期刊
BIOCHEMICAL JOURNAL
卷 411, 期 -, 页码 581-591出版社
PORTLAND PRESS LTD
DOI: 10.1042/BJ20071312
关键词
armadillo-repeat-containing protein 8 (ARMc8); alpha-catenin; C-terminal to lissencephaly type-1-like homology motif (CTLH) complex; proteasome; glucose-induced degradation (Gid)-protein complex; vacuole import and degradation (Vid)
ARMc8 (armadillo-repeat-containing protein 8) is a key component of the CTLH (C-terminal to lissencephaly type-1-like homology motif) complex in mammalian cells. This complex is well conserved in Saccharomyces cerevisiae and has been characterized as a FBPase (fructose-1, 6-bisphosphatase)degrading complex. The yeast homologue of ARMc8, Gid (glucose-induced degradation) 5p, plays an essential role in the ubiquitin- and proteasome-dependent degradation of FBPase. To elucidate the function of ARMc8, we used a yeast two-hybrid system to screen a human skeletal muscle cDNA library. alpha-Catenin was isolated as a binding protein of ARMc8 alpha. This association was confirmed by co-immunoprecipitation assay using MDCK (Madin-Darby canine kidney) cells in which exogenous alpha-catenin and ARMc8 alpha were overexpressed. The association was also confirmed by co-immunoprecipitation assay using endogenous proteins in untransfected MDCK cells. We then used immunofluorescence microscopy of MDCK cells and C2Cl2 cells to investigate the intracellular distribution of ARMc8. Exogenously expressed ARMc8 was co-localized with alpha-catenin and beta-catenin along the cell membrane, suggesting an association between alpha-catenin and ARMc8 in the cells. To compare the binding domain of alpha-catenin with ARMc8 alpha with that of beta-catenin, we performed a co-immunoprecipitation assay, again using 5'- and 3'-deletion constructs of alpha-catenin. The N-terminal sequence (amino acids 82-148) of alpha-catenin was sufficient to bind to both ARMc8 alpha and beta-catenin. Next, we investigated the proteasome-dependent degradation of alpha-catenin by immunoblotting using proteasome inhibitors. Co-expression of ARMc8 alpha with alpha-catenin resulted in rapid degradation of the exogenous alpha-catenin. Furthermore, ARMc8 knockdown inhibited alpha-catenin degradation and prolonged the half-life of alpha-catenin. We conclude that ARMc8 alpha associates with alpha-catenin and upregulates its degradation.
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