MK2不同突变体构建、表达及细胞内定位探究.docVIP

MK2不同突变体构建、表达及细胞内定位探究 　 作者：魏洁 龚小卫 明小燕 王旭 王达安 邓 鹏 姜勇 【摘要】 目的： 为了研究MAPK激活蛋白激酶2 (MAPK-activated protein kinase 2, MK2)细胞内定位与其功能的关系，构建了MK2不同突变体的绿色荧光蛋白融合表达载体并观察这些突变体在细胞内的定位情况。 方法： 将克隆在绿色荧光蛋白载体pEGFP-C2上的野生型MK2(WT)通过定点突变技术构建其无活性突变体MK2(320A)和活性突变体MK2(320E)，转染NIH3T3细胞，并在荧光显微镜下观察其细胞内定位情况。结果： 各种MK2不同突变体经测序鉴定正确后，在NIH3T3细胞中得到高量表达。融合蛋白发出的绿色荧光表明，在静息状态下，MK2(WT)主要分布于细胞核内，MK2(320E)主要分布于细胞质内，而MK2(320A)在细胞核和细胞质中都有分布。在受到紫外线（UV）刺激后，MK2(WT)和MK2(320A)移位出核，而MK2(320E)的细胞内定位则没有发生明显的改变。结论： 成功构建了绿色荧光蛋白标记的MK2不同突变体并在真核细胞中进行了表达，观察到不同突变体对MK2细胞内定位的影响。 【关键词】 蛋白激酶类； 突变； 基因表达； 细胞内定位； p38丝裂原活化蛋白激酶； MAPK激活蛋白激酶2 ［Abstract］ Objective: To study the intracellular localization of MAPK-activated protein kinase 2 (MAPKAPK2/MK2) and its relation with the function. Methods: Eukaryotic cell expression vectors of green fluorescent protein fused with MAPK-activated protein kinase 2 (MK2) mutants were constructed. Wild-type MK2 cloned in the green fluorescent protein vector, pEGFP-C2, was site-mutated to produce dominant negative mutant MK2(320A) and constitutively active mutant MK2(320E). Then the mutants were transfected into NIH3T3 cells, and the intracellular localizations of these mutants were observed with fluorescence microscope. Results: After identification by sequencing, the GFP-fused MK2 mutants were highly expressed in NIH3T3 cells. The green fluorescence of fusion proteins showed that in resting cells, MK2(WT) were mainly in the nuclei, and MK2 (320E) was mainly in the cytosol, while MK2(320A) dispersed all over the cell. Once the cells were stimulated by UV, MK2 (WT) translocated into the cytosol, while the intracellular localizations of MK2 (320E) showed no stress-dependent redistribution. Conclusions: Expression vectors with different mutants of MK2 fused with GFP are successfully constructed and highly expressed in eukaryotic cells. Some functional point mutations have effects on the intracellular localizations of MK2. ［Key words］ protein kinases; mutation; gene expression; intracellular localizat