Imaging protein phosphorylation by fluorescence in single living cells.
Protein phosphorylation by intracellular kinases plays one of the most pivotal roles in signaling pathways within cells. To reveal the biological issues 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.
Sato
Moritoshi
M
Department of Chemistry, School of Science, The University of Tokyo, Japan Science and Technology Agency, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Umezawa
Yoshio
Y
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D000081247:CSK Tyrosine-Protein Kinase; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D006224:Cricetinae; D003412:Cricetulus; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010770:Phosphotransferases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism; D051025:bcl-Associated Death Protein; D019061:src-Family Kinases
D001426:Bacterial Proteins; D002352:Carrier Proteins; C485182:Cyan Fluorescent Protein; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D008164:Luminescent Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D051025:bcl-Associated Death Protein; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D004958:Estradiol; D004815:Epidermal Growth Factor; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D010770:Phosphotransferases; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt