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 +> V（CAM激酶IV）作为模型底物。当将重组的CAM激酶IV蛋白注入Hela细胞（人宫颈癌）或COS7细胞的细胞质时肾细胞）表明，CAM激酶IV以细胞类型的特定方式从细胞质转移到细胞核，我们试图通过使用通透性细胞 - 弗雷伊系统来确定CAM激酶IV的核进口所需的因素。大脑提取物支持支持CAM激酶IV的支持比Ehrlich腹水肿瘤细胞提取物更多的支持更多的IV，此外，通过添加IBB（importinβ结合）importinα和N-N-terminal NPC的IBB（importinβ结合）结构抑制了进口（核N-末端NPC））部分β，这意味着CAM激酶IV的核迁移不是由常规进口途径介导的，因此发现脑提取物介导的核能不是通过胚芽凝集素的治疗而介导的。屁股是肿瘤细胞提取物，可以将CAM激酶IV转运为独立途径。