Molecular mechanism of extracellular dependent nuclear import of STAT1

到目前为止，大量的研究主要集中在对含有底物的SV40 T-NLS （nuclear localization signal，核定位信号）不明确输入的分子机制的理解上，并获得了许多重要的发现。SV40 t抗原是一个很好的候选亲核蛋白，在合成后立即在细胞质中的自由核糖体上运输。然而，稳态存在于细胞质中的蛋白质是如何响应胞外信号迁移到细胞核中的，目前尚不清楚。为了回答这个问题，我们使用了一个转录因子，aSTAT（信号转导和转录激活因子）蛋白作为模型底物。在干扰素γ (ifn - γ)的作用下，STAT1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STAT1通过NPI-1家族与核孔靶向复合物的β亚基相关，而不是通过α亚基的Rch -1家族。抗NPI-1或β亚基的抗体抑制活细胞中ifn - γ依赖性STAT1的核输入。NPI-1缺失突变体的溶液结合实验表明，NPI-1的stat1结合域位于羧基端，这与SV40 T-NLS明显不同。这些结果表明STAT1的细胞外信号依赖核转运是由NPI-1介导的，而不是Rch1与β亚基联合介导的。此外，我们发现通过微量注射针对小GTP酶Ran和两个突变Ran蛋白的抗体，STAT1的核输入被抑制，其中一个在GTP水解中有缺陷（G19V），另一个很少或不与GTP结合（T24N），这两种蛋白都是已知的核输入的主要阴性抑制剂。这些结果表明STAT1的条件核导入需要Ran。

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.