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

Eukaryotic translation initiation factor 4E (eIF4E) is essential for effici ent 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 t he assembly of the 48S preinitiation complex. eIF4E is phosphorylated in vi vo at residue 209 of the human sequence. The phosphorylated form is often r egarded as the active state of the protein, with ribosome-associated eIF4E enriched for the phosphorylated form and increased phosphorylation often co rrelated with upregulation of rates of protein synthesis. However, the only reported measured effect attributable to phosphorylation at the physiologi cal site has been a relatively small increase in the affinity of eIF4E for the mRNA m(7)GTP cap structure. Here, we provide data to suggest that phosp horylation of eIF4E at Ser209 is not required for translation. eIF4E that i s modified such that it cannot be phosphorylated (Ser209 --> Ala), is unimp aired in its ability to restore translation to an eIF4E-dependent in vitro translation system. In addition, both the wild-type and mutant forms of eIF 4E 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.