Abstract
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.
Highlights
The canonical Wnt/b-catenin signaling is crucial for normal embryonic development, patterning, and its abnormal expression is associated with many forms of oncogenesis [1,2]
We wondered whether the effect of human KCTD1 on Wnt signaling pathway, a TOPFLASH reporter construct containing seven copies of the TCF binding site was employed to report the activity of Wnt/b-catenin signaling
Five mutants of KCTD1 in SEN syndrome were constructed to identify critical amino acid residues of the BTB domain in KCTD1, we found that three KCTD1 mutants (P20S, H33P and P31R) still inhibited the TOPFLASH reporter activities to the same extent as wild-type KCTD1 did, but mutant KCTD1 with A30E or H33Q had no effect on the TOPFLASH reporter transcription activities as truncated KCTD1C did, indicating that alanine at position 30 and histidine at position 33 are critical amino acid residues of KCTD1 in the regulation of Wnt signaling
Summary
The canonical Wnt/b-catenin signaling is crucial for normal embryonic development, patterning, and its abnormal expression is associated with many forms of oncogenesis [1,2]. In the presence of Wnt ligands, the ligands bind to a Frizzled/LRP complex to activate the cytoplasmic protein Disheveled (Dvl) [3,4]. Dvl inhibits the activity of the destruction complex including CK1, axin, GSK-3b and APC, which phosphorylates b-catenin for ubiquitin-mediated proteasomal degradation. In the absence of Wnt ligands, cytoplasmic b-catenin is constitutively phosphorylated by CK1 and GSK-3b. Phosphorylated b-catenin is polyubiquitinated by E3 ubiquitin ligase b-TrCP and degraded by the 26 S proteasome [7,8]. Inappropriate activation of the Wnt pathway is observed in several human cancers, such as colorectal carcinoma, hepatocellular carcinoma, melanoma and ovarian cancer [9,10,11]
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