Identification and Analysis of proteins which intreact with CRM1

与 CRM1 相互作用的蛋白质的鉴定和分析

• 批准号: 10680668
• 负责人: FUKUDA Makoto
• 金额: $ 2.43万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680668/
• 关键词: Nuclear Sport; CRM1; NES; サイクリンB

CRM1, an evolutionarily conserved protein, is shown to be a receptor for leucine-rich nuclear export signal (NES)-dependent protein transport, but its role in mRNA export has not been established in higher eukaryotes. We have found that treatment of mammalian cells with leptomycin B (LMB), a specific inhibitor of CRM1, induces nuclear accumulation of endogenous mRNA probably due to the inhibition of its export. In fission yeast, nuclear accumulation of mRNA also occurred in cells treated with LMB or in a temperature-sensitive crm1 mutant at a restrictive temperature. A synthetic mRNA that was injected into the nucleus of mammalian cultured cells was exported from the nucleus within 5 h. This export was inhibited by wheat germ agglutinin or at 40C. Importantly, this mRNA export was inhibited by LMB or by an excess amount of the NES peptideconjugates. These results suggest that CRM1 is involved in mRNA export in eukaryotic cells.We previously demonstrated that MAPKK(MAPK kinase),a direct activator for MAPK, as an NES and acts as a cytoplasmic anchor of MAPK. In this project, we revealed the regulatory mechanisms of the dissociation and/or the association of the MAPKK/MAPK complex. Tyrosine(Tyr190 in Xenopus MAPK)phosphorylation of MAPK by MAPKK is necessary and sufficient for the dissociation of the complex.

CRM1 是一种进化上保守的蛋白质，被证明是富含亮氨酸的核输出信号 (NES) 依赖性蛋白质转运的受体，但其在 mRNA 输出中的作用尚未在高等真核生物中确定。我们发现，用 CRM1 的特异性抑制剂瘦霉素 B (LMB) 处理哺乳动物细胞，可能会由于抑制其输出而诱导内源 mRNA 的核积累。在裂殖酵母中，用 LMB 处理的细胞或在限制温度下的温度敏感 crm1 突变体中也发生 mRNA 的核积累。将合成的 mRNA 注射到哺乳动物培养细胞的细胞核中，5 小时内即可从细胞核中输出。这种输出受到麦芽凝集素或 40°C 的抑制。重要的是，这种 mRNA 输出受到 LMB 或过量 NES 肽缀合物的抑制。这些结果表明CRM1参与真核细胞的mRNA输出。我们之前证明MAPKK（MAPK激酶）是MAPK的直接激活剂，作为NES并充当MAPK的细胞质锚。在这个项目中，我们揭示了 MAPKK/MAPK 复合物解离和/或缔合的调节机制。 MAPKK 对 MAPK 的酪氨酸（爪蟾 MAPK 中的 Tyr190）磷酸化对于复合物的解离是必要且充分的。

项目成果

Watanabe,M.,Fukuda,M.,Yoshida,M.,Yanagida,M.and Nishida,E.: "Inovolvement of CRM1,a nuclear export receptor,in mRNA export in mammalian cells and fission yeast"Genes Cells.. 4. 291-297 (1999)

渡边，M.，福田，M.，吉田，M.，柳田，M.和西田，E.：“CRM1，一种核输出受体，在哺乳动物细胞和裂殖酵母的 mRNA 输出中的参与”基因细胞.. 4

Adachi,M., Fukuda,M. et al.: "Two co-existing mechanisms for nuclear import of MAP Kinase : passive diffusion of a monomer and active transport of a dimer"EMBO J.. 18. 5437-5358 (1999)

安达，M.，福田，M.