Activation of NF-κB signalling by fusicoccin-induced dimerization
Activation of NF-κB signalling by fusicoccin-induced dimerization
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DOI：
10.1073/pnas.1212990110
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发表时间：
2013-01-29
影响因子：
11.1
通讯作者：
Ottmann, Christian
中科院分区：
文献类型：
--
作者：
Skwarczynska, Malgorzata;Molzan, Manuela;Ottmann, Christian
化学诱导二聚化是化学生物学中分析细胞内蛋白质功能的重要工具。在这里，我们报告了使用天然产物梭菌素(FC)来诱导14-3-3-融合目标蛋白与标记在H+-ATPase PIVIA2的C末端(CT)的蛋白的二聚化。为了防止14-3-3蛋白和CT融合与内源细胞蛋白的非生产性或有害相互作用，它们的相互作用表面被设计成促进Fc诱导的二聚化，仅在引入的蛋白质结构之间。活细胞成像记录了可逆的FC诱导的14-3-3和CT移位到不同的细胞室，这取决于融合到它们的二聚化伙伴蛋白的定位序列。该系统的功能通过FC诱导的核因子-kappa B-CT进入核内而得到证明。在HeLa细胞中，Fc介导的NF-kappa B-CT与核定位的14-3-3蛋白的二聚体通过诱导IL-8的分泌而导致NF-kappa B特异性的细胞反应。
Chemically induced dimerization is an important tool in chemical biology for the analysis of protein function in cells. Here we report the use of the natural product fusicoccin (FC) to induce dimerization of 14-3-3-fused target proteins with proteins tagged to the C terminus (CT) of the H+-ATPase PIVIA2. To prevent nonproductive or detrimental interactions of the 14-3-3 proteins and CT fusions with endogenous cell proteins, their interaction surface was engineered to facilitate FC-induced dimerization exclusively between the introduced protein constructs. Live-cell imaging documented the reversible FC-induced translocation of 14-3-3 and CT to different cell compartments depending on localization sequences fused to their dimerization partner protein. The functionality of this system was demonstrated by the FC-induced importation of the NF-kappa B-CT into the nucleus. In He La cells, FC-mediated dimerization of the NF-kappa B-CT with a constitutively nuclear-localized 14-3-3 protein led to an NF-kappa B-specific cellular response by inducing IL-8 secretion.