1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) induces phosphorylation of eukaryotic elongation factor-2 (eEF2): a cautionary note on the anticancer mechanism of an eEF2 kinase inhibitor

Eukaryotic elongation factor-2 kinase (eEF2K) relays growth and stress signals to protein synthesis through phosphorylation and inactivation of eukaryotic elongation factor 2 (eEF2). 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) is a widely accepted inhibitor of mammalian eEF2K and an efficacious anti-proliferation agent against different cancer cells. It implied that eEF2K could be an efficacious anticancer target. However, eEF2K siRNA was ineffective against cancer cells including those sensitive to NH125. To test if pharmacological intervention differs from siRNA interference, we identified a highly selective small molecule eEF2K inhibitor A-484954. Like siRNA, A-484954 had little effect on cancer cell growth. We carefully examined the effect of NH125 and A-484954 on phosphorylation of eEF2, the known cellular substrate of eEF2K. Surprisingly, NH125 increased eEF2 phosphorylation, whereas A-484954 inhibited the phosphorylation as expected for an eEF2K inhibitor. Both A-484954 and eEF2K siRNA inhibited eEF2K and reduced eEF2 phosphorylation with little effect on cancer cell growth. These data demonstrated clearly that the anticancer activity of NH125 was more correlated with induction of eEF2 phosphorylation than inhibition of eEF2K. Actually, induction of eEF2 phosphorylation was reported to correlate with inhibition of cancer cell growth. We compared several known inducers of eEF2 phosphorylation including AMPK activators and an mTOR inhibitor. Interestingly, stronger induction of eEF2 phosphorylation correlated with more effective growth inhibition. We also explored signal transduction pathways leading to NH125-induced eEF2 phosphorylation. Preliminary data suggested that NH125-induced eEF2 phosphorylation was likely mediated through multiple pathways. These observations identified an opportunity for a new multipathway approach to anticancer therapies.     

Activation of AMP-activated protein kinase leads to the phosphorylation of elongation factor 2 and an inhibition of protein synthesis

Protein synthesis, in particular peptide-chain elongation, consumes cellular energy. Anoxia activates AMP-activated protein kinase (AMPK, see ), resulting in the inhibition of biosynthetic pathways to conserve ATP. In anoxic rat hepatocytes or in hepatocytes treated with 5-aminoimidazole-4-carboxamide (AICA) riboside, AMPK was activated and protein synthesis was inhibited. The inhibition of protein synthesis could not be explained by changes in the phosphorylation states of initiation factor 4E binding protein-1 (4E-BP1) or eukaryotic initiation factor 2alpha (eIF2alpha). However, the phosphorylation state of eukaryotic elongation factor 2 (eEF2) was increased in anoxic and AICA riboside-treated hepatocytes and in AICA riboside-treated CHO-K1 cells, and eEF2 phosphorylation is known to inhibit its activity. Incubation of CHO-K1 cells with increasing concentrations of 2-deoxyglucose suggested that the mammalian target of the rapamycin (mTOR) signaling pathway did not play a major role in controlling the level of eEF2 phosphorylation in response to mild ATP depletion. In HEK293 cells, transfection of a dominant-negative AMPK construct abolished the oligomycin-induced inhibition of protein synthesis and eEF2 phosphorylation. Lastly, eEF2 kinase, the kinase that phosphorylates eEF2, was activated in anoxic or AICA riboside-treated hepatocytes. Therefore, the activation of eEF2 kinase by AMPK, resulting in the phosphorylation and inactivation of eEF2, provides a novel mechanism for the inhibition of protein synthesis.     

Analysis of interaction partners for eukaryotic translation elongation factor 1A M-domain by functional proteomics

The eukaryotic translation elongation factor 1A (eEF1A), besides to its canonical role in protein synthesis, is also involved in several other cellular processes, depending on changes in cellular location, cell type, concentration of ligands, substrates or cofactors. Therefore eEF1A is a moonlighting protein that participates to a network of molecular interactions involving its structural domains. Since the identification of novel protein–protein interactions represents important tasks in post-genomic era, the interactome of eEF1A1 M-domain was investigated by using a proteomic approach. To this purpose, the eEF1A1 M-domain was fused with glutathione-S-transferase (GST) and Strep-tag (ST) at it’s N- and C-terminal, respectively. The recombinant protein (GST-M-ST) was purified and incubated with a mouse embryo lysate by applying an affinity chromatography strategy. The interacting proteins were separated by SDS-PAGE and identified by peptide mass fingerprinting using MALDI-TOF mass spectrometry. Besides the known partners, the pool of interacting proteins contained sorbin, a polypeptide of 153 amino acids present in SH3 domain-containing adaptor proteins, such as SORBS2. This interaction was also assessed by Western blot on immunoprecipitate from mouse embryo or H1355 cell lysates with anti-eEF1A or anti-SORBS2 antibodies and on eEF1A1-His pull-down from H1355 cell lysate with antibody anti-SORBS2. Furthermore, the interaction between eEF1A and SORBS2 was also confirmed by confocal microscopy and FRET analysis. Interestingly, a co-localization of SORBS2 and eEF1A was evidenced at level of plasma membrane, thus suggesting the involvement of eEF1A1 in novel key signal transduction complexes.     

Avian reovirus influences phosphorylation of several factors involved in host protein translation including eukaryotic translation elongation factor 2 (eEF2) in Vero cells

Viral infection usually influences cellular protein synthesis either actively or passively via modification of various translation initiation factors. Here we demonstrated that infection with avian reovirus (ARV) interfered with cellular protein synthesis. This study demonstrated for the first time that ARV influenced the phosphorylation of translation initiation factors including eIF4E and eIF-4G. Interestingly, ARV also induced phosphorylation of eukaryotic translation elongation factor (eEF2) in a time- and dose-dependent manner. Inhibition of mTOR by rapamycin notably increased the level of phosphorylated eEF2 in infected cells. However, rapamycin did not show any negative effects on ARV replication, suggesting that phosphorylation of eEF2 in infected cells did not reduce ARV propagation. These results demonstrated for the first time that ARV promotes phosphorylation of eEF2 which in turn influenced host protein production not simply by modulating the function of translation initiation factors but also by regulating elongation factor eEF2.     

Stimulation of the AMP-activated protein kinase leads to activation of eukaryotic elongation factor 2 kinase and to its phosphorylation at a novel site, serine 398

Protein synthesis consumes a high proportion of the metabolic energy of mammalian cells, and most of this is used by peptide chain elongation. An important regulator of energy supply and demand in eukaryotic cells is the AMP-activated protein kinase (AMPK). The rate of peptide chain elongation can be modulated through the phosphorylation of eukaryotic elongation factor (eEF) 2, which inhibits its activity and is catalyzed by a specific calcium/calmodulin-dependent protein kinase termed eEF2 kinase. Here we show that AMPK directly phosphorylates eEF2 kinase, and we identify the major site of phosphorylation as Ser-398 in a regulatory domain of eEF2 kinase. AMPK also phosphorylates two other sites (Ser-78 and Ser-366) in eEF2 kinase in vitro. We develop appropriate phosphospecific antisera and show that phosphorylation of Ser-398 in eEF2 kinase is enhanced in intact cells under a range of conditions that activate AMPK and increase the phosphorylation of eEF2. Ser-78 and Ser-366 do not appear to be phosphorylated by AMPK within cells. Although cardiomyocytes appear to contain a distinct isoform of eEF2 kinase, it also contains a site corresponding to Ser-398 that is phosphorylated by AMPK in vitro. Stimuli that activate AMPK and increase eEF2 phosphorylation within cells increase the activity of eEF2 kinase. Thus, AMPK and eEF2 kinase may provide a key link between cellular energy status and the inhibition of protein synthesis, a major consumer of metabolic energy.     

TGFβ enforces activation of eukaryotic elongation factor-2 (eEF2) via inactivation of eEF2 kinase by p90 ribosomal S6 kinase (p90Rsk) to induce mesangial cell hypertrophy

eEF2 phosphorylation is under tight control to maintain mRNA translation elongation. We report that TGFβ activates eEF2 by decreasing eEF2 phosphorylation and simultaneously increasing eEF2 kinase phosphorylation. Remarkably, inhibition of Erk1/2 blocked the TGFβ-induced dephosphorylation and phosphorylation of eEF2 and eEF2 kinase. TGFβ increased phosphorylation of p90Rsk in an Erk1/2-dependent manner. Inactive p90Rsk reversed TGFβ-inhibited phosphorylation of eEF2 and suppressed eEF2 kinase activity. Finally, inactive p90Rsk significantly attenuated TGFβ-induced protein synthesis and hypertrophy of mesangial cells. These results present the first evidence that TGFβ utilizes the two layered kinase module Erk/p90Rsk to activate eEF2 for increased protein synthesis during cellular hypertrophy.     

cdc2-cyclin B regulates eEF2 kinase activity in a cell cycle- and amino acid-dependent manner

The calcium/calmodulin-dependent kinase that phosphorylates and inactivates eukaryotic elongation factor 2 (eEF2 kinase; eEF2K) is subject to multisite phosphorylation, which regulates its activity. Phosphorylation at Ser359 inhibits eEF2K activity even at high calcium concentrations. To identify the kinase that phosphorylates Ser359 in eEF2K, we developed an extensive purification protocol. Tryptic mass fingerprint analysis identified it as cdc2 (cyclin-dependent kinase 1). cdc2 co-purifies with Ser359 kinase activity and cdc2-cyclin B complexes phosphorylate eEF2K at Ser359. We demonstrate that cdc2 contributes to controlling eEF2 phosphorylation in cells. cdc2 is activated early in mitosis. Kinase activity against Ser359 in eEF2K also peaks at this stage of the cell cycle and eEF2 phosphorylation is low in mitotic cells. Inactivation of eEF2K by cdc2 may serve to keep eEF2 active during mitosis (where calcium levels rise) and thereby permit protein synthesis to proceed in mitotic cells. Amino-acid starvation decreases cdc2's activity against eEF2K, whereas loss of TSC2 (a negative regulator of mammalian target of rapamycin complex 1(mTORC1)) increases it. These data closely match the control of Ser359 phosphorylation and indicate that cdc2 may be regulated by mTORC1.     

ATP depletion increases phosphorylation of elongation factor eEF2 in adult cardiomyocytes independently of inhibition of mTOR signalling

Translation elongation consumes a high proportion of cellular energy and can be regulated by phosphorylation of elongation factor eEF2 which inhibits its activity. We have studied the effects of ATP depletion on the phosphorylation of eEF2 in adult rat ventricular cardiomyocytes. Energy depletion rapidly leads to inhibition of protein synthesis and increased phosphorylation of eEF2. Stimulation of the AMP-activated protein kinase also causes increases eEF2 phosphorylation. Only at later times is an effect on mTOR signalling observed. These data suggest that energy depletion leads to inhibition of protein synthesis through phosphorylation of eEF2 independently of inhibition of mTOR signalling.     

Regulation of translation elongation and phosphorylation of eEF2 in rat pancreatic acini

While pancreatic protein synthesis and the initiation of translation are regulated by hormones and neurotransmiters, whether the elongation process is also regulated is unknown. Stimulatory doses of cholecystokinin (CCK) (100 pM), bombesin (10 nM), and carbachol (10 microM) increased elongation rates (measured as ribosomal half-transit time) in pancreatic acini in vitro. At the same time these secretagogues reduced elongation factor 2 (eEF2) phosphorylation, the main factor known to regulate elongation, and increased the phosphorylation of the eEF2 kinase. The mTOR inhibitor rapamycin reversed the dephosphorylation of eEF2 induced by CCK, as did treatment with the p38 MAPK inhibitor SB202190, the MEK inhibitor PD98059, and the phosphatase inhibitor calyculin A. Neither rapamycin, SB202190, PD98059 nor calyculin A had an effect on CCK mediated eEF2 kinase phosphorylation. Translation elongation in pancreatic acinar cells is likely regulated by eEF2 through the mTOR, p38, and MEK pathways, and modulated through PP2A.     

Ser/Thr kinases and polyamines in the regulation of non-canonical functions of elongation factor 1A

The link between eukaryotic translation elongation factor 1A (eEF1A) and signal transduction pathways through the regulatory mechanism of phosphorylation has never been considered. In this review, we focus on the different kinases that recognize the Ser and Thr residues of the eEF1A1 and eEF1A2 isoforms and regulate their involvement in different cellular processes like cell survival and apoptosis. In this context, polyamines seem to play a role in the regulation of the translation elongation process by modulating the Ser/Thr kinases involved in the phosphorylation of translation elongation factors.     

A non-canonical function of eukaryotic elongation factor 1A1: Regulation of interleukin-6 expression

Interleukin-6 is one of the most prominent triggers of inflammatory processes. We have shown recently that heteroarylketones (HAKs) interfere with stimulated interleukin-6 expression in astrocytes by suppression of STAT3 phosphorylation at serine 727. Surprisingly, this effect is not based on the inhibition of STAT3-relevant kinases. Therefore, we here used the structurally modified HAK compound biotin-HAK-3 in a reverse chemical approach to identify the relevant molecular target in UV-mediated cross-linking experiments. Employing streptavidin-specific 2D-immunoblotting followed by mass spectrometry we identified nine proteins putatively interacting with biotin-HAK-3. After co-immunoprecipitation, co-immunofluorescence, surface plasmon resonance analyses and RNAi-mediated knock-down, the eukaryotic elongation factor 1A1 (eEF1A1) was verified as the relevant target of HAK bioactivity. eEF1A1 forms complexes with STAT3 and PKCδ, which are crucial for STAT3^S727 phosphorylation and for NF-κB/STAT3-enhanced interleukin-6 expression. Furthermore, the intracellular HAK accumulation is strongly dependent on eEF1A1 expression. Taken together, the results reveal a novel molecular mechanism for a non-canonical role of eEF1A1 in signal transduction via direct modulation of kinase-dependent phosphorylation events.     

Enhancement of translation elongation in neurons by brain-derived neurotrophic factor: implications for mammalian target of rapamycin signaling

The effects and signaling mechanisms of brain-derived neurotrophic factor (BDNF) on translation elongation were investigated in cortical neurons. BDNF increased the elongation rate approximately twofold, as determined by measuring the ribosomal transit time. BDNF-accelerated elongation was inhibited by rapamycin, implicating the mammalian target of rapamycin (mTOR). To explore the mechanisms underlying these effects, we examined the protein phosphorylation cascades that lead to the activation of translation elongation in neurons. BDNF increased eukaryote elongation factor 1A (eEF1A) phosphorylation and decreased eEF2 phosphorylation. Whereas eEF2 phosphorylation levels altered by BDNF were inhibited by rapamycin, eEF1A phosphorylation was not affected by rapamycin or PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor. BDNF induced phosphorylation of eEF2 kinase (Ser366), as well as decreased its kinase activity. All these events were inhibited by rapamycin. Furthermore, mTOR siRNA, which reduced mTOR levels up to 50%, inhibited the BDNF-induced enhancement in elongation rate and decrease in eEF2 phosphorylation. These results strongly suggest that BDNF enhances translation elongation through the activation of the mTOR-eEF2 pathway.     

Eukaryotic Elongation Factor 2 Kinase Activity Is Controlled by Multiple Inputs from Oncogenic Signaling

Eukaryotic elongation factor 2 kinase (eEF2K), an atypical calmodulin-dependent protein kinase, phosphorylates and inhibits eEF2, slowing down translation elongation. eEF2K contains an N-terminal catalytic domain, a C-terminal α-helical region and a linker containing several regulatory phosphorylation sites. eEF2K is expressed at high levels in certain cancers, where it may act to help cell survival, e.g., during nutrient starvation. However, it is a negative regulator of protein synthesis and thus cell growth, suggesting that cancer cells may possess mechanisms to inhibit eEF2K under good growth conditions, to allow protein synthesis to proceed. We show here that the mTORC1 pathway and the oncogenic Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway cooperate to restrict eEF2K activity. We identify multiple sites in eEF2K whose phosphorylation is regulated by mTORC1 and/or ERK, including new ones in the linker region. We demonstrate that certain sites are phosphorylated directly by mTOR or ERK. Our data reveal that glycogen synthase kinase 3 signaling also regulates eEF2 phosphorylation. In addition, we show that phosphorylation sites remote from the N-terminal calmodulin-binding motif regulate the phosphorylation of N-terminal sites that control CaM binding. Mutations in the former sites, which occur in cancer cells, cause the activation of eEF2K. eEF2K is thus regulated by a network of oncogenic signaling pathways.     

Direct and biochemical interaction between dopamine D3 receptor and elongation factor-1Bbetagamma

Novel signaling components of dopamine D3 receptor (D3R) were searched using yeast two-hybrid system, and the gamma subunit of elongation Factor-1B (eEF1Bgamma) was found to interact with D3R. This interaction was observed specifically between eEF1Bgamma and D3R but not with D2R or D4R. Immunocytochemical studies showed that D3R and eEF1Bgamma form clusters on the plasma membrane and their co-localization was evident in these clusters. The beta subunit of eEF1B (eEF1Bbeta), which forms a tight complex with eEF1Bgamma, was phosphorylated on serine residues in response to the stimulation of D3R. Phosphorylation of eEF1Bbeta was insensitive to pertussis toxin or wortmannin, however, stimulation of cellular protein kinase C (PKC) directly phosphorylated eEF1Bbeta and depletion of PKC abolished D3R-mediated phosphorylation of eEF1Bbeta. These results suggest the involvement of PKC, but not Gi/o proteins or phosphatidylinositol 3-kinase, in D3R-mediated phosphorylation of eEF1Bbeta. Stimulation of D3R did not activate PKC, but the activation of PKC resulted in the phosphorylation of D3R. These results show that PKC has a permissive role for the D3R-mediated phosphorylation of eEF1Bbeta, and suggest that PKC could modulate the mutual interaction between two protein by phosphorylating both D3R and eEF1Bbeta. Therefore, the cellular PKC level would be important for the D3R-mediated modulation of eEF1B, and for their cellular regulations such as protein synthesis or cellular proliferation.     

Cyclic AMP inhibits translation of cyclin D3 in T lymphocytes at the level of elongation by inducing eEF2-phosphorylation

The purpose of the present study was to understand the mechanism by which activated protein kinase A (PKA) leads to down-regulation of cyclin D3 in lymphocytes. By using Jurkat cells as a model system, we have been able to demonstrate that cyclin D3 is reduced at the level of translation by inhibition of elongation. One of the important factors involved in translational elongation is the eukaryotic elongation factor 2 (eEF2). eEF2 promotes translation in its unphosphorylated form, and we observed a rapid phosphorylation of the eEF2-protein upon forskolin treatment. When using specific inhibitors of the eEF2-kinase prior to forskolin treatment, we were able to inhibit the increased phosphorylation of eEF2. Furthermore, inhibition of eEF2-kinase prevented the forskolin-mediated down-regulation of cyclin D3. Taken together, it appears that activation of PKA in Jurkat cells reduces the expression of cyclin D3 at the level of translational elongation by increasing the phosphorylation of eEF2 and thereby inhibiting its activity.     

Activation of AMP-activated protein kinase inhibits protein synthesis associated with hypertrophy in the cardiac myocyte

A necessary mediator of cardiac myocyte enlargement is protein synthesis, which is controlled at the levels of both translation initiation and elongation. Eukaryotic elongation factor-2 (eEF2) mediates the translocation step of peptide-chain elongation and is inhibited through phosphorylation by eEF2 kinase. In addition, p70S6 kinase can regulate protein synthesis by phosphorylating eEF2 kinase or via phosphorylation of ribosomal protein S6. We have recently shown that eEF2 kinase is also controlled by phosphorylation by AMP-activated protein kinase (AMPK), a key regulator of cellular energy homeostasis. Moreover, the mammalian target of rapamycin has also been shown to be inhibited, indirectly, by AMPK, thus leading to the inhibition of p70S6 kinase. Although AMPK activation has been shown to modulate protein synthesis, it is unknown whether AMPK could also be a regulator of cardiac hypertrophic growth. Therefore, we investigated the role of AMPK activation in regulating protein synthesis during both phenylephrine- and Akt-induced cardiac hypertrophy. Metformin and 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside were used to activate AMPK in neonatal rat cardiac myocytes. Activation of AMPK significantly decreased protein synthesis induced by phenylephrine treatment or by expression of constitutively active Akt. Activation of AMPK also resulted in decreased p70S6 kinase phosphorylation and increased phosphorylation of eEF2, suggesting that inhibition of protein synthesis involves the eEF2 kinase/eEF2 axis and/or the p70S6 kinase pathway. Together, our data suggest that the inhibition of protein synthesis by pharmacological activation of AMPK may be a key regulatory mechanism by which hypertrophic growth can be controlled.     

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.     

Phosphorylation of eukaryotic elongation factor 2 can be regulated by phosphoinositide 3-kinase in the early stages of myoblast differentiation

We have previously reported that phosphorylation of eukaryotic elongation factor 2 (eEF2) is related to the differentiation of chick embryonic muscle cells in culture. In the present study, we found that eEF2 phosphorylation declined shortly after induction of differentiation of L6 myoblasts, when the cells prepare for terminal differentiation by withdrawing from the cell cycle. This decrease in phosphorylation was prevented by inhibitors of phosphoinositide 3-kinase (PI3-kinase) that strongly inhibit myoblast differentiation. We hypothesized that PI3-kinase plays an important role in myoblast differentiation by regulating eEF2 phosphorylation in the early stages of differentiation. To test this hypothesis, myoblasts were synchronized at in G2/M and cultured in fresh differentiation medium (DM) or growth medium (GM). In DM the released cells accumulated in G0/G1 while in GM they progressed to S phase. In addition, cyclin D1 was more rapidly degraded in DM than in GM, and eEF2 phosphorylation decreased more. Inhibitors of PI3-kinase increased eEF2 phosphorylation, but PI3-kinase became more activated when eEF2 phosphorylation declined. These results suggest that the regulation of L6 myoblast differentiation by PI3-kinase is related to eEF2 phosphorylation.     

Regulation of peptide-chain elongation in mammalian cells.

The elongation phase of mRNA translation is the stage at which the polypeptide is assembled and requires a substantial amount of metabolic energy. Translation elongation in mammals requires a set of nonribosomal proteins called eukaryotic elongation actors or eEFs. Several of these proteins are subject to phosphorylation in mammalian cells, including the factors eEF1A and eEF1B that are involved in recruitment of amino acyl-tRNAs to the ribosome. eEF2, which mediates ribosomal translocation, is also phosphorylated and this inhibits its activity. The kinase acting on eEF2 is an unusual and specific one, whose activity is dependent on calcium ions and calmodulin. Recent work has shown that the activity of eEF2 kinase is regulated by MAP kinase signalling and by the nutrient-sensitive mTOR signalling pathway, which serve to activate eEF2 in response to mitogenic or hormonal stimuli. Conversely, eEF2 is inactivated by phosphorylation in response to stimuli that increase energy demand or reduce its supply. This likely serves to slow down protein synthesis and thus conserve energy under such circumstances.