Ras oncogene induced protein SUMOylation changes

通过RNAi合成致死筛选，我们已经鉴定了Ras突变癌细胞生存所需的蛋白SUMO化途径。在这个项目中，我们的目标是解决以下问题：1。Ras突变抑制SUMO化途径的机制; 2.哪些细胞蛋白质在Ras突变细胞中被差异SUMO化; 3.在Ras突变的背景下，这些细胞中SUMO化状态的变化如何影响它们的功能。靶向SUMO E1和E2连接酶的shRNA。2.表达6x His标记蛋白的稳定细胞系。3.我们已经验证了靶向SUMO E1和E2连接酶的shRNA。我们还产生了稳定表达His标记的SUMO 1或SUMO 2蛋白的细胞系。我们已经进行了蛋白质组学筛选，并确定了一些候选蛋白质，这些蛋白质在Ras WT和突变细胞之间差异SUMO化。

BACKGROUND Through a RNAi synthetic lethal screen we have identified the protein SUMOylation pathway to be required for the viability of Ras mutant cancer cells. PURPOSE In this project we aim to address the following questions: 1. the mechanism by which the inhibition of SUMOylation pathway is synthetically lethal with Ras mutation; 2. which cellular proteins are differentially SUMOylated in Ras mutant cells; 3. How the changes in SUMOylation status in these cells affect their function in the context of Ras mutation SIGNIFICANT MATERIALS AND METHODS 1. shRNAs that target the SUMO E1 and E2 ligases. 2. Stable cell lines that express 6x His tagged proteins. 3. mass-spectrometry protocol for protein ID FY2010 ACCOMPLISHMENT We have validated shRNAs that target the SUMO E1 and E2 ligases. We have also generated cell lines stably expressing His-tagged SUMO1 or SUMO2 proteins. We have carried out a proteomics screen and identified a number of candidate proteins that are differentially SUMOylated between Ras WT and mutant cells.