A-Raf-S214C突变体的构建、表达及其对MAPK信号通路的影响

doi:10.13560/j.cnki.biotech.bull.1985.2017-0445

生物技术通报 ›› 2017, Vol. 33 ›› Issue (12): 185-190.doi: 10.13560/j.cnki.biotech.bull.1985.2017-0445

• 研究报告 • 上一篇

A-Raf-S214C突变体的构建、表达及其对MAPK信号通路的影响

李安毅, 杨洋, 刘莹, 郭晓汐, 郝倩, 徐天瑞, 安输

昆明理工大学生命科学与技术学院云南省高校靶点药物筛选与利用重点实验室，昆明 650500

收稿日期:2017-05-30 出版日期:2017-12-25 发布日期:2017-12-21
作者简介:李安毅，女，硕士研究生，研究方向：细胞信号传导；E-mail：82291898@qq.com
基金资助:
国家自然科学基金项目（81460253，U1302225），云南省人才培养项目（KK23201426014）

Construction and Expression of Mutant A-Raf-S214C and Studies on Its Role in MAPK Signaling Pathway

LI An-yi, YANG Yang, LIU Ying, GUO Xiao-xi, HAO Qian, XU Tian-rui, AN Shu

University Based Provincial Key Laboratory of Screening and Utilization of Targeted Drugs，Faculty of Life Science and Technology，Kunming University of Science and Technology，Kunming 650500

Received:2017-05-30 Published:2017-12-25 Online:2017-12-21

摘要/Abstract

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。

关键词: A-Raf激酶, MAPK信号通路, 信号传导

Abstract: 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.

Key words: A-Raf kinase, MAPK signaling pathway, signal transduction

李安毅, 杨洋, 刘莹, 郭晓汐, 郝倩, 徐天瑞, 安输. A-Raf-S214C突变体的构建、表达及其对MAPK信号通路的影响[J]. 生物技术通报, 2017, 33(12): 185-190.

LI An-yi, YANG Yang, LIU Ying, GUO Xiao-xi, HAO Qian, XU Tian-rui, AN Shu. Construction and Expression of Mutant A-Raf-S214C and Studies on Its Role in MAPK Signaling Pathway[J]. Biotechnology Bulletin, 2017, 33(12): 185-190.

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A-Raf-S214C突变体的构建、表达及其对MAPK信号通路的影响

Construction and Expression of Mutant A-Raf-S214C and Studies on Its Role in MAPK Signaling Pathway

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