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.
Highlights
Phosphorylation of eukaryotic initiation factor1-4E, which binds to the 5Ј-cap structure on eukaryotic mRNAs, has been shown to correlate positively with changes in the rate of translation under a wide range of conditions. eIF-4E is the least abundant of the components of the eIF-4F complex [4], which contains the RNA helicase eIF-4A and eIF-4␥, and translation of messages with a high degree of secondary structure is enhanced by overexpression of eIF-4E [5]
As we have previously reported,4 isoelectric focusing analysis reveals the existence of only two distinct species of eIF-4E in Chinese hamster ovary (CHO) cells as detected by immunoblotting, and treatment of isolated eIF-4E with alkaline phosphatase causes conversion of all the factor to the more basic form
These findings are consistent with the data from a range of other cell types indicating that eIF-4E contains one major phosphorylation site [20]
Summary
Phosphorylation of eukaryotic initiation factor (eIF)1-4E ( known as eIF-4␣), which binds to the 5Ј-cap structure on eukaryotic mRNAs, has been shown to correlate positively with changes in the rate of translation under a wide range of conditions (for reviews, see Refs. 1–3). eIF-4E is the least abundant of the components of the eIF-4F complex [4], which contains the RNA helicase eIF-4A and eIF-4␥, and translation of messages with a high degree of secondary structure is enhanced by overexpression of eIF-4E [5]. Rhoads (Shreveport, LA) had obtained data indicating that Ser-53 was not the major phosphorylation site in eIF-4E.
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