Abstract
The advent of genetically encoded FRET-based kinase activity reporters has ushered in a new era of signal transduction research. Such reporters allow the direct monitoring of kinase activity in live cells at specific locations, providing unprecedented information on the spatiotemporal dynamics of kinase signaling. Specifically, FRET-sensitive conformational changes in the reporters following phosphorylation serve as a direct readout of kinase activity. These genetically encoded reporters allow not only temporal resolution of kinase activity, but also spatial resolution: by fusing appropriate targeting sequences, reporters can be positioned at specific subcellular locations. Herein we present a strategy to generate and target kinase activity reporters to discrete intracellular regions to measure kinase signaling in live cells.