Genetically Encoded Fluorescent Indicators to Visualize Protein Phosphorylation in Living Cells.
Protein phosphorylation by intracellular kinases plays one of the most pivotal roles in signaling pathways within cells. To reveal the biological processes related to the kinase proteins, electrophoresis, immunocytochemistry, and in vitro kinase assay have been used. However, these conventional methods do not provide enough information about spatial and temporal dynamics of the signal transduction based on protein phosphorylation and dephosphorylation in living cells. To overcome the limitation for investigating the kinase signaling, we developed genetically encoded fluorescent indicators for visualizing the protein phosphorylation in living cells. Using these indicators, we visualized under a fluorescence microscope when, where, and how the protein kinases are activated in single living cells.
Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo, 153-8902, Japan.
Moritoshi
M
Sato
Department of Chemistry, School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan. umezawa@chem.s.u-tokyo.ac.jp.
Yoshio
Y
Umezawa
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics*; D015374:Biosensing Techniques; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005813:Genes, Synthetic; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics*; D006801:Humans; D000069416:Intravital Microscopy / Q000379:methods*; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics*; D008856:Microscopy, Fluorescence / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011972:Receptor, Insulin / Q000037:antagonists & inhibitors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics*; D015398:Signal Transduction
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C527701:enhanced cyan fluorescent protein; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D011494:Protein Kinases; D011972:Receptor, Insulin
Fluorescence resonance energy transfer (FRET); Fluorescent indicators; Live cell imaging; Protein kinases; Protein phosphorylation reactions; Subcellular dynamic s