Ni Qiang, Titov Denis V, Zhang Jin
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Methods. 2006 Nov;40(3):279-86. doi: 10.1016/j.ymeth.2006.06.013.
Genetically encoded reporters based on fluorescence resonance energy transfer (FRET) are being developed for analyzing spatiotemporal dynamics of kinase activities in living cells, as the activities of this class of enzymes are often dynamically regulated and spatially compartmentalized within specific signaling context. Here we describe a general modular design and engineering strategies for the development of activity reporters for kinases of interest, using A-kinase activity reporter (AKAR) as an illustrative example. Discussed here are basic structure of such reporters, design considerations, reporter gene construction, cellular and in vitro characterization. Strategies for improving specificity, dynamic range or sensitivity, reversibility and integrity of the reporter as well as basic methods for live-cell time-lapse imaging using these reporters are summarized. Discussion of using this approach in the study of MAPK cascades is also provided. These FRET-based kinase activity reporters, along with analogous probes based on alternative designs, provide real-time tracking of kinase dynamics with subcellular resolution, which should complement other methods and offer great opportunities to delineate the molecular mechanisms underlying the complex regulation of kinases.
基于荧光共振能量转移（FRET）的基因编码报告分子正被开发用于分析活细胞中激酶活性的时空动态，因为这类酶的活性在特定信号背景下通常受到动态调节且在空间上进行分隔。在此，我们以A激酶活性报告分子（AKAR）为例，描述用于开发感兴趣激酶活性报告分子的通用模块化设计和工程策略。本文讨论了此类报告分子的基本结构、设计考量、报告基因构建、细胞和体外特性。总结了提高报告分子特异性、动态范围或灵敏度、可逆性和完整性的策略以及使用这些报告分子进行活细胞延时成像的基本方法。还提供了在丝裂原活化蛋白激酶（MAPK）级联反应研究中使用该方法的讨论。这些基于FRET的激酶活性报告分子，以及基于其他设计的类似探针，能够以亚细胞分辨率实时追踪激酶动态，这应能补充其他方法，并为阐明激酶复杂调控背后的分子机制提供绝佳机会。
