Visualization of Kinase Activity with FRET‐Based Activity Biosensors
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- Abstract
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Abstract
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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PDF or HTML at Wiley Online LibraryTable 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
Materials
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PDF or HTML at Wiley Online LibraryFigures
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Figure 18.15.1 General design and mechanism of kinase activity reporters. When incorporated between a FRET pair, the conformational change of the molecular switch, which is induced via kinase‐mediated phosphorylation and subsequent intramolecular binding between the phosphorylated substrate and phosphoamino acid‐binding domain (PAABD), results in a change in FRET. View Image -
Figure 18.15.2 Diffusible (left) and plasma membrane‐targeted (right) AKAR. View Image -
Figure 18.15.3 Excitation and emission spectra for CFP and YFP. View Image -
Figure 18.15.4 Plasma membrane–targeted AKAR responses to PKA stimulation. HEK 293 cells transfected with either pmAKAR or pmAKAR‐T/A (negative control) generate different responses upon forskolin (Fsk), an AC activator, which demonstrates that the pmAKAR response is phosphorylation dependent. View Image
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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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