Structure of the complex between calmodulin and a functional construct of eukaryotic elongation factor 2 kinase bound to an ATP-competitive inhibitor.

The calmodulin-activated α-kinase, eukaryotic elongation factor 2 kinase (eEF-2K), serves as a master regulator of translational elongation by specifically phosphorylating and reducing the ribosome affinity of the guanosine triphosphatase, eukaryotic elongation factor 2 (eEF-2). Given its critical role in a fundamental cellular process, dysregulation of eEF-2K has been implicated in several human diseases, including those of the cardiovascular system, chronic neuropathies, and many cancers, making it a critical pharmacological target. In the absence of high-resolution structural information, high-throughput screening efforts have yielded small-molecule candidates that show promise as eEF-2K antagonists. Principal among these is the ATP-competitive pyrido-pyrimidinedione inhibitor, A-484954, which shows high specificity toward eEF-2K relative to a panel of "typical" protein kinases. A-484954 has been shown to have some degree of efficacy in animal models of several disease states. It has also been widely deployed as a reagent in eEF-2K-specific biochemical and cell-biological studies. However, given the absence of structural information, the precise mechanism of the A-484954-mediated inhibition of eEF-2K has remained obscure. Leveraging our identification of the calmodulin-activatable catalytic core of eEF-2K, and our recent determination of its long-elusive structure, here we present the structural basis for its specific inhibition by A-484954. This structure, which represents the first for an inhibitor-bound catalytic domain of a member of the α-kinase family, enables rationalization of the existing structure-activity relationship data for A-484954 variants and lays the groundwork for further optimization of this scaffold to attain enhanced specificity/potency against eEF-2K.

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.

Eukaryotic elongation factor-2 kinase (eEF2K) signaling in tumor and microenvironment as a novel molecular target.

Eukaryotic elongation factor-2 kinase (eEF2K), an atypical member of alpha-kinase family, is highly overexpressed in breast, pancreatic, brain, and lung cancers, and associated with poor survival in patients. eEF2K promotes cell proliferation, survival, and aggressive tumor characteristics, leading to tumor growth and progression. While initial studies indicated that eEF2K acts as a negative regulator of protein synthesis by suppressing peptide elongation phase, later studies demonstrated that it has multiple functions and promotes cell cycle, angiogenesis, migration, and invasion as well as induction of epithelial-mesenchymal transition through induction of integrin ß1, SRC/FAK, PI3K/AKT, cyclin D1, VEGF, ZEB1, Snail, and MMP-2. Under stress conditions such as hypoxia and metabolic distress, eEF2K is activated by several signaling pathways and slows down protein synthesis and helping cells to save energy and survive. In vivo therapeutic targeting of eEF2K by genetic methods inhibits tumor growth in various tumor models, validating it as a potential molecular target. Recent studies suggest that eEF2K plays a role in tumor microenvironment cells by monocyte chemoattractant protein-1 (MCP-1) and accumulation of tumor-associated macrophages. Due to its clinical significance and the pivotal role in tumorigenesis and progression, eEF2K is considered as an important therapeutic target in solid tumors. However, currently, there is no specific and potent inhibitor for translation into clinical studies. Here, we aim to systematically review current knowledge regarding eEF2K in tumor biology, microenvironment, and development of eEF2K targeted inhibitors and therapeutics.

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.

Protein Domain Level Cancer Drug Targets in the Network of MAPK Pathways.

Proteins in the MAPK pathways considered as potential drug targets for cancer treatment. Pathways along with the cross-talks increase their scope to view them as a network of MAPK pathways. Side effect causing targeted domains act as a proxy for drug targets due to its structural similarity and frequent reuse of their variants. We proposed to identify non-repeatable protein domains as the drug targets to disrupt the signal transduction than targeting the whole protein. Network based approach is used to understand the contribution of 52 domains in non-hub, non-essential, and intra-pathway cancerous nodes and to identify potential drug target domains. 34 distinct domains in the cancerous proteins are playing vital roles in making cancer as a complex disease and pose challenges to identify potential drug targets. Distribution of domain families follows the power law in the network. Single promiscuous domains are contributing to the formation of hubs like Pkinease, Pkinease Tyr, and Ras. Hub nodes are positively correlated with the domain coverage and targeting them would disrupt functional properties of the proteins. EIF 4EBP, alpha Kinase, Sel1, ROKNT, and KH 1 are the domains identified as potential domain targets for the disruption of the signaling mechanism involved in cancer.

Discovery of new substrates of the elongation factor-2 kinase suggests a broader role in the cellular nutrient response.

Elongation Factor-2 Kinase (eEF2K) in an unusual mammalian enzyme that has one known substrate, elongation factor-2. It belongs to a class of kinases, called alpha kinases, that has little sequence identity to the >500 conventional protein kinases, but performs the same reaction and has similar catalytic residues. The phosphorylation of eEF2 blocks translation elongation, which is thought to be critical to regulating cellular energy usage. Here we report a system for discovering new substrates of alpha kinases and identify the first new substrates of eEF2K including AMPK and alpha4, and determine a sequence motif for the kinase that shows a requirement for threonine residues as the target of phosphorylation. These new substrates suggest that eEF2K has a more diverse role in regulating cellular energy usage that involves multiple pathways and regulatory feedback.

Structural Basis for the Recognition of Eukaryotic Elongation Factor 2 Kinase by Calmodulin.

Binding of Ca(2+)-loaded calmodulin (CaM) activates eukaryotic elongation factor 2 kinase (eEF-2K) that phosphorylates eEF-2, its only known cellular target, leading to a decrease in global protein synthesis. Here, using an eEF-2K-derived peptide (eEF-2KCBD) that encodes the region necessary for its CaM-mediated activation, we provide a structural basis for their interaction. The striking feature of this association is the absence of Ca(2+) from the CaM C-lobe sites, even under high Ca(2+) conditions. eEF-2KCBD engages CaM largely through the C lobe of the latter in an anti-parallel 1-5-8 hydrophobic mode reinforced by a pair of unique electrostatic contacts. Sparse interactions of eEF-2KCBD with the CaM N lobe results in persisting inter-lobe mobility. A conserved eEF-2K residue (W85) anchors it to CaM by inserting into a deep hydrophobic cavity within the CaM C lobe. Mutation of this residue (W85S) substantially weakens interactions between full-length eEF-2K and CaM in vitro and reduces eEF-2 phosphorylation in cells.

Eukaryotic elongation factor 2 kinase as a drug target in cancer, and in cardiovascular and neurodegenerative diseases.

Eukaryotic elongation factor 2 kinase (eEF2K) is an unusual protein kinase that regulates the elongation stage of protein synthesis by phosphorylating and inhibiting its only known substrate, eEF2. Elongation is a highly energy-consuming process, and eEF2K activity is tightly regulated by several signaling pathways. Regulating translation elongation can modulate the cellular energy demand and may also control the expression of specific proteins. Growing evidence links eEF2K to a range of human diseases, including cardiovascular conditions (atherosclerosis, via macrophage survival) and pulmonary arterial hypertension, as well as solid tumors, where eEF2K appears to play contrasting roles depending on tumor type and stage. eEF2K is also involved in neurological disorders and may be a valuable target in treating depression and certain neurodegenerative diseases. Because eEF2K is not required for mammalian development or cell viability, inhibiting its function may not elicit serious side effects, while the fact that it is an atypical kinase and quite distinct from the vast majority of other mammalian kinases suggests the possibility to develop it into compounds that inhibit eEF2K without affecting other important protein kinases. Further research is needed to explore these possibilities and there is an urgent need to identify and characterize potent and specific small-molecule inhibitors of eEF2K. In this article we review the recent evidence concerning the role of eEF2K in human diseases as well as the progress in developing small-molecule inhibitors of this enzyme.

Elongation Factor 2 Kinase Is Regulated by Proline Hydroxylation and Protects Cells during Hypoxia.

Protein synthesis, especially translation elongation, requires large amounts of energy, which is often generated by oxidative metabolism. Elongation is controlled by phosphorylation of eukaryotic elongation factor 2 (eEF2), which inhibits its activity and is catalyzed by eEF2 kinase (eEF2K), a calcium/calmodulin-dependent α-kinase. Hypoxia causes the activation of eEF2K and induces eEF2 phosphorylation independently of previously known inputs into eEF2K. Here, we show that eEF2K is subject to hydroxylation on proline-98. Proline hydroxylation is catalyzed by proline hydroxylases, oxygen-dependent enzymes which are inactivated during hypoxia. Pharmacological inhibition of proline hydroxylases also stimulates eEF2 phosphorylation. Pro98 lies in a universally conserved linker between the calmodulin-binding and catalytic domains of eEF2K. Its hydroxylation partially impairs the binding of calmodulin to eEF2K and markedly limits the calmodulin-stimulated activity of eEF2K. Neuronal cells depend on oxygen, and eEF2K helps to protect them from hypoxia. eEF2K is the first example of a protein directly involved in a major energy-consuming process to be regulated by proline hydroxylation. Since eEF2K is cytoprotective during hypoxia and other conditions of nutrient insufficiency, it may be a valuable target for therapy of poorly vascularized solid tumors.

Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival.

Acidification of the extracellular and/or intracellular environment is involved in many aspects of cell physiology and pathology. Eukaryotic elongation factor 2 kinase (eEF2K) is a Ca(2+)/calmodulin-dependent kinase that regulates translation elongation by phosphorylating and inhibiting eEF2. Here we show that extracellular acidosis elicits activation of eEF2K in vivo, leading to enhanced phosphorylation of eEF2. We identify five histidine residues in eEF2K that are crucial for the activation of eEF2K during acidosis. Three of them (H80, H87, and H94) are in its calmodulin-binding site, and their protonation appears to enhance the ability of calmodulin to activate eEF2K. The other two histidines (H227 and H230) lie in the catalytic domain of eEF2K. We also identify His108 in calmodulin as essential for activation of eEF2K. Acidification of cancer cell microenvironments is a hallmark of malignant solid tumors. Knocking down eEF2K in cancer cells attenuated the decrease in global protein synthesis when cells were cultured at acidic pH. Importantly, activation of eEF2K is linked to cancer cell survival under acidic conditions. Inhibition of eEF2K promotes cancer cell death under acidosis.

Eukaryotic elongation factor-2 kinase (eEF2K): a potential therapeutic target in cancer.

Eukaryotic elongation factor-2 kinase (eEF2K), encoded by the EEF2K gene, is well-known to be a Ca(2+)/calmodulin (CaM)-dependent kinase which can negatively modulate protein synthesis. It is highly conserved among eukaryotes from mammals to invertebrates, of which human and mouse may have 99 % overall amino acid identity. This kinase can phosphorylate eukaryotic elongation factor-2 (eEF2) or undergo the process of autophosphorylation at multiple sites to inhibit its function in translation elongation. Due to the fact that regulation of eEF2 by eEF2K is an evolutionarily conserved mechanism, eEF2K activity may confer tumor cell adaption to metabolic stress under acute nutrient depletion, and the high expressed level of eEF2K has been found in several types of malignancies. eEF2K may modulate the expression of some apoptotic proteins such as XIAP, c-FLIPL, Bcl-XL, PI3KCI and p70(S6K) to inhibit apoptotic process in cancer. On the other hand, it plays a regulatory role in autophagy involved in mTORC1, AMPK and Atg8, thereby promoting cancer cell survival. Additionally, eEF2K may play a crucial role in the crosstalk between apoptosis and autophagy in cancer. Collectively, these findings have led to the conclusions that eEF2K may contribute to carcinogenesis, and thus being utilized as a potential target for future cancer therapy.

The molecular mechanism of eukaryotic elongation factor 2 kinase activation.

Calmodulin (CaM)-dependent eukaryotic elongation factor 2 kinase (eEF-2K) impedes protein synthesis through phosphorylation of eukaryotic elongation factor 2 (eEF-2). It is subject to complex regulation by multiple upstream signaling pathways, through poorly described mechanisms. Precise integration of these signals is critical for eEF-2K to appropriately regulate protein translation rates. Here, an allosteric mechanism comprising two sequential conformations is described for eEF-2K activation. First, Ca(2+)/CaM binds eEF-2K with high affinity (Kd(CaM)(app) = 24 ± 5 nm) to enhance its ability to autophosphorylate Thr-348 in the regulatory loop (R-loop) by > 10(4)-fold (k(auto) = 2.6 ± 0.3 s(-1)). Subsequent binding of phospho-Thr-348 to a conserved basic pocket in the kinase domain potentially drives a conformational transition of the R-loop, which is essential for efficient substrate phosphorylation. Ca(2+)/CaM binding activates autophosphorylated eEF-2K by allosterically enhancing k(cat)(app) for peptide substrate phosphorylation by 10(3)-fold. Thr-348 autophosphorylation results in a 25-fold increase in the specificity constant (k(cat)(app)/K(m)(Pep-S) (app)), with equal contributions from k(cat)(app) and K(m)(Pep-S)(app), suggesting that peptide substrate binding is partly impeded in the unphosphorylated enzyme. In cells, Thr-348 autophosphorylation appears to control the catalytic output of active eEF-2K, contributing more than 5-fold to its ability to promote eEF-2 phosphorylation. Fundamentally, eEF-2K activation appears to be analogous to an amplifier, where output volume may be controlled by either toggling the power switch (switching on the kinase) or altering the volume control (modulating stability of the active R-loop conformation). Because upstream signaling events have the potential to modulate either allosteric step, this mechanism allows for exquisite control of eEF-2K output.

Eukaryotic elongation factor 2 kinase, an unusual enzyme with multiple roles.

Eukaryotic elongation factor 2 kinase (eEF2K) is a member of the small group of atypical 'α-kinases'. It phosphorylates and inhibits eukaryotic elongation factor 2, to slow down the elongation stage of protein synthesis, which normally consumes a great deal of energy and amino acids. The activity of eEF2K is normally dependent on calcium ions and calmodulin. eEF2K is also regulated by a plethora of other inputs, including inhibition by signalling downstream of anabolic signalling pathways such as the mammalian target of rapamycin complex 1. Recent data show that eEF2K helps to protect cancer cells against nutrient starvation and is also cytoprotective in other settings, including hypoxia. Growing evidence points to roles for eEF2K in neurological processes such as learning and memory and perhaps in depression.

A conserved loop in the catalytic domain of eukaryotic elongation factor 2 kinase plays a key role in its substrate specificity.

Eukaryotic elongation factor 2 kinase (eEF2K) is the best-characterized member of the α-kinase family. Within this group, only eEF2K and myosin heavy chain kinases (MHCKs) have known substrates. Here we have studied the roles of specific residues, selected on the basis of structural data for MHCK A and TRPM7, in the function of eEF2K. Our data provide the first information regarding the basis of the substrate specificity of α-kinases, in particular the roles of residues in the so-called N/D loop, which appears to occupy a position in the structure of α-kinases similar to that of the activation loop in other kinases. Several mutations in the EEF2K gene occur in tumors, one of which (Arg303Cys) is at a highly conserved residue in the N/D loop. This mutation greatly enhances eEF2K activity and may be cytoprotective. Our data support the concept that the major autophosphorylation site (Thr348 in eEF2K) docks into a binding pocket to help create the kinase-competent conformation. This is similar to the situation for MHCK A and is consistent with this being a common feature of α-kinases.

The role of the diphthamide-containing loop within eukaryotic elongation factor 2 in ADP-ribosylation by Pseudomonas aeruginosa exotoxin A.

eEF2 (eukaryotic elongation factor 2) contains a post-translationally modified histidine residue, known as diphthamide, which is the specific ADP-ribosylation target of diphtheria toxin, cholix toxin and Pseudomonas aeruginosa exotoxin A. Site-directed mutagenesis was conducted on residues within the diphthamide-containing loop (Leu693-Gly703) of eEF2 by replacement with alanine. The purified yeast eEF2 mutant proteins were then investigated to determine the role of this loop region in ADP-ribose acceptor activity of elongation factor 2 as catalysed by exotoxin A. A number of single alanine substitutions in the diphthamide-containing loop caused a significant reduction in the eEF2 ADP-ribose acceptor activities, including two strictly conserved residues, His694 and Asp696. Analysis by MS revealed that all of these mutant proteins lacked the 2'-modification on the His699 residue and that eEF2 is acetylated at Lys509. Furthermore, it was revealed that the imidazole ring of Diph699 (diphthamide at position 699) still functions as an ADP-ribose acceptor (albeit poorly), even without the diphthamide modification on the His699. Therefore, this diphthamide-containing loop plays an important role in the ADP-ribosylation of eEF2 catalysed by toxin and also for modification of His699 by the endogenous diphthamide modification machinery.