Serine 209, not serine 53, is the major site of phosphorylation in initiation factor eIF-4E in serum-treated Chinese hamster ovary cells

Ser-53 has previously been considered the major phosphorylation site in eukaryotic initiation factor (eIF)-4E, and this appeared to be supported by studies using a S53A mutant. Recently, however, several lines of evidence have indicated that Ser-53 might not be the true phosphorylation site. This prompted us to re-examine the phosphorylation site in eIF-4E using factor purified from ³²P-labeled, serum-treated Chinese hamster ovary cells. Isoelectric focusing and phosphoamino acid analysis indicated the existence of a single phosphorylated serine. Edman degradation of the major radiolabeled tryptic product from ³²P-labeled eIF-4E showed that the phosphorylated site was positioned three residues from the N terminus of this peptide. There are three serines in the sequence of eIF-4E that are three residues away from a tryptic cleavage site (i.e. lysine or arginine). ³²P- Labeled eIF-4E was digested with trypsin, Lys-C, or trypsin followed by Glu- C and subjected to two-dimensional mapping; the data obtained eliminated two of these potential sites, leaving Ser-209. Comigration of the synthetic peptide SGS(P)²⁰⁹TTK with the radiolabeled tryptic product on (i) reverse- phase chromatography and (ii) two-dimensional mapping at different pH values confirmed that Ser-209 is the major phosphorylation site in eIF-4E in serum- stimulated Chinese hamster ovary cells.