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PMID: 15003608 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Imaging protein phosphorylation by fluorescence in single living cells.

Methods (San Diego, Calif.) ·Vol. 32 ·No. 4 ·2004-04-00 ·Pages 451-5

Sato M, Umezawa Y

Abstract

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.

MeSH Terms

Animals Bacterial Proteins/genetics,metabolism CHO Cells CSK Tyrosine-Protein Kinase Carrier Proteins/genetics,metabolism Catalysis Cricetinae Cricetulus Epidermal Growth Factor/pharmacology Estradiol/pharmacology Fluorescence Resonance Energy Transfer/methods Green Fluorescent Proteins/genetics,metabolism Insulin/pharmacology Insulin Receptor Substrate Proteins Luminescent Proteins/genetics,metabolism Microscopy, Fluorescence/methods Nitric Oxide Synthase/genetics,metabolism Nitric Oxide Synthase Type III Peptide Fragments/genetics,metabolism Phosphoproteins/genetics,metabolism Phosphorylation/drug effects Phosphotransferases/metabolism Protein Serine-Threonine Kinases/genetics,metabolism Protein-Tyrosine Kinases/metabolism Proteins/genetics,metabolism Proto-Oncogene Proteins/genetics,metabolism Proto-Oncogene Proteins c-akt Receptor, Insulin/genetics,metabolism bcl-Associated Death Protein src-Family Kinases

Chemicals

Bacterial Proteins Carrier Proteins Cyan Fluorescent Protein Insulin Insulin Receptor Substrate Proteins Luminescent Proteins Peptide Fragments Phosphoproteins Proteins Proto-Oncogene Proteins bcl-Associated Death Protein yellow fluorescent protein, Bacteria Green Fluorescent Proteins Estradiol Epidermal Growth Factor Nitric Oxide Synthase Nitric Oxide Synthase Type III Phosphotransferases Protein-Tyrosine Kinases Receptor, Insulin CSK Tyrosine-Protein Kinase src-Family Kinases Protein Serine-Threonine Kinases Proto-Oncogene Proteins c-akt

Authors & Affiliations

2 authors, click to expand affiliations / ORCID

Sato Moritoshi

Department of Chemistry, School of Science, The University of Tokyo, Japan Science and Technology Agency, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Umezawa Yoshio

Article Info

Journal

Methods (San Diego, Calif.)

Abbr.

Methods

ISSN

1046-2023

DOI

10.1016/j.ymeth.2003.10.013

Published

2004-04-00

Pages

451-5

Language

English

Region

United States

NLM ID

9426302

Subset

External Links

PubMed source DOI: 10.1016/j.ymeth.2003.10.013 View journal details

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