Phosphorylation of eukaryotic initiation factor 4E (eIF4E) at Ser209 is not required for protein synthesis in vitro and in vivo

Eur J Biochem. 2001 Oct;268(20):5375-85. doi: 10.1046/j.0014-2956.2001.02478.x.

Abstract

Eukaryotic translation initiation factor 4E (eIF4E) is essential for efficient translation of the vast majority of capped cellular mRNAs; it binds the 5'-methylated guanosine cap of mRNA and serves as a nucleation point for the assembly of the 48S preinitiation complex. eIF4E is phosphorylated in vivo at residue 209 of the human sequence. The phosphorylated form is often regarded as the active state of the protein, with ribosome-associated eIF4E enriched for the phosphorylated form and increased phosphorylation often correlated with upregulation of rates of protein synthesis. However, the only reported measured effect attributable to phosphorylation at the physiological site has been a relatively small increase in the affinity of eIF4E for the mRNA m7GTP cap structure. Here, we provide data to suggest that phosphorylation of eIF4E at Ser209 is not required for translation. eIF4E that is modified such that it cannot be phosphorylated (Ser209-->Ala), is unimpaired in its ability to restore translation to an eIF4E-dependent in vitro translation system. In addition, both the wild-type and mutant forms of eIF4E interact equally well with eIF4G, with the phosphorylation of eIF4E not required to effect the change in conformation of eIF4G that is required for efficient cleavage of eIF4G by L-protease. Furthermore, we show that wild-type and phosphorylation-site variants of eIF4E protein are equally able to rescue the lethal phenotype of eIF4E deletion in S. cerevisiae.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Blotting, Western
  • Cell Extracts
  • Endopeptidases / metabolism
  • Eukaryotic Initiation Factor-4E
  • Eukaryotic Initiation Factor-4G
  • Humans
  • Luciferases / genetics
  • Luciferases / metabolism
  • Mutation / genetics
  • Peptide Initiation Factors / chemistry*
  • Peptide Initiation Factors / genetics
  • Peptide Initiation Factors / metabolism*
  • Phosphorylation
  • Polymerase Chain Reaction
  • Polyribosomes / chemistry
  • Polyribosomes / metabolism
  • Protein Biosynthesis*
  • Rabbits
  • Recombinant Proteins / metabolism
  • Reticulocytes / cytology
  • Reticulocytes / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / growth & development
  • Serine / metabolism*

Substances

  • Cell Extracts
  • Eukaryotic Initiation Factor-4E
  • Eukaryotic Initiation Factor-4G
  • Peptide Initiation Factors
  • Recombinant Proteins
  • Serine
  • Luciferases
  • Endopeptidases