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底物的SV40 T-NLS（核定位信号）的不清楚输入的分子机制的理解上，并因此获得了许多重要的发现。 SV40 T 抗原是亲核蛋白的良好候选者，该蛋白在细胞质中的游离核糖体上合成后立即组成型转运。然而，以稳态预先存在于细胞质中的蛋白质如何响应细胞外信号迁移到细胞核中仍然未知。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剂）蛋白作为模型底物。响应干扰素-γ (IFN-γ)，STAT1 被酪氨酸磷酸化并易位至细胞核。在这项研究中，我们发现酪氨酸磷酸化的STAT1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 亚基的抗体可抑制活细胞中 STAT1 的 IFN-γ 依赖性核输入。 NPI-1 缺失突变体的溶液结合分析表明，NPI-1 的 STAT1 结合域位于羧基末端区域，这与 SV40 T-NLS 明显不同。这些结果表明，细胞外信号依赖性STAT1 的核转运由 NPI-1（而非 Rch1）与 β 亚基结合介导。此外，我们发现，通过显微注射抗小 GTP 酶 Ran 和两种突变 Ran 蛋白的抗体，可以抑制 STAT1 的核输入，其中一种蛋白有 GTP 水解缺陷 (G19V)，另一种与 GTP 结合很少或没有结合 (T24N) ，两者都被认为是核输入的显性负抑制剂。这些结果表明STAT1的条件性核输入需要Ran。