Genetically encoded fluorescent indicators to visualize protein phosphorylation by extracellular signal-regulated kinase in single living cells.

Extracellular signal-regulated kinase (ERK) is a serine/threonine protein kinase that regulates a wide variety of cell functions, such as cell growth and differentiation. To study the spatiotemporal dynamics of protein phosphor-ylation by activated ERK in living cells, we have developed genetically encoded fluorescent indicators for ERK. The present indicators are based on fluorescence resonance energy transfer (FRET) between two green fluorescent protein mutants. Phosphorylation of the indicators by activated ERK changes the FRET efficiency due to their conformational alterations. We visualized the cytosolic and nuclear activity of ERK using the present indicators. We thus found that the activation duration of ERK is considerably different between the cytosol and nucleus in living cells. The subcellular difference in the ERK activity may be fundamental to the regulation of cell functions by ERK. The present fluorescent indicators provide a powerful tool to reveal the spatiotemporal dynamics of protein phosphorylation by ERK in single living cells.