Characterization of wild-type and Ser53 mutant eukaryotic initiation factor 4E overexpression in mammalian cells.
Eukaryotic translation initiation factor 4E (eIF-4E) is one component of the m7G-cap-binding protein complex eIF-4F and is required for cap-dependent translation initiation. The phosphorylation state of eIF-4E correlates with increased activity and a major phosphorylation site resides at serine 53. To further evaluate the role of eIF-4E phosphorylation, eIF-4E wild-type and two Ser53 mutants, Ser53Ala and Ser53Asp, were expressed at high level, representing almost 2% of the total cell protein, by transient transfection of COS-1 monkey cells. 32PO4 metabolic labeling of transfected cells demonstrated both Ser53 mutants were phosphorylated at an alternate serine residue. [35S]Methionine pulse-labeling demonstrated that the wild-type and both Ser53 mutants were equally incorporated into the eIF-4F complex. The effect of wild-type and Ser53 mutant overexpression on cap-dependent translation initiation and internal translation initiation was monitored by cotransfection with an expression vector encoding a dicistronic mRNA for which the 5' cistron is translated in a cap-dependent manner, and the 3' cistron is translated by internal ribosome binding. Unexpectedly, overexpression of the wild-type or either mutant did not affect the efficiency of either cap-dependent or internal initiation. These results demonstrate that phosphorylation of eIF-4E at residue 53 is not required for interaction with p220 and suggest that Ser53 phosphorylation may not be required for cap-dependent translation initiation in this system.
Genetics Institute, Cambridge, Massachusetts 02140.
R J
RJ
Kaufman
P
P
Murtha-Riel
D D
DD
Pittman
M V
MV
Davies
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D004591:Electrophoresis, Polyacrylamide Gel; D039561:Eukaryotic Initiation Factor-4E; D039562:Eukaryotic Initiation Factor-4F; D015870:Gene Expression; D005822:Genetic Vectors; D007525:Isoelectric Focusing; D007668:Kidney; D046911:Macromolecular Substances; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010448:Peptide Initiation Factors / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012694:Serine; D014162:Transfection
D039561:Eukaryotic Initiation Factor-4E; D039562:Eukaryotic Initiation Factor-4F; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D010448:Peptide Initiation Factors; D011994:Recombinant Proteins; D012694:Serine; D008715:Methionine