Optimization of ERK Activity Biosensors for both Ratiometric and Lifetime FRET Measurements

Sensors

Pauline Vandame¹  Corentin Spriet²  Franck Riquet¹  Dave Trinel²  Katia Cailliau-Maggio¹ 

[1] Laboratoire de Régulation des Signaux de division, EA4479, Institut Fédératif de Recherche (IFR) 147, Site de Recherche Intégré en Cancérologie (SIRIC) ONCOLILLE, University Lille1, Villeneuve d'Ascq F-59655, France; E-Mails:;Interdisciplinary Research Institute, USR3078 CNRS, Université Lille Nord de France, 50 avenue de Halley, Villeneuve d'Ascq Cedex F-59558, France; E-Mails:

关键词: genetically-encoded biosensor;    ERK;    FRET;   

DOI  :  10.3390/s140101140

来源: mdpi

【 摘 要 】

Among biosensors, genetically-encoded FRET-based biosensors are widely used to localize and measure enzymatic activities. Kinases activities are of particular interest as their spatiotemporal regulation has become crucial for the deep understanding of cell fate decisions. This is especially the case for ERK, whose activity is a key node in signal transduction pathways and can direct the cell into various processes. There is a constant need for better tools to analyze kinases in vivo, and to detect even the slightest variations of their activities. Here we report the optimization of the previous ERK activity reporters, EKAR and EKAREV. Those tools are constituted by two fluorophores adapted for FRET experiments, which are flanking a specific substrate of ERK, and a domain able to recognize and bind this substrate when phosphorylated. The latter phosphorylation allows a conformational change of the biosensor and thus a FRET signal. We improved those biosensors with modifications of: (i) fluorophores and (ii) linkers between substrate and binding domain, resulting in new versions that exhibit broader dynamic ranges upon EGF stimulation when FRET experiments are carried out by fluorescence lifetime and ratiometric measurements. Herein, we characterize those new biosensors and discuss their observed differences that depend on their fluorescence properties.

【 授权许可】

CC BY   
© 2014 by the authors; licensee MDPI, Basel, Switzerland.