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  

Amino acids 395-416 in DNA binding domain of STAT4 is involved in IL-12-induced nuclear import of STAT4

作　　者：黄玉梅[1] 文亚平[1] 李轩岸[2] 袁媛[1] 罗奇志[1] 黎明[1] 

机构地区：[1]中南大学湘雅医学院·基础医学院免疫学教研室,长沙410078 [2]中南大学湘雅医学院,长沙410078

出　　处：《生理学报》2012年第4期372-378,共7页Acta Physiologica Sinica

基　　金：supported by the National Natural Scientific Foundation of China (No. 30771122);National Undergraduate Innovation Training Project of Ministry of Education,China (No.AE11528)

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(in-terleukin-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leptomycinB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。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 pEGFPSTAT4.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 bothcytoplasm 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

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：R341[医药卫生—基础医学]