On the Inference of ERK Signaling Dynamics from Protein Biosensor Measurements

The extracellular signal-regulated kinase (ERK) signaling pathway plays prominent roles in cell growth, proliferation, and differentiation. ERK signaling is dynamic, involving phosphorylation/dephosphorylation, nucleocytoplasmic shuttling, and interactions with scores of protein substrates in the cytosol and in the nucleus. Live-cell fluorescence microscopy using genetically encoded ERK biosensors offers the potential to infer those dynamics in individual cells. In this study, we have monitored ERK signaling using four commonly used translocation- and Förster resonance energy transfer-based biosensors in a common cell stimulation context. Consistent with previous reports, we found that each biosensor responds with unique kinetics; it is clear that there is not a single dynamic signature characterizing the complexity of ERK phosphorylation, translocation, and kinase activity. In particular, the widely adopted ERK Kinase Translocation Reporter (ERKKTR) gives a readout that reflects ERK activity in both compartments. Mathematical modeling offers an interpretation of the measured ERKKTR kinetics, in relation to cytosolic and nuclear ERK activity, and suggests that biosensor-specific dynamics substantially influence the measured output.