| Budget Amount *help |
¥14,900,000 (Direct Cost: ¥14,900,000)
Fiscal Year 2006: ¥7,200,000 (Direct Cost: ¥7,200,000)
Fiscal Year 2005: ¥7,700,000 (Direct Cost: ¥7,700,000)
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| Research Abstract |
Cellular FLIP (cFLIP) is a homologue of caspase-8 without protease activity, and inhibits the apoptosis signaling initiated by death receptor ligation. We previously reported that a long form of cFLIP (cFLIP-L) inhibits ubiquitylation of β-catenin, and enhances Wnt signaling. We showed the followings in this study.
1. cFLIP-L impairs the function of the ubiquitin-proteasome system (UPS), and increases the accumulation of various short-lived proteins, such as GFP conjugated with destabilization sequence, β-catenin and HIF1a, that are subjected to rapid ubiquitylation and degradation by proteasomes. Accordingly, β-catenin- and HIF1a-mediated gene expression are induced in the cFLIP-L-expressing cells. Exogenously expressed cFLIP-L accumulates in aggregates at the peri-nuclear region in the cells, and the cFLIP-L aggregates are refractory to solubilization. Like exogenously expressed cFLIP-L, the endogenous cFLIP in A549 lung cancer cells displays particulate distribution in the cells, and more than 60% of cFLIP-L is refractory to solubilization. Downregulation of cFLIP in A549 cells by RNA-mediated interference reduced β-catenin- and HIF1α-mediated gene expression. These results suggest that cFLIP-L is prone to aggregate and impairs UPS function, which could be involved in the pathological function of cFLIP-L expressed in certain cancer cells.
2. cFLIP-L contains a nuclear localization signal (NLS) on its carboxy terminus, and it shuttles between cytosol and nuclei. cFLIP NLS mutants and cFLIP lacking carboxy terminal 42 as predominantly localize to cytosol, and increase the accumulation of β-catenin but not enhance Wnt signaling. cFLIP-L conjugated with authentic nuclear export signal (NES) also localizes to cytosol and has lost the activity to enhance Wnt signaling. These results suggest that cFLIP-L in nuclei plays a role in Wnt signaling.
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