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Using FRET-Based Reporters to Visualize Subcellular Dynamics of Protein Kinase A Activity

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 756)

Abstract

The ubiquitous Protein Kinase A (PKA) signaling pathway is responsible for the regulation of numerous processes including gene expression, metabolism, cell growth, and cell proliferation. This method details how to monitor real-time PKA activity dynamics in mammalian cells using fluorescence resonance energy transfer (FRET)-based reporters.

Key words

Protein kinase A Kinase activity reporter Fluorescence resonance energy transfer Live-cell imaging 

References

  1. 1.
    Tasken, K., and Aandahl, E. M. (2004) Localized Effects of cAMP Mediated by Distinct Routes of Protein Kinase A. Physiol Rev 84, 137–167.PubMedCrossRefGoogle Scholar
  2. 2.
    Taylor, S. S., Yang, J., Wu, J., Haste, N. M., Radzio-Andzelm, E., and Anand, G. (2004) PKA: A Portrait of Protein Kinase Dynamics. Biochim Biophys Acta 1697, 259–269.PubMedCrossRefGoogle Scholar
  3. 3.
    Skalhegg, B. S., and Tasken, K. (2000) Specificity in the cAMP/PKA Signaling Pathway. Differential Expression,Regulation, and Subcellular Localization of Subunits of PKA. Front Biosci 5, D678–93.PubMedCrossRefGoogle Scholar
  4. 4.
    Wong, W., and Scott, J. D. (2004) AKAP Signalling Complexes: Focal Points in Space and Time. Nat Rev Mol Cell Biol 5, 959–970.PubMedCrossRefGoogle Scholar
  5. 5.
    Smith, F. D., and Scott, J. D. (2002) Signaling Complexes: Junctions on the Intracellular Information Super Highway. Curr Biol 12, R32–40.PubMedCrossRefGoogle Scholar
  6. 6.
    Beene, D. L., and Scott, J. D. (2007) A-Kinase Anchoring Proteins Take Shape. Curr Opin Cell Biol 19, 192–198.PubMedCrossRefGoogle Scholar
  7. 7.
    Ananthanarayanan, B., Ni, Q., and Zhang, J. (2008) Chapter 2: Molecular Sensors Based on Fluorescence Resonance Energy Transfer to Visualize Cellular Dynamics. Methods Cell Biol 89, 37–57.PubMedCrossRefGoogle Scholar
  8. 8.
    Miyawaki, A., and Tsien, R. Y. (2000) Monitoring Protein Conformations and Interactions by Fluorescence Resonance Energy Transfer between Mutants of Green Fluorescent Protein. Methods Enzymol 327, 472–500.PubMedCrossRefGoogle Scholar
  9. 9.
    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.PubMedCrossRefGoogle Scholar
  10. 10.
    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.PubMedCrossRefGoogle Scholar
  11. 11.
    Lim, C. J., Kain, K. H., Tkachenko, E., Goldfinger, L. E., Gutierrez, E., Allen, M. D., Groisman, A., Zhang, J., and Ginsberg, M. H. (2008) Integrin-Mediated Protein Kinase A Activation at the Leading Edge of Migrating Cells. Mol Biol Cell 19, 4930–4941.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Pharmacology and Molecular SciencesThe Johns Hopkins University School of MedicineBaltimoreUSA
  2. 2.Solomon H. Snyder Department of NeuroscienceThe Johns Hopkins University School of MedicineBaltimoreUSA
  3. 3.Department of OncologyThe Johns Hopkins University School of MedicineBaltimoreUSA

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