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Molecular mechanism of extracellular dependent nuclear import of STAT1

STAT1细胞外依赖性核输入的分子机制

基本信息

批准号：
08458229
负责人：
YONEDA Yoshihiro
金额：
$ 5.38万
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：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08458229/
关键词：
nuclear pore-nuclear protein nuclear localization signal signal transduction nuclear pore complex a small G protein transcription factor

项目摘要

Thus far, extensive studies have been mainly concentrated on developing an understanding of the molecular mechanism of unclear import of SV40 T-NLS (nuclear localization signal) containing substrate, and as a result, many significant findings have been obtained. The SV40 T-antigen is a good candidate for karyophilic proteins which are transported constitutively and immediately after synthesis on free ribosomes in the cytoplasm. However, how proteins, which preexist in the cytoplasm at steady state, migrate into the nucleus in response to extracellular signal, remains unknown. To answer this question, we used a transcription factor, aSTAT (signal transducers and activators of transcription) protein, as a model substrate. In response to interferon-gamma (IFN-gamma), STAT1 is tyrosine phosphorylated and translocates to the nucleus. In this study, we found that tyrosine-phosphorylated STAT1 associated with the beta subunit of the nuclear pore-targeting complex via the NPI-1 family, but not the Rch 1 family, of the alpha subunit. Antibodies against NPI-1 or beta subunit inhibited the IFN-gamma-dependent nuclear import of STAT1 in living cells. Solution binding assays with deletion mutants of NPI-1 showed that the STAT1-binding domain of NPI-1 was located in the carboxy-terminal region, which is clearly distinct from the SV40 T-NLS.These results indicate that the extracellular signal-dependent nuclear transport of STAT1 is mediated by NPI-1, but not Rch1, in conjunction with beta subunit. Moreover, we found that nuclear import of STAT1 was suppressed by microinjection of the antibody against a small GTPase, Ran, and two mutant Ran proteins, one defective in GTP hydrolysis (G19V) and the other with little or no binding to GTP (T24N), both of which are known to act as dominant negative inhibitors of nuclear import. These results indicate that the conditional nuclear import of STAT1 requires Ran.

迄今为止，广泛的研究主要集中在对含有SV 40 T-NLS（核定位信号）的底物的不清楚输入的分子机制的理解上，结果，已经获得了许多重要的发现。SV 40 T抗原是嗜核蛋白的良好候选者，这些蛋白在细胞质中的游离核糖体上合成后立即组成性转运。然而，如何蛋白质，在稳定状态下存在于细胞质中，迁移到细胞核中的细胞外信号的反应，仍然是未知的。为了回答这个问题，我们使用转录因子aSTAT（信号转导和转录激活因子）蛋白作为模型底物。作为对干扰素-γ（IFN-γ）的应答，STAT 1被酪氨酸磷酸化并易位到细胞核。在这项研究中，我们发现酪氨酸磷酸化的STAT 1通过α亚基的NPI-1家族而不是Rch 1家族与核孔靶向复合物的β亚基相关。针对NPI-1或β亚基的抗体抑制活细胞中STAT 1的IFN-γ依赖性核输入。NPI-1缺失突变体的溶液结合试验表明，NPI-1的STAT 1结合结构域位于羧基末端区域，与SV 40 T-NLS明显不同。这些结果表明，STAT 1的细胞外信号依赖性核转运是由NPI-1介导的，而不是Rch 1。与β亚基一起。此外，我们发现，STAT 1的核输入被抑制的抗体对一个小的GTP酶，Ran，和两个突变的Ran蛋白，一个有缺陷的GTP水解（G19 V）和其他很少或没有绑定到GTP（T24 N），这两个都是已知的作为显性负抑制剂的核输入。这些结果表明，STAT 1的条件性核输入需要Ran。