| Wnt/β-Catenin signaling pathway plays key roles in the regulation of embryogenesis, homeostasis, regeneration, and stem cell pluripotency. Many diseases, including cancers, are correlated to the mis-regulation of Wnt/β-Catenin activity in cells. Lots of transcription factors and signal transducers had been shown to be involved in Wnt/β-Catenin pathway. Protein lysine demethylases (KDMs) are known as a class of chromatin modifying enzymes that influence many aspects of cellular functions involved in development, physiology and diseases. However, it is unclear whether there is correlation between KDMs and Wnt/β-Catenin pathway. We think it is important to understand detailed mechanisms of control the balance of Wnt pathway in vivo.We tested whether KDMs might regulate early embryonic development via mediating Wnt/β-Catenin pathway. Among these demethylase, Kdm2a and Kdm2b strongly repressed Wnt/β-Catenin pathway. It is unexpected that Kdm2a overexpression did not cause significant change in H3K36me1, H3K36me2, and H3K36me3, which are considered to be its substrates. Further studies showed that Kdm2a regulates the degradation of P-Catenin. We conclude that Kdm2a/b are involved in the regulation of P-Catenin stability via the ubiquitin-proteasome pathway and the catalytic activity is essential.Next, we studied the mechanism of regulation of β-Catenin stability by Kdm2a/b. we found that KDM2A inhibition led to up-regulation of total and non-phosphorylated β-Catenin, but not the phosphorylated form. Upon Wnt3a treatment, non-phosphorylated β-Catenin was upregulated significantly in all three fractions. However, only the nuclear P-Catenin was downregulated in presence of Kdm2a transfection, while the cytoplasmic and membranous fractions were not affected. This indicates that Kdm2a/b induces specifically the degradation of P-Catenin within cell nucleus upon Wnt activation and the regulation is independent of the N-terminal phosphorylation of p-Catenin.Then, we subsequently have tested whether Kdm2a/b and P-Catenin interact directly. By deletion mutant and immunoprecipitation, we found that the region around the arm repeats four and five is necessary for the interaction with and induced degradation by Kdm2a. We deduced that P-Catenin could be another nonhistone substrate for Kdm2a, and we successfully detected the methylation signal by a pan-methyl lysine antibody, we first discovered methylation modification for β-Catenin. Further studies demonstrate that methylation can occur in both phosphorylated and non-phosphorylated β-Catenin, and in not only nuclei but also cytosol. In summary, β-Catenin stabilization causes methylation of nuclear β-Catenin, and Kdm2a/b mediated demethylation is required for the degradation of β-Catenin in nucleus.We have explored further whether Kdm2a disrupts the formation of β-Catenin/Tcf711 complex. It was interesting that Tcf711 showed interaction with either the wild type Kdm2a or the mutants. This means that Tcf711 or Kdm2a binds to the same or overlapping regions of β-Catenin, hence β-Catenin/Tcf711 complex formation is interrupted when Kdm2a is present.We thus propose a novel model for Kdm2a/b mediated regulation of β-Catenin. The protein lysine demethylases Kdm2a/b regulate the turnover of non-phosphorylated P-Catenin specifically within the nucleus via direct interaction with the fourth and fifth armadillo repeats. The lysine residues within this region are required for the methylation of non-phosphorylated β-Catenin, which is demethylated by Kdm2a/b and subsequently ubiquitylated. During Xenopus embryogenesis, kdm2a/b genes are transcribed during early embryogenesis and are required for the specification of the body axis. Kdm2a/b knockdown in Xenopus embryos leads to increases in non-phosphorylated and methylated (3-Catenin, concurrent with the upregulation of β-Catenin target genes. This mechanism is required for controlling the output of the Wnt/β-Catenin signaling pathway to maintain normal cellular functions. |
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