title: Fluorescent indicators for imaging protein phosphorylation in single living cells.
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.
authors.affiliation: Department of Chemistry, School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Moritoshi
authors.initials: M
authors.lastname: Sato
authors.forename: Takeaki
authors.initials: T
authors.lastname: Ozawa
authors.forename: Kouichi
authors.initials: K
authors.lastname: Inukai
authors.forename: Tomoichiro
authors.initials: T
authors.lastname: Asano
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Umezawa
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D008957:Models, Genetic; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
chemical_list: D008164:Luminescent Proteins; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins