丝氨酸526位点在MEKK3介导的IL-1信号途径中的作用

Role of Ser 526 site in 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-κB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-κB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-κB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.

AIM： To determine the amino acid residue which is essential for MEKK3 mediated IL-1 signaling. METHODS： Ser residue at position 526 of the activation loop of MEKK3 was re- placed with alanine （ Ser 526 to Ala mutant, 526A ） by using site-directed mutagenesis. Wild type-MEKK3, and 526A were then transiently transfected or co-transfected with TRAF6 expres- sion vector into GES-1 cells ; then, the 526A expression level and its effect on IL-1 and TRAF6 mediated MEKK3-dependent NF-KB reporter gene expression, and MEKK3 interaction with TRAF6 were detected by Western-blot, NF-KB luciferase assay, and immunoprecipitation assays, respectively. RESULTS ： 526A blocked IL-1 and TRAF6 mediated NF-KB reporter gene expres- sion, 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 me- diated IL-1 signaling.