Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells.

Détails

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Etat: Serval
Version: de l'auteur
ID Serval
serval:BIB_7502
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells.
Périodique
Journal of Cell Biology
Auteur(s)
Oancea E., Teruel M.N., Quest A.F., Meyer T.
ISSN
0021-9525
Statut éditorial
Publié
Date de publication
1998
Volume
140
Numéro
3
Pages
485-498
Langue
anglais
Notes
Publication types: Journal Article
Résumé
Cysteine-rich domains (Cys-domains) are approximately 50-amino acid-long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-gamma (Cys1-GFP). Strikingly, stimulation of G-protein or tyrosine kinase-coupled receptors induced a transient translocation of cytosolic Cys1-GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1-GFP in the membrane, whereas DiC8 left Cys1-GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1-GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-gamma also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2-GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester-mediated translocation of proteins to selective lipid membranes.
Mots-clé
Animals, Arachidonic Acid/pharmacology, Cell Membrane/enzymology, Cell Membrane/metabolism, Cloning, Molecular, Cytosol/enzymology, Cytosol/metabolism, Diffusion, Diglycerides/metabolism, Diglycerides/pharmacology, Green Fluorescent Proteins, Isoenzymes/chemistry, Isoenzymes/metabolism, Luminescent Proteins/metabolism, Nuclear Envelope/enzymology, Nuclear Envelope/metabolism, Platelet Activating Factor, Platelet Membrane Glycoproteins/metabolism, Protein Kinase C/chemistry, Protein Kinase C/metabolism, Rats, Receptors, Cell Surface, Receptors, G-Protein-Coupled, Receptors, IgE/metabolism, Recombinant Fusion Proteins/metabolism, Second Messenger Systems, Signal Transduction, Tetradecanoylphorbol Acetate/pharmacology, Transfection, Tumor Cells, Cultured
Pubmed
Web of science
Open Access
Oui
Création de la notice
19/11/2007 13:45
Dernière modification de la notice
08/05/2019 20:29
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