Phosphorylation of eukaryotic protein synthesis initiation factor 4E at Ser-209.
Initiation factor 4E (eIF-4E) binds to the m7GTP-containing cap of eukaryotic mRNA and facilitates the entry of mRNA into the initiation cycle of protein synthesis. eIF-4E is a phosphoprotein, and the phosphorylated form binds to mRNA caps 3-4-fold more tightly than the nonphosphorylated form. A previous study indicated that the major phosphorylation site was Ser-53 (Rychlik, W., Russ, M. A., and Rhoads, R. E. (1987) J. Biol. Chem. 262, 10434-10437). In the present study, we synthesized the phosphopeptide expected to result from tryptic digestion of eIF-4E, O-phosphoseryllysine. Surprisingly, the tryptic and synthetic phosphopeptides did not comigrate electrophoretically. Accordingly, we redetermined the phosphorylation site by isolating a chymotryptic phosphopeptide on reverse phase high performance liquid chromatography. The peptide was sequenced by Edman degradation and corresponded to 198QSHADTATKSGSTTKNRF215. The site of phosphorylation was determined to be Ser-209 by four methods: the increase in the ratio of dehydroalanine to serine derivatives during Edman degradation, the release of 32P, the further digestion of the chymotryptic phosphopeptide with trypsin, Glu-C, and Asp-N, and site-directed mutagenesis of eIF-4E cDNA. The S209A variant was not phosphorylated in a rabbit reticulocyte lysate system, whereas the wild-type, S53A, and S207A variants were. This site falls within the consensus sequence for phosphorylation by protein kinase C.
Joshi
B
B
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport 71130-3932, USA.
Cai
A L
AL
Keiper
B D
BD
Minich
W B
WB
Mendez
R
R
Beach
C M
CM
Stepinski
J
J
Stolarski
R
R
Darzynkiewicz
E
E
Rhoads
R E
RE
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D017931:DNA Primers; D039561:Eukaryotic Initiation Factor-4E; D006801:Humans; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism*
D017931:DNA Primers; D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors; D012694:Serine; D002918:Chymotrypsin