丝氨酸526位点在MEKK3介导的IL-1信号途径中的作用

Ser 526 of MEKK3 is essential for MEKK3 mediated IL-1 signaling

目的:确定MEKK3分子中介导IL-1信号途径的关键性氨基酸。方法:对哺乳动物MEKK3活性环526位点的丝氨酸进行人工诱变后,用瞬时转染的方法分别将526A基因单独及与TRAF6基因同时导入GES-1细胞中;用免疫印迹、NF-kB荧光素酶报告基因测定及免疫沉淀实验检测了526A突变体的表达水平、对IL-1及TRAF6介导的NF-kB基因的表达及MEKK3与TRAF6相互作用的影响。结果:通过与野生型MEKK3的对比,观察到526A不但阻断了IL-1及TRAF6介导的依赖于MEKK3的NF-kB基因的激活,同时阻断了TRAF6与MEKK3的相互作用。结论:526位点的丝氨酸在MEKK3介导的IL-1信号途径中起关键性的作用,是不可缺少的关键性氨基酸。

Objective： AIM： To determine the amino acid residue which is essential for MEKK3 mediated IL - 1 signaling. Method： Ser residue at position 526 of the activation loop of MEKK3 was replaced with ahnine （ Ser 526 to Ala mutant, 526A） by using site - directed mutagenesis. Wild type- MEKK3, and 526A were then transiently transfected or cotransfected with TRAF6 expression vector into GES- 1 ceils ; then, the 526A expression level and its effect on IL- 1 and TRAF6 mediated MEKK3 - dependent NF-κB reporter gene expression, and MEKK3 interaction with TRAF6 were detected by Wastem - blot, NF-κB luciferase assay, and immunoprecipitation assays, respectively. Results： 526A blocked IL - 1 and TRAF6 mediated NF-κB reporter gene expression, and interfered the interaction of MEKK3 with TRAF6. Conclusion： The crucial Ser residue at position 526 of the activation loop of MEKK3 plays essential role in the MEKK3 mediated IL- 1 signaling.