基于荧光共振能量转移（FRET）的报告基因可视化观察蛋白激酶A活性的亚细胞动力学。

Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters.

作者信息

Allen Michael D, Zhang Jin

机构信息

Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

出版信息

Biochem Biophys Res Commun. 2006 Sep 22;348(2):716-21. doi: 10.1016/j.bbrc.2006.07.136. Epub 2006 Jul 31.

DOI:10.1016/j.bbrc.2006.07.136

PMID:16895723

Abstract

cAMP-dependent protein kinase (PKA) mediates key cellular processes via compartmentalized activity, and the ability to track its activity in living cells should help increase our understanding of this precise regulation. Here, through systematic testing of new fluorescent proteins, we developed a new FRET-based A-kinase activity reporter (AKAR), AKAR3, with a dynamic range of 31-41%, twice that of predecessors. Visualization of PKA activity at plasma membrane, cytoplasm, nucleus, and mitochondria was achieved. Targeting AKAR3 to outer mitochondrial membrane revealed that basal PKA activity at mitochondria differs from that in the cytoplasm, indicating differential regulation of PKA activity at different subcellular locations.

摘要

环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）通过分区化活性介导关键的细胞过程，而在活细胞中追踪其活性的能力应有助于增进我们对这种精确调控的理解。在此，通过对新型荧光蛋白的系统测试，我们开发了一种基于荧光共振能量转移（FRET）的新型A激酶活性报告分子（AKAR）——AKAR3，其动态范围为31% - 41%，是先前版本的两倍。实现了对质膜、细胞质、细胞核和线粒体中PKA活性的可视化。将AKAR3靶向线粒体外膜显示，线粒体处的基础PKA活性与细胞质中的不同，表明PKA活性在不同亚细胞位置存在差异调控。