title: Raf kinases mediate the phosphorylation of eukaryotic translation elongation factor 1A and regulate its stability in eukaryotic cells.
abstract: We identified eukaryotic translation elongation factor 1A (eEF1A) Raf-mediated phosphorylation sites and defined their role in the regulation of eEF1A half-life and of apoptosis of human cancer cells. Mass spectrometry identified in vitro S21 and T88 as phosphorylation sites mediated by B-Raf but not C-Raf on eEF1A1 whereas S21 was phosphorylated on eEF1A2 by both B- and C-Raf. Interestingly, S21 belongs to the first eEF1A GTP/GDP-binding consensus sequence. Phosphorylation of S21 was strongly enhanced when both eEF1A isoforms were preincubated prior the assay with C-Raf, suggesting that the eEF1A isoforms can heterodimerize thus increasing the accessibility of S21 to the phosphate. Overexpression of eEF1A1 in COS 7 cells confirmed the phosphorylation of T88 also in vivo. Compared with wt, in COS 7 cells overexpressed phosphodeficient (A) and phospho-mimicking (D) mutants of eEF1A1 (S21A/D and T88A/D) and of eEF1A2 (S21A/D), resulted less stable and more rapidly proteasome degraded. Transfection of S21 A/D eEF1A mutants in H1355 cells increased apoptosis in comparison with the wt isoforms. It indicates that the blockage of S21 interferes with or even supports C-Raf induced apoptosis rather than cell survival. Raf-mediated regulation of this site could be a crucial mechanism involved in the functional switching of eEF1A between its role in protein biosynthesis and its participation in other cellular processes.
authors.affiliation: Department of Biochemistry and Medical Biotechnology, University of Naples Federico II, Naples, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Sanges
authors.forename: C
authors.initials: C
authors.lastname: Scheuermann
authors.forename: R P
authors.initials: RP
authors.lastname: Zahedi
authors.forename: A
authors.initials: A
authors.lastname: Sickmann
authors.forename: A
authors.initials: A
authors.lastname: Lamberti
authors.forename: N
authors.initials: N
authors.lastname: Migliaccio
authors.forename: A
authors.initials: A
authors.lastname: Baljuls
authors.forename: M
authors.initials: M
authors.lastname: Marra
authors.forename: S
authors.initials: S
authors.lastname: Zappavigna
authors.forename: J
authors.initials: J
authors.lastname: Reinders
authors.forename: U
authors.initials: U
authors.lastname: Rapp
authors.forename: A
authors.initials: A
authors.lastname: Abbruzzese
authors.forename: M
authors.initials: M
authors.lastname: Caraglia
authors.forename: P
authors.initials: P
authors.lastname: Arcari
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D019556:COS Cells; D002522:Chlorocebus aethiops; D005786:Gene Expression Regulation; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D020648:Peptide Elongation Factor 1 / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D055503:Protein Multimerization; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
chemical_list: C094814:EEF1A1 protein, human; C503234:EEF1A2 protein, human; D020648:Peptide Elongation Factor 1; D010750:Phosphoproteins; D011994:Recombinant Proteins; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf