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A fluorescent indicator for visualizing cAMP-induced phosphorylation in vivo

Nature Biotechnology volume 18, pages 313–316 (2000)Cite this article

Abstract

We have developed a method for visualizing phosphorylation of proteins in living cells using a novel fluorescent indicator composed of two green fluorescent protein (GFP) variants joined by the kinase-inducible domain (KID) of the transcription factor cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB). Phosphorylation of KID by the cAMP-dependent protein kinase A (PKA) decreased the fluorescence resonance energy transfer (FRET) among the flanking GFPs. By transfecting COS-7 cells with an expression vector encoding this indicator protein (termed ART for cAMP-responsive tracer), we were able to visualize activation dynamics of PKA in living cells.

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Figure 1: Phosphorylation of ART by PKA in vitro.
Figure 2: Phosphorylation-induced change of FRET.
Figure 3: Imaging of FRET of ART in COS-7 cells.
Figure 4: Effect of PKA inhibitor on the FRET in the cells.

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Acknowledgements

We thank Dr. Anthony Persechini (University of Rochester) for pETIC-1 plasmid and Grace M. Lopresto-Mizuno for English correction. This work was supported by Research for the Future Program, the Inamori Foundation, Nissan Science Foundation, The Mitsubishi Foundation, Project Research Program (Cell Fate Modulation Research Unit) of the Medical Research Institute, Tokyo Medical and Dental University, and grants from the Ministry of Education, Science and Culture in Japan.

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  1. Satoshi Inouye

    Present address: Yokohama Research Center, Chisso Corp., Yokohama, Kanagawa, 236-8605, Japan

Authors and Affiliations

  1. Department of Functional Genomics, Medical Research Institute, Tokyo Medical and Dental University, Yushima, Tokyo, 113-8510, Japan

    Yasuo Nagai, Masami Miyazaki, Ryoko Aoki, Takeru Zama & Masatoshi Hagiwara

  2. Scripps Institution of Oceanography, University of California at San Diego, La Jolla, 92093-0202 , CA, USA

    Satoshi Inouye

  3. Department of Pharmacology, Faculty of Medicine, University of Tokyo, Hongo, Tokyo, 113-0033, Japan

    Kenzo Hirose & Masamitsu Iino

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Correspondence to Masatoshi Hagiwara.

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Nagai, Y., Miyazaki, M., Aoki, R. et al. A fluorescent indicator for visualizing cAMP-induced phosphorylation in vivo. Nat Biotechnol 18, 313–316 (2000). https://doi.org/10.1038/73767

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