Abstract
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.
Highlights
How a cell can integrate numerous external signals to elicit a specific and adapted response with only a limited number of signaling effectors remains a puzzling question
As the FRET pair used in the second construction gives us good results, we tried to optimize it with an improved linker named EV-linker contained in another Mitogen Activated Protein Kinase (MAPK) biosensor EKAREV [16]
Concerning the PKA activity biosensors, the optimization from A Kinase Activity Reporter 3 (AKAR3) to AKAR4 consisted in a swapping of the donor fluorophores eCFP for the Cerulean FP in combination with a circularly permutated Venus [18,19]
Summary
How a cell can integrate numerous external signals to elicit a specific and adapted response with only a limited number of signaling effectors remains a puzzling question. Those fluorophores are flanking a specific peptide substrate of ERK and a domain recognizing and binding this peptide substrate when phosphorylated The latter recognition allows a conformational change of the biosensor and a FRET signal. When the MAPK/ERK pathway cascade is recruited by an external signal, activated MEK binds to endogenous ERK as well as the ERK enclosed within the sensor in order to achieve phosphorylation This binding lead to a modification of ERK conformation within Miu, bringing closer the two fluorophores and noticeably increasing the FRET signal. Upon ERK activation, the substrate is phosphorylated and recognized by the phospho-aminoacid binding domain (PAABD), leading to a conformational change that allows FRET phenomenon between the fluorophores Such a process remains reversible upon the action of specific phosphatases or inhibition of kinase activity [13]. Those new probes have been tested and compared with two different FRET techniques, fluorescence intensity and lifetime based measurements, in order to provide the most versatile ERK activity biosensor
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