Real time analysis of STAT3 nucleocytoplasmic shuttling
Real time analysis of STAT3 nucleocytoplasmic shuttling
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DOI：
10.1074/jbc.m312530200
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发表时间：
2004-04-09
影响因子：
4.8
通讯作者：
Müller-Newen, G
中科院分区：
文献类型：
--
作者：
Pranada, AL;Metz, S;Müller-Newen, G
转录因子STAT 3在白细胞介素-6及其相关细胞因子的信号转导中起重要作用。在刺激时，细胞质STAT 3在酪氨酸705处磷酸化，易位到细胞核中，并诱导靶基因。值得注意的是，STAT蛋白在未刺激的细胞的细胞核中也是可检测的。在这份报告中，我们介绍了一种新的方法的真实的时间分析的STAT 3核质穿梭在活细胞中，这是基于最近建立的荧光定位后光漂白（FLAP）的方法。STAT 3与绿色荧光蛋白的青色（CFP）和黄色（YFP）变体在C末端融合。在所得的STAT 3-CFP-YFP（STAT 3-CY）融合蛋白中，可以使用共聚焦显微镜的514-nm激光选择性地漂白YFP。该设置允许通过监测荧光标记和选择性漂白的STAT 3-CY的亚细胞分布来研究STAT 3核质转运的动力学。通过这种方法，我们证明了STAT 3-CY在未受刺激的细胞中在胞质溶胶和细胞核之间连续穿梭。这种组成型穿梭不依赖于酪氨酸705的磷酸化，因为STAT 3（Y 705 F）-CY突变体的穿梭程度与STAT 3-CY相同。缺失突变体的实验表明，STAT 3的N-末端部分是必不可少的穿梭。进一步的研究表明，STAT 3核输出的减少有助于STAT 3响应于细胞因子刺激的核积累。在这项研究中提出的新方法通常适用于任何感兴趣的蛋白质，用于分析核质转运机制在真实的时间。
The transcription factor STAT3 is most important for the signal transduction of interleukin-6 and related cytokines. Upon stimulation cytoplasmic STAT3 is phosphorylated at tyrosine 705, translocates into the nucleus, and induces target genes. Notably, STAT proteins are also detectable in the nuclei of unstimulated cells. In this report we introduce a new method for the real time analysis of STAT3 nucleocytoplasmic shuttling in living cells which is based on the recently established fluorescence localization after photobleaching (FLAP) approach. STAT3 was C-terminally fused with the cyan (CFP) and yellow (YFP) variants of the green fluorescent protein. In the resulting STAT3-CFP-YFP (STAT3-CY) fusion protein the YFP can be selectively bleached using the 514-nm laser of a confocal microscope. This setting allows studies on the dynamics of STAT3 nucleocytoplasmic transport by monitoring the subcellular distribution of fluorescently labeled and selectively bleached STAT3-CY. By this means we demonstrate that STAT3-CY shuttles continuously between the cytosol and the nucleus in unstimulated cells. This constitutive shuttling does not depend on the phosphorylation of tyrosine 705 because a STAT3(Y705F)-CY mutant shuttles to the same extent as STAT3-CY. Experiments with deletion mutants reveal that the N-terminal moiety of STAT3 is essential for shuttling. Further studies suggest that a decrease in STAT3 nuclear export contributes to the nuclear accumulation of STAT3 in response to cytokine stimulation. The new approach presented in this study is generally applicable to any protein of interest for analyzing nucleocytoplasmic transport mechanisms in real time.