Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A genetically encoded, fluorescent indicator for cyclic AMP in living cells

Nature Cell Biology volume 2, pages 25–29 (2000)Cite this article

Abstract

Cyclic AMP controls several signalling cascades within cells, and changes in the amounts of this second messenger have an essential role in many cellular events. Here we describe a new methodology for monitoring the fluctuations of cAMP in living cells. By tagging the cAMP effector protein kinase A with two suitable green fluorescent protein mutants, we have generated a probe in which the fluorescence resonance energy transfer between the two fluorescent moieties is dependent on the levels of cAMP. This new methodology opens the way to the elucidation of the biochemistry of cAMP in vivo.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Construction and expression of GFP-tagged PKA.
Figure 2: Fluorescence changes induced by increasing cAMP levels in cells transfected with GFP-tagged PKA.
Figure 3: Kinetics of changes in the fluorescence emission ratios from GFP-tagged PKA subunits.
Figure 4: Effect of norepinephrine on COS-7 cells co-transfected with C-S65T and RII-EBFP.

Similar content being viewed by others

References

  1. Simonds, W. F. G protein regulation of adenylate cyclase. Trends Pharmacol. Sci. 20, 66–73 (1999).

    Article  CAS  Google Scholar 

  2. Brandon, E. P., Idzerda, R. L. & McKnight, G. S. PKA isoforms, neural pathways, and behaviour: making the connection. Curr. Opin. Neurobiol. 7, 397–403 (1997).

    Article  CAS  Google Scholar 

  3. Walsh, D. A. & Van-Patten, S. M. Multiple pathway signal transduction by the cAMP-dependent protein kinase. FASEB J. 8, 1227–1236 (1994).

    Article  CAS  Google Scholar 

  4. Meinkoth, J. L. et al. Signal transduction through the cAMP-dependent protein kinase. Mol. Cell. Biochem. 127, 179–187 (1993).

    Article  Google Scholar 

  5. Lee, K. A. Transcriptional regulation by cAMP. Curr. Opin. Cell Biol. 3, 953–959 (1991).

    Article  CAS  Google Scholar 

  6. Clegg, R. M. in Fluorescence Imaging Spectroscopy and Microscopy (eds Wang, X. F. & Herman, B.) 172–252 (Wiley, New York, 1996).

    Google Scholar 

  7. Adams, S. R., Harootunian, A. T., Buechler, Y. J., Taylor, S. S. & Tsien, R. Y. Fluorescence ratio imaging of cyclic AMP in single cells. Nature 349, 694–697 (1991).

    Article  CAS  Google Scholar 

  8. Tsien, R. Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    Article  CAS  Google Scholar 

  9. Adams, S. R., Bacskai, B. J., Taylor, S. S. & Tsien, R. Y. in Fluorescence and Luminescent Probes for Biological Activity 2nd edn (ed. Mason, W. T.) 156–172 (Academic, San Diego, 1999).

    Book  Google Scholar 

  10. Zlokarnik, G. et al. Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter. Science 279, 84–88 (1998).

    Article  CAS  Google Scholar 

  11. Knighton, D. R. et al. Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science 253, 407–414 (1991).

    Article  CAS  Google Scholar 

  12. Biondi, R. M., Baehler, P. J., Reymond, C. D. & Veron, M. Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum. Nucleic Acids Res. 26, 4946–4952 (1998).

    Article  CAS  Google Scholar 

  13. Colledge, M. & Scott, J. D. AKAPs: from structure to function. Trends Cell Biol. 9, 216–221 (1999).

    Article  CAS  Google Scholar 

  14. Griffin, B. A., Adams, S. R. & Tsien, R. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–272 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank G. Ronconi and M. Santato for technical assistance. This work was supported by grants to T.P. from the Italian Association for Cancer Research (AIRC; grant number 98), from Telethon (grant 1226), from the CNR target project Biotechnology (grant 049), from the EU Programs Biotechnology (grants BIO4CT960382 and TMR FMRXCT960382), and from the Armenise Harvard Foundation, and to C.Y.C. and R.Y.T. from the Howard Hughes Medical Institute.

Correspondence and requests for materials should be addressed to T.P.

Author information

Author notes

  1. Manuela Zaccolo, Francesca De Giorgi, Luxin Feng, Tom Knapp and Paul A. Negulescu: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Experimental Biomedical Sciences, University of Padova, Padova, 35121, Italy

    Manuela Zaccolo, Francesca De Giorgi & Tullio Pozzan

  2. Howard Hughes Medical Institute, University of California, San Diego, 92093, California, USA

    Charles Y. Cho, Susan S. Taylor & Roger Y. Tsien

  3. Department of Pharmacology 0647, University of California, San Diego, 92093, California, USA

    Charles Y. Cho & Roger Y. Tsien

  4. Department of Chemistry and Biochemistry, University of California, San Diego, 92093, California, USA

    Susan S. Taylor & Roger Y. Tsien

  5. Aurora Biosciences Corporation, 11010 Torreyana Road, San Diego, 92121, California, USA

    Luxin Feng, Tom Knapp & Paul A. Negulescu

Authors
  1. Manuela Zaccolo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Francesca De Giorgi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Charles Y. Cho
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Luxin Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tom Knapp
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Paul A. Negulescu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Susan S. Taylor
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Roger Y. Tsien
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Tullio Pozzan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tullio Pozzan.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zaccolo, M., De Giorgi, F., Cho, C. et al. A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat Cell Biol 2, 25–29 (2000). https://doi.org/10.1038/71345

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/71345

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing