Visualization of Kinase Activity with FRET‐Based Activity Biosensors

Charlene Depry1, Jin Zhang2

1 Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, 2 The Solomon H. Snyder Department of Neuroscience and Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 18.15
DOI:  10.1002/0471142727.mb1815s91
Online Posting Date:  July, 2010
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Genetically encodable FRET‐based kinase activity reporters (KARs) enable real‐time monitoring of kinase activity dynamics in living cells with high spatiotemporal resolution. This unit describes a general protocol for utilizing KARs to visualize kinase activity in living mammalian cells with fluorescence microscopy. Curr. Protoc. Mol. Biol. 91:18.15.1‐18.15.9. © 2010 by John Wiley & Sons, Inc.

Keywords: kinase activity reporter; FRET; live‐cell imaging

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Measuring Protein Kinase Activity Using Genetically Encoded Reporters
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Measuring Protein Kinase Activity Using Genetically Encoded Reporters

  • Cells of interest
  • Kinase activity reporter (KAR) plasmid DNA
  • Hanks' balanced salt solution (HBSS; see appendix 22)
  • Immersion oil
  • Stimulant/inhibitor, optional
  • 35‐mm glass‐bottom imaging dishes (MatTek or World Precision Instruments)
  • Inverted fluorescence microscope with appropriate objective, filters/mirrors, camera, and acquisition software; the following are some examples:
    • Light source
    • Zeiss Axiovert 200 M microscope
    • 40×/1.3‐NA oil‐immersion objective lens
    • 420DF20 excitation filter, 450DRLP dichroic mirror, and two emission filters: 475DF40 for cyan fluorescent protein (CFP) and 535DF25 for yellow fluorescent protein (YFP) (Chroma Technology)
    • Lambda 10‐2 filter changer (Sutter Instruments)
    • Cooled charge‐coupled device (CCD) camera (MicroMAX BFT512, Roper Scientific)
    • METAFLUOR 6.2 imaging software (Molecular Devices)
  • PC to run microscope
  • Additional reagents and equipment for culture techniques ( appendix 3F) and transfection (units 9.1 9.4)
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Literature Cited

   Allen, M.D. and Zhang, J. 2006. Subcellular dynamics of protein kinase A activity visualized by FRET‐based reporters. Biochem. Biophys. Res. Commun. 348:716‐721.
   Ananthanarayanan, B., Ni, Q., and Zhang, J. 2008. Molecular sensors based on fluorescence resonance energy transfer to visualize cellular dynamics. In Methods in Cell Biology, Vol. 89: Biophysical Tools for Biologists, Volume 2 (J.J Correia and H.W. Detrich III, eds.) pp. 37‐57. Academic Press, Cambridge, Mass.
   DiPilato, L.M., Cheng, X., and Zhang, J. 2004. Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments. Proc. Natl. Acad. Sci. U.S.A. 101:16513‐16518.
   Gallegos, L., Kunkel, M.T., and Newton, A.C. 2006. Targeting protein kinase C activity reporter to discrete intracellular regions reveals spatiotemporal differences in agonist‐dependent signaling. J. Biol. Chem. 281:30947‐30956.
   Gao, X. and Zhang, J. 2008. Spatiotemporal analysis of differential Akt regulation in plasma membrane microdomains. Mol. Biol. Cell. 19:4366‐4373.
   Gordon, G.W., Berry, G., Liang, X.H., Levine, B., and Herman, B. 1998. Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J. 2702‐2713.
   Lim, C.J., Kain, K.H., Tkachenko, E., Goldfinger, L.E., Gutierrez, E., Allen, M.D., Groisman, A., Zhang, J., and Ginsgerb, M.H. 2008. Integrin‐mediated protein kinase A activation at the leading edge of migrating cells. Mol. Biol. Cell 19:4930‐4941.
   Miyawaki, A. and Tsien, R.Y. 2000. Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. In Methods in Enzymology, Vol. 327: Part B: Applications of Chimeric Genes and Hybrid Proteins (J. Thorner, S.D. Emr, and J.N. Abelson, eds.) pp. 472‐500. Academic Press, San Diego.
   Verveer, P.J., Harpur, A.G., and Bastiaens, P.I.H. 2002. Imaging protein interactions by FRET microscopy. In Protein‐Protein Interactions: A Molecular Cloning Manual (E. Golemis, ed.) pp. 181‐214. Cold Spring Harbor Laboratory Press, New York.
   Zhang, J., Hupfeld, C.J., Taylor, S.S., Olefsky, J.M., and Tsien, R.Y. 2005. Insulin disrupts beta‐adrenergic signalling to protein kinase A in adipocytes. Nature 437:569‐573.
Key References
   Ni, Q., Titov, D.V., and Zhang, J. 2006. Analyzing protein kinase dynamics in living cells with FRET reporters. Methods 40:279‐286.
  This paper provides detailed information on how to design and characterize FRET‐based kinase activity biosensors.
   Zhang, J. and Allen, M.D. 2007. FRET‐based biosensors for protein kinases: Illuminating the kinome. Mol. Biosyst. 3:759‐765.
  This paper provides information on the design of genetically encodable, FRET‐based kinase activity biosensors and several examples of their use in studying kinase regulation in living cells with high spatiotemporal resolution.
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