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Analysis of neuron-specific nuclear protein transport by using CaM kinase IV as a substrate.

使用 CaM 激酶 IV 作为底物分析神经元特异性核蛋白转运。

基本信息

批准号：
10480200
负责人：
YONEDA Yoshihiro
金额：
$ 8.19万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
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-10480200/
关键词：
nuclear protein import nuclear localization signal neuron CaM kinase semi-intact cell importin cell nucleus

项目摘要

In order to know the molecular mechanism of neuron-specific nuclear protein transport, we used Ca^<2+>/calmodulin-dependent protein kinase type IV (CaM kinase IV) as a model substrate. CaM kinase IV is known to be localized in the nucleus of neuronal cells, while throughout the cytoplasm of non-neuronal cells such as parathyroid cells. Further, the nuclear localization signal of CaM kinase IV has not yet been identified. When the recombinant CaM kinase IV proteins were injected into the cytoplasm of HeLa cells (human cervical cancer cells) or COS7 cells (African green monkey kidney cells), they migrated into the nuclei of COS7 cells but not those of HeLa cells, suggesting that CaM kinase IV is translocated from cytoplasm to the nucleus in a cell-type specific manner. Next, we tried to identify factors required for the nuclear import of CaM kinase IV by using a permeabilized cell-free system. It was demonstrated that brain extracts support the nuclear import of CaM kinase IV more efficiently than Ehrlich ascites tumor cell extracts. Moreover, the import was not inhibited by the addition of IBB (importin β-binding) domain of importin α and the N-terminal NPC (nuclear pore complex)-binding portion of importin β, meaning that the nuclear migration of CaM kinase IV is not mediated by conventional importin α/β pathway. More interestingly, it was found that the nuclear import mediated by brain extracts was not inhibited by the treatment with wheat germ agglutinin, whereas was that by Ehrlich ascites tumor cell extracts, suggesting that CaM kinase IV may be transported into the nucleus through at least two independent pathways. In the near future, factors involved in these reactions should be isolated and characterized.

为了了解神经元特异性核蛋白转运的分子机制，我们使用Ca^2+/钙调蛋白依赖性蛋白激酶IV型(CaM激酶IV)作为模型底物。已知 CaM 激酶 IV 位于神经元细胞的细胞核中，而遍及非神经元细胞（例如甲状旁腺细胞）的细胞质。此外，CaM 激酶 IV 的核定位信号尚未确定。当重组CaM激酶IV蛋白被注射到HeLa细胞（人宫颈癌细胞）或COS7细胞（非洲绿猴肾细胞）的细胞质中时，它们迁移到COS7细胞的细胞核中，而不是HeLa细胞的细胞核中，这表明CaM激酶IV以细胞类型特异性的方式从细胞质易位到细胞核。接下来，我们尝试使用透化无细胞系统来鉴定 CaM 激酶 IV 核输入所需的因子。研究表明，脑提取物比艾氏腹水肿瘤细胞提取物更有效地支持 CaM 激酶 IV 的核输入。此外，导入蛋白α的IBB（导入蛋白β结合）结构域和导入蛋白β的N末端NPC（核孔复合物）结合部分的添加并没有抑制导入，这意味着CaM激酶IV的核迁移不是由传统的导入蛋白α/β途径介导的。更有趣的是，我们发现，麦芽凝集素处理不会抑制脑提取物介导的核输入，而艾氏腹水肿瘤细胞提取物则不会抑制，这表明CaM激酶IV可能通过至少两条独立的途径转运到细胞核中。在不久的将来，应该对参与这些反应的因素进行分离和表征。