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 m 7GTP-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 Ser 53, although results potentially inconsistent with phosphorylation of this site have been reported. To resolve whether Ser 53 is phosphorylated, and if so whether it regulates eIF-4E activity, we directly examined whether Ser 53 is a site for phophorylation of mammalian eIF-4E in human and yeast cells. Wild-type (wt) human eIF-4E protein variants, Ser 53→Asp 53 or Ser 53→Ala 53, 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 Ser 53 mutants functioned equally well in protein synthesis in both systems, and were phosphorylated to the same extent. Most importantly, the wt and Ser 53 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 Ser 53 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.