Elongation factor-2 kinase (eEF-2 kinase, also known as calmodulin-dependent protein kinase III), is a unique calcium/calmodulin-dependent enzyme that inhibits protein synthesis by phosphorylating and inactivating elongation factor-2 (eEF-2). We previously reported that expression/activity of eEF-2 kinase was up-regulated in several types of malignancies including Gliomas, and was associated with response of tumor cells to certain therapeutic stress. In the current study, we sought to determine whether eEF-2 kinase expression affected sensitivity of glioma cells to treatment with tumor the necrosis factor-related apoptosis-inducing ligand (TRAIL), a targeted therapy able to induce apoptosis in cancer cells but causes no toxicity in most normal cells. We found that inhibition of eEF-2 kinase by RNA interference (RNAi) or by a pharmacological inhibitor (NH125) enhanced TRAIL-induced apoptosis in the human glioma cells, as evidenced by an increase in apoptosis in the tumor cells treated with eEF-2 kinase siRNA or the eEF-2 kinase inhibitor. We further demonstrated that sensitization of tumor cells to TRAIL was accompanied by a down-regulation of the anti-apoptotic protein, Bcl-xL, and that overexpression of Bcl-xL could abrogate the sensitizing effect of inhibiting eEF-2 kinase on TRAIL. The results of this study may help devise a new therapeutic strategy for enhancing the efficacy of TRAIL against malignant glioma by targeting eEF-2 kinase.
eEF-2 kinase; TRAIL; Bcl-xl; apoptosis; glioblastoma
Inhibition of the survival kinase Akt can trigger apoptosis but also has been found to activate autophagy, which may confound tumor attack. In this study, we investigated regulatory mechanisms through which apoptosis and autophagy were modulated in tumor cells subjected to Akt inhibition by MK-2206, the first allosteric small molecule inhibitor of Akt to enter clinical development. In human glioma cells, Akt inhibition by MK-2206 or siRNA-mediated attenuation strongly activated autophagy, whereas silencing of eukaryotic elongation factor-2 (eEF-2) kinase, a protein synthesis regulator, blunted this autophagic response. Suppression of MK-2206-induced autophagy by eEF-2 silencing was accompanied by a promotion of apoptotic cell death. Similarly, siRNA-mediated inhibition of eEF-2 kinase potentiated the efficacy of MK-2206 against glioma cells. Together, these results demonstrated that blunting autophagy and augmenting apoptosis by inhibition of eEF-2 kinase could modulate the sensitivity of glioma cells to Akt inhibition. Our findings suggest that targeting eEF-2 kinase may reinforce the anti-tumor efficacy of Akt inhibitors such as MK-2206.
Elongation factor-2 kinase; Akt; Autophagy; Apoptosis; MK-2206; Glioblastoma
Autophagy is a highly conserved and regulated cellular process employed by living cells to degrade proteins and organelles as a response to metabolic stress. We have previously reported that eukaryotic elongation factor-2 kinase (eEF-2 kinase, also known as Ca2+/calmodulin-dependent protein kinase III) can positively modulate autophagy and negatively regulate protein synthesis. The purpose of the current study was to determine the role of the eEF-2 kinase-regulated autophagy in the response of breast cancer cells to inhibitors of growth factor signaling.
We found that nutrient depletion or growth factor inhibitors activated autophagy in human breast cancer cells, and the increased activity of autophagy was associated with a decrease in cellular ATP and an increase in activities of AMP kinase and eEF-2 kinase. Silencing of eEF-2 kinase relieved the inhibition of protein synthesis, led to a greater reduction of cellular ATP, and blunted autophagic response. We further showed that suppression of eEF-2 kinase-regulated autophagy impeded cell growth in serum/nutrient-deprived cultures and handicapped cell survival, and enhanced the efficacy of the growth factor inhibitors such as trastuzumab, gefitinib, and lapatinib.
The results of this study provide new evidence that activation of eEF-2 kinase-mediated autophagy plays a protective role for cancer cells under metabolic stress conditions, and that targeting autophagic survival may represent a novel approach to enhancing the effectiveness of growth factor inhibitors.
Eukaryotic elongation factor 2 (eEF2) kinase is an unusual calcium- and calmodulin-dependent protein kinase that is regulated by insulin through the rapamycin-sensitive mTOR pathway. Here we show that insulin decreases the ability of eEF2 kinase to bind calmodulin in a rapamycin-sensitive manner. We identify a novel phosphorylation site in eEF2 kinase (Ser78) that is located immediately next to its calmodulin-binding motif. Phosphorylation of this site is increased by insulin in a rapamycin-sensitive fashion. Regulation of the phosphorylation of Ser78 also requires amino acids and the protein kinase phosphoinositide-dependent kinase 1. Mutation of this site to alanine strongly attenuates the effects of insulin and rapamycin both on the binding of calmodulin to eEF2 kinase and on eEF2 kinase activity. Phosphorylation of Ser78 is thus likely to link insulin and mTOR signaling to the control of eEF2 phosphorylation and chain elongation. This site is not a target for known kinases in the mTOR pathway, e.g., the S6 kinases, implying that it is phosphorylated by a novel mTOR-linked protein kinase that serves to couple hormones and amino acids to the control of translation elongation. eEF2 kinase is thus a target for mTOR signaling independently of previously known downstream components of the pathway.
Eukaryotic elongation factor-2 kinase (eEF-2K) is a Ca2+/calmodulin-dependent enzyme that negatively regulates protein synthesis. eEF-2K has been shown to be up-regulated in cancer, and to play an important role in cell survival through inhibition of protein synthesis. Post-translational modification of protein synthesis machinery is important for its regulation and could be critical for survival of cancer cells encountering stress. The purpose of our study was to examine the regulation of eEF-2K during stress with a focus on the roles of phosphorylation in determining the stability of eEF-2K. We found that stress conditions (nutrient deprivation and hypoxia) increase eEF-2K protein. mRNA levels are only transiently increased and shortly return to normal, while eEF-2K protein levels continue to increase after further exposure to stress. A seemingly paradoxical decrease in eEF-2K stability was found when glioma cells were subjected to stress despite increased protein expression. We further demonstrated that phosphorylation of eEF-2K differentially affects the enzyme’s turnover under both normal and stress conditions, as evidenced by the different half-lives of phosphorylation-defective mutants of eEF-2K. We further found that the eEF-2K site (Ser398) phosphorylated by AMPK is pivotal to the protein’s stability, as the half-life of S398A mutant increases to greater than 24 h under both normal and stress conditions. These data indicate that eEF-2K is regulated at multiple levels with phosphorylation playing a critical role in the enzyme’s turnover under stressful conditions. The complexity of eEF-2K phosphorylation highlights the intricacies of protein synthesis control during cellular stress.
eEF-2K; Phosphorylation; Enzyme stability; Protein synthesis; Glioblastoma; AMPK
Eukaryotic elongation factor 2 kinase (eEF-2K), through its phosphorylation of elongation factor 2 (eEF2), provides a mechanism by which cells can control the rate of the elongation phase of protein synthesis. The activity of eEF-2K is increased in rapidly proliferating malignant cells, is inhibited during mitosis, and may contribute to the promotion of autophagy in response to anti-cancer therapies. The purpose of this study was to examine the therapeutic potential of targeting eEF-2K in breast cancer tumors. Through the systemic administration of liposomal eEF-2K siRNA (twice a week, i.v. 150 µg/kg), the expression of eEF-2K was down-regulated in vivo in an orthotopic xenograft mouse model of a highly aggressive triple negative MDA-MB-231 tumor. This targeting resulted in a substantial decrease in eEF2 phosphorylation in the tumors, and led to the inhibition of tumor growth, the induction of apoptosis and the sensitization of tumors to the chemotherapy agent doxorubicin. eEF-2K down-modulation in vitro resulted in a decrease in the expression of c-Myc and cyclin D1 with a concomitant increase in the expression of p27Kip1. A decrease in the basal activity of c-Src (phospho-Tyr-416), focal adhesion kinase (phospho-Tyr-397), and Akt (phospho-Ser-473) was also detected following eEF-2K down-regulation in MDA-MB-231 cells, as determined by Western blotting. Where tested, similar results were seen in ER-positive MCF-7 cells. These effects were also accompanied by a decrease in the observed invasive phenotype of the MDA-MB-231 cells. These data support the notion that the disruption of eEF-2K expression in breast cancer cells results in the down-regulation of signaling pathways affecting growth, survival and resistance and has potential as a therapeutic approach for the treatment of breast cancer.
The p53 family of transcription factors is a key regulator of cell proliferation and death. In this report we identify the eukaryotic translation elongation factor 1-alpha 1 (eEF1A1) to be a novel p53 and p73 interacting protein. Previous studies have demonstrated that eEF1A1 has translation-independent roles in cancer. We report that overexpression of eEF1A1 specifically inhibits p53-, p73- and chemotherapy-induced apoptosis resulting in chemoresistance. Short-interfering RNA-mediated silencing of eEF1A1 increases chemosensitivity in cell lines bearing wild type p53, but not in p53 null cells. Furthermore, silencing of eEF1A1 partially rescues the chemoresistance observed in response to p53 or p73 knockdown, suggesting that eEF1A1 is a negative regulator of the pro-apoptotic function of p53 and p73. Thus, in the context of p53-family signaling, eEF1A1 has anti-apoptotic properties. These findings identify a novel mechanism of regulation of the p53 family of proteins by eEF1A1 providing additional insight into potential targets to sensitize tumors to chemotherapy.
Protein synthesis is highly regulated via both initiation and elongation. One mechanism that inhibits elongation is phosphorylation of eukaryotic elongation factor 2 (eEF2) on threonine 56 (T56) by eEF2 kinase (eEF2K). T56 phosphorylation inactivates eEF2 and is the only known normal eEF2 functional modification. In contrast, eEF2K undergoes extensive regulatory phosphorylations that allow diverse pathways to impact elongation. We describe a new mode of eEF2 regulation and show that its phosphorylation by cyclin A–cyclin-dependent kinase 2 (CDK2) on a novel site, serine 595 (S595), directly regulates T56 phosphorylation by eEF2K. S595 phosphorylation varies during the cell cycle and is required for efficient T56 phosphorylation in vivo. Importantly, S595 phosphorylation by cyclin A-CDK2 directly stimulates eEF2 T56 phosphorylation by eEF2K in vitro, and we suggest that S595 phosphorylation facilitates T56 phosphorylation by recruiting eEF2K to eEF2. S595 phosphorylation is thus the first known eEF2 modification that regulates its inhibition by eEF2K and provides a novel mechanism linking the cell cycle machinery to translational control. Because all known eEF2 regulation is exerted via eEF2K, S595 phosphorylation may globally couple the cell cycle machinery to regulatory pathways that impact eEF2K activity.
The phosphorylation of the subunit α of eukaryotic translation initiation factor 2 (eIF2α), a critical regulatory event in controlling protein translation, has recently been found to mediate the induction of autophagy. However, the mediators of autophagy downstream of eIF2α remain unknown. Here, we provide evidence that eIF2α phosphorylation is required for phosphorylation of eukaryotic elongation factor 2 (eEF-2) during nutrient starvation. In addition, we show that eukaryotic elongation factor 2 kinase (eEF-2K) is also required for autophagy signaling during ER stress, suggesting that phosphorylation of eEF-2 may serve as an integrator of various cell stresses for autophagy signaling. On the other hand, although the activation of eEF-2K in response to starvation requires the phosphorylation of eIF2α, additional pathways relying partly on Ca2+ flux may control eEF-2K activity during ER stress, as eIF2α phosphorylation is dispensable for both eEF-2 phosphorylation and autophagy in this context.
2-Deoxy-D-glucose (2-DG), a synthetic glucose analog that acts as a glycolytic inhibitor, is currently being evaluated in the clinic as an anticancer agent. In this study, we observed that treatment of human glioma cells with 2-DG activated autophagy, a highly conserved cellular response to metabolic stress and a catabolic process of self-digestion of intracellular organelles for energy utilization and survival in stressed cells. The induction of autophagy by 2-DG was associated with activation of elongation factor-2 kinase (eEF-2 kinase), a structurally and functionally unique enzyme that phosphorylates eEF-2 leading to loss of affinity of this elongation factor for the ribosome and to termination of protein elongation. We also showed that inhibition of eEF-2 kinase by RNA interference blunted the 2-DG-induced autophagic response, resulted in a greater reduction of cellular ATP contents, and increased the sensitivity of tumor cells to the cytotoxic effect of 2-DG. Furthermore, the blunted autophagy and enhanced 2-DG cytotoxicity were accompanied by augmentation of apoptosis in cells in which eEF-2 kinase expression was knocked down. The results of this study indicate that the energy stress and cytotoxicity caused by 2-DG can be accelerated by inhibition of eEF-2 kinase, and suggest that targeting eEF-2 kinase – regulated autophagic survival pathway may represent a novel approach to sensitizing cancer cells to glycolytic inhibitors.
Elongation factor-2 kinase; 2-Deoxy-D-Glucose; Glycolysis; Autophagy; Protein synthesis; Glioblastoma
The eukaryotic elongation factor 2 kinase (eEF-2K) modulates the rate of protein synthesis by impeding the elongation phase of translation by inactivating the eukaryotic elongation factor 2 (eEF-2) via phosphorylation. eEF-2K is known to be activated by calcium and calmodulin, whereas the mTOR and MAPK pathways are suggested to negatively regulate kinase activity. Despite its pivotal role in translation regulation and potential role in tumor survival, the structure, function and regulation of eEF-2K have not been described in detail. This deficiency may result from the difficulty of obtaining the recombinant kinase in a form suitable for biochemical analysis. Here we report the purification and characterization of recombinant human eEF-2K expressed in the Escherichia coli strain Rosetta-gami 2(DE3). Successive chromatography steps utilizing Ni-NTA affinity, anion-exchange and gel filtration columns accomplished purification. Cleavage of the thioredoxin-His6-tag from the N-terminus of the expressed kinase with TEV protease yielded 9 mg of recombinant (G-D-I)-eEF-2K per liter of culture. Light scattering shows that eEF-2K is a monomer of ~ 85 kDa. In vitro kinetic analysis confirmed that recombinant human eEF-2K is able to phosphorylate wheat germ eEF-2 with kinetic parameters comparable to the mammalian enzyme.
Elongation factor 2 kinase; eEF-2K; calmodulin
eEF2K (eukaryotic elongation factor 2 kinase) is a Ca2+/CaM (calmodulin)-dependent protein kinase which regulates the translation elongation machinery. eEF2K belongs to the small group of so-called ‘α-kinases’ which are distinct from the main eukaryotic protein kinase superfamily. In addition to the α-kinase catalytic domain, other domains have been identified in eEF2K: a CaM-binding region, N-terminal to the kinase domain; a C-terminal region containing several predicted α-helices (resembling SEL1 domains); and a probably rather unstructured ‘linker’ region connecting them. In the present paper, we demonstrate: (i) that several highly conserved residues, implicated in binding ATP or metal ions, are critical for eEF2K activity; (ii) that Ca2+/CaM enhance the ability of eEF2K to bind to ATP, providing the first insight into the allosteric control of eEF2K; (iii) that the CaM-binding/α-kinase domain of eEF2K itself possesses autokinase activity, but is unable to phosphorylate substrates in trans; (iv) that phosphorylation of these substrates requires the SEL1-like domains of eEF2K; and (v) that highly conserved residues in the C-terminal tip of eEF2K are essential for the phosphorylation of eEF2, but not a peptide substrate. On the basis of these findings, we propose a model for the functional organization and control of eEF2K.
calmodulin (CaM); eukaryotic elongation factor 2 (eEF2); α-kinase; SEL1 domain; ATP-γS, adenosine 5′-[γ-thio]triphosphate; CaM, calmodulin; eEF2, eukaryotic elongation factor 2; eEF2K, eEF2 kinase; GST, glutathione transferase; HEK, human embryonic kidney; HRP, horseradish peroxidase; LB, Luria–Bertani; MHCKA, myosin heavy-chain kinase A; mTORC1, mammalian target of rapamycin, complex 1; Ni-NTA, Ni2+-nitrilotriacetic acid; STD, saturation transfer difference; TEV, tobacco etch virus; TRPM, transient receptor potential melastatin-like
eEF2K [eEF2 (eukaryotic elongation factor 2) kinase] phosphorylates and inactivates the translation elongation factor eEF2. eEF2K is not a member of the main eukaryotic protein kinase superfamily, but instead belongs to a small group of so-called α-kinases. The activity of eEF2K is normally dependent upon Ca2+ and calmodulin. eEF2K has previously been shown to undergo autophosphorylation, the stoichiometry of which suggested the existence of multiple sites. In the present study we have identified several autophosphorylation sites, including Thr348, Thr353, Ser366 and Ser445, all of which are highly conserved among vertebrate eEF2Ks. We also identified a number of other sites, including Ser78, a known site of phosphorylation, and others, some of which are less well conserved. None of the sites lies in the catalytic domain, but three affect eEF2K activity. Mutation of Ser78, Thr348 and Ser366 to a non-phosphorylatable alanine residue decreased eEF2K activity. Phosphorylation of Thr348 was detected by immunoblotting after transfecting wild-type eEF2K into HEK (human embryonic kidney)-293 cells, but not after transfection with a kinase-inactive construct, confirming that this is indeed a site of autophosphorylation. Thr348 appears to be constitutively autophosphorylated in vitro. Interestingly, other recent data suggest that the corresponding residue in other α-kinases is also autophosphorylated and contributes to the activation of these enzymes [Crawley, Gharaei, Ye, Yang, Raveh, London, Schueler-Furman, Jia and Cote (2011) J. Biol. Chem. 286, 2607–2616]. Ser366 phosphorylation was also detected in intact cells, but was still observed in the kinase-inactive construct, demonstrating that this site is phosphorylated not only autocatalytically but also in trans by other kinases.
calmodulin; eukaryotic elongation factor 2 (eEF2); elongation; α-kinase; mass spectrometry (MS); translation; 2D, two-dimensional; CaM, calmodulin; eEF2, eukaryotic elongation factor 2; eEF2K, eEF2 kinase; ESI, electrospray ionization; GST, glutathione transferase; HEK, human embryonic kidney; LC, liquid chromatography; MHCKA, myosin heavy chain kinase A; TRP, transient receptor potential
Eukaryotic elongation factor 2 kinase (eEF-2K) is an atypical protein kinase regulated by Ca2+ and calmodulin (CaM). Its only known substrate is eukaryotic elongation factor 2 (eEF-2), whose phosphorylation by eEF-2K impedes global protein synthesis. To date, the mechanism of eEF-2K autophosphorylation has not been fully elucidated. To investigate the mechanism of autophosphorylation, human eEF-2K was co-expressed with λ-phosphatase, and purified from bacteria in a three-step protocol using a calmodulin-affinity column. Purified eEF-2K was induced to autophosphorylate by incubation with Ca2+/CaM in the presence of MgATP. Analyzing tryptic or chymotryptic peptides by mass spectrometry monitored the autophosphorylation over 0–180 minutes. The following five major autophosphorylation sites were identified, Thr-348, Thr-353, Ser-445, Ser-474 and Ser-500. In the presence of Ca2+/CaM, robust phosphorylation of Thr-348 occurs within seconds of adding MgATP. Mutagenesis studies suggest that phosphorylation of Thr-348 is required for substrate (eEF-2 or a peptide substrate) phosphorylation, but not self-phosphorylation. Phosphorylation of Ser-500 lags behind the phosphorylation of Thr-348, and is associated with calcium-independent activity of eEF-2K. Mutation of Ser-500 to Asp, but not Ala, renders eEF-2K calcium-independent. Surprisingly, this calcium-independent activity requires the presence of calmodulin.
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.
Diabetic nephropathy; Receptor serine/threonine kinase; mRNA translation
Transforming growth factor-β (TGFβ) signaling pathways regulate a wide array of cellular activities that are crucial for cell proliferation, apoptosis, migration and differentiation. TGFβ signaling pathways are initiated by ligand-activated TGFβ receptors, with type I TGFβ receptors (TβR-I) kinase being essential for phosphorylation of downstream targets. Until now, a prevalent view was that the TGFβ intracellular signaling targets would regulate transcription. Recently, we uncovered a novel TGFβ signaling pathway that exerts a direct regulatory effect on mRNA translation and protein synthesis. Eukaryotic elongation factor eEF1A1 is a GTP-binding protein that plays a central role in protein synthesis. By using a screening method for kinase substrate that was developed in our laboratory, we identified eEF1A1 as a novel substrate of TβR-I. This shed a new light on the convergence of TGFβ signaling and protein synthesis. We also showed phosphorylation of eEF1A1 at Ser300 by TβR-I prevents aa-tRNA binding to eEF1A1. As a consequence, TGFβ-dependent phosphorylation of eEF1A1 has an inhibitory effect on protein synthesis and cell proliferation. Therefore, we unveiled a novel regulatory mechanism of cellular proliferation by TGFβ at the translational level. Here we discuss this finding in the context of its potential role in the multiplicity of TGFβ signaling, and in the regulation of fundamental cellular functions, such as proliferation.
TGFβ; elongation; protein; translation; eEF1A1; cancer
HIV anti-retroviral drugs decrease protein synthesis, although the underlying regulatory mechanisms of this process are not fully established. Therefore, we investigated the effects of the HIV protease inhibitor lopinavir (LPV) on protein metabolism. We also characterized the mechanisms that mediate the effects of this drug on elongation factor-2 (eEF2), a key component of the translational machinery. Treatment of C2C12 myocytes with LPV produced a dose-dependent inhibitory effect on protein synthesis. This effect was observed at 15 min and was maintained for at least 4 h. Mechanistically, LPV increased the phosphorylation of eEF2 and thereby decreased the activity of this protein. Increased phosphorylation of eEF2 was associated with increased activity of its upstream regulators AMP-activated protein kinase (AMPK) and eEF2 kinase (eEF2K). Both AMPK and eEF2K directly phosphorylated eEF2 in an in vitro kinase assay suggesting two distinct paths lead to eEF2 phosphorylation. To verify this connection, myocytes were treated with the AMPK inhibitor compound C. Compound C blocked eEF2K and eEF2 phosphorylation, demonstrating that LPV affects eEF2 activity via an AMPK-eEF2K dependent pathway. In contrast, incubation of myocytes with rottlerin suppressed eEF2K, but not eEF2 phosphorylation, suggesting that eEF2 can be regulated independent of eEF2K. Finally, LPV did not affect PP2A activity when either eEF2 or peptide was used as the substrate. Collectively, these results indicate that LPV decreases protein synthesis, at least in part, via inhibition of eEF2. This appears regulated by AMPK which can act directly on eEF2 or indirectly via the action of eEF2K.
AMPK; eEF2K; HIV antiretroviral drugs
To survive starvation and other forms of stress, eukaryotic cells undergo a lysosomal process of cytoplasmic degradation known as autophagy. Autophagy has been implicated in a number of cellular and developmental processes, including cell growth control and programmed cell death. However, direct evidence of a causal role for autophagy in these processes is lacking, due in part to the pleiotropic effects of signaling molecules such as TOR that regulate autophagy. Here, we circumvent this difficulty by directly manipulating autophagy rates in Drosophila through the autophagy-specific protein kinase Atg1.
We find that overexpression of Atg1 is sufficient to induce high levels of autophagy, the first such demonstration among wild type Atg proteins. In contrast to findings in yeast, induction of autophagy by Atg1 is dependent on its kinase activity. We find that cells with high levels of Atg1-induced autophagy are rapidly eliminated, demonstrating that autophagy is capable of inducing cell death. However, this cell death is caspase dependent and displays DNA fragmentation, suggesting that autophagy represents an alternative induction of apoptosis, rather than a distinct form of cell death. In addition, we demonstrate that Atg1-induced autophagy strongly inhibits cell growth, and that Atg1 mutant cells have a relative growth advantage under conditions of reduced TOR signaling. Finally, we show that Atg1 expression results in negative feedback on the activity of TOR itself.
Our results reveal a central role for Atg1 in mounting a coordinated autophagic response, and demonstrate that autophagy has the capacity to induce cell death. Furthermore, this work identifies autophagy as a critical mechanism by which inhibition of TOR signaling leads to reduced cell growth.
autophagy; cell growth; programmed cell death; Target of Rapamycin (TOR); Drosophila
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.
EF-1A; Raf kinases; signal transduction; apoptosis; ubiquitin; mass spectrometry
To determine whether elongation factor-2 kinase (eEF-2 kinase) contributes to the malignant phenotype of glioblastoma multiforme by promoting the migration and invasion of glioma cells. The mechanism involved was also explored.
Human glioma cell lines T98G and LN-229 were used. The expression of eEF-2 kinase was silenced using siRNA, and the invasive potential of tumor cells was assessed using a wound-healing assay and a Matrigel invasion assay. Apoptosis was determined using propidium iodide (PI) staining and western blot analysis of cleaved caspase-3.
Silencing the expression of eEF-2 kinase by siRNA significantly suppressed both the migration and invasion of human glioma cells. Silencing eEF-2 kinase expression also sensitized glioma cells to anoikis, thereby decreasing tumor cell viability in the absence of attachment. Treatment of tumor cells with the caspase inhibitor z-VAD-fmk down-regulated Bim accumulation and abolished glioma cell sensitivity to anoikis.
The results suggest that the expression of eEF-2 kinase contributes to migration and invasion of human glioma cells by protecting them from anoikis. eEF-2 kinase expression may serve as a prognostic marker and a novel target for cancer therapy.
eEF-2 kinase; migration; invasion; anoikis; glioma
Autophagy proteins are normally involved in the formation of double-membrane autophagosomes that mediate bulk cytoplasmic and organelle degradation. Here we report the modification of single-membrane vacuoles in cells by autophagy proteins. Light Chain 3 (LC3), a component of autophagosomes, is recruited to single-membrane entotic vacuoles, macropinosomes, and phagosomes harboring apoptotic cells, in a manner dependent on lipidation machinery including Atg5 and Atg7, and the class III PI-3-kinase Vps34. These downstream components of autophagy machinery, but not the upstream mTor-regulated Ulk- Atg13-Fip200 complex, facilitate lysosome fusion to single membranes and the degradation of internalized cargo. For entosis, a live cell engulfment program, the autophagy protein-dependent fusion of lysosomes to vacuolar membranes leads to the death of internalized cells, which are killed by their hosts. As pathogen-containing phagosomes can be targeted in a similar manner, the death of epithelial cells by this mechanism mimics pathogen destruction. These data define the targeting of single-membrane vacuoles as a property of autophagy pathway proteins in cells in the absence of pathogenic organisms.
Autophagy represents an alternative tumor-suppressing mechanism that overcomes the dramatic resistance of malignant gliomas to radiotherapy and proapoptotic-related chemotherapy. This study reports that valproic acid (VPA), a widely used anti-epilepsy drug, induces autophagy in glioma cells. Autophagy, crucial for VPA-induced cell death, is independent of apoptosis, even though apoptotic machinery is proficient. Oxidative stress induced by VPA occurs upstream of autophagy. Oxidative stress also activates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, whereas blocking this pathway inhibits autophagy and induces apoptosis. VPA-induced autophagy cannot be alleviated by inositol, suggesting a mechanism different from that for lithium. Moreover, VPA potentiates autophagic cell death, but not apoptosis, when combined with other autophagy inducers such as rapamycin, Ly294002, and temozolomide in glioma cells both in vitro and in vivo, which may warrant further investigation toward possible clinical application in patients with malignant gliomas.
autophagy; glioma; oxidative stress; valproic acid
Legionella pneumophila, which is the causative organism of Legionnaireś disease, translocates numerous effector proteins into the host cell cytosol by a type IV secretion system during infection. Among the most potent effector proteins of Legionella are glucosyltransferases (lgt's), which selectively modify eukaryotic elongation factor (eEF) 1A at Ser-53 in the GTP binding domain. Glucosylation results in inhibition of protein synthesis. Here we show that in vitro glucosylation of yeast and mouse eEF1A by Lgt3 in the presence of the factors Phe-tRNAPhe and GTP was enhanced 150 and 590-fold, respectively. The glucosylation of eEF1A catalyzed by Lgt1 and 2 was increased about 70-fold. By comparison of uncharged tRNA with two distinct aminoacyl-tRNAs (His-tRNAHis and Phe-tRNAPhe) we could show that aminoacylation is crucial for Lgt-catalyzed glucosylation. Aminoacyl-tRNA had no effect on the enzymatic properties of lgt's and did not enhance the glucosylation rate of eEF1A truncation mutants, consisting of the GTPase domain only or of a 5 kDa peptide covering Ser-53 of eEF1A. Furthermore, binding of aminoacyl-tRNA to eEF1A was not altered by glucosylation. Taken together, our data suggest that the ternary complex, consisting of eEF1A, aminoacyl-tRNA and GTP, is the bona fide substrate for lgt's.
The deleterious consequences of fatty acid (FA) and neutral lipid accumulation in nonadipose tissues, such as the heart, contribute to the pathogenesis of type 2 diabetes. To elucidate mechanisms of FA-induced cell death, or lipotoxicity, we generated Chinese hamster ovary (CHO) cell mutants resistant to palmitate-induced death and isolated a clone with disruption of eukaryotic elongation factor (eEF) 1A-1. eEF1A-1 involvement in lipotoxicity was confirmed in H9c2 cardiomyoblasts, in which small interfering RNA-mediated knockdown also conferred palmitate resistance. In wild-type CHO and H9c2 cells, palmitate increased reactive oxygen species and induced endoplasmic reticulum (ER) stress, changes accompanied by increased eEF1A-1 expression. Disruption of eEF1A-1 expression rendered these cells resistant to hydrogen peroxide- and ER stress-induced death, indicating that eEF1A-1 plays a critical role in the cell death response to these stressors downstream of lipid overload. Disruption of eEF1A-1 also resulted in actin cytoskeleton defects under basal conditions and in response to palmitate, suggesting that eEF1A-1 mediates lipotoxic cell death, secondary to oxidative and ER stress, by regulating cytoskeletal changes critical for this process. Furthermore, our observations of oxidative stress, ER stress, and induction of eEF1A-1 expression in a mouse model of lipotoxic cardiomyopathy implicate this cellular response in the pathophysiology of metabolic disease.
Calmodulin-dependent protein kinase III (CaM kinase III, elongation factor-2 kinase) is a unique member of the Ca2+/CaM-dependent protein kinase family. Activation of CaM kinase III leads to the selective phosphorylation of elongation factor 2 (eEF-2) and transient inhibition of protein synthesis. Recent cloning and sequencing of CaM kinase III revealed that this enzyme represents a new superfamily of protein kinases. The activity of CaM kinase III is selectively activated in proliferating cells; inhibition of the kinase blocked cells in G0/G1-S and decreased viability. To determine the significance of CaM kinase III in breast cancer, we measured the activity of the kinase in human breast cancer cell lines as well as in fresh surgical specimens. The specific activity of CaM kinase III in human breast cancer cell lines was equal to or greater than that seen in a variety of cell lines with similar rates of proliferation. The specific activity of CaM kinase III was markedly increased in human breast tumour specimens compared with that of normal adjacent breast tissue. The activity of this enzyme was regulated by breast cancer mitogens. In serum-deprived MDA-MB-231 cells, the combination of insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) stimulated cell proliferation and activated CaM kinase III to activities observed in the presence of 10% serum. Inhibition of enzyme activity blocked cell proliferation induced by growth factors. In MCF-7 cells separated by fluorescence-activated cell sorting, CaM kinase III was increased in S-phase over that of other phases of the cell cycle. In summary, the activity of Ca2+/CaM-dependent protein kinase III is controlled by breast cancer mitogens and appears to be constitutively activated in human breast cancer. These results suggest that CaM kinase III may contribute an important link between growth factor/receptor interactions, protein synthesis and the induction of cellular proliferation in human breast cancer. © 1999 Cancer Research Campaign
Ca2+/calmodulin-dependent protein kinase III (elongation factor-2 kinase); breast cancer; breast cancer mitogens; cell cycle; protein synthesis