Maryu Gembu, Miura Haruko, Uda Youichi, Komatsubara Akira T, Matsuda Michiyuki, Aoki Kazuhiro
Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University.
Division of Quantitative Biology, Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences.
Cell Struct Funct. 2018 Apr 25;43(1):61-74. doi: 10.1247/csf.18003. Epub 2018 Mar 17.
Protein kinases play pivotal roles in intracellular signal transduction, and dysregulation of kinases leads to pathological results such as malignant tumors. Kinase activity has hitherto been measured by biochemical methods such as in vitro phosphorylation assay and western blotting. However, these methods are less useful to explore spatial and temporal changes in kinase activity and its cell-to-cell variation. Recent advances in fluorescent proteins and live-cell imaging techniques enable us to visualize kinase activity in living cells with high spatial and temporal resolutions. Several genetically encoded kinase activity reporters, which are based on the modes of action of kinase activation and phosphorylation, are currently available. These reporters are classified into single-fluorophore kinase activity reporters and Förster (or fluorescence) resonance energy transfer (FRET)-based kinase activity reporters. Here, we introduce the principles of genetically encoded kinase activity reporters, and discuss the advantages and disadvantages of these reporters.Key words: kinase, FRET, phosphorylation, KTR.
蛋白激酶在细胞内信号转导中起关键作用，激酶的失调会导致诸如恶性肿瘤等病理结果。迄今为止，激酶活性一直通过体外磷酸化测定和蛋白质免疫印迹等生化方法来测量。然而，这些方法在探索激酶活性的时空变化及其细胞间差异方面用处较小。荧光蛋白和活细胞成像技术的最新进展使我们能够以高时空分辨率在活细胞中可视化激酶活性。目前有几种基于激酶激活和磷酸化作用模式的基因编码激酶活性报告分子。这些报告分子分为单荧光团激酶活性报告分子和基于荧光共振能量转移（FRET）的激酶活性报告分子。在这里，我们介绍基因编码激酶活性报告分子的原理，并讨论这些报告分子的优缺点。关键词：激酶；FRET；磷酸化；激酶活性报告分子
