Nagai Y, Miyazaki M, Aoki R, Zama T, Inouye S, Hirose K, Iino M, Hagiwara M
Department of Functional Genomics, Medical Research Institute, Tokyo Medical and Dental University, Yushima, Tokyo 113-8510, Japan.
Nat Biotechnol. 2000 Mar;18(3):313-6. doi: 10.1038/73767.
We have developed a method for visualizing phosphorylation of proteins in living cells using a novel fluorescent indicator composed of two green fluorescent protein (GFP) variants joined by the kinase-inducible domain (KID) of the transcription factor cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB). Phosphorylation of KID by the cAMP-dependent protein kinase A (PKA) decreased the fluorescence resonance energy transfer (FRET) among the flanking GFPs. By transfecting COS-7 cells with an expression vector encoding this indicator protein (termed ART for cAMP-responsive tracer), we were able to visualize activation dynamics of PKA in living cells.
我们开发了一种方法，可利用一种新型荧光指示剂来可视化活细胞中蛋白质的磷酸化过程。该指示剂由两个绿色荧光蛋白（GFP）变体组成，它们通过转录因子环磷酸腺苷（cAMP）反应元件结合蛋白（CREB）的激酶诱导结构域（KID）连接在一起。cAMP依赖性蛋白激酶A（PKA）使KID发生磷酸化，降低了侧翼GFP之间的荧光共振能量转移（FRET）。通过用编码这种指示剂蛋白（称为cAMP反应示踪剂ART）的表达载体转染COS-7细胞，我们能够可视化活细胞中PKA的激活动态。
