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首页> 外文期刊>Journal of Cell Science >The COOH-terminal nuclear localization sequence of interferon gamma regulates STAT1 alpha nuclear translocation at an intracellular site
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The COOH-terminal nuclear localization sequence of interferon gamma regulates STAT1 alpha nuclear translocation at an intracellular site

机译：干扰素γ的COOH末端核定位序列调节细胞内位点的STAT1α核易位

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We have recently shown that the nuclear localization of IFN gamma is mediated by a polybasic nuclear localization sequence (NLS) in its C terminus. This NLS is required for the full expression of biological activity of IFN gamma, both extracellularly and intracellularly. We now show that this NLS plays an integral intracellular role in the nuclear translocation of the transcription factor STAT1 alpha activated by IFN gamma. Treatment of IFN gamma with antibodies to the C-terminal region (95-133) containing the NLS blocked the induction of STAT1 alpha nuclear translocation. The antibodies had no effect on nuclear translocation of STAT1 alpha in IFN alpha treated cells. A deletion mutant of human IFN gamma, IFN gamma(1-123), which is devoid of the C-terminal NLS region was found to be biologically inactive, but was still able to bind to the IFN gamma receptor complex on cells with a K-d similar to that of the wild-type protein. Deletion of the NLS specifically abolished the ability of IFN gamma(1-123) to initiate the nuclear translocation of STAT1 alpha, which is required for the biological activities of IFN gamma following binding to the IFN gamma receptor complex. Thus, the NLS region appears to contribute minimally to extracellular high-affinity receptor-ligand binding, yet exerts a strong functional role in STAT1 alpha nuclear localization. A high-affinity site for the interaction of the C-terminal NLS domain of IFN gamma with a K-d approx. 3x10(-8) M-1 has been described by previous studies on the intracellular cytoplasmic domain of the IFN gamma receptor alpha-chain. To examine the role of the NLS at the intracellular level, we microinjected neutralizing antibodies raised against the C-terminal NLS domain of IFN gamma into the cytoplasm of cells before treatment of cells with IFN gamma. These intracellular antibodies specifically blocked the nuclear translocation of STAT1 alpha following the subsequent treatment of these cells extracellularly with IFN gamma. These data show that the NLS domain of IFN gamma interacts at an intracellular site to regulate STAT1 alpha nuclear import. A C-terminal peptide of murine IFN gamma, IFN gamma(95-133), that contains the NLS motif, induced nuclear translocation of STAT1 alpha when taken up intracellularly by a murine macrophage cell line. Deletion of the NLS moth specifically abrogated the ability of this intracellular peptide to cause STAT1 alpha nuclear translocation. In cells activated with IFN gamma, IFN gamma was found to as part of a complex that contained STAT1 alpha and the importin-alpha analog Npi-1, which mediates STAT1 alpha nuclear import. The tyrosine phosphorylation of STAT1 alpha, the formation of the complex IFN gamma/Npi-1/STAT1 alpha complex and the subsequent nuclear translocation of STAT1 alpha were all found to be dependent on the presence of the IFN gamma NLS. Thus, the NLS of IFN gamma functions intracellularly to directly regulate the activation and ultimate nuclear translocation STAT1 alpha. [References: 27]
机译：我们最近显示，IFNγ的核定位是由其C末端的多核核定位序列（NLS）介导的。该NLS是细胞外和细胞内完整表达IFNγ生物学活性所必需的。现在我们显示该NLS在由IFNγ激活的转录因子STAT1α的核易位中起着不可或缺的细胞内作用。用含有NLS的C端区域（95-133）抗体处理IFN gamma可以阻止STAT1α核易位的诱导。抗体对IFNα处理的细胞中STAT1α的核易位没有影响。发现缺少C端NLS区的人IFNγ缺失突变体IFNγ（1-123）具有生物学活性，但仍能与Kd细胞上的IFNγ受体复合物结合。与野生型蛋白质相似。 NLS的删除特别消除了IFNγ（1-123）启动STAT1α的核易位的能力，这是IFNγ与IFNγ受体复合物结合后的生物学活性所必需的。因此，NLS区似乎对细胞外高亲和力受体-配体结合的贡献最小，但在STAT1α核定位中发挥了强大的功能。一个高亲和力位点，用于干扰素γ的C末端NLS结构域与约K-d的相互作用。先前对IFNγ受体α链的胞质域的研究已经描述了3x10（-8）M-1。为了检查NLS在细胞内水平上的作用，我们在用γ干扰素处理细胞之前，将针对IFNγ的C端NLS结构域产生的中和抗体显微注射到细胞质中。在随后用IFNγ对这些细胞进行细胞外处理后，这些细胞内抗体特异性阻断STAT1α的核易位。这些数据表明，IFNγ的NLS结构域在细胞内位点相互作用以调节STAT1α核输入。包含NLS基序的鼠IFNγ的C末端肽IFNγ（95-133）在被鼠巨噬细胞细胞系吸收后被诱导诱导STAT1α的核易位。 NLS蛾的删除特别废除了这种细胞内肽引起STAT1α核易位的能力。在被IFNγ激活的细胞中，发现IFNγ是含有STAT1α和importin-alpha类似物Npi-1的复合物的一部分，后者介导STAT1α的核输入。发现STAT1α的酪氨酸磷酸化，复合IFNγ/ Npi-1 /STAT1α复合物的形成以及随后的STAT1α的核易位均取决于IFNγNLS的存在。因此，IFNγ的NLS在细胞内起作用以直接调节活化和最终核易位STAT1α。 [参考：27]

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