Abstract
Adenomatous polyposis coli (APC) protein and Axin form a complex that mediates the down-regulation of beta-catenin, a key effector of Wnt signaling. Truncation mutations in APC are responsible for familial and sporadic colorectal tumors due to failure in the down-regulation of beta-catenin. While the regulation of beta-catenin by APC has been extensively studied, the regulation of APC itself has received little attention. Here we show that the level of APC is down-regulated by the ubiquitin-proteasome pathway and that Wnt signaling inhibits the process. The domain responsible for the down-regulation and direct ubiquitination was identified. We also show an unexpected role for Axin in facilitating the ubiquitination-proteasome-mediated down-regulation of APC through the oligomerization of Axin. Our results suggest a new mechanism for the regulation of APC by Axin and Wnt signaling.
Highlights
Adenomatous polyposis coli (APC) protein and Axin form a complex that mediates the down-regulation of -catenin, a key effector of Wnt signaling
It is still controversial whether APC contains real nuclear export sequences, it has been claimed that truncation of APC abolishes specific nuclear export sequences, which blocks the APC-dependent export of -catenin from the nucleus, resulting in constitutive activation of downstream target genes, which leads to tumor formation [19]
The Level of APC Is Up-regulated by Wnt3a—A previous study demonstrated that the expression of Wnt-1 resulted in an increased steady-state level of APC protein as well as an increase in the amount of -catenin and suggested that APC might be regulated by Wnt-1 at the post-translational level in a fashion similar to -catenin [25]
Summary
Construction of Plasmids—Full-length Xenopus VSV-G epitopetagged APC and Myc-epitope tagged Axin and deletion constructs and pCS2MT-GFP have been described previously [10]. Nusse), these cells were cultured in 175T flask containing DMEM supplemented with 10% fetal bovine serum. CM derived from L cells that had been transfected with PGK-neo was used as a control for Wnt3a-conditioned medium. Pulse-Chase Analysis—Myc-tagged hAPC- and HA-ubiquitin-expressing plasmids were transiently transfected into 293T cells in 60-mm dishes. Confluent cells from 100-mm dishes were harvested, washed twice with phosphate-buffered saline, and suspended in 1 ml of hypotonic buffer (10 mM Tris-Cl, pH 6.7, 20 mM NaF, 1 mM Na3VO4, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 1 g/ml each leupeptin, aprotinin, and pepstatin) on ice for 15 min to swell. Cells were treated with Wnt3a-conditioned or control medium for 24 h, fixed with 4% paraformaldehyde in phosphate-buffered saline for 15 min, and subsequently permeabilized and blocked with buffer A at room temperature for 15 min. Signal was visualized by epifluorescence using a confocal laser scanning microscopy (MRC1024, Bio-Rad)
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