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Dynamics of nuclear shuttling with MAP kinase

MAP 激酶的核穿梭动力学

基本信息

批准号：
12480202
负责人：
NAKANISHI Mamoru
金额：
$ 4.42万
依托单位：
Nagoya City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12480202/
关键词：
MAP kinase MAP kinase cascade GFP Confocal microscope Fluorescent protein Raf-1 Nuclear shuttling Basophile キメラ蛋白質核シヤトル

项目摘要

The mitogen-activated protein (MAP) kinase cascade consists of MAP kinase (MAP; ERK2) and its activator, MAP kinase (MAPKK; MEK). However, the mechanisms for activation of ERK2 have not been defined yet in cells. Here, we used fluorescent-tagged ERK2 and MEK to examine the localization of ERK2 and MEK in living rat basophilic leukemia (RBL-2H3) cells. ERK2 was mainly in the cytoplasm in resting cells, but translocated into the nucleus after the ligation of IgE receptors. The import of ERK2 reached the maximum at 6-7 min, and then the imported ERK2 was exported from the nucleus. MEK mainly resided in the cytoplasm, and no significant MEK translocation was detected after ligation of IgE receptors. The data suggested that the sustained calcium increase was required for the optimal translocation of ERK2 into the nucleus in RBL cells. However, analysis of the dynamics of ERK2 and MEK suggested that both of them rapidly shuttle between the cytoplasm and the nucleus, and that MEK regulate the … More nuclear shuttling of ERK2 while MEK remain mainly in the cytoplasm. Pretreatment of RBL-2H3 cells with cytochalasin D and Latrunculin A, inhibitors of actin polymerization, prolonged the nuclear translocation of ERK2 after antigen stimulation. Western blotting analysis revealed that these drugs enhanced the phosphorylation of both ERK2 and MEK after antigen stimuation. To the contrary, nocodazole, an inhibitor of tubulin polymerization, caused neither prolongation of nuclear translocation of ERK2 nor enhancement of phosphorylation of ERK2 and MEK. Dissociation of cross-linked receptors by the addition of excess amount of monovalent hapten halted translocation of ERK2 even in cells pretreated with cytochalasin D. Taken together, these results indicate that actin polymerization affects the nuclear shuttling of ERK2 by the regulation of IgE receptor cross-linking. These results gave a new insight of the dynamics of ERK2 and MEK in the nuclear shuttling of RBL-2H3 cells after Ag stimulation. Less

丝裂原活化蛋白(MAP)信号通路由MAP(MAP；ERK2)及其激活物MAP(MAPKK；MEK)组成。然而，ERK2的激活机制在细胞中还没有明确。在这里，我们使用荧光标记的ERK2和MEK来检测ERK2和MEK在活的大鼠嗜碱粒细胞白血病(RBL-2H3)细胞中的定位。静息细胞中ERK2主要分布在胞浆中，而结扎IgE受体后ERK2主要分布于胞核。ERK2的输入在6-7min达到高峰，然后从胞核输出。结扎IgE受体后，MEK主要分布在胞浆内，未检测到明显的MEK易位。这些数据表明，在RBL细胞中，ERK2的最佳核内转位需要持续的钙增加。然而，对ERK2和MEK的动力学分析表明，它们都在细胞质和细胞核之间快速穿梭，并且MEK调节…ERK2有较多的核穿梭，而MEK主要留在胞浆内。在RBL-2H3细胞中，用肌动蛋白聚合抑制剂松胞素D和乳突菌素A处理后，ERK2的核转位时间延长。Western blotting分析表明，这些药物在抗原刺激后均能增强ERK2和MEK的磷酸化。相反，微管蛋白聚合的抑制剂诺可达唑既没有延长ERK2的核转位，也没有增加ERK2和MEK的磷酸化。在细胞松弛素D处理的细胞中，加入过量的单价半抗原使交联型受体解离，阻止ERK2的转位。综上所述，这些结果表明，肌动蛋白聚合通过调节IgE受体的交联性影响ERK2的核穿梭。这些结果为进一步了解抗原刺激后RBL-2H3细胞核穿梭过程中ERK2和MEK的动态变化提供了新的思路。较少