferation and growth, and its aberrant activity results in human carcinogenesis. The core component in regulation of this pathway is β-catenin, but molecular regulation mechanisms of β-catenin stability are not completely known. Here, our recent studies have shown that KCTD1 strongly inhibits TCF/LEF reporter activity. Moreover, KCTD1 interacted with β-catenin both in vivo by co-immunoprecipitation as well as in vitro through GST pull-down assays. We further mapped the interaction regions to the 1-9 armadillo repeats of β-catenin and the BTB domain of KCTD1, especially Position Ala-30 and His-33. Immunofluorescence analysis indicated that KCTD1 promotes the cytoplasmic accumulation of β-catenin. Furthermore, protein stability assays revealed that KCTD1 enhances the ubiquitination/degradation of β-catenin in a concentration-dependent manner in HeLa cells. And the degradation of β-catenin mediated by KCTD1 was alleviated by the proteasome inhibitor, MG132. In addition, KCTD1-mediated β-catenin degradation was dependent on casein kinase 1 (CK1)- and glycogen synthase kinase-3β (GSK-3β)-mediated phosphorylation and enhanced by the E3 ubiquitin ligase β-transducin repeat-containing protein (β-TrCP). Moreover, KCTD1 suppressed the expression of endogenous Wnt downstream genes and transcription factor AP-2α. Finally, we found that Wnt pathway member APC and tumor suppressor p53 influence KCTD1-mediated downregulation of β-catenin. These results suggest that KCTD1 functions as a novel inhibitor of Wnt signaling pathway.

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The root cap and root border cells (RBCs) of most plant species produced pectinaceous mucilage, which can bind metal cations. In order to evaluate the potential role of root mucilage on aluminum (Al) resistance, two soybean cultivars differing in Al resistance were aeroponic cultured, the effects of Al on root mucilage secretion, root growth, contents of mucilage-bound Al and root tip Al, and the capability of mucilage to bind Al were investigated. Increasing Al concentration and exposure time significantly enhanced mucilage excretion from both root caps and RBCs, decreased RBCs viability and relative root elongation except roots exposed to 400 μM Al for 48 h in Al-resistant cultivar. Removal of root mucilage from root tips resulted in a more severe inhibition of root elongation. Of the total Al accumulated in root, mucilage accounted 48-72 and 12-27 %, while root tip accounted 22-52 and 73-88 % in Al-resistant and Al-sensitive cultivars, respectively. A (27)Al nuclear magnetic resonance spectrum of the Al-adsorbed mucilage showed Al tightly bound to mucilage. Higher capacity to exclude Al in Al-resistant soybean cultivar is related to the immobilization and detoxification of Al by the mucilage secreted from root cap and RBCs.

The broad-complex, tramtrack, and bric-a-brac/poxvirus and zinc finger domain-containing protein tumor necrosis factor, alpha-induced protein 1 (TNFAIP1) was first identified as a gene whose expression can be induced by the tumor necrosis factor alpha. Some studies showed that TNFAIP1 may function in DNA replication, apoptosis and human diseases. However, the definite functions and the mechanisms of TNFAIP1 are poorly known. In this study, we performed a yeast two-hybrid assay and used TNFAIP1 as the bait to screen human brain cDNA library. Potassium channel tetramerisation domain containing 10 (KCTD10) was identified as TNFAIP1-interacting partner. The KCTD10-TNFAIP1 interaction was then confirmed by the in vitro GST pull-down assays and the in vivo co-immunoprecipitation and colocalization assays. In addition, protein degradation and ubiquitin assays revealed TNFAIP1 overexpression resulted in ubiquitin-mediated degradation of KCTD10 proteins, which was significantly alleviated with the proteasome inhibitor MG132 treatment. Furthermore, transient transfection assays with two reporters showed that TNFAIP1 and KCTD10 inhibited the transcriptional activities of nuclear factor kappa B (NF-κB) and activating protein-1 reporters. Taken together, our results indicated the novel interaction and function between KCTD10 and TNFAIP1 in human PDIP1 family.

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Transcription factor AP-2β mediates the transcription of a number of genes implicated in mammalian development, cell proliferation, and carcinogenesis. Although the expression pattern of AP-2β has been analyzed in cervical cancer cell lines, the functions and molecular mechanism of AP-2β are unknown. Here, we found that AP-2β significantly inhibits TCF/LEF reporter activity. Moreover, AP-2β and β-catenin interact both in vitro through GST pull-down assays and in vivo by co-immunoprecipitation. We further identified the interaction regions to the DNA-binding domain of AP-2β and the 1-9 Armadillo repeats of β-catenin. Moreover, AP-2β binds with β-TrCP and promotes the degradation of endogenous β-catenin via the proteasomal degradation pathway. Immunohistochemistry analysis revealed a negative correlation between the two proteins in cervical cancer tissues and cell lines. Finally, functional analysis showed that AP-2β suppresses cervical cancer cell growth in vitro and in vivo by inhibiting the expression of Wnt downstream genes. Taken together, these findings demonstrated that AP-2β functions as a novel inhibitor of the Wnt/β-catenin signaling pathway in cervical cancer.

Eps8 was initially identified as a substrate of the epidermal growth factor receptor. Overexpression of Eps8 leads to increased mitogenic signaling and malignant transformation. However, little is known concerning the importance of Eps8 in human gliomas. In this study, we found that Eps8 was overexpressed in 56.6% of human gliomas (WHO grades III and IV) compared with adjacent normal brain tissues by immunohistochemical analysis. The U251 human glioma cell line stably expressing Eps8 was established by G418 screening, and the ectopic expression of Eps8 enhanced U251 glioma cell growth and survival by cell survival, MTT and liquid colony formation assays. By contrast, the lentiviral expression of Eps8 siRNA in SHG-44 cells resulted in a significant reduction in cellular growth and proliferation. Furthermore, Eps8 modulated the levels of phosphorylated extracellular signal-regulated protein kinase (ERK), phosphorylated serine-threonine protein kinase Akt and β-catenin expression in glioma cell lines and tissues. These results suggest that Eps8 is overexpressed in human gliomas, and affects glioma cell growth possibly by regulating ERK and Akt/β-catenin signaling. Therefore, Eps8 may represent a novel potential target in human glioma therapy.

Participates in actin remodeling through Rac and receptor endocytosis via Rab5. Here, we used yeast two-hybrid system with Eps8 as bait to screen a human brain cDNA library. ITSN2 was identified as the novel binding factor of Eps8. The interaction between ITSN2 and Eps8 was demonstrated by the in vivo co-immunoprecipitation and colocalization assays and the in vitro GST pull-down assays. Furthermore, we mapped the interaction domains to the region between amino acids 260-306 of Eps8 and the coiled-coil domain of ITSN2. In addition, protein stability assays and immunofluorescence analysis showed ITSN2 overexpression induced the degradation of Eps8 proteins, which was markedly alleviated with the lysosome inhibitor NH4Cl treatment. Taken together, our results suggested ITSN2 interacts with Eps8 and stimulates the degradation of Eps8 proteins. [BMB reports 2012; 45(3): 183-188]