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 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.
Tschopp
C
C
NOVARTIS Pharma AG, Basel, CH-4002, Switzerland.
Knauf
U
U
Brauchle
M
M
Zurini
M
M
Ramage
P
P
Glueck
D
D
New
L
L
Han
J
J
Gram
H
H
D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D002474:Cell-Free System; D002478:Cells, Cultured; D016207:Cytokines / Q000276:immunology / Q000494:pharmacology; D039561:Eukaryotic Initiation Factor-4E; D005347:Fibroblasts; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D007249:Inflammation / Q000276:immunology; D047908:Intracellular Signaling Peptides and Proteins; D020935:MAP Kinase Signaling System / Q000187:drug effects; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008934:Mitogens / Q000494:pharmacology*; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000276:immunology* / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D012867:Skin / Q000166:cytology / Q000201:enzymology; D019311:Staurosporine / Q000031:analogs & derivatives / Q000494:pharmacology; D013997:Time Factors; D048051:p38 Mitogen-Activated Protein Kinases
D000906:Antibodies; D016207:Cytokines; D039561:Eukaryotic Initiation Factor-4E; D047908:Intracellular Signaling Peptides and Proteins; D008934:Mitogens; D010448:Peptide Initiation Factors; D010768:Phosphoserine; C491395:MKNK1 protein, human; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D019311:Staurosporine