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STAT4的DNA结合域中395~416位氨基酸残基序列参与IL-12介导的STAT4入核

黄玉梅, 文亚平, 李轩岸, 袁媛, 罗奇志, 黎明

中南大学湘雅医学院1基础医学院免疫学教研室；临床医学五年制07级教改班，长沙 410078

摘要

本研究旨在探讨信号转导和转录活化蛋白4 (signal transducer and activator of transcription 4, STAT4)受白介素-12 (interleukin-12, IL-12)刺激后移位入细胞核的机制。对STATs家族成员进行同源性比对分析结果显示，STAT4的DNA结合域的395~416位氨基酸残基序列可能具有二聚体特异性核定位信号(dimer-specific nuclear localization signal, dsNLS)功能。有鉴于此，本研究以pEGFP-C1为表达载体，分别构建了pEGFP-STAT4质粒、缺失395~416位氨基酸残基序列的缺失型STAT4质粒(pEGFP-STAT4-Del)、将SV40大T抗原上经典的NLS核酸序列插入表达载体的阳性对照质粒(pEGFP-NLS)和将缺失型STAT4插入pEGFP-NLS的pEGFP-NLS-STAT4-Del质粒。将这些质粒瞬时转染宫颈癌腺癌细胞系Caski细胞，经过IL-12刺激，发现野生型STAT4移位入细胞核，而缺失型STAT4分布在细胞浆中；进一步用leptomycin B处理，IL-12再刺激后野生型STAT4被滞留于细胞核中，而缺失型STAT4仍然分布在胞浆中；将NLS插入缺失型STAT4，能恢复缺失型STAT4的核移位。以上结果说明野生型STAT4在IL-12刺激下能移位入细胞核，其入核机制是在其DNA结合域的395~416位氨基酸残基序列具有dsNLS功能，能介导活化的STAT4移位入细胞核。

关键词： 核移位; 核定位信号; 信号转导和转录活化蛋白4

分类号：R3

[Amino acids 395–416 in DNA binding domain of STAT4 is involved in IL-12-induced nuclear import of STAT4.] [Article in Chinese]

HUANG Yu-Mei, WEN Ya-Ping, LI Xuan-An, YUAN Yuan, LUO Qi-Zhi, LI Ming

1Department of Immunology, Basic Medical College； Teaching Innovation Class of Clinical Medicine Five-year Program 07 Grade, Xiangya Medical College, Central South University, Changsha 410078, China

Abstract

The purpose of the present study is to explore the mechanism of IL-12-induced nuclear import of signal transducer and activator of transcription 4 (STAT4). Assayed by analyses of homology alignment of STATs, amino acids 395-416 in DNA binding domain was found to be a potential dimer-specific nuclear localization signal (dsNLS) of STAT4. Therefore, several plasmids were constructed. Wild-type STAT4 was inserted into the SalI and BamHI sites of pEGFP-C1 for the construction of plasmid pEGFP-STAT4. The DNA fragment of STAT4 with the deletion of amino acids 395-416 was amplified by RCR and introduced into the SalI and BamHI sites of pEGFP-C1 which was named pEGFP-STAT4-Del. Classic NLS DNA sequence of SV40 T antigen was inserted into the XhoI and HindIII sites of pEGFP-C1. This plasmid was named as pEGFP-NLS and used as a positive control. Plasmid pEGFP-NLS-STAT4-Del was constructed by inserting STAT4-Del into SalI and BamHI sites of pEGFP-NLS. These plasmids were transiently transfected into Caski cells, respectively. The results showed that, after these transfected cells were stimulated by IL-12, wild type STAT4 existed in the cytoplasm at 0 min, and was predominantly localized to the nucleus at 45 min, and distributed in both cytoplasm and nucleus at 60 min, suggesting that STAT4 translocates from cytoplasm into nucleus and finally re-entries into the cytoplasm during the stimulation of IL-12. However, deletion mutant of STAT4 was arrested in cytoplasm during the IL-12 stimulation. Leptomycin B, which specifically blocks protein export from nucleus into cytoplasm, was used to further demonstrate whether STAT4-Del is transferred into nucleus even with stimulation of IL-12. After the transfected cells were pre-treated by leptomycin B, the wild type STAT4 was mainly localized in nucleus after the IL-12 stimulation, suggesting that STAT4 was translocated from cytoplasm into nucleus by the stimulation of IL-12. On the other hand, the deletion mutant of STAT4 distributed in cytoplasm throughout, implying that the mutant STAT4 lacking of amino acids 395-416 cannot move into nucleus. Furthermore, the insertion of classic NLS into EGFP-STAT4-Del restored nuclear import of STAT4-Del. These results suggest the amino acids 395-416 is a dsNLS mediating IL-12-stimulated nuclear import of activated STAT4.

Key words: nuclear import; nuclear localization signal; signal transducer and activator of transcription 4

收稿日期：2012-04-26　　录用日期：2012-07-02

通讯作者：黎明　　E-mail: liming@csu.edu.cn

引用本文：

黄玉梅, 文亚平, 李轩岸, 袁媛, 罗奇志, 黎明. STAT4的DNA结合域中395~416位氨基酸残基序列参与IL-12介导的STAT4入核[J]. 生理学报 2012; 64 (4): 372-378.

HUANG Yu-Mei, WEN Ya-Ping, LI Xuan-An, YUAN Yuan, LUO Qi-Zhi, LI Ming. [Amino acids 395–416 in DNA binding domain of STAT4 is involved in IL-12-induced nuclear import of STAT4.] [Article in Chinese]. Acta Physiol Sin 2012; 64 (4): 372-378 (in Chinese with English abstract).