title: In vitro activity of human translation initiation factor eIF4B is not affected by phosphomimetic amino acid substitutions S422D and S422E.
abstract: Eukaryotic translation initiation factor eIF4B is necessary for ribosomal scanning through structured mRNA leaders. In higher eukaryotes, eIF4B serves as a downstream effector of several signaling pathways. In response to mitogenic stimuli, eIF4B undergoes multiple phosphorylations which are thought to regulate its activity. Recently, Ser422 was identified as a predominant site for human eIF4B phosphorylation via several signaling pathways, and phosphomimetic amino acid substitutions S422D or S422E were shown to activate eIF4B in living cells. However, stimulatory role of these modifications has never been analyzed directly. Here, using both mammalian reconstituted translation initiation assay and complete cell-free translation system, we perform a comparison of recombinant eIF4B derivatives with the wild type recombinant protein, and do not find any difference in their activities. On the contrary, native eIF4B purified from HeLa cells reveals significantly higher activity in both assays. Thus, the effects of S422D and S422E substitutions on eIF4B activity in living cells observed previously either require some other protein modification(s), or may only be manifested in an intact cell. Our study raises the question on whether the phosphorylation of Ser422 is sufficient for eIF4B activation observed upon mitogenic stimulation.
authors: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Bldg. A, Leninskie Gory, Moscow 119234, Russia.
authors: Lev I
authors: LI
authors: Shagam
authors: Ilya M
authors: IM
authors: Terenin
authors: Dmitri E
authors: DE
authors: Andreev
authors: Jacov E
authors: JE
authors: Dunaevsky
authors: Sergey E
authors: SE
authors: Dmitriev
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002474:Cell-Free System; D039642:Eukaryotic Initiation Factors / Q000235:genetics* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008934:Mitogens / Q000494:pharmacology; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D014176:Protein Biosynthesis / Q000235:genetics*; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics
chemical_list: D039642:Eukaryotic Initiation Factors; D008934:Mitogens; C031799:eIF-4B; D012694:Serine