12414
2012
eng
e276
3
article
1
2016-01-13
--
--
Raf kinases mediate the phosphorylation of eukaryotic translation elongation factor 1A and regulate its stability in eukaryotic cells
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.
Cell Death and Disease
10.1038/cddis.2012.16
urn:nbn:de:bvb:20-opus-124149
Cell Death and Disease (2012) 3, e276; doi:10.1038/cddis.2012.16
Corrigendum: Cell Death and Disease (2012) 3, e317; doi:10.1038/cddis.2012.67
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
eng
uncontrolled
EF-1A
eng
uncontrolled
Raf kinases
eng
uncontrolled
signal transduction
eng
uncontrolled
apoptosis
eng
uncontrolled
ubiquitin
eng
uncontrolled
mass spectrometry
Medizin und Gesundheit
open_access
Institut für Medizinische Strahlenkunde und Zellforschung
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/12414/Sanges_cddis201216a(1).pdf
https://opus.bibliothek.uni-wuerzburg.de/files/12414/Sanges_Corrigendum_cddis201267a.pdf
13467
2012
eng
e276
3
article
1
2016-06-10
--
--
Raf kinases mediate the phosphorylation of eukaryotic translation elongation factor 1A and regulate its stability in eukaryotic cells
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.
Cell Death & Disease
10.1038/cddis.2012.16
urn:nbn:de:bvb:20-opus-134673
Cell Death and Disease (2012) 3, e276; doi:10.1038/cddis.2012.16
C. Sanges
C. Scheuermann
R. P. Zahedi
A. Sickmann
A. Lamberti
N. Migliaccio
A. Baljuls
M. Marra
S. Zappavigna
U. Rapp
A. Abbruzzese
M. Caraglia
P. Arcari
eng
uncontrolled
signal transduction
eng
uncontrolled
mass spectrometry
eng
uncontrolled
elongation
eng
uncontrolled
protein docking
eng
uncontrolled
factor EEF1A2
eng
uncontrolled
cancer-cells
eng
uncontrolled
lung cancer
eng
uncontrolled
EF-1A
eng
uncontrolled
Raf kinases
eng
uncontrolled
aminoacyl-transfer-RNA
eng
uncontrolled
tyrosine phosphorylation
eng
uncontrolled
factor 1-alpha
eng
uncontrolled
nucleotide exchange
eng
uncontrolled
polyarcylamide gels
eng
uncontrolled
chain
eng
uncontrolled
apoptosis
eng
uncontrolled
ubiquitin
Medizin und Gesundheit
open_access
Institut für Medizinische Strahlenkunde und Zellforschung
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/13467/Sanges_CellDeathDisease.pdf