Phosphorylation of eukaryotic initiation factor EIF-4E

Eukaryotic protein synthesis initiation factor eIF-4E is a 25-kDa protein which binds to the cap structure of mRNA and is involved in the entry of mRNA into the initiation of protein synthesis. EIF-4E is isolated as a mixture of phosphorylated and nonphosphorylated forms. The site of phosphorylation was determined to be serine-53 of the eIF-4E sequence. The amino acid sequence surrounding the phosphorylated serine is 40-QNRWALWFFKNDKSKTWQANLRL-62. Tryptic digestion of eIF-4E, labeled with /sup 32/P in intact rabbit reticulocytes, yielded one labeled peptide whose amino acid composition was consistent with the structure SK. After treatment of the protein with citraconic anhydride to block lysyl residues, tryptic digestion yielded a labeled peptide whose composition was consistent with the structure WALWFFKNDKSKTWQANLR. A protein kinase was purified from rabbit reticulocyte lysate which specifically phosphorylated eIF-4E and not other initiation factors. Peptide analysis of the in vitro-phosphorylated protein revealed that a single site was phosphorylated and that it was the same as the in vivo-phosphorylated site. Comparison of eIF-4E and eIF-4F as substrates for the kinase revealed that the K/sub s/ was 15-fold lower for eIF-4F, indicating that the latter is the preferred substrate.