title: Role of Ser-53 phosphorylation in the activity of human translation initiation factor eIF-4E in mammalian and yeast cells.
abstract: Eukaryotic translation initiation factor eIF-4E is essential for protein synthesis and cell viability. eIF-4E participates in formation of an m7GTP-cap binding protein complex that mediates association of 40S ribosomal subunits with mRNAs, which occurs only when eIF-4E is phosphorylated. Regulation of eIF-4E by phosphorylation was thought to occur on Ser53, although results potentially inconsistent with phosphorylation of this site have been reported. To resolve whether Ser53 is phosphorylated, and if so whether it regulates eIF-4E activity, we directly examined whether Ser53 is a site for phosphorylation of mammalian eIF-4E in human and yeast cells. Wild-type (wt) human eIF-4E protein variants, Ser53-->Asp53 or Ser53-->Ala53, were constructed and analyzed by overproduction in transfected human 293/T-Ag cells, or in Saccharomyces cerevisiae in which the endogenous eIF-4E gene was disrupted. Wt eIF-4E and Ser53 mutants functioned equally well in protein synthesis in both systems, and were phosphorylated to the same extent. Most importantly, the wt and Ser53 mutants of human eIF-4E produced identical tryptic phophopeptide patterns in human cells, and identical but more complicated patterns in yeast. These data demonstrate that Ser53 is not a requisite activating site for phosphorylation of mammalian eIF-4E in human or yeast cells, under conditions in which it participates in protein synthesis.
authors.affiliation: Department of Biochemistry, New York University Medical Center, NY 10016, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Zhang
authors.forename: H L
authors.initials: HL
authors.lastname: Klein
authors.forename: R J
authors.initials: RJ
authors.lastname: Schneider
mesh_terms: D002460:Cell Line; D018014:Gene Transfer Techniques; D006801:Humans; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D012441:Saccharomyces cerevisiae; D012694:Serine / Q000378:metabolism
chemical_list: D010448:Peptide Initiation Factors; C075833:eIF-4; D012694:Serine