Silhan Jan, Vacha Petr, Strnadova Pavla, Vecer Jaroslav, Herman Petr, Sulc Miroslav, Teisinger Jan, Obsilova Veronika, Obsil Tomas
Departments of Physical and Macromolecular Chemistry, Charles University, 12843 Prague.
J Biol Chem. 2009 Jul 17;284(29):19349-60. doi: 10.1074/jbc.M109.002725. Epub 2009 May 5.
The role of 14-3-3 proteins in the regulation of FOXO forkhead transcription factors is at least 2-fold. First, the 14-3-3 binding inhibits the interaction between the FOXO and the target DNA. Second, the 14-3-3 proteins prevent nuclear reimport of FOXO factors by masking their nuclear localization signal. The exact mechanisms of these processes are still unclear, mainly due to the lack of structural data. In this work, we used fluorescence spectroscopy to investigate the mechanism of the 14-3-3 protein-dependent inhibition of FOXO4 DNA-binding properties. Time-resolved fluorescence measurements revealed that the 14-3-3 binding affects fluorescence properties of 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acid moiety attached at four sites within the forkhead domain of FOXO4 that represent important parts of the DNA binding interface. Observed changes in 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acid fluorescence strongly suggest physical contacts between the 14-3-3 protein and labeled parts of the FOXO4 DNA binding interface. The 14-3-3 protein binding, however, does not cause any dramatic conformational change of FOXO4 as documented by the results of tryptophan fluorescence experiments. To build a realistic model of the FOXO4.14-3-3 complex, we measured six distances between 14-3-3 and FOXO4 using Förster resonance energy transfer time-resolved fluorescence experiments. The model of the complex suggests that the forkhead domain of FOXO4 is docked within the central channel of the 14-3-3 protein dimer, consistent with our hypothesis that 14-3-3 masks the DNA binding interface of FOXO4.
14-3-3蛋白在调控FOXO叉头转录因子方面的作用至少有两方面。其一，14-3-3蛋白的结合抑制了FOXO与靶DNA之间的相互作用。其二，14-3-3蛋白通过掩盖FOXO因子的核定位信号来阻止其重新导入细胞核。这些过程的确切机制仍不清楚，主要是由于缺乏结构数据。在这项研究中，我们使用荧光光谱法来研究14-3-3蛋白依赖性抑制FOXO4 DNA结合特性的机制。时间分辨荧光测量结果表明，14-3-3蛋白的结合影响了连接在FOXO4叉头结构域内四个位点上的5-(((乙酰氨基)乙基)氨基)萘-1-磺酸部分的荧光特性，这些位点代表了DNA结合界面的重要部分。5-(((乙酰氨基)乙基)氨基)萘-1-磺酸荧光的观察变化强烈表明14-3-3蛋白与FOXO4 DNA结合界面的标记部分之间存在物理接触。然而，色氨酸荧光实验结果表明，14-3-3蛋白的结合并未引起FOXO4的任何显著构象变化。为了构建FOXO4-14-3-3复合物的真实模型，我们使用福斯特共振能量转移时间分辨荧光实验测量了14-3-3与FOXO4之间的六个距离。复合物模型表明，FOXO4的叉头结构域对接在14-3-3蛋白二聚体的中央通道内，这与我们提出的14-3-3掩盖FOXO4的DNA结合界面的假设一致。
