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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 aim of this study was to investigate the mechanism by which signal transducer and activator of transcription 4 (STAT4) translocates into the nucleus upon stimulation with interleukin-12 (IL-12). The homology alignment of STATs family members showed that the 395-416 amino acid residues of DNA binding domain of STAT4 may have the function of dimer-specific nuclear localization signal (dsNLS). In view of this, we used pEGFP-C1 as the expression vector to construct the pEGFP-STAT4 plasmid and the deletion STAT4 plasmid (pEGFP-STAT4-Del) with the 395- 416 amino acid residue sequence, respectively. SV40 large T antigen The classical NLS nucleic acid sequence was inserted into the expression vector positive control plasmid (pEGFP-NLS) and the deletion STAT8 was inserted into pEGFP-NLS-STAT4-Del plasmid. These plasmids were transiently transfected into cervical cancer cell line Caski cells. After stimulation with IL-12, wild-type STAT4 was translocated into the nucleus and missing STAT4 was distributed in the cytoplasm. After further treatment with leptomycin B, IL-12 restimulated After the wild-type STAT4 was retained in the nucleus, and missing STAT4 is still distributed in the cytoplasm; the NLS insertion of missing STAT4, can restore missing nuclear translocation of STAT4. The above results indicate that wild-type STAT4 can translocate into the nucleus under the stimulation of IL-12. The mechanism of its entry into the nucleus is that it has dsNLS function in the 395-416 amino acid residues of its DNA binding domain and can mediate the activation of STAT4 translocation Nucleus.