ABSTRACT
Protein kinases catalyze the phosphorylation of proteins most commonly on Ser, Thr, and Tyr residues and regulate many cellular events in eukaryotic cells, such as cell cycle progression, transcription, metabolism, and apoptosis. Protein kinases each have a conserved ATP-binding site and one or more substrate-binding site(s) that exhibit recognition features for different protein substrates. By bringing ATP and a substrate into proximity, each protein kinase can transfer the γ phosphate of the ATP molecule to a hydroxyl group of the target residue on the substrate. In such a way, signaling pathways downstream from the substrate can be regulated based on the phosphorylated versus dephosphorylated status of the substrate. Although there are a number of ways to assay the activity of protein kinases, most of them are technically cumbersome and/or are indirect or based on quenched reactions. This protocol describes an assay employing a fluorescent peptide substrate to detect phosphorylation by protein kinases in real time. The assay is based on the principle that the phosphorylation of the peptide substrate leads to an increase in the fluorescence emission intensity of an appended fluorophore. We extend the application of this assay to an example of how to assess time-dependent covalent inhibition of kinases as well. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Measuring protein kinase activity using fluorescent peptides Alternate Protocol: Measuring protein kinase activity using a fluorescence plate reader Support Protocol: Labeling peptides with sox fluorophore Basic Protocol 2: Measuring time-dependent ATP-competitive inhibition of protein kinases using fluorescent peptides.
Subject(s)
Peptides , Protein Kinases , Phosphorylation , Fluorescent Dyes , Adenosine TriphosphateABSTRACT
Hypoxic solid tumors induce the stabilization of hypoxia-inducible factor 1 alpha (HIF1α), which stimulates the expression of many glycolytic enzymes and hypoxia-responsive genes. A high rate of glycolysis supports the energetic and material needs for tumors to grow. Fructose-1,6-bisphosphate aldolase A (ALDOA) is an enzyme in the glycolytic pathway that promotes the expression of HIF1α. Therefore, inhibition of ALDOA activity represents a potential therapeutic approach for a range of cancers by blocking two critical cancer survival mechanisms. Here, we present a luminescence-based strategy to determine ALDOA activity. The assay platform was developed by integrating a previously established ALDOA activity assay with a commercial NAD/NADH detection kit, resulting in a significant (>12-fold) improvement in signal/background (S/B) compared with previous assay platforms. A screening campaign using a mixture-based compound library exhibited excellent statistical parameters of Z' (>0.8) and S/B (~20), confirming its robustness and readiness for high-throughput screening (HTS) application. This assay platform provides a cost-effective method for identifying ALDOA inhibitors using a large-scale HTS campaign.
Subject(s)
Fructose-Bisphosphate Aldolase/isolation & purification , High-Throughput Screening Assays , Cost-Benefit Analysis , Fructose/genetics , Fructose/metabolism , Fructose-Bisphosphate Aldolase/genetics , Glycolysis/genetics , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , LuminescenceABSTRACT
Iron is an essential requirement for the survival and virulence for bacteria. The bacterial ferrous iron transporter protein B (FeoB) functions as a major iron transporter in prokaryotes and has an N-terminal domain (NFeoB) with homology to eukaryotic G-proteins. Its GTPase activity is required for ferrous iron uptake, making it a potential target for antivirulence therapies. Here, two assay strategies relying on different spectroscopic readouts are described to monitor NFeoB GTPase activity. The first one is the colorimetric-based platform that utilizes a malachite green reagent to monitor phosphate production from GTP hydrolysis. The absorbance change directly relates to the GTPase activity of NFeoB. The assay was further improved by the addition of Tween-20 and miniaturized in a 384-well plate format with a 10 µL assay volume. The second format is a luminescence-based platform, measuring the GTP depletion by using a modified GTPase-Glo assay from Promega. In this platform, the luminescence signal correlates to the amount of GTP remaining, allowing for the direct calculation of GTP hydrolysis by NFeoB. The colorimetric platform was tested in a high-throughput manner against a custom-assembled library of a~2000 small molecules and was found to be simple, cost-effective, and robust. Additionally, the luminescence-based platform demonstrated its capability as an orthogonal assay to monitor GTPase activity, providing a valid and convenient method to filter false hits. These two assay platforms are proven to offset the limitations of each platform while enhancing overall quality and success rates.
Subject(s)
Cation Transport Proteins/isolation & purification , Escherichia coli Proteins/isolation & purification , Escherichia coli/enzymology , GTP Phosphohydrolases/isolation & purification , High-Throughput Screening Assays , Amino Acid Sequence , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Colorimetry/methods , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Guanosine Triphosphate/metabolism , Hydrolysis , Luminescence , Models, Molecular , Protein Domains/genetics , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacologyABSTRACT
Lysyl hydroxylase-2 (LH2) catalyzes the hydroxylation of telopeptidyl lysine residues on collagen, leading to the formation of stable collagen cross-links that connect collagen molecules and stabilize the extracellular matrix. High levels of LH2 have been reported in the formation and stabilization of hydroxylysine aldehyde-derived collagen cross-links (HLCCs), leading to fibrosis and cancer metastasis in certain tissues. Identification of small-molecule inhibitors targeting LH2 activity requires a robust and suitable assay system, which is currently lacking. Thus, despite being a promising target for these diseases, small-molecule inhibitors for LH2 have yet to be reported. Therefore, we developed a luminescence-based strategy to monitor LH activity and validated its ability to identify new inhibitors in a screen of approximately 65,000 compounds against LH2. Primary hits were confirmed using the same LH assay against mimiviral L230. This newly developed LH assay is robust, suitable for high-throughput screening, and able to identify potent specific inhibitors of LH2.
Subject(s)
Enzyme Inhibitors/pharmacology , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/antagonists & inhibitors , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/metabolism , Small Molecule Libraries/pharmacology , High-Throughput Screening Assays/methodsABSTRACT
The use of AlphaScreen® detection has allowed researchers to examine a wide variety of molecular interactions for use in high-throughput screening. However, the cost of Alpha reagents can often be prohibitory for extended screening campaigns or for young investigators with limited funding. To reduce assay costs, many labs have focused on miniaturization, while there have been limited efforts to scale down Alpha reagents. Thus, we describe the optimization of an AlphaScreen detection platform by systematically reducing the Alpha reagents down to 2.5 µg/ml beads, without compromising assay integrity. We demonstrate that reducing bead concentration reduces detection costs substantially while yielding robust statistics. We believe this simple new protocol will enhance the future utilization of AlphaScreen technology in high-throughput screening.
Subject(s)
High-Throughput Screening Assays/economics , Animals , Carbohydrate Metabolism , Cost-Benefit Analysis , DNA/metabolism , High-Throughput Screening Assays/methods , Humans , Indicators and Reagents/economics , Luminescent Measurements/economics , Luminescent Measurements/methods , Peptides/metabolism , Proteins/metabolism , RNA/metabolismABSTRACT
A high rate of glycolysis, which supplies energy and materials for anabolism, is observed in a wide range of tumor cells, making it a potential pathway to control cancer growth. ALDOA is a multifunctional enzyme in the glycolytic pathway and also promotes HIF-1α, which is of importance in hypoxic solid tumors. The current method for assaying ALDOA activity involves monitoring the consumption of NADH in vitro using absorbance or intrinsic fluorescence via a coupled enzymatic reaction. Here, we report the development of a homogeneous biochemical assay that can overcome limitations of current methods, in particular for the application of high-throughput drug screening. The assay utilizes the commercially available Elite NADH Assay Kit, which incorporates an enzymatic reaction to measure the level of NADH using a fluorescent probe. Assay optimization and validation are discussed. Its feasibility for high-throughput screening (HTS) was demonstrated by screening 65,000 compounds for the identification of small molecules that inhibit ALDOA. Through a validation screen and dose-response evaluation, four inhibitors with IC50 below 10 µM were identified. In conclusion, we demonstrate that a traditional ALDOA assay can be transformed readily into a fluorescence-based assay utilizing a commercial NADH detection kit that is rapid, sensitive, inexpensive, and HTS friendly.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Screening Assays, Antitumor/methods , Fructose-Bisphosphate Aldolase/antagonists & inhibitors , High-Throughput Screening Assays , Fluorescent Dyes , Glycolysis/drug effects , Humans , Reproducibility of Results , Small Molecule LibrariesABSTRACT
Serotonin (5-hydroxytryptamine, 5-HT) is a critical local regulator of epithelial homeostasis in the breast and exerts its actions through a number of receptors. Dysregulation of serotonin signaling is reported to contribute to breast cancer pathophysiology by enhancing cell proliferation and promoting resistance to apoptosis. Preliminary analyses indicated that the potent 5-HT1B/1D serotonin receptor agonist 5-nonyloxytryptamine (5-NT), a triptan-like molecule, induced cell death in breast cancer cell lines. Thus, we synthesized a series of novel alkyloxytryptamine analogues, several of which decreased the viability of various human cancer cell lines. Proteomic and metabolomic analyses showed that compounds 6 and 10 induced apoptosis and interfered with signaling pathways that regulate protein translation and survival, such as the Akt/mTOR pathway, in triple-negative breast cancer cells.
ABSTRACT
Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated using E coli- or insect-based expression systems is either insoluble or enzymatically unstable, and the LH2 enzymatic activity assays that are currently available measure radioactive CO2 released from 14C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems.
Subject(s)
Ketoglutaric Acids/chemistry , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/biosynthesis , Animals , CHO Cells , Carbon/chemistry , Carbon Dioxide/chemistry , Chromatography, Liquid , Collagen/chemistry , Cricetulus , Culture Media, Conditioned/chemistry , Glycosylation , Humans , Luciferases/chemistry , Lysine/chemistry , Recombinant Proteins/biosynthesis , Succinic Acid/chemistry , Tandem Mass SpectrometryABSTRACT
Apyrase is a calcium-activated enzyme that catalyzes the conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP), adenosine monophosphate (AMP), and Pi. It is currently used in studies involving cancer and platelet aggregation in humans, as well as herbicide resistance in plants. Inhibitors of apyrase are being investigated for their use to suppress tumors and combat herbicide resistance. Only a few inhibitors of apyrase have been reported, many of which were identified through automated screening using a 96-well plate format and colorimetric phosphate detection. However, these screens have had limitations, including large volumes, inconsistent reproducibility, high incidence of false hits, and lack of higher-throughput compatibility. A luciferin/luciferase-based detection system has been reported to examine potential inhibitors of apyrase; however, these reactions were performed in tubes with the assay completion in seconds, which necessitate the development of a high-throughput screening (HTS)-compatible format for screening. Therefore, a more cost-effective biochemical assay that improved the limitations of the previous assay formats using a commercially available luminescence-based detection system was developed. This new robust mix-and-read platform incorporates a low-volume luminescence-based protocol, formatted for use in 384-well microplates. This new format provides a simple and cost-effective method to screen for apyrase inhibitors and will facilitate larger HTS efforts to identify potent inhibitors of apyrase.
Subject(s)
Apyrase/metabolism , High-Throughput Screening Assays/methods , Luminescence , Drug Evaluation, Preclinical , Enzyme Inhibitors/analysis , Enzyme Inhibitors/pharmacology , Enzyme Stability , KineticsABSTRACT
The hypoxia-inducible transcription factor HIF1α drives expression of many glycolytic enzymes. Here, we show that hypoxic glycolysis, in turn, increases HIF1α transcriptional activity and stimulates tumor growth, revealing a novel feed-forward mechanism of glycolysis-HIF1α signaling. Negative regulation of HIF1α by AMPK1 is bypassed in hypoxic cells, due to ATP elevation by increased glycolysis, thereby preventing phosphorylation and inactivation of the HIF1α transcriptional coactivator p300. Notably, of the HIF1α-activated glycolytic enzymes we evaluated by gene silencing, aldolase A (ALDOA) blockade produced the most robust decrease in glycolysis, HIF-1 activity, and cancer cell proliferation. Furthermore, either RNAi-mediated silencing of ALDOA or systemic treatment with a specific small-molecule inhibitor of aldolase A was sufficient to increase overall survival in a xenograft model of metastatic breast cancer. In establishing a novel glycolysis-HIF-1α feed-forward mechanism in hypoxic tumor cells, our results also provide a preclinical rationale to develop aldolase A inhibitors as a generalized strategy to treat intractable hypoxic cancer cells found widely in most solid tumors. Cancer Res; 76(14); 4259-69. ©2016 AACR.
Subject(s)
Fructose-Bisphosphate Aldolase/antagonists & inhibitors , Glycolysis , Hypoxia-Inducible Factor 1, alpha Subunit/physiology , Neoplasms/drug therapy , Signal Transduction/physiology , AMP-Activated Protein Kinases/physiology , Animals , Cell Hypoxia , Cell Line, Tumor , E1A-Associated p300 Protein/physiology , Humans , Mice , Neoplasms/metabolism , Xenograft Model Antitumor AssaysABSTRACT
We have recently demonstrated that the undifferentiated PSA-/lo prostate cancer (PCa) cell population harbors self-renewing long-term tumor-propagating cells that are refractory to castration, thus representing a therapeutic target. Our goals here are, by using the same lineage-tracing reporter system, to track the dynamic changes of PSA-/lo and PSA+ cells upon castration in vitro, investigate the molecular changes accompanying persistent castration, and develop large numbers of PSA-/lo PCa cells for drug screening. To these ends, we treated LNCaP cells infected with the PSAP-GFP reporter with three regimens of castration, i.e., CDSS, CDSS plus bicalutamide, and MDV3100 continuously for up to ~21 months. We observed that in the first ~7 months, castration led to time-dependent increases in PSA-/lo cells, loss of AR and PSA expression, increased expression of cancer stem cell markers, and many other molecular changes. Meanwhile, castrated LNCaP cells became resistant to high concentrations of MDV3100, chemotherapeutic drugs, and other agents. However, targeted and medium-throughput library screening identified several kinase (e.g., IGF-1R, AKT, PI3K/mTOR, Syk, GSK3) inhibitors as well as the BCL2 inhibitor that could effectively sensitize the LNCaP-CRPC cells to killing. Of interest, LNCaP cells castrated for >7 months showed evidence of cyclic changes in AR and the mTOR/AKT signaling pathways potentially involving epigenetic mechanisms. These observations indicate that castration elicits numerous molecular changes and leads to enrichment of PSA-/lo PCa cells. The ability to generate large numbers of PSA-/lo PCa cells should allow future high-throughput screening to identify novel therapeutics that specifically target this population.
Subject(s)
Antineoplastic Agents/pharmacology , Biomarkers, Tumor/genetics , Prostate-Specific Antigen/metabolism , Prostatic Neoplasms, Castration-Resistant/drug therapy , Protein Kinase Inhibitors/pharmacology , Apoptosis/drug effects , Biomarkers, Tumor/metabolism , Cell Cycle/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Humans , Longitudinal Studies , Male , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/metabolism , Prostatic Neoplasms, Castration-Resistant/pathology , Tumor Cells, CulturedABSTRACT
Protein kinases have emerged as an important class of therapeutic targets, as they are known to be involved in pathological pathways linked to numerous human disorders. Major efforts to discover kinase inhibitors in both academia and pharmaceutical companies have centered on the development of robust assays and cost-effective approaches to isolate them. Drug discovery procedures often start with hit identification for lead development, by screening a library of chemicals using an appropriate assay in a high-throughput manner. Considering limitations unique to each assay technique and screening capability, intelligent integration of various assay schemes and level of throughput, in addition to the choice of chemical libraries, is the key to success of this initial step. Here, we describe the purification of the protein kinase, eEF-2K, and the utilization of three biochemical assays in the course of identifying small molecules that block its enzymatic reaction.
Subject(s)
Antineoplastic Agents/pharmacology , Elongation Factor 2 Kinase/antagonists & inhibitors , Fluorometry/methods , High-Throughput Screening Assays/methods , Luminescent Measurements/methods , Neoplasm Proteins/antagonists & inhibitors , Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Radiometry/methods , Antineoplastic Agents/isolation & purification , Elongation Factor 2 Kinase/biosynthesis , Elongation Factor 2 Kinase/isolation & purification , Gamma Rays , Humans , Indicators and Reagents , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/isolation & purification , Neoplasms/enzymology , Phosphorus Radioisotopes/analysis , Protein Kinase Inhibitors/isolation & purification , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purification , Scintillation CountingABSTRACT
eEF-2K is a potential target for treating cancer. However, potent specific inhibitors for this enzyme are lacking. Previously, we identified 2,6-diamino-4-(2-fluorophenyl)-4H-thiopyran-3,5-dicarbonitrile (DFTD) as an inhibitor of eEF-2K. Here we describe its mechanism of action against eEF-2K, on the basis of kinetic, mutational, and docking studies, and use chemoinformatic approaches to identify a similar class of carbonitrile-containing compounds that exhibit the same mechanism of action. We show that DFTD behaves as a reversible covalent inhibitor of eEF-2K with a two-step mechanism of inhibition: a fast initial binding step, followed by a slower reversible inactivation step. Molecular docking suggests that a nitrile group of DFTD binds within 4.5 Å of the active site Cys146 to form a reversible thioimidate adduct. Because Cys146 is not conserved amongst other related kinases, targeting this residue holds promise for the development of selective covalent inhibitors of eEF-2K.
Subject(s)
Elongation Factor 2 Kinase/antagonists & inhibitors , Nitriles/chemistry , Amino Acid Sequence , Binding Sites , Catalytic Domain , Elongation Factor 2 Kinase/genetics , Elongation Factor 2 Kinase/metabolism , Humans , Kinetics , Molecular Docking Simulation , Molecular Sequence Data , Mutagenesis, Site-Directed , Nitriles/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Sequence AlignmentABSTRACT
PERK, as one of the principle unfolded protein response signal transducers, is believed to be associated with many human diseases, such as cancer and type-II diabetes. There has been increasing effort to discover potent PERK inhibitors due to its potential therapeutic interest. In this study, a computer-based virtual screening approach is employed to discover novel PERK inhibitors, followed by experimental validation. Using a focused library, we show that a consensus approach, combining pharmacophore modeling and docking, can be more cost-effective than using either approach alone. It is also demonstrated that the conformational flexibility near the active site is an important consideration in structure-based docking and can be addressed by using molecular dynamics. The consensus approach has further been applied to screen the ZINC lead-like database, resulting in the identification of 10 active compounds, two of which show IC50 values that are less than 10 µM in a dose-response assay.
Subject(s)
Protein Kinase Inhibitors/pharmacology , eIF-2 Kinase/antagonists & inhibitors , Animals , Catalytic Domain , Databases, Pharmaceutical , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , High-Throughput Screening Assays , Inhibitory Concentration 50 , Mice , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Kinase Inhibitors/metabolism , Reproducibility of Results , User-Computer Interface , eIF-2 Kinase/chemistry , eIF-2 Kinase/metabolismABSTRACT
eEF-2 kinase is a potential therapeutic target for breast cancer, gliomas, and depression. No potent inhibitors of eEF-2K have been reported, and thus development of high-throughput assay systems may expedite the process. Two high-throughput assays are described for eEF-2K using recombinant, tag-free enzyme purified from bacteria. The first is a fluorescence-based assay that uses the phosphorylation of a Sox-based peptide substrate by eEF-2K, which results in a 5-fold increase in fluorescence emission, allowing for continuous monitoring of the kinase activity. The second is a luminescence-based assay that produces a luminescence signal, which correlates with the amount of adenosine triphosphate remaining in the kinase reaction. Both assays have been optimized and miniaturized for a 384-well plate format and validated in screens. In conclusion, we demonstrated that a traditional radiolabeled assay can be readily transferred to universal spectroscopic assays that are robust and will facilitate high-throughput screening of larger size libraries for the identification of small-molecule inhibitors and significantly contribute to the development of therapies for targeting eEF2K.
Subject(s)
Elongation Factor 2 Kinase/metabolism , High-Throughput Screening Assays , Antineoplastic Agents/pharmacology , Drug Screening Assays, Antitumor , Elongation Factor 2 Kinase/antagonists & inhibitors , Luminescent Measurements/methods , Peptides/chemistry , Peptides/metabolism , Protein Kinase Inhibitors/pharmacology , Reproducibility of Results , Substrate SpecificityABSTRACT
The JNK-JIP1 interaction represents an attractive target for the selective inhibition of JNK-mediated signaling. We report a virtual screening (VS) workflow, based on a combination of three-dimensional shape and electrostatic similarity to discover novel scaffolds for the development of non-ATP competitive inhibitors of JNK targeting the JNK-JIP interaction. Of 352 (0.13%) compounds selected from the NCI diversity set more than 22% registered as hits in a biochemical kinase assay. Several compounds discovered to inhibit JNK activity under standard kinase assay conditions also impeded JNK activity in HEK293 cells. These studies led to the discovery that the lignan (-)-zuonin A inhibits JNK-protein interactions with a selectivity of 100-fold over ERK2 and p38 MAPKα. These results demonstrate the utility of a virtual screening protocol to identify novel scaffolds for highly selective, cell-permeable inhibitors of JNK-protein interactions.
ABSTRACT
Eukaryotic elongation factor 2 kinase (eEF-2K) is an atypical protein kinase regulated by Ca(2+) 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 coexpressed with λ-phosphatase and purified from bacteria in a three-step protocol using a CaM affinity column. Purified eEF-2K was induced to autophosphorylate by incubation with Ca(2+)/CaM in the presence of MgATP. Analyzing tryptic or chymotryptic peptides by mass spectrometry monitored the autophosphorylation over 0-180 min. The following five major autophosphorylation sites were identified: Thr-348, Thr-353, Ser-445, Ser-474, and Ser-500. In the presence of Ca(2+)/CaM, robust phosphorylation of Thr-348 occurs within seconds of addition of 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 the Ca(2+)-independent activity of eEF-2K. Mutation of Ser-500 to Asp, but not Ala, renders eEF-2K Ca(2+)-independent. Surprisingly, this Ca(2+)-independent activity requires the presence of CaM.
Subject(s)
Calcium/metabolism , Calmodulin/metabolism , Elongation Factor 2 Kinase/metabolism , Serine/genetics , Threonine/genetics , Amino Acid Sequence , Binding Sites , Elongation Factor 2 Kinase/genetics , Humans , Mass Spectrometry , Molecular Sequence Data , Phosphorylation , Threonine/metabolismABSTRACT
Evidence that elongation factor 2 kinase (eEF-2K) has potential as a target for anticancer therapy and possibly for the treatment of depression is emerging. Here the steady-state kinetic mechanism of eEF-2K is presented using a peptide substrate and is shown to conform to an ordered sequential mechanism with ATP binding first. Substrate inhibition by the peptide was observed and revealed to be competitive with ATP, explaining the observed ordered mechanism. Several small molecules are reported to inhibit eEF-2K activity with the most notable being the histidine kinase inhibitor NH125, which has been used in a number of studies to characterize eEF-2K activity in cells. While NH125 was previously reported to inhibit eEF-2K in vitro with an IC(50) of 60 nM, its mechanism of action was not established. Using the same kinetic assay, the ability of an authentic sample of NH125 to inhibit eEF-2K was assessed over a range of substrate and inhibitor concentrations. A typical dose-response curve for the inhibition of eEF-2K by NH125 is best fit to an IC(50) of 18 ± 0.25 µM and a Hill coefficient of 3.7 ± 0.14, suggesting that NH125 is a weak inhibitor of eEF-2K under the experimental conditions of a standard in vitro kinase assay. To test the possibility that NH125 is a potent inhibitor of eEF2 phosphorylation, we assessed its ability to inhibit the phosphorylation of eEF2. Under standard kinase assay conditions, NH125 exhibits a similar weak ability to inhibit the phosphorylation of eEF2 by eEF-2K. Notably, the activity of NH125 is severely abrogated by the addition of 0.1% Triton to the kinase assay through a process that can be reversed upon dilution. These studies suggest that NH125 is a nonspecific colloidal aggregator in vitro, a notion further supported by the observation that NH125 inhibits other protein kinases, such as ERK2 and TRPM7 in a manner similar to that of eEF-2K. As NH125 is reported to inhibit eEF-2K in a cellular environment, its ability to inhibit eEF2 phosphorylation was assessed in MDA-MB-231 breast cancer, A549 lung cancer, and HEK-293T cell lines using a Western blot approach. No sign of a decrease in the level of eEF2 phosphorylation was observed up to 12 h following addition of NH125 to the media. Furthermore, contrary to the previously reported literatures, NH125 induced the phosphorylation of eEF-2.
Subject(s)
Elongation Factor 2 Kinase/antagonists & inhibitors , Imidazoles/pharmacology , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Artifacts , Cell Line , Dose-Response Relationship, Drug , Elongation Factor 2 Kinase/genetics , Elongation Factor 2 Kinase/metabolism , HEK293 Cells , Humans , Imidazoles/administration & dosage , In Vitro Techniques , Kinetics , Molecular Sequence Data , Peptides/chemistry , Peptides/metabolism , Phosphorylation , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/pharmacology , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
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-His(6)-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 â¼85 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.
Subject(s)
Cloning, Molecular/methods , Elongation Factor 2 Kinase/metabolism , Peptide Elongation Factor 2/metabolism , Plasmids/genetics , Protein Biosynthesis/genetics , Recombinant Proteins/metabolism , Amino Acid Sequence , Calcium/metabolism , Calmodulin/genetics , Calmodulin/metabolism , Chromatography, Affinity , Chromatography, Gel , Chromatography, Ion Exchange , Elongation Factor 2 Kinase/genetics , Elongation Factor 2 Kinase/isolation & purification , Endopeptidases/metabolism , Escherichia coli , Histidine/metabolism , Humans , Kinetics , Molecular Sequence Data , Oligopeptides/metabolism , Phosphorylation , Plasmids/chemistry , Plasmids/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Thioredoxins/metabolism , Transformation, BacterialABSTRACT
The mitogen-activated protein (MAP) kinase ERK2 contains recruitment sites that engage canonical and noncanonical motifs found in a variety of upstream kinases, regulating phosphatases and downstream targets. Interactions involving two of these sites, the D-recruitment site (DRS) and the F-recruitment site (FRS), have been shown to play a key role in signal transduction by ERK/MAP kinases. The dynamic nature of these recruitment events makes NMR uniquely suited to provide significant insight into these interactions. While NMR studies of kinases in general have been greatly hindered by their large size and complex dynamic behavior leading to the suboptimal performance of standard methodologies, we have overcome these difficulties for inactive full-length ERK2 and obtained an acceptable level of backbone resonance assignments. This allowed a detailed investigation of the structural perturbations that accompany interactions involving both canonical and noncanonical recruitment events. No crystallographic information exists for the latter. We found that the chemical shift perturbations in inactive ERK2, indicative of structural changes in the presence of canonical and noncanonical motifs, are not restricted to the recruitment sites but also involve the linker that connects the N- and C-lobes and, in most cases, a gatekeeper residue that is thought to exert allosteric control over catalytic activity. We also found that, even though the canonical motifs interact with the DRS utilizing both charge-charge and hydrophobic interactions, the noncanonical interactions primarily involve the latter. These results demonstrate the feasibility of solution NMR techniques for a comprehensive analysis of docking interactions in a full-length ERK/MAP kinase.