␦-Catenin was first identified because of its interaction with presenilin-1, and its aberrant expression has been reported in various human tumors and in patients with Cri-du-Chat syndrome, a form of mental retardation. However, the mechanism whereby ␦-catenin is regulated in cells has not been fully elucidated. We investigated the possibility that glycogen-synthase kinase-3 (GSK-3) phosphorylates ␦-catenin and thus affects its stability. Initially, we found that the level of ␦-catenin was greater and the half-life of ␦-catenin was longer in GSK-3
fibroblasts than those in GSK-3؉/؉ fibroblasts. Furthermore, four different approaches designed to specifically inhibit GSK-3 activity, i.e. GSK-3-specific chemical inhibitors, Wnt-3a conditioned media, small interfering RNAs, and GSK-3␣ and -3 kinase dead constructs, consistently showed that the levels of endogenous ␦-catenin in CWR22Rv-1 prostate carcinoma cells and primary cortical neurons were increased by inhibiting GSK-3 activity. In addition, it was found that both GSK-3␣ and -3 interact with and phosphorylate ␦-catenin. The phosphorylation of ⌬C207-␦-catenin (lacking 207 C-terminal residues) and T1078A ␦-catenin by GSK-3 was noticeably reduced compared with that of wild type ␦-catenin, and the data from liquid chromatography-tandem mass spectrometry analyses suggest that the Thr 1078 residue of ␦-catenin is one of the GSK-3 phosphorylation sites. Treatment with MG132 or ALLN, specific inhibitors of proteosome-dependent proteolysis, increased ␦-catenin levels and caused an accumulation of ubiquitinated ␦-catenin. It was also found that GSK-3 triggers the ubiquitination of ␦-catenin. These results suggest that GSK-3 interacts with and phosphorylates ␦-catenin and thereby negatively affects its stability by enabling its ubiquitination/proteosome-mediated proteolysis.
Sensitive and safe methods for visualization of DNA in agarose gels are described. 0.001% crystal violet dissolved in distilled water was used for DNA staining on agarose gels and it could detect as little as 16 ng of DNA (3 kb, pGem-7Zf/EcoRI) without destaining procedure. The detection limit is four times lower than that of ethidium bromide. To improve the sensitivity, we studied a counterion-dye staining method using methyl orange as a counterion-dye which contributes to reduce excessive background staining by crystal violet. Dye concentration, pH of staining solution, mixing molar ratio of two dyes, and staining times were optimized for the counterion-dye staining. By the staining with a mixed solution of 0.0025% crystal violet and 0.0005% methyl orange in distilled water, 8 ng of the 3 kb DNA in an agarose gel was detected within 30 min.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.