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.