Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM

Claire Demeautis,Franck B Riquet,Marc Tramier,Sergi Padilla-Parra,Julien Roul,François Sipieter,Catherine Chapuis

doi:10.1038/srep41026

Abstract

Monitoring of different signalling enzymes in a single assay using multiplex biosensing provides a multidimensional workspace to elucidate biological processes, signalling pathway crosstalk, and determine precise sequence of events at the single living cell level. In this study, we interrogate the complexity in cAMP/PKA-MAPK/ERK1&2 crosstalk by using multi-parameter biosensing experiments to correlate biochemical activities simultaneously in time and space. Using a single excitation wavelength dual colour FLIM method we are able to detect fluorescence lifetime images of two donors to simultaneously measure PKA and ERK1&2 kinase activities in the same cellular localization by using FRET biosensors. To this end, we excite two FRET donors mTFP1 and LSSmOrange with a 440 nm wavelength and we alleviate spectral bleed-through associated limitations with the very dim-fluorescent acceptor ShadowG for mTFP1 and the red-shifted mKate2 for LSSmOrange. The simultaneous recording of PKA and ERK1&2 kinase activities reveals concomitant EGF-mediated activations of both kinases in HeLa cells. Under these conditions the subsequent Forskolin-induced cAMP release reverses the transient increase of EGF-mediated ERK1&2 kinase activity while reinforcing PKA activation. Here we propose a validated methodology for multiparametric kinase biosensing in living cells using FRET-FLIM.

Highlights

Binding of cAMP to the two regulatory subunits, the two catalytic subunits dissociate from the holoenzyme and become active[9]
We needed to determine whether LSSmOrange was suitable for Förster Resonance Energy Transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM) experiments
Fluorescence decay measurements in U2OS cells expressing LSSmOrange were performed by time correlated single photon counting (TCSPC)

Summary

Introduction

Binding of cAMP to the two regulatory subunits, the two catalytic subunits dissociate from the holoenzyme and become active[9]. The best-characterized connections are PKA-dependent ERK activity modulation mediated by different Raf isoforms in different cell types[11] These differential effects are explained by the fact that Raf kinase family comprises different isoforms. PDEs that are directly targeted by ERK1&217 were recently found to bind to and to regulate Raf-1 kinase[18] Such a level of complexity in cAMP/PKA-MAPK/ERK1&2 crosstalk calls for novel approaches. Taking advantage of the long stoke shift of Large Stoke Shift mOrange (LSSmOrange)[25], we used a 440 nm single excitation wavelength for both donors, monomeric teal fluorescent protein (mTFP1) and LSSmOrange, and a dual colour fluorescence lifetime imaging microscopy (FLIM) to simultaneously measure signals from two genetically encoded FRET biosensors. MAPK/ERK1&2 and cAMP/PKA interplay in living HeLa cells was examined using EKAR2G28 and AKAR429 biosensors respectively modified with mTFP1/ShadowG and LSSmOrange/mKate[2] fluorescent protein pairs. Using these conditions we could simultaneously record cAMP inhibitory effect on EGF-mediated ERK1&2 activity level together with its reinforcing of PKA activity

Methods

Results

Conclusion