Nature Biotechnology
20, 287 - 294 (2002)
doi:10.1038/nbt0302-287
Fluorescent indicators for imaging protein phosphorylation in single living cellsMoritoshi Sato1, Takeaki Ozawa1, Kouichi Inukai2, Tomoichiro Asano2
& Yoshio Umezawa11
Department of Chemistry, School of Science, The University of Tokyo, and Japan Science and Technology Corporation (JST), Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 2
Third Department of Internal Medicine, School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Correspondence should be addressed to Yoshio Umezawa umezawa@chem.s.u-tokyo.ac.jpTo 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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