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PNAS 105 (49): 19264-19269

Copyright © 2008 by the National Academy of Sciences.

BIOLOGICAL SCIENCES / BIOPHYSICS

A genetically encoded fluorescent sensor of ERK activity

Christopher D. Harveya,b, Anka G. Ehrhardtc, Cristina Celluralec, Haining Zhonga, Ryohei Yasudad, Roger J. Davisc, and Karel Svobodaa,b,1

aJanelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147; bWatson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724; cHoward Hughes Medical Institute and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605; and dNeurobiology Department, Duke University, Durham, NC 27710

Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved October 16, 2008

Received for publication May 13, 2008.

Abstract: The activity of the ERK has complex spatial and temporal dynamics that are important for the specificity of downstream effects. However, current biochemical techniques do not allow for the measurement of ERK signaling with fine spatiotemporal resolution. We developed a genetically encoded, FRET-based sensor of ERK activity (the extracellular signal-regulated kinase activity reporter, EKAR), optimized for signal-to-noise ratio and fluorescence lifetime imaging. EKAR selectively and reversibly reported ERK activation in HEK293 cells after epidermal growth factor stimulation. EKAR signals were correlated with ERK phosphorylation, required ERK activity, and did not report the activities of JNK or p38. EKAR reported ERK activation in the dendrites and nucleus of hippocampal pyramidal neurons in brain slices after theta-burst stimuli or trains of back-propagating action potentials. EKAR therefore permits the measurement of spatiotemporal ERK signaling dynamics in living cells, including in neuronal compartments in intact tissues.

Key Words: fluorescence lifetime imaging microscopy • FRET • MAPK

Author contributions: C.D.H., A.G.E., C.C., R.J.D., and K.S. designed research; C.D.H., A.G.E., and C.C. performed research; C.D.H., H.Z., R.Y., R.J.D., and K.S. contributed new reagents/analytic tools; C.D.H., A.G.E., and C.C. analyzed data; and C.D.H. and K.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0804598105/DCSupplemental.

1To whom correspondence should be addressed. E-mail: svobodak{at}janelia.hhmi.org

© 2008 by The National Academy of Sciences of the USA

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