Phosphorylation of eIF-4E on Ser 209 in Response to Mitogenic and Inflammatory Stimuli Is Faithfully Detected by Specific Antibodies

Phosphorylation of Ser 209 is thought to modulate the activity of the cap-binding factor eIF-4E which is a crucial component in the initiation complex for cap-dependent translation of mRNA. We report here the full reconstitution of the p38 Map kinase cascade leading to phosphorylation of eIF-4E in v...

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Bibliographic Details
Published in:Molecular cell biology research communications 2000-04, Vol.3 (4), p.205-211
Main Authors: Tschopp, C., Knauf, U., Brauchle, M., Zurini, M., Ramage, P., Glueck, D., New, L., Han, J., Gram, H.
Format: Article
Language:English
Subjects:
Antibodies - immunology
Antibody Specificity - immunology
Cell-Free System
Cells, Cultured
Cytokines - immunology
Cytokines - pharmacology
Eukaryotic Initiation Factor-4E
Fibroblasts
Fluorescent Antibody Technique
HeLa Cells
Humans
Inflammation - immunology
Intracellular Signaling Peptides and Proteins
MAP Kinase Signaling System - drug effects
Mitogen-Activated Protein Kinases - metabolism
Mitogens - pharmacology
p38 Mitogen-Activated Protein Kinases
Peptide Initiation Factors - metabolism
Phosphorylation - drug effects
Phosphoserine - immunology
Phosphoserine - metabolism
Protein-Serine-Threonine Kinases - antagonists & inhibitors
Protein-Serine-Threonine Kinases - metabolism
Skin - cytology
Skin - enzymology
Staurosporine - analogs & derivatives
Staurosporine - pharmacology
Time Factors
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Summary:Phosphorylation of Ser 209 is thought to modulate the activity of the cap-binding factor eIF-4E which is a crucial component in the initiation complex for cap-dependent translation of mRNA. We report here the full reconstitution of the p38 Map kinase cascade leading to phosphorylation of eIF-4E in vitro and the generation of antibodies specific for phospho-serine 209 in eIF-4E. These antibodies were used to probe the phosphorylation of eIF-4E in mammalian cells stimulated with mitogens and pro-inflammatory cytokines. Treatment of human dermal fibroblasts with FCS led to a transient hyperphosphorylation, followed by hypophosphorylation and return to normal state phosphorylation at 16 h after the initial stimulation. By using a potent small molecular weight inhibitor of Mnk1, the upstream kinase for eIF-4E, we observed a rapid dephosphorylation of eIF-4E within 45 min after addition of the inhibitor, suggesting a high turnover of phosphate on eIF-4E mediated by Mnk1 and a yet unidentified phosphatase.
ISSN:1522-4724
1522-4732
DOI:10.1006/mcbr.2000.0217