Dynamics of nuclear shuttling with MAP kinase

丝裂原活化蛋白（MAP）激酶级联由MAP激酶（MAP; ERK 2）及其激活剂MAP激酶（MAPKK; MEK）组成。然而，ERK 2的激活机制尚未在细胞中确定。在这里，我们使用荧光标记的ERK 2和MEK来检查ERK 2和MEK在活的大鼠嗜碱性白血病（RBL-2 H3）细胞中的定位。ERK 2在静息状态下主要定位于细胞质，而在IgE受体连接后则定位于细胞核。ERK 2的输入在6-7 min达到最大值，随后ERK 2从细胞核输出。MEK主要存在于细胞质中，连接IgE受体后，未检测到明显的MEK易位。这些数据表明，在RBL细胞中，持续的钙增加是ERK 2最佳易位到细胞核中所必需的。然而，对ERK 2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调节细胞的增殖。 关于我们 ERK 2的核穿梭，而MEK主要保留在细胞质中。用细胞松弛素D和Latrunculin A（肌动蛋白聚合的抑制剂）预处理RBL-2 H3细胞，延长了抗原刺激后ERK 2的核转位。Western blotting分析表明，这些药物增强了ERK 2和MEK的磷酸化抗原刺激后。相反，微管蛋白聚合抑制剂诺考达唑既不引起ERK 2核转位的延长，也不引起ERK 2和MEK磷酸化的增强。通过加入过量的单价半抗原使交联的受体解离，即使在用细胞松弛素D预处理的细胞中也停止了ERK 2的易位。总而言之，这些结果表明肌动蛋白聚合通过调节IgE受体交联来影响ERK 2的核穿梭。这些结果为研究ERK 2和MEK在Ag刺激后RBL-2 H3细胞核穿梭中的动力学提供了新的见解。少

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