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.
Sensors (Basel). 2014 Jan 10;14(1):1140-54. doi: 10.3390/s140101140.
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.
在生物传感器中，基于基因编码的 FRET 生物传感器被广泛用于定位和测量酶活性。激酶活性尤其受到关注，因为它们的时空调节对于深入了解细胞命运决定变得至关重要。ERK 的活性就是一个关键的信号转导途径节点，它可以引导细胞进入各种过程。因此，人们一直需要更好的工具来在体内分析激酶，并检测其活性的哪怕最微小的变化。在这里，我们报告了先前 ERK 活性报告器 EKAR 和 EKAREV 的优化。这些工具由两个适用于 FRET 实验的荧光团组成，它们侧翼是 ERK 的特定底物，以及一个能够识别和结合该底物的结构域，当该底物被磷酸化时。这种磷酸化允许传感器发生构象变化，从而产生 FRET 信号。我们通过对：（i）荧光团和（ii）底物和结合域之间的连接体进行修饰，改进了这些生物传感器，结果在进行 FRET 实验时，通过荧光寿命和比率测量，新的版本在 EGF 刺激下表现出更宽的动态范围。在这里，我们对这些新的生物传感器进行了表征，并讨论了它们观察到的差异，这些差异取决于它们的荧光特性。
