TI - Phosphorylation of eukaryotic initiation factor 4E markedly reduces its affinity for capped mRNA . AB - In eukaryotes , a key step in the initiation of translation is the binding of the eukaryotic initiation factor 4E ( eIF4E ) to the cap structure of the mRNA . Subsequent recruitment of several components , including the small ribosomal subunit , is thought to allow migration of initiation complexes and recognition of the initiation codon . Mitogens and cytokines stimulate the phosphorylation of eIF4E at Ser(209) , but the functional consequences of this modification have remained a major unresolved question . Using fluorescence spectroscopy and surface plasmon resonance techniques , we show that phosphorylation of eIF4E markedly reduces its affinity for capped RNA , primarily due to an increased rate of dissociation . Variant eIF4E proteins harboring negatively charged acidic residues at position 209 also showed decreased binding to capped RNA . Furthermore , a basic residue at position 159 was shown to be essential for cap binding . Although eIF4E -binding protein 1 greatly stabilized binding of phosphorylated eIF4E to capped RNA , in the presence of eIF4E -binding protein 1 the phosphorylated form still dissociated faster compared with nonphopshorylated eIF4E . The implications of our findings for the mechanism of translation initiation are discussed .