Green, Harry M. and Alberola-Ila, Jose (2005) Development of ERK Activity Sensor, an in vitro, FRET-based sensor of Extracellular Regulated Kinase activity. BMC Chemical Biology, 5 (1). pp. 1-8. ISSN 1472-6769 http://resolver.caltech.edu/CaltechAUTHORS:GREbmccb05
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Background: Study of ERK activation has thus far relied on biochemical assays that are limited to the use of phospho-specific antibodies and radioactivity in vitro, and analysis of whole cell populations in vivo. As with many systems, fluorescence resonance energy transfer (FRET) can be utilized to make highly sensitive detectors of molecular activity. Here we introduce FRET-based ERK Activity Sensors, which utilize variants of Enhanced Green Fluorescent Protein fused by an ERK-specific peptide linker to detect ERK2 activity. Results: ERK Activity Sensors display varying changes in FRET upon phosphorylation by active ERK2 in vitro depending on the composition of ERK-specific peptide linker sequences derived from known in vivo ERK targets, Ets1 and Elk1. Analysis of point mutations reveals specific residues involved in ERK binding and phosphorylation of ERK Activity Sensor 3. ERK2 also shows high in vitro specificity for these sensors over two other major MAP Kinases, p38 and pSAPK/JNK. Conclusion: EAS's are a convenient, non-radioactive alternative to study ERK dynamics in vitro. They can be utilized to study ERK activity in real-time. This new technology can be applied to studying ERK kinetics in vitro, analysis of ERK activity in whole cell extracts, and high-throughput screening technologies.
|Additional Information:||© 2005 Green and Alberola-Ila; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 5 January 2005; Accepted 5 July 2005; Published 5 July 2005. Authors' contributions: HMG performed all experimental work. JAI provided advice, funding and supervision for the work. Both authors have read and approved the final manuscript. Acknowledgements: We would like to thank Dr. Kornfeld for providing us with GST-Elk1 cDNA and Dr. Sharrocks for providing us with Ets1 cDNA, from which EAS linkers were derived. We also thank Dr. Richard Roberts for use of his fluorimetry equipment. This work was supported by the NIH (AI45072) and the Keck Foundation.|
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|Deposited On:||25 Aug 2005|
|Last Modified:||26 Dec 2012 08:40|
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