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ACS Chem. Biol. 1 (6), 371–376
10.1021/cb600202f
Web Release Date: July 21,
2006
Copyright © 2006 American Chemical Society
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Reading Dynamic Kinase Activity in Living Cells for High-Throughput Screening
Michael D. Allen†, Lisa M. DiPilato†,
Meghdad Rahdar†,
Yunzhao R. Ren†,
Curtis Chong†,
Jun O. Liu†,‡, and
Jin Zhang†,‡,*
†Department of Pharmacology and Molecular Sciences, and ‡The Solomon H. Snyder Department of Neuroscience and Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Received for review May 15, 2006 and accepted June 28, 2006
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*To whom correspondence should be addressed. E-mail: jzhang32#jhmi.edu.
Protein kinases, as crucial signaling molecules, represent an emerging class of drug targets, and the ability to assay their activities in living cells with high-throughput screening should provide exciting opportunities for drug discovery and chemical and functional genomics. Here, we describe a general method for high-throughput reading of dynamic kinase activities using ratiometric fluorescent sensors, and showcase an example of reading intracellular activities of protein kinase A (PKA) and the cyclic adenosine monophosphate (cAMP)/PKA pathway downstream of many G-protein coupled receptors (GPCRs). We further demonstrate the first compound screen based on the ability of compounds to modulate dynamic kinase activities in living cells and show that such screening of a collection of clinical compounds has successfully identified modulators of the GPCR/cAMP/PKA pathway.
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