K-ras第12密码子突变对K-ras蛋白自身活性及下游信号通路的影响

目的 探讨K-ras第12密码子突变对K-ras蛋白自身活性及其下游信号通路的影响。方法 选用本室已构建好的稳定高表达WT、G12A、G12C、G12D、G12R、G12S和G12V型K-ras蛋白的cos7同源细胞株作为研究对象，通过GST-Raf-1 Ras结合域特异性结合Ras-GTP空间构象，免疫沉淀细胞内具有活性的K-ras蛋白，利用Western blot技术检测沉淀下来的Ras-GTP水平并进行比较分析；通过Western blot法检测K-ras下游信号通路中MER、ERK、ATK蛋白分子的磷酸化水平。结果 饥饿处理后表达野生型K-ras蛋白的cos7同源细胞株内RAS-GTP水平较低，而表达G12A、G12C、G12D、G12R、G12S和G12V型K-ras蛋白的cos7同源细胞株内RAS-GTP水平活性为WT型K-ras蛋白3.5~40倍不等；表达K-ras突变蛋白的cos7同源细胞株内MEK、ERK和AKT的磷酸化水平比表达野生型K-ras蛋白的cos7同源细胞高，差异有统计学意义（P<0.05）。结论 K-ras第12密码子突变导致K-ras蛋白自身永久活化，且不需要外源性生长因子的刺激；突变K-ras可以持续激活K-ras蛋白下游效应因子，使K-ras信号通路持久活化。

Effects of K-ras Codon 12 Mutation on the Activity of K-ras Protein and Its Down Stream Signal Pathway

Objective To explore the effects of K-ras codon 12 mutation on the activity of K-ras protein and its down stream signal pathway. Methods We chosed the cos7 isogenic cell lines that we established before,which stably expressing the WT, G12A, G12C, G12D, G12R, G12S and G12V K-ras protein,as the study object.And we used the GST-Raf-1 Ras-binding domain to specifically bind to the Ras-GTP space structure to immunoprecipitate the above wild and mutant K-ras proteins with activity. Then the levels of Ras-GTP precipitated in cos7 isogenic cell lines were detected and compared by Western Blot. The phosphorylations of MEK, ERK and ATK protein in K-ras downstream signaling pathway were further detected by Western blot. Results After starvation treatment, the level of Ras-GTP in the wildtype cos7 isogenic cell line was lower, and there were still higher levels of Ras-GTP in Kras-mutant cos7 isogenic cell lines. The phosphorylation levels of MEK, ERK and AKT in the K-ras mutants were higher than that of the cos7 cell lines expressing the wild type K-ras protein,and the difference was statistically significant(P<0.05). Conclusion K-ras codon 12 mutation leads to the permanent activation of the K-ras protein itself, which does not require exogenous growth factor''s stimulation. Mutant K-ras can continue to activate the K-ras downstream effector factors, resulting in K-ras signaling pathway''s persistent activation.