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: C Sanges, C Scheuermann, R P Zahedi, A Sickmann, A Lamberti, N Migliaccio, A Baljuls, M Marra, S Zappavigna, J Reinders, U Rapp, A Abbruzzese, M Caraglia, P Arcari
• Journal: Cell death & disease (Cell Death Dis) Vol. 3 Pg. e276 (Mar 01 2012) ISSN: 2041-4889 [Electronic] England
• PMID: 22378069 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• EEF1A1 protein, human
• EEF1A2 protein, human
• Peptide Elongation Factor 1
• Phosphoproteins
• Recombinant Proteins
• BRAF protein, human
• Proto-Oncogene Proteins B-raf
• Proto-Oncogene Proteins c-raf

Topics

• Animals
• Apoptosis (genetics)
• COS Cells
• Chlorocebus aethiops
• Gene Expression Regulation
• Humans
• Models, Molecular
• Mutation
• Peptide Elongation Factor 1 (genetics, metabolism)
• Phosphoproteins (genetics, metabolism)
• Phosphorylation
• Protein Binding
• Protein Multimerization
• Protein Stability
• Protein Structure, Tertiary
• Proto-Oncogene Proteins B-raf (genetics, metabolism)
• Proto-Oncogene Proteins c-raf (genetics, metabolism)
• Recombinant Proteins (genetics, metabolism)
• Signal Transduction
• Transfection