Fluorescent indicators for imaging protein phosphorylation in single living cells.
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.
Sato
Moritoshi
M
Department of Chemistry, School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Ozawa
Takeaki
T
Inukai
Kouichi
K
Asano
Tomoichiro
T
Umezawa
Yoshio
Y
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
D008164:Luminescent Proteins; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins