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.

New insights on the interaction between the isoforms 1 and 2 of human translation elongation factor 1A

“…eEF1A1 and eEF1A2 were phosphorylated in vitro by B-Raf and C-Raf on S21, while T88 phosphorylation, observed only on eEF1A1, suggested that this post-translational modification was due to structural differences between the two isoforms. In addition, S21 phosphorylation of eEF1A by C-Raf required the presence of both eEF1A isoforms [24]. On the basis of these findings, we proposed that the formation in cancer cells of a possible heterodimer allowed eEF1A phosphorylation (S21) and this event might represents a switch from the canonical (protein synthesis) to non-canonical (such as tumorigenesis) functions of eEF1A.…”

Overexpression of eukaryotic elongation factor 1 alpha-2 is associated with poorer prognosis in patients with gastric cancer

“…Although eEF1A2 maps on chromosome 20q13, a region suspected to harbor oncogenes and related to gene amplification in many tumors, Anand et al and Grassi et al reported that overexpression of eEF1A2 did not completely depend on the genomic status of this locus (Anand et al 2002;Grassi et al 2007), suggesting the existence of alternative mechanisms. Furthermore, phosphorylation and expression of eEF1A2 were reported to be modulated by Raf, an important oncogene involved in the Ras/Raf/ERK1/2 signaling pathway (Lamberti et al 2007;Olson and Hallahan 2004;Sanges et al 2012). Besides, the molecular mechanisms of the association of eEF1A2 overexpression with patients' prognosis were also far from elucidation.…”

Raf kinases in signal transduction and interaction with translation machinery

“…Further analyzed, phosphoThr88 seems to stabilize the elongation complex in vivo . In contrast, the phosphorylation of Ser21 -residue that belongs to the first GTP/GDP-binding consensus sequence (G14HVDSGKST in both eEF1A1 and eEF1A2) -could potentially prevent the binding of eEF1A to guanine nucleotides, thus allowing a switch of eEF1A activity from protein biosynthesis to different non-canonical functions (74) . These findings are also supported by other evidence.…”

“…7 -8 eEF1A1 overexpressed in COS7 cells. Phosphorylated eEF1As isolated by SDS-PAGE were then analyzed by mass spectrometry for the presence of phosphopeptides (Sanges et al 2012) (74) .…”