The space between notes: emerging roles for translationally silent ribosomes.

In addition to their central functions in translation, ribosomes can adopt inactive structures that are fully assembled yet devoid of mRNA. We describe how the abundance of idle eukaryotic ribosomes is influenced by a broad range of biological conditions spanning viral infection, nutrient deprivation, and developmental cues. Vacant ribosomes may provide a means to exclude ribosomes from translation while also shielding them from degradation, and the variable identity of factors that occlude ribosomes may impart distinct functionality. We propose that regulated changes in the balance of idle and active ribosomes provides a means to fine-tune translation. We provide an overview of idle ribosomes, describe what is known regarding their function, and highlight questions that may clarify their biological roles.

Ribosomal dormancy at the nexus of ribosome homeostasis and protein synthesis.

Dormancy or hibernation is a non-proliferative state of cells with low metabolic activity and gene expression. Dormant cells sequester ribosomes in a translationally inactive state, called dormant/hibernating ribosomes. These dormant ribosomes are important for the preservation of ribosomes and translation shut-off. While recent studies attempted to elucidate their modes of formation, the regulation and roles of the diverse dormant ribosomal populations are still largely understudied. The mechanistic details of the formation of dormant ribosomes in stress and especially their disassembly during recovery remain elusive. In this review, we discuss the roles of dormant ribosomes and their potential regulatory mechanisms. Furthermore, we highlight the paradigms that need to be answered in the field of ribosomal dormancy.

Research advances on CaMKs-mediated neurodevelopmental injury.

Calcium/calmodulin-dependent protein kinases (CaMKs) are important proteins in the calcium signaling cascade response pathway, which can broadly regulate biological functions in vivo. Multifunctional CaMKs play key roles in neural development, including neuronal circuit building, synaptic plasticity establishment, and neurotrophic factor secretion. Currently, four familial proteins, calcium/calmodulin-dependent protein kinase I (CaMKI), calcium/calmodulin-dependent protein kinase II (CaMKII), eukaryotic elongation factor 2 kinase (eEF2K, popularly known as CaMKIII) and calcium/calmodulin-dependent protein kinase IV (CaMKIV), are thought to have been the most extensively studied during neurodevelopment. Although their spatial structures are extremely similar, as well as the initial starting point of activation, both require the activation of calcium and calmodulin (CaM) complexes to be involved in the process, and the phosphorylation sites and modes of each member are different. Furthermore, due to the high structural similarity of CaMKs, their members may play synergistic roles in the regulation of neural development, but different CaMKs also have their own means of regulating neural development. In this review, we first describe the visualized protein structural forms of CaMKI, CaMKII, eEF2K and CaMKIV, and then describe the functions of each kinase in neurodevelopment. After that, we focus on four main mechanisms of neurodevelopmental damage caused by CaMKs: CaMKI/ERK/CREB pathway inhibition leading to dendritic spine structural damage; Ca2+/CaM/CaMKII through induction of mitochondrial kinetic disorders leading to neurodevelopmental damage; CaMKIII/eEF2 hyperphosphorylation affects the establishment of synaptic plasticity; and CaMKIV/JNK/NF-κB through induction of an inflammatory response leading to neurodevelopmental damage. In conclusion, we briefly discuss the pathophysiological significance of aberrant CaMK family expression in neurodevelopmental disorders, as well as the protective effects of conventional CaMKII and CaMKIII antagonists against neurodevelopmental injury.

eEF2K Activity Determines Synergy to Cotreatment of Cancer Cells With PI3K and MEK Inhibitors.

PI3K-mammalian target of rapamycin and MAPK/ERK kinase (MEK)/mitogen-activated protein kinase (MAPK) are the most frequently dysregulated signaling pathways in cancer. A problem that limits the success of therapies that target individual PI3K-MAPK members is that these pathways converge to regulate downstream functions and often compensate each other, leading to drug resistance and transient responses to therapy. In order to overcome resistance, therapies based on cotreatments with PI3K/AKT and MEK/MAPK inhibitors are now being investigated in clinical trials, but the mechanisms of sensitivity to cotreatment are not fully understood. Using LC-MS/MS-based phosphoproteomics, we found that eukaryotic elongation factor 2 kinase (eEF2K), a key convergence point downstream of MAPK and PI3K pathways, mediates synergism to cotreatment with trametinib plus pictilisib (which target MEK1/2 and PI3Kα/δ, respectively). Inhibition of eEF2K by siRNA or with a small molecule inhibitor reversed the antiproliferative effects of the cotreatment with PI3K plus MEK inhibitors in a cell model-specific manner. Systematic analysis in 12 acute myeloid leukemia cell lines revealed that eEF2K activity was increased in cells for which PI3K plus MEKi cotreatment is synergistic, while PKC potentially mediated resistance to such cotreatment. Together, our study uncovers eEF2K activity as a key mediator of responses to PI3Ki plus MEKi and as a potential biomarker to predict synergy to cotreatment in cancer cells.

Translational control by ketamine and its implications for comorbid cognitive deficits in depressive disorders.

Ketamine has shown antidepressant effects in patients with major depressive disorder (MDD) resistant to first-line treatments and approved for use in this patient population. Ketamine induces several forms of synaptic plasticity, which are proposed to underlie its antidepressant effects. However, the molecular mechanism of action directly responsible for ketamine's antidepressant effects remains under active investigation. It was recently demonstrated that the effectors of the mammalian target of rapamycin complex 1 (mTORC1) signalling pathway, namely, eukaryotic initiation factor 4E (eIF4E) binding proteins 1 and 2 (4E-BP1 and 4E-BP2), are central in mediating ketamine-induced synaptic plasticity and behavioural antidepressant-like effect. 4E-BPs are a family of messenger ribonucleic acid (mRNA) translation repressors inactivated by mTORC1. We observed that their expression in inhibitory interneurons mediates ketamine's effects in the forced swim and novelty suppressed feeding tests and the long-lasting inhibition of GABAergic neurotransmission in the hippocampus. In addition, another effector pathway that regulates translation elongation downstream of mTORC1, the eukaryotic elongation factor 2 kinase (eEF2K), has been implicated in ketamine's behavioural effects. We will discuss how ketamine's rapid antidepressant effect depends on the activation of neuronal mRNA translation through 4E-BP1/2 and eEF2K. Furthermore, given that these pathways also regulate cognitive functions, we will discuss the evidence of ketamine's effect on cognitive function in MDD. Overall, the data accrued from pre-clinical research have implicated the mRNA translation pathways in treating mood symptoms of MDD. However, it is yet unclear whether the pro-cognitive potential of subanesthetic ketamine in rodents also engages these pathways and whether such an effect is consistently observed in the treatment-resistant MDD population.

Roles of eukaryotic elongation factor 2 kinase (eEF2K) in neuronal plasticity, cognition, and Alzheimer disease.

Understanding the molecular signaling mechanisms underlying cognition and neuronal plasticity would provide insights into the pathogenesis of neuronal disorders characterized by cognitive syndromes such as Alzheimer disease (AD). Phosphorylation of the mRNA translational factor eukaryotic elongation factor 2 (eEF2) by its specific kinase eEF2K is critically involved in protein synthesis regulation. In this review, we discussed recent studies on the roles of eEF2K/eEF2 signaling in the context of regulation/dysregulation of cognitive function and synaptic plasticity. We specifically focus on the discussion of recent evidence indicating suppression of eEF2K signaling as a potential novel therapeutic avenue for AD and related dementias (ADRDs).

Can endocan serve as a molecular "hepatostat" in liver regeneration?

BACKGROUND: Intriguingly, liver regeneration after injury does not induce uncontrolled growth and the underlying mechanisms of such a "hepatostat" are still not clear. Endocan, a proteoglycan, was implicated in liver regeneration. It can support the function of hepatocyte growth factor/scatter factor in tissue repair after injury. Endostatin, a 20 kDa C-terminal fragment of collagen XVIII, may modulate the cessation of liver regeneration. eEF2K, a protein kinase that regulates protein synthesis, can regulate angiogenesis. Thus, we investigated the role of endocan, endostatin and eEF2K during normal liver regeneration. METHODS: Serum samples and regenerating remnant liver tissues were obtained on various days after partial hepatectomy in rats. mRNA expression levels of Vegf and Pcna were analyzed in addition to immunohistochemical evaluations. Liver tissue protein levels of endostatin, endocan and p-eEF2K/eEF2K were determined with Western blot. Serum levels of endostatin and endocan were assessed with ELISA. RESULTS: Pcna expression level in residual liver tissues peaked on day-1, while Vegf expression reached its highest level on days 1-3 after partial hepatectomy (70%). Endocan activity declined gradually on days 1-7. The decrease in liver endocan expression was accompanied by an increase in serum endocan levels. Partial hepatectomy induced a rapid increase in liver endostatin levels. Following its surge on day-1, endostatin expression gradually declined, which was accompanied by a peak in serum endostatin. Finally, partial hepatectomy was shown to regulate eEF2K; thus, increasing protein translation. CONCLUSIONS: We revealed possible mechanistic insights into liver regeneration by examining the associations of Pcna, Vegf, endocan, endostatin, eEF2K with hepatic regeneration after partial hepatectomy. Indeed, endocan might serve as a useful biomarker to monitor clinical prognosis in a plethora of conditions such as recovery of donor's remaining liver after living-donor liver transplant. Whether endocan might represent a strategy to optimize liver regeneration when given therapeutically needs to be investigated in future studies.

Eukaryotic elongation factor 2 kinase regulates foam cell formation via translation of CD36.

Eukaryotic elongation factor 2 kinase (eEF2K) is an atypical protein kinase that controls protein synthesis in cells under stress. Although well studied in cancer, less is known about its roles in chronic inflammatory diseases. Here, we examined its regulation of macrophage cholesterol handling in the context of atherosclerosis. eEF2K mRNA expression and protein activity were upregulated in murine bone marrow-derived macrophages (BMDMs) exposed to oxidized low-density lipoprotein cholesterol (oxLDL). When incubated with oxLDL, BMDMs from eEF2K knockout (Eef2k-/- ) mice formed fewer Oil Red O+ foam cells than Eef2k+/+ BMDMs (12.5% ± 2.3% vs. 32.3% ± 2.0%, p < .01). Treatment with a selective eEF2K inhibitor, JAN-384, also decreased foam cell formation for C57BL/6J BMDMs and human monocyte-derived macrophages. Disabling eEF2K selectively decreased protein expression of the CD36 cholesterol uptake receptor, mediated by a reduction in the proportion of translationally active Cd36 mRNA. Eef2k-/- mice bred onto the Ldlr-/- background developed aortic sinus atherosclerotic plaques that were 30% smaller than Eef2k+/+ -Ldlr-/- mice after 16 weeks of high cholesterol diet (p < .05). Although accompanied by a reduction in plaque CD36+ staining (p < .05) and lower CD36 expression in circulating monocytes (p < .01), this was not associated with reduced lipid content in plaques as measured by oil red O staining. Finally, EEF2K and CD36 mRNA levels were higher in blood mononuclear cells from patients with coronary artery disease and recent myocardial infarction compared to healthy controls without coronary artery disease. These results reveal a new role for eEF2K in translationally regulating CD36 expression and foam cell formation in macrophages. Further studies are required to explore therapeutic targeting of eEF2K in atherosclerosis.

Impact of silencing eEF2K expression on the malignant properties of chordoma.

BACKGROUND: Eukaryotic elongation factor 2 kinase (eukaryotic elongation factor 2 kinase, eEF2K) is a calcium calmodulin dependent protein kinase that keeps the highest energy consuming cellular process of protein synthesis under check through negative regulation. eEF2K pauses global protein synthesis rates at the translational elongation step by phosphorylating its only kown substrate elongation factor 2 (eEF2), a unique translocase activity in ekaryotic cells enabling the polypeptide chain elongation. Therefore, eEF2K is thought to preserve cellular energy pools particularly upon acute development of cellular stress conditions such as nutrient deprivation, hypoxia, or infections. Recently, high expression of this enzyme has been associated with poor prognosis in an array of solid tumor types. Therefore, in a growing number of studies tremendous effort is being directed to the development of treatment methods aiming to suppress eEF2K as a novel therapeutic approach in the fight against cancer. METHODS: In our study, we aimed to investigate the changes in the tumorigenicity of chordoma cells in presence of gene silencing for eEF2K. Taking a transient gene silencing approach using siRNA particles, eEF2K gene expression was suppressed in chordoma cells. RESULTS: Silencing eEF2K expression was associated with a slight increase in cellular proliferation and a decrease in death rates. Furthermore, no alteration in the sensitivity of chordoma cells to chemotherapy was detected in response to the decrease in eEF2K expression which intriguingly promoted suppression of cell migratory and invasion related properties. CONCLUSION: Our findings indicate that the loss of eEF2K expression in chordoma cell lines results in the reduction of metastatic capacity.

eEF2K Inhibitor Design: The Progression of Exemplary Structure-Based Drug Design.

The α-kinase, eEF2K, phosphorylates the threonine 56 residue of eEF2 to inhibit global peptide elongation (protein translation). As a master regulator of protein synthesis, in combination with its unique atypical kinase active site, investigations into the targeting of eEF2K represents a case of intense structure-based drug design that includes the use of modern computational techniques. The role of eEF2K is incredibly diverse and has been scrutinized in several different diseases including cancer and neurological disorders-with numerous studies inhibiting eEF2K as a potential treatment option, as described in this paper. Using available crystal structures of related α-kinases, particularly MHCKA, we report how homology modeling has been used to improve inhibitor design and efficacy. This review presents an overview of eEF2K related drug discovery efforts predating from the 1990's, to more recent in vivo studies in rat models. We also provide the reader with a basic introduction to several approaches and software programs used to undertake such drug discovery campaigns. With the recent exciting publication of an eEF2K crystal structure, we present our view regarding the future of eEF2K drug discovery.

Antagonists targeting eEF2 kinase rescue multiple aspects of pathophysiology in Alzheimer's disease model mice.

It is imperative to develop novel therapeutic strategies for Alzheimer's disease (AD) and related dementia syndromes based on solid mechanistic studies. Maintenance of memory and synaptic plasticity relies on de novo protein synthesis, which is partially regulated by phosphorylation of eukaryotic elongation factor 2 (eEF2) via its kinase eEF2K. Abnormally increased eEF2 phosphorylation and impaired mRNA translation have been linked to AD. We recently reported that prenatal genetic suppression of eEF2K is able to prevent aging-related cognitive deficits in AD model mice, suggesting the therapeutic potential of targeting eEF2K/eEF2 signaling in AD. Here, we tested two structurally distinct small-molecule eEF2K inhibitors in two different lines of AD model mice after the onset of cognitive impairments. Our data revealed that treatment with eEF2K inhibitors improved AD-associated synaptic plasticity impairments and cognitive dysfunction, without altering brain amyloid ß (Aß) and tau pathology. Furthermore, eEF2K inhibition alleviated AD-associated defects in dendritic spine morphology, post-synaptic density formation, protein synthesis, and dendritic polyribosome assembly. Our results may offer critical therapeutic implications for AD, and the proof-of-principle study indicates translational implication of inhibiting eEF2K for AD and related dementia syndromes. Cover Image for this issue: https://doi.org/10.1111/jnc.15392.

Eukaryotic Extension Factor 2 Kinase may Affect the Occurrence and Development of Glioblastoma Through Immune Cell Infiltration.

Glioblastoma (GBM) is one of the most common malignancies among primary brain tumors in adults, featuring a poor prognosis and a high recurrence rate. Eukaryotic elongation factor 2 kinase (eEF2K) is a calcium/calmodulin-dependent protein kinase that is involved in promoting tumor cell proliferation, migration, invasion, and survival. However, its expression level in GBM, its prognostic impact and correlation with immune infiltration are not yet known. In this study, we used The Cancer Genome Atlas (TCGA) database to explore the potential molecular mechanisms of eEF2K in GBM development and clinical prognosis in terms of gene expression, survival status, immune infiltration, and associated cellular pathways. We found that eEF2K expression levels were elevated in GBM, but eEF2K was not associated with the prognosis of GBM patients; eEF2K expression in GBM was associated with multiple immune cell infiltrations. These results show a statistical correlation between eEF2K expression and the development of GBM and immune cell infiltration, which helps us to understand the roles of eEF2K in GBM from different perspectives.

Repression of eEF2K transcription by NF-κB tunes translation elongation to inflammation and dsDNA-sensing.

Gene expression is rapidly remodeled by infection and inflammation in part via transcription factor NF-κB activation and regulated protein synthesis. While protein synthesis is largely controlled by mRNA translation initiation, whether cellular translation elongation factors are responsive to inflammation and infection remains poorly understood. Here, we reveal a surprising mechanism whereby NF-κB restricts phosphorylation of the critical translation elongation factor eEF2, which catalyzes the protein synthesis translocation step. Upon exposure to NF-κB-activating stimuli, including TNFα, human cytomegalovirus infection, or double-stranded DNA, eEF2 phosphorylation on Thr56, which slows elongation to limit protein synthesis, and the overall abundance of eEF2 kinase (eEF2K) are reduced. Significantly, this reflected a p65 NF-κB subunit-dependent reduction in eEF2K pre-mRNA, indicating that NF-κB activation represses eEF2K transcription to decrease eEF2K protein levels. Finally, we demonstrate that reducing eEF2K abundance regulates protein synthesis in response to a bacterial toxin that inactivates eEF2. This establishes that NF-κB activation by diverse physiological effectors controls eEF2 activity via a transcriptional repression mechanism that reduces eEF2K polypeptide abundance to preclude eEF2 phosphorylation, thereby stimulating translation elongation and protein synthesis. Moreover, it illustrates how nuclear transcription regulation shapes translation elongation factor activity and exposes how eEF2 is integrated into innate immune response networks orchestrated by NF-κB.

Targeting Protein Translation in Melanoma by Inhibiting EEF-2 Kinase Regulates Cholesterol Metabolism though SREBP2 to Inhibit Tumour Development.

Decreasing the levels of certain proteins has been shown to be important for controlling cancer but it is currently unknown whether proteins could potentially be targeted by the inhibiting of protein synthesis. Under this circumstance, targeting protein translation could preferentially affect certain pathways, which could then be of therapeutic advantage when treating cancer. In this report, eukaryotic elongation factor-2 kinase (EEF2K), which is involved in protein translation, was shown to regulate cholesterol metabolism. Targeting EEF2K inhibited key parts of the cholesterol pathway in cancer cells, which could be rescued by the addition of exogenous cholesterol, suggesting that it is a potentially important pathway modulated by targeting this process. Specifically, targeting EEF2K significantly suppressed tumour cell growth by blocking mRNA translation of the cholesterol biosynthesis transcription factor, sterol regulatory element-binding protein (SREBP) 2, and the proteins it regulates. The process could be rescued by the addition of LDL cholesterol taken into the cells via non-receptor-mediated-uptake, which negated the need for SREBP2 protein. Thus, the levels of SREBP2 needed for cholesterol metabolism in cancer cells are therapeutically vulnerable by targeting protein translation. This is the first report to suggest that targeting EEF2K can be used to modulate cholesterol metabolism to treat cancer.

Discovery of New Inhibitors of eEF2K from Traditional Chinese Medicine Based on In Silico Screening and In Vitro Experimental Validation.

Eukaryotic elongation factor 2 kinase (eEF2K) is a highly conserved α kinase and is increasingly considered as an attractive therapeutic target for cancer as well as other diseases. However, so far, no selective and potent inhibitors of eEF2K have been identified. In this study, pharmacophore screening, homology modeling, and molecular docking methods were adopted to screen novel inhibitor hits of eEF2K from the traditional Chinese medicine database (TCMD), and then cytotoxicity assay and western blotting were performed to verify the validity of the screen. Resultantly, after two steps of screening, a total of 1077 chemicals were obtained as inhibitor hits for eEF2K from all 23,034 compounds in TCMD. Then, to verify the validity, the top 10 purchasable chemicals were further analyzed. Afterward, Oleuropein and Rhoifolin, two reported antitumor chemicals, were found to have low cytotoxicity but potent inhibitory effects on eEF2K activity. Finally, molecular dynamics simulation, pharmacokinetic and toxicological analyses were conducted to evaluate the property and potential of Oleuropein and Rhoifolin to be drugs. Together, by integrating in silico screening and in vitro biochemical studies, Oleuropein and Rhoifolin were revealed as novel eEF2K inhibitors, which will shed new lights for eEF2K-targeting drug development and anticancer therapy.

Bicuculline regulated protein synthesis is dependent on Homer1 and promotes its interaction with eEF2K through mTORC1-dependent phosphorylation.

The regulation of protein synthesis is a vital and finely tuned process in cellular physiology. In neurons, this process is very precisely regulated, as which mRNAs undergo translation is highly dependent on context. One of the most prominent regulators of protein synthesis is the enzyme eukaryotic elongation factor kinase 2 (eEF2K) that regulates the elongation stage of protein synthesis. This kinase and its substrate, eukaryotic elongation factor 2 (eEF2) are important in processes such as neuronal development and synaptic plasticity. eEF2K is regulated by multiple mechanisms including Ca2+ -ions and the mTORC1 signaling pathway, both of which play key roles in neurological processes such as learning and memory. In such settings, the localized control of protein synthesis is of crucial importance. In this work, we sought to investigate how the localization of eEF2K is controlled and the impact of this on protein synthesis in neuronal cells. In this study, we used both SH-SY5Y neuroblastoma cells and mouse cortical neurons, and pharmacologically and/or genetic approaches to modify eEF2K function. We show that eEF2K activity and localization can be regulated by its binding partner Homer1b/c, a scaffolding protein known for its participation in calcium-regulated signaling pathways. Furthermore, our results indicate that this interaction is regulated by the mTORC1 pathway, through a known phosphorylation site in eEF2K (S396), and that it affects rates of localized protein synthesis at synapses depending on the presence or absence of this scaffolding protein.

Novel inhibitors of eukaryotic elongation factor 2 kinase: In silico, synthesis and in vitro studies.

Eukaryotic elongation factor 2 kinase (eEF2K) is an unusual alpha kinase whose expression is highly upregulated in various cancers and contributes to tumor growth, metastasis, and progression. More importantly, eEF2K expression is associated with poor clinical outcome and shorter patient survival in breast, lung and ovarian cancers. Therefore, eEF2K is an emerging molecular target for development of novel targeted therapeutics and precision medicine in solid cancers. Currently, there are not any available potent and specific eEF2K inhibitors for clinical translation. In this study, we designed and synthesized a series of novel compounds with coumarin scaffold with various substitutions and investigated their effects in inhibiting eEF2K activity using in silico approaches and in vitro studies in breast cancer cells. We utilized an amide substitution at position 3 on the coumarin ring with their pharmacologically active groups containing pyrrolidine, piperidine, morpholine and piperazine groups with (CH2)2 bridged for aliphatic amides. Due to their ability to form covalent binding to the target enzyme, we also investigated the effects of boron containing groups on functionalized coumarin ring (3 compounds) and designed novel aliphatic and aromatic derivatives of coumarin scaffolds (10 compounds) and phenyl ring with boron groups (4 compounds). The Glide/SP module of the Maestro molecular modeling package was used to perform in silico analysis and molecular docking studies. According to our combined results, structure activity relationship (SAR) was performed in detail. Among the newly designed, synthesized, and tested compounds, our in vitro findings revealed that several compounds displayed a highly effective eEF2K inhibition at submicromolar concentrations in in vitro breast cancer cells. In conclusion, we identified novel compounds that can be used as eEF2K inhibitors and that they should be further evaluated by in vivo preclinical tumor models studies for antitumor efficacy and clinical translation.

Progress in the Development of Eukaryotic Elongation Factor 2 Kinase (eEF2K) Natural Product and Synthetic Small Molecule Inhibitors for Cancer Chemotherapy.

Eukaryotic elongation factor 2 kinase (eEF2K or Ca2+/calmodulin-dependent protein kinase, CAMKIII) is a new member of an atypical α-kinase family different from conventional protein kinases that is now considered as a potential target for the treatment of cancer. This protein regulates the phosphorylation of eukaryotic elongation factor 2 (eEF2) to restrain activity and inhibit the elongation stage of protein synthesis. Mounting evidence shows that eEF2K regulates the cell cycle, autophagy, apoptosis, angiogenesis, invasion, and metastasis in several types of cancers. The expression of eEF2K promotes survival of cancer cells, and the level of this protein is increased in many cancer cells to adapt them to the microenvironment conditions including hypoxia, nutrient depletion, and acidosis. The physiological function of eEF2K and its role in the development and progression of cancer are here reviewed in detail. In addition, a summary of progress for in vitro eEF2K inhibitors from anti-cancer drug discovery research in recent years, along with their structure-activity relationships (SARs) and synthetic routes or natural sources, is also described. Special attention is given to those inhibitors that have been already validated in vivo, with the overall aim to provide reference context for the further development of new first-in-class anti-cancer drugs that target eEF2K.

Ablation of elongation factor 2 kinase enhances heat-shock protein 90 chaperone expression and protects cells under proteotoxic stress.

Eukaryotic elongation factor 2 kinase (eEF2K) negatively regulates the elongation stage of mRNA translation and is activated under different stress conditions to slow down protein synthesis. One effect of eEF2K is to alter the repertoire of expressed proteins, perhaps to aid survival of stressed cells. Here, we applied pulsed stable isotope labeling with amino acids in cell culture (SILAC) to study changes in the synthesis of specific proteins in human lung adenocarcinoma (A549) cells in which eEF2K had been depleted by an inducible shRNA. We discovered that levels of heat-shock protein 90 (HSP90) are increased in eEF2K-depleted human cells as well as in eEF2K-knockout (eEF2K-/-) mouse embryonic fibroblasts (MEFs). This rise in HSP90 coincided with an increase in the fraction of HSP90 mRNAs associated with translationally active polysomes, irrespective of unchanged total HSP90 levels. These results indicate that blocking eEF2K function can enhance expression of HSP90 chaperones. In eEF2K-/- mouse embryonic fibroblasts (MEFs), inhibition of HSP90 by its specific inhibitor AUY922 promoted the accumulation of ubiquitinated proteins. Notably, HSP90 inhibition promoted apoptosis of eEF2K-/- MEFs under proteostatic stress induced by the proteasome inhibitor MG132. Up-regulation of HSP90 likely protects cells from protein folding stress, arising, for example, from faster rates of polypeptide synthesis due to the lack of eEF2K. Our findings indicate that eEF2K and HSPs closely cooperate to maintain proper proteostasis and suggest that concomitant inhibition of HSP90 and eEF2K could be a strategy to decrease cancer cell survival.

Eukaryotic elongation factor 2 kinase promotes angiogenesis in hepatocellular carcinoma via PI3K/Akt and STAT3.

Hepatocellular carcinoma (HCC) is an aggressive malignancy with increasing mortality in China. Angiogenesis is crucial for tumor formation, development and metastasis in HCC. Previous studies indicated that high expression levels of elongation factor 2 kinase (eEF2K), a protein kinase that negatively regulates the elongation stage of translation, were associated with poor prognosis of HCC. Here, we show that pharmacological inhibition or knockdown of eEF2K in highly metastatic liver cancer cells inhibits their colony forming and migratory capacities, as well as reducing their invasiveness. Importantly, knocking down eEF2K by lentiviral directed shRNA prevented tumor growth and angiogenesis of HCC in mice. Silencing of eEF2K in endothelial cells (HUVECs) led to a reduction in vascularization, evidenced by a decrease in capillary-like structures in the matrigel. Notably, knocking down eEF2K reduced the expression of angiogenesis-related growth factors in liver cancer cells and the expression of growth factor receptors on HUVECs, and thus restricted signaling crosstalk that promotes angiogenesis between HCC cells and endothelial cells. We also showed that silencing of eEF2K effectively reduced protein levels of SP1/KLF5 transcription factors and hence decreased the levels of bound SP1/KLF5 to the VEGF promoter, resulted in a decrease in VEGF mRNA expression. Knocking down eEF2K also led to a striking decrease in the phosphorylation of PI3K/Akt and STAT3, indicating inactivation of these tumorigenic pathways. Taken together, our data suggest that eEF2K contributes to angiogenesis and tumor progression in HCC via SP1/KLF5-mediated VEGF expression, as well as the subsequent stimulation of PI3K/Akt and STAT3 signaling.