Real-Time Multiplex Kinase Phosphorylation Sensors in Living Cells.

Phosphorylation is an important post-translational modification implicated in cellular signaling and regulation. However, current methods to study protein phosphorylation by various kinases lack spatiotemporal resolution or the ability to simultaneously observe in real time the activity of multiple kinases in live cells. We present a peptide biosensor strategy with time correlated single photon counting-fluorescence lifetime imaging (TCSPC-FLIM) to interrogate the spatial and temporal dynamics of VEGFR-2 and AKT phosphorylation activity in real time in live 2D and 3D cell culture models at single cell resolution. By recording the increase in fluorescence lifetime due to a change in the solvatochromic environment of the sensor upon phosphorylation, we demonstrate that spatiotemporal maps of protein kinase activity can be obtained. Our results suggest that fluorescence lifetime imaging of peptide biosensors can be effectively and specifically used to monitor and quantify phosphorylation of multiple kinases in live cells.