Phosphorylation of eIF-4E on Ser 209 in response to mitogenic and inflammatory stimuli is faithfully detected by specific antibodies

Mol Cell Biol Res Commun. 2000 Apr;3(4):205-211. doi: 10.1006/mcbr.2000.0217.

Abstract

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.

MeSH terms

  • 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*
  • 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
  • p38 Mitogen-Activated Protein Kinases

Substances

  • Antibodies
  • Cytokines
  • Eukaryotic Initiation Factor-4E
  • Intracellular Signaling Peptides and Proteins
  • Mitogens
  • Peptide Initiation Factors
  • Phosphoserine
  • MKNK1 protein, human
  • Protein Serine-Threonine Kinases
  • Mitogen-Activated Protein Kinases
  • p38 Mitogen-Activated Protein Kinases
  • Staurosporine