Editors' ChoiceBiophysics

Sensitive Fluorescence

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Science Signaling  16 Dec 2008:
Vol. 1, Issue 50, pp. ec432
DOI: 10.1126/scisignal.150ec432

Members of the extracellular signal–regulated kinase (ERK) subfamily of mitogen-activated protein kinases (MAPKs) are important in such processes as cellular proliferation and differentiation. The consequences of ERK activation depend on its subcellular localization (for example, cytoplasmic versus nuclear) and the duration of its active state; however, analysis of ERK activity in live cells is technically challenging. Harvey et al. built upon previously described fluorescence resonance energy transfer (FRET)–based reporter systems to design an ERK activity sensor (EKAR). One member of a donor-acceptor pair of fluorescent molecules is found at each end of EKAR. The central portions consist of the phospho-binding WW domain, a linker sequence, an ERK-specific substrate peptide, and an ERK-docking site. When ERK phosphorylates the substrate peptide, the WW domain shifts to bind to the phosphorylated site, which causes a conformational change in EKAR that brings the two fluorescent moieties sufficiently close to one another for FRET to occur. When expressed in human embryonic kidney (HEK) 293 cells, EKAR was localized to the nucleus; the addition of a nuclear export sequence to EKAR generated a cytoplasmic form, EKARcyto. Two-photon fluorescence lifetime imaging (2pFLIM) demonstrated that EKARcyto responded to activation of ERK but not to that of other MAPKs such as JNK and p38, that both the ERK-docking site and the substrate peptide were required for sensing ERK activation, and that EKAR signals were reversible. Finally, the authors successfully used EKAR to monitor both cytoplasmic and nuclear ERK signaling in organotypic hippocampal brain slices and in pyramidal neurons. This new reagent now enables the monitoring of ERK activity in live cells and presumably could form the basis of sensors for other MAPKs.

C. D. Harvey, A. G. Ehrhardt, C. Cellurale, H. Zhong, R. Yasuda, R. J. Davis, K. Svoboda, A genetically encoded fluorescent sensor of ERK activity. Proc. Natl. Acad. Sci. U.S.A. 105, 19264–19269 (2008). [Abstract] [Full Text]