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Fluorescent indicators for imaging protein phosphorylation in single living cells

Nature Biotechnology volume 20, pages 287–294 (2002)Cite this article

Abstract

To visualize signal transduction based on protein phosphorylation in living cells, we have developed genetically encoded fluorescent indicators, named phocuses. Two different color mutants of green fluorescent protein (GFP) were joined by a tandem fusion domain composed of a substrate domain for the protein kinase of interest, a flexible linker sequence, and a phosphorylation recognition domain that binds with the phosphorylated substrate domain. Intramolecular interaction of the substrate domain and the adjacent phosphorylation recognition domain within a phocus was dependent upon phosphorylation of the substrate domain by protein kinase, which influenced the efficiency of fluorescence resonance energy transfer (FRET) between the GFPs within a phocus. In the present study, we employed phocuses composed of insulin signaling proteins to visualize protein phosphorylation by the insulin receptor. This method may provide a general approach for studying the dynamics of protein phosphorylation–based signal transduction in living cells.

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Figure 1: Fluorescent indicators for protein phosphorylation in living cells.
Figure 2: Fluorescence imaging of phocus-2nes upon insulin stimulation.
Figure 3: FRET in phocus-2nes.
Figure 4: Dephosphorylation of phocuses.
Figure 5: Fluorescence imaging with phocus-2ppnes. (A) CHO-IR cells expressing phocus-2ppnes were starved with a serum-free medium and stimulated with 100 nM insulin for 20 min at 25°C.
Figure 6: Response of phocuses in a differentiated L6 myotube.
Figure 7: Selectivity of phocus-2ppnes for tyrosine kinase receptors.

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Acknowledgements

This work has been supported by CREST (Core Research for Evolutional Science and Technology) of JST (Japan Science and Technology) and grants to Y.U. from the Ministry of Education, Science and Culture, Japan. We thank Prof. Y. Ebina for providing CHO-EGFR and CHO-PDGFR cell. We thank Y. Imai for her experimental help.

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Authors and Affiliations

  1. Department of Chemistry, School of Science, The University of Tokyo, and Japan Science and Technology Corporation (JST), Hongo, Bunkyo-ku, 113-0033, Tokyo, Japan

    Moritoshi Sato, Takeaki Ozawa & Yoshio Umezawa

  2. Third Department of Internal Medicine, School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, 113-0033, Tokyo, Japan

    Kouichi Inukai & Tomoichiro Asano

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  1. Moritoshi Sato
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Correspondence to Yoshio Umezawa.

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Sato, M., Ozawa, T., Inukai, K. et al. Fluorescent indicators for imaging protein phosphorylation in single living cells. Nat Biotechnol 20, 287–294 (2002). https://doi.org/10.1038/nbt0302-287

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