Gus Yael, Karni Rotem, Levitzki Alexander
The Hebrew University, Department of Biological Chemistry, Silberman Institute, Givat Ram, Jerusalem 91904, Israel.
FEBS J. 2007 Jun;274(11):2815-31. doi: 10.1111/j.1742-4658.2007.05815.x. Epub 2007 Apr 25.
We performed a functional genetic screen to find novel antiapoptotic genes that are under the regulation of the oncoprotein c-Src. Several clones were identified, including subunit S5a of the 26S proteasome. We found that S5a rescued Saos-2 cells from apoptosis induced by Src inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1). S5a mRNA and protein levels were downregulated as a result of Src inhibition, either by siRNA or PP1. In cell lines that possess high activity of Src S5a levels were elevated. Cloning of the S5a promoter region showed that S5a transcription responds to several stimuli. Analysis of the promoter sequence revealed a binding site for Tcf/Lef-1 transcription factor. Indeed, beta-catenin significantly induced transcription from the S5a promoter, whereas EMSA studies showed that Lef-1 binds the S5a promoter-binding site. Furthermore, we also found that PP1 and LY294002, but not PD98059 inhibit the S5a promoter activity. These results suggest that S5a is regulated during apoptosis at the transcriptional level and that S5a upregulation by antiapoptotic signals can contribute to cell survival.
我们进行了一项功能基因筛选，以寻找受癌蛋白c-Src调控的新型抗凋亡基因。鉴定出了几个克隆，包括26S蛋白酶体的亚基S5a。我们发现S5a可使Saos-2细胞免受Src抑制剂4-氨基-5-（4-甲基苯基）-7-（叔丁基）吡唑并[3,4-d]嘧啶（PP1）诱导的凋亡。由于通过siRNA或PP1抑制Src，S5a的mRNA和蛋白质水平下调。在具有高Src活性的细胞系中，S5a水平升高。S5a启动子区域的克隆表明S5a转录对多种刺激有反应。对启动子序列的分析揭示了Tcf/Lef-1转录因子的结合位点。实际上，β-连环蛋白可显著诱导S5a启动子的转录，而电泳迁移率变动分析（EMSA）研究表明Lef-1与S5a启动子结合位点结合。此外，我们还发现PP1和LY294002可抑制S5a启动子活性，但PD98059则不能。这些结果表明，S5a在凋亡过程中在转录水平受到调控，抗凋亡信号上调S5a有助于细胞存活。
