A-Raf-S214C突变体的构建、表达及其对MAPK信号r通路的影响

摘要

为了观察A-Raf的突变体A-Raf-S214C对MAPK(mitogen-activated protein kinase,丝裂原活化蛋白激酶)信号通路的影响,并探讨其诱发肺癌和朗格汉斯增生症的可能分子机制.通过定点突变构建pcDNA5-FRT-TO-A-Raf-S214C突变质粒,并利用该质粒构建稳定表达A-Raf-S214C突变体的HEK293细胞系,之后通过检测下游ERK磷酸化程度,确定该突变体对MAPK信号通路的影响.结果显示,通过实验成功构建了稳定表达A-Raf-S214C突变体的细胞系,在不同浓度的多西环素诱导下,其表达量有所不同.与野生型相比,A-Raf-S214C突变体可明显增强MAPK信号通路中下游信号分子ERK的磷酸化.结果表明A-Raf-S214C突变体是A-Raf激酶的激活突变体,可以导致MAPK信号通路下游信号分子ERK磷酸化明显增强.%This study is to observe the influence of A-Raf mutant A-Raf-S214C on MAPK(mitogen-activated protein kinase)signal pathway and to explore the molecular mechanism of lung cancer and Langerhans cell histiocytosis caused by A-Raf-S214C. The pcDNA5-FRT-TO-A-Raf-S214C plasmid was constructed through point mutation,and then it was used to establish HEK293 cell lines stably expressing A-Raf-S214C. Further,the influence of A-Raf-S214C on MAPK signaling pathway was determined by detecting the level of ERK phosphorylation. Results showed that HEK293 cell line with stable expression of A-Raf-S214C was successfully constructed,and A-Raf-S214C expression levels increased under induction with increasing concentrations of doxycycline. In comparison with wild type A-Raf,mutant A-Raf-S214C significantly enhanced the phosphorylation level of ERK . Conclusively,A-Raf-S214C mutant is an activating mutant of A-Raf kinase, resulting in significantly increased phosphorylation level of ERK,a downstream signaling molecule in MAPK pathway.