| Project/Area Number |
12480202
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biophysics
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| Research Institution | Nagoya City University |
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Principal Investigator |
NAKANISHI Mamoru Nagoya City University, Graduate School of Pharmaceutical Sciences, Professor, 大学院・薬学研究科, 教授 (90090472)
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| Co-Investigator(Kenkyū-buntansha) |
FURUNO Tadahide Nagoya City University, Graduate School of Pharmaceutical Sciences, Assistant Professor, 大学院・薬学研究科, 講師 (80254308)
HIRASHIMA Naohide Nagoya City University, Graduate School of Pharmaceutical Sciences, Assistant Professor, 大学院・薬学研究科, 助教授 (10192296)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2001: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2000: ¥2,900,000 (Direct Cost: ¥2,900,000)
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| Keywords | 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 |
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| Research Abstract |
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
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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
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