| Summary: | 碩士 === 長庚大學 === 基礎醫學研究所 === 94 === Nucleophosmin B23 (NPM/B23), a nucleolar phosphoprotein, is more abundant in cancer cells than in normal cells. NPM/B23 plays a potential role as a positive regulator of cell proliferation and regulated cell growth. Previous study found Gadd45α nuclear localization is mediated through a NPM/B23 chaperone and is required for its role in the control of cell cycle progression. Here we show that NPM/B23 over-expression can up-regulate Gadd45α promoter activity and RNA expression in HeLa Tet-Off cells. These data suggest that NPM/B23 plays a critical role in transcriptional regulation of Gadd45α gene expression. To further study how NPM/B23 phosphorylation regulates Gadd45α promoter activity, we constructed five phosphorylation site point mutated NPM/B23 expression clones (S125A, T199A, S227A, T234A/T237A, S227A/ T234A/T237A). The results exhibit wild type FLAG-B23 had ability to induce Gadd45α promoter activity up to 4 fold. However, five phosphorylation site point mutated clones all bear lower activity ranging from 2.5 fold to 3.5 fold.
To determine whether NPM/B23 associate with Egr-1, immunoprecipitation experiment was carried out. Egr-1 protein was detected in the NPM/B23 immunoprecipitates, and no Egr-1 protein was detected in the negative controls. The results showed that B23 and Egr-1 made up a complex in HL-60 cells. Moreover, the binding of Egr-1 to the Gadd45 promoter are increased by NPM/B23 overexpression and UV treatment. Our study demonstrates NPM/B23 may regulate Gadd45α promoter through interacting with Egr-1 and enhancing its cis-element binding.
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