Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.


Logo for

PNAS 105 (49): 19264-19269

Copyright © 2008 by the National Academy of Sciences.


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

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

© 2008 by The National Academy of Sciences of the USA

Visualizing and Manipulating Temporal Signaling Dynamics with Fluorescence-Based Tools.
D. P. Doupe and N. Perrimon (2014)
Science Signaling 7, re1
   Abstract »    Full Text »    PDF »
Data-driven modeling reconciles kinetics of ERK phosphorylation, localization, and activity states.
S. Ahmed, K. G. Grant, L. E. Edwards, A. Rahman, M. Cirit, M. B. Goshe, and J. M. Haugh (2014)
Mol Syst Biol 10, 718
   Abstract »    Full Text »    PDF »
Genetically encoded molecular probes to visualize and perturb signaling dynamics in living biological systems.
V. Sample, S. Mehta, and J. Zhang (2014)
J. Cell Sci. 127, 1151-1160
   Abstract »    Full Text »    PDF »
Long-Distance Integration of Nuclear ERK Signaling Triggered by Activation of a Few Dendritic Spines.
S. Zhai, E. D. Ark, P. Parra-Bueno, and R. Yasuda (2013)
Science 342, 1107-1111
   Abstract »    Full Text »    PDF »
A Versatile Toolkit to Produce Sensitive FRET Biosensors to Visualize Signaling in Time and Space.
R. D. Fritz, M. Letzelter, A. Reimann, K. Martin, L. Fusco, L. Ritsma, B. Ponsioen, E. Fluri, S. Schulte-Merker, J. van Rheenen, et al. (2013)
Science Signaling 6, rs12
   Abstract »    Full Text »    PDF »
Engineering A-kinase Anchoring Protein (AKAP)-selective Regulatory Subunits of Protein Kinase A (PKA) through Structure-based Phage Selection.
M. G. Gold, D. M. Fowler, C. K. Means, C. T. Pawson, J. J. Stephany, L. K. Langeberg, S. Fields, and J. D. Scott (2013)
J. Biol. Chem. 288, 17111-17121
   Abstract »    Full Text »    PDF »
The Temporal Pattern of Stimulation Determines the Extent and Duration of MAPK Activation in a Caenorhabditis elegans Sensory Neuron.
T. Tomida, S. Oda, M. Takekawa, Y. Iino, and H. Saito (2012)
Science Signaling 5, ra76
   Abstract »    Full Text »    PDF »
Studying Signal Transduction in Single Dendritic Spines.
R. Yasuda (2012)
Cold Spring Harb Perspect Biol 4, a005611
   Abstract »    Full Text »    PDF »
Forster resonance energy transfer-based sensor targeting endoplasmic reticulum reveals highly oxidative environment.
V. L. Kolossov, M. T. Leslie, A. Chatterjee, B. M. Sheehan, P. J. A. Kenis, and H. R. Gaskins (2012)
Experimental Biology and Medicine 237, 652-662
   Abstract »    Full Text »    PDF »
A Cre-Dependent GCaMP3 Reporter Mouse for Neuronal Imaging In Vivo.
H. A. Zariwala, B. G. Borghuis, T. M. Hoogland, L. Madisen, L. Tian, C. I. De Zeeuw, H. Zeng, L. L. Looger, K. Svoboda, and T.-W. Chen (2012)
J. Neurosci. 32, 3131-3141
   Abstract »    Full Text »    PDF »
Development of an optimized backbone of FRET biosensors for kinases and GTPases.
N. Komatsu, K. Aoki, M. Yamada, H. Yukinaga, Y. Fujita, Y. Kamioka, and M. Matsuda (2011)
Mol. Biol. Cell 22, 4647-4656
   Abstract »    Full Text »    PDF »
Spatiotemporally Regulated Protein Kinase A Activity Is a Critical Regulator of Growth Factor-Stimulated Extracellular Signal-Regulated Kinase Signaling in PC12 Cells.
K. J. Herbst, M. D. Allen, and J. Zhang (2011)
Mol. Cell. Biol. 31, 4063-4075
   Abstract »    Full Text »    PDF »
Imaging CREB Activation in Living Cells.
M. W. Friedrich, G. Aramuni, M. Mank, J. A. G. Mackinnon, and O. Griesbeck (2010)
J. Biol. Chem. 285, 23285-23295
   Abstract »    Full Text »    PDF »
Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues.
D. M. Chudakov, M. V. Matz, S. Lukyanov, and K. A. Lukyanov (2010)
Physiol Rev 90, 1103-1163
   Abstract »    Full Text »    PDF »
Visualization of JNK activity dynamics with a genetically encoded fluorescent biosensor.
M. Fosbrink, N.-N. Aye-Han, R. Cheong, A. Levchenko, and J. Zhang (2010)
PNAS 107, 5459-5464
   Abstract »    Full Text »    PDF »
Bistable Switches for Synaptic Plasticity.
H. Ogasawara and M. Kawato (2009)
Science Signaling 2, pe7
   Abstract »    Full Text »    PDF »

To Advertise     Find Products

Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882