Department of Biological Sciences, Graduate School of Nanoscience & Technology, Daejeon 305-701, Korea.
Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3412-7. doi: 10.1073/pnas.0911262107. Epub 2010 Feb 3.
A new visualizing method was developed for monitoring protein-protein (P-P) interactions in live mammalian cells. P-P interactions are visualized by directing localization of a bait protein to endosomes. This method is sufficiently robust to analyze signal-dependent P-P interactions such as calcium-dependent protein interactions. We visualized interactions between activated calmodulin and calmodulin-binding proteins, and observed oscillatory interactions via time-lapse imaging. In addition, this new method can simultaneously monitor multiple P-P interactions in a single live cell, which allows comparison of interactions between several prey proteins and a single bait protein. We observed that CaMKK1 and CaMKIIalpha bind calmodulin with distinct binding affinities in live cell, which indicates that calcium signaling is fine-tuned by distinct activation patterns of CaM kinases. This method will enable investigation of cellular processes based on dynamic P-P interactions.
一种新的可视化方法被开发用于监测活哺乳动物细胞中的蛋白质-蛋白质（P-P）相互作用。通过将诱饵蛋白定向定位到内体中来可视化 P-P 相互作用。该方法足够强大，可以分析信号依赖性的 P-P 相互作用，如钙依赖性蛋白相互作用。我们可视化了激活钙调蛋白和钙调蛋白结合蛋白之间的相互作用，并通过延时成像观察到了振荡相互作用。此外，这种新方法可以在单个活细胞中同时监测多个 P-P 相互作用，这允许比较几个猎物蛋白和一个诱饵蛋白之间的相互作用。我们观察到 CaMKK1 和 CaMKIIalpha 在活细胞中与钙调蛋白具有不同的结合亲和力，这表明钙信号通过 CaM 激酶的不同激活模式进行微调。该方法将能够基于动态 P-P 相互作用研究细胞过程。
