Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Mar 1;3(3):e276.
doi: 10.1038/cddis.2012.16.

Raf kinases mediate the phosphorylation of eukaryotic translation elongation factor 1A and regulate its stability in eukaryotic cells

C Sanges  1 , 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
Affiliations

Raf kinases mediate the phosphorylation of eukaryotic translation elongation factor 1A and regulate its stability in eukaryotic cells

C Sanges et al. Cell Death Dis. .

Erratum in

  • Cell Death Dis. 2012;3:e317. Reinders, J [added]

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
B-Raf kinase assay of eEF1A1-His and eEF1A2-His. (a) Autoradiography of the assay on eEF1A1-His in the presence of B-Raf wt (10 ng/μl) and B-Raf V600E (10 ng/μl), respectively. Lanes: 1, B-Raf wt (diluted 1 : 5) + eEF1A1-His; 2, B-Raf wt + eEF1A1-His; 3, B-Raf V600E (diluted 1 : 5) + eEF1A1-His; 4, B-Raf V600E + eEF1A1-His; 5, B-Raf wt; 6, eEF1A1-His and 7, B-Raf wt + Mek + ERK. (b and c) Immunoblotting against antibody (Ab) anti-eEF1A and anti-B-Raf, respectively. (d) Autoradiography of the assay on eEF1A2-His in the presence of B-Raf wt (10 ng/μl). Lanes: 1, B-Raf wt (diluted 1 : 5) + eEF1A2-His; 2, B-Raf wt + eEF1A2-His; 3, B-Raf wt; 4 and B-Raf wt + Mek + ERK. (e and f) Immunoblotting against Ab anti-eEF1A and anti-B-Raf, respectively. (g) Autoradiography of the assay on eEF1A1-His in the presence of B-Raf wt (20 ng/μl). Lanes: 1, B-Raf wt (diluted 1 : 5) + eEF1A1-His; 2, B-Raf wt + eEF1A1-His; 3, eEF1A1-His; 4, B-Raf wt and 5, B-Raf wt + Mek + ERK. (h and i) Immunoblotting against Ab anti-eEF1A and anti-B-Raf, respectively. (k) Autoradiography of the assay on eEF1A2-His in the presence of B-Raf wt (20 ng/μl). Lanes: 1, B-Raf wt (diluted 1 : 5) + eEF1A2-His; 2, B-Raf wt + eEF1A2-His; 3, B-Raf wt + Mek + ERK; 4, B-Raf wt and 5, eEF1A2-His
Figure 2
Figure 2
C-Raf DD kinase assay of eEF1A1-His and eEF1A2-His. (a) Autoradiography. Lanes: 1, C-Raf DD; 2, C-Raf DD + eEF1A1-His; 3, C-Raf DD + eEF1A2-His; 4, C-Raf DD + eEF1A1-His + eEF1A2-His and 5, positive control C-Raf DD + Mek + ERK. (b) Relative densitometric evaluation of C-Raf DD signal intensity. (c) Western blot with anti-eEF1A antibody. Each measurement and western blot was carried out in triplicate. Error bars indicate the maximum deviation from the mean value of two independent experiments
Figure 3
Figure 3
Expression levels of GST-eEF1A1, eEF1A1-HIS and eEF1A2-HIS and corresponding mutants in COS 7 cells. (a, g and d) Western blots of cell lysates with antibody anti-His and anti-GST, respectively. (a) Lanes: 1, eEF1A1-HIS wt; 2, eEF1A1-HIS S21A; 3, eEF1A1-HIS S21D; 4, eEF1A1-HIS T88A; 5, eEF1A1-HIS T88D. (d) Lanes: 1, GST-eEF1A1 wt; 2, GST-eEF1A1 S21A; 3, GST-eEF1A1 S21D; 4, GST-eEF1A1 T88A; 5, GST-eEF1A1 T88D. (g) Lanes: 1. eEF1A2-HIS wt; 2, eEF1A2-HIS S21A and 3, eEF1A2-HIS S21D. (b, e and h) Relative band intensity evaluation. (c, i and f) Western blots of cell lysates with antibody anti-β-actin and anti-GAPDH, respectively. Each measurement and western blot was carried out in triplicate. Error bars indicate the maximum deviation from the mean value of two independent experiments
Figure 4
Figure 4
Ubiquitination assay of eEF1A1-HIS and eEF1A2-HIS and their mutants. After cotransfection of COS 7 cells with wt eEF1A-HISs and corresponding mutants with HA-ubiquitin wt and treatment of cells in the absence (−) or in presence (+) of MG132, cell extracts were analyzed after pull-down for eEF1A1-HIS (a, b, i) and eEF1A2-HIS (e, f, k), respectively, by immunoblotting with anti-HA (a, b, e, f) and anti-His (i, k) antibodies. (a, b, i) Lanes: 1, eEF1A1-HIS wt; 2, eEF1A1-HIS S21A; 3, eEF1A1-HIS S21D; 4, eEF1A1-HIS T88A and 5, eEF1A1-HIS T88D. (e, f, k) Lanes: 1, eEF1A2-HIS wt; 2, eEF1A2-HIS S21A and 3, eEF1A2-HIS S21D. Band intensities were analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA) (c, d, g, h, j and l)
Figure 5
Figure 5
Effects of transfection with eEF1A-HIS isoforms and their S21 mutants on apoptosis in H1355 cells. FACS analysis after double labelling with annexin V and propidium iodide of H1355 cells transfected with eEF1A-HIS constructs for 72 h as described in Materials and Methods and Supplementary Information. White bars: percentage of annexin V-FITC and PI positive (late apoptosis). Black bars: percentage of annexin V-FITC positive (early apoptosis). Samples: 1, control cells; 2, cells treated with lipophectamine; 3, cells transfected with void vector; 4, eEF1A1-HIS wt, 5, eEF1A1-HIS S21A; 6, eEF1A1-HIS S21D; 7, eEF1A2-HIS wt, 8, eEF1A2-HIS S21A and 9, eEF1A2-HIS S21D. Error bars indicate the maximum deviation from the mean value of two independent experiments
Figure 6
Figure 6
Cotransfection of GST-eEF1A1 and eEF1A2-HIS in Cos 7 cells. GST-eEF1A1 wt and mutants were cotransfected with eEF1A2-HIS wt in COS 7 cells. After 24 h, the cells were harvested, lysed and analyzed after GST pull-down with antibody anti-GST (GST Ab, a) and anti-His (His Ab, c). Lanes: 1, cells transfected with GST-eEF1A1 wt and eEF1A2-HIS wt; 2, cells transfected with GST-eEF1A1 S21D and eEF1A2-HIS wt; 3. Cells transfected with GST-eEF1A1 T88D and eEF1A2-HIS wt. Band intensities were evaluated with Image J software (b and d)
Figure 7
Figure 7
Imitation of a 3D model of an eEF1A1·eEF1A2 heterodimer. The heterodimer representation was obtained from the molecular docking pdb file (r-1.pdb) using PyMol software (DeLano Scientific LLC, San Carlos, CA, USA). In both eEF1A isoforms, the position of S21 is highlighted
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Axelos M, Bardet C, Liboz T, Van Thai AL, Curie C, Lescure B. The gene family encoding the Arabidopsis thaliana translation elongation factor EF-1a: molecular cloning, characterization and expression. Mol Gen Genet. 1989;219:106–112. - PubMed
    1. Andersen GR, Nissen P, Nyborg J. Elongation factors in protein biosynthesis. Trends Biochem Sci. 2003;28:434–441. - PubMed
    1. Lund A, Knudsen SM, Vissing H, Clark B, Tommerup N. Assignment of human elongation factor 1alpha genes: EEF1A maps to chromosome 6q14 and EEF1A2 to 20q13.3. Genomics. 1996;36:359–361. - PubMed
    1. Stark H, Rodnina MV, Rinke-Appel J, Brimacombe R, Wintermey W, van Heel M. Visualization of the elongation factor Tu on the E. Coli Ribosome Nat. 1997;389:403–406. - PubMed
    1. Pape T, Wintermayer W, Rodnina M. Induced fit in initial selection and proofreading of ee-tRNA on the ribosome. EMBO J. 1999;18:3800–3807. - PMC - PubMed

Publication types

MeSH terms