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

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

Abstract

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

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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