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

A. Flynn, C. G. Proud

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Abstract

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.

Original languageEnglish
Pages (from-to)21684-21688
Number of pages5
JournalJournal of Biological Chemistry
Volume270
Issue number37
DOIs
Publication statusPublished or Issued - 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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