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.
Flynn
A
A
Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, United Kingdom.
Proud
C G
CG
D000595:Amino Acid Sequence; D000818:Animals; D001769:Blood; D016466:CHO Cells; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003470:Culture Media; D039561:Eukaryotic Initiation Factor-4E; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D012694:Serine; D014357:Trypsin
D003470:Culture Media; D039561:Eukaryotic Initiation Factor-4E; D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D010748:Phosphopeptides; D010768:Phosphoserine; D012694:Serine; D014357:Trypsin