Analysis of the domain structure of elongation factor-2 kinase by mutagenesis.

A number of elongation factor-2 kinase (eEF-2K) mutants were constructed to investigate features of this kinase that may be important in its activity. Typical protein kinases possess a highly conserved lysine residue in subdomain II which follows the GXGXXG motif of subdomain I. Mutation of two lysine residues, K340 and K346, which follow the GXGXXG motif in eEF-2K had no effect on activity, showing that such a lysine residue is not important in eEF-2K activity. Mutation of a conserved pair of cysteine residues C-terminal to the GXGXXG sequence, however, completely inactivated eEF-2K. The eEF-2K CaM binding domain was localised to residues 77-99 which reside N-terminal to the catalytic domain. Tryptophan 84 is an important residue within this domain as mutation of this residue completely abolishes CaM binding and eEF-2K activity. Removal of approximately 130 residues from the C-terminus of eEF-2K completely abolished autokinase activity; however, removal of only 19 residues inhibited eEF-2 kinase activity but not autokinase activity, suggesting that a short region at the C-terminal end may be important in interacting with eEF-2. Likewise, removal of between 75 and 100 residues from the N-terminal end completely abolished eEF-2K activity.     

Endogenous ADP-ribosylation of elongation factor-2 by interleukin-1��

Eukaryotic elongation factor-2 (eEF-2) catalyses the motion of the growing peptide chain relative to the mRNA at the ribosomes during protein synthesis. This highly conserved G-protein is the specific target of two lethal bacterial toxins, Pseudomonas aeruginosa exotoxin A and diphtheria toxin. These toxins exert their detrimental action by ADP-ribosylating a biologically unique posttranslationally modified histidine residue (diphthamide(715)) within eEF-2, thus inactivating the enzyme. Diphthamide(715) is also the target of endogenous (mono) ADP-ribosyl transferase activity. In this article, we report the first known activator of endogenous ADP-ribosylation of eEF-2, interleukin-1β (IL-1β). Thereby, systemic inflammatory processes may link to protein synthesis regulation.     

eEF-2 kinase,another meddler in the “Yin and Yang” of Akt–mediated cell fate?

Eukaryotic elongation factor-2 (eEF-2) kinase, also known as calmodulin-dependent protein kinase III, is a unique calcium/calmodulin-dependent enzyme. eEF-2 kinase can act as a negative regulator of protein synthesis and a positive regulator of autophagy under environmental or metabolic stresses. Akt, a key downstream effector of the PI3K signaling pathway that regulates cell survival and proliferation, is an attractive therapeutic target for anticancer treatment. Akt inhibition leads to activation of both apoptosis, type I programmed cell death and autophagy, a cellular degradation process via lysosomal machinery (also termed type II programmed cell death). However, the underlying mechanisms that dictate functional relationship between autophagy and apoptosis in response to Akt inhibition remain to be delineated. Our recent study demonstrated that inhibition of eEF-2 kinase can suppress autophagy but promote apoptosis in tumor cells subjected to Akt inhibition, indicating a role of eEF-2 kinase as a controller in the crosstalk between autophagy and apoptosis. Furthermore, inhibition of eEF-2 kinase can reinforce the efficacy of a novel Akt inhibitor, MK-2206, against human glioma. These findings may help optimize the use of Akt inhibitors in the treatment of cancer and other diseases.     

eEF-2 kinase: Another meddler in the “yin and yang” of Akt-mediated cell fate?

Eukaryotic elongation factor-2 (eEF-2) kinase, also known as calmodulin-dependent protein kinase III, is a unique calcium/calmodulin-dependent enzyme. eEF-2 kinase can act as a negative regulator of protein synthesis and a positive regulator of autophagy under environmental or metabolic stresses. Akt, a key downstream effector of the PI3K signaling pathway that regulates cell survival and proliferation, is an attractive therapeutic target for anticancer treatment. Akt inhibition leads to activation of both apoptosis, type I programmed cell death and autophagy, a cellular degradation process via lysosomal machinery (also termed type II programmed cell death). However, the underlying mechanisms that dictate functional relationship between autophagy and apoptosis in response to Akt inhibition remain to be delineated. Our recent study demonstrated that inhibition of eEF-2 kinase can suppress autophagy but promote apoptosis in tumor cells subjected to Akt inhibition, indicating a role of eEF-2 kinase as a controller in the crosstalk between autophagy and apoptosis. Furthermore, inhibition of eEF-2 kinase can reinforce the efficacy of a novel Akt inhibitor, MK-2206, against human glioma. These findings may help optimize the use of Akt inhibitors in the treatment of cancer and other diseases.     

Phosphorylation of elongation factor 2: a key mechanism regulating gene expression in vertebrates

Elongation factor 2 (eEF-2) is a 100-kD protein that catalyzes the ribosomal translocation reaction, resulting in the movement of ribosomes along mRNA. eEF-2 is the target for a very specific Ca2+/calmodulin-dependent eEF-2 kinase. Phosphorylation of eEF-2 makes it inactive in translation, which suggests that protein synthesis can be regulated by Ca2+ through eEF-2 phosphorylation. Recent data demonstrate that eEF-2 phosphorylation can be involved in cell-cycle regulation and other processes where changes of intracellular Ca2+ concentration induce a new physiological state of a cell. The main role of eEF-2 phosphorylation in these processes is temporary inhibition of overall translation in response to transient elevation of the Ca2+ concentrations in the cytoplasm. Temporary inhibition of translation may trigger the transition of a cell from one physiologic state into another because of the disappearance of short-lived repressors and thus the activation of expression of new genes.     

A pharmacoproteomic approach implicates eukaryotic elongation factor 2 kinase in ER stress-induced cell death

Apoptosis triggered by endoplasmic reticulum (ER) stress has been implicated in many diseases but its cellular regulation remains poorly understood. Previously, we identified salubrinal (sal), a small molecule that protects cells from ER stress-induced apoptosis by selectively activating a subset of endogenous ER stress-signaling events. Here, we use sal as a probe in a proteomic approach to discover new information about the endogenous cellular response to ER stress. We show that sal induces phosphorylation of the translation elongation factor eukaryotic translation elongation factor 2 (eEF-2), an event that depends on eEF-2 kinase (eEF-2K). ER stress itself also induces eEF-2K-dependent eEF-2 phosphorylation, and this pathway promotes translational arrest and cell death in this context, identifying eEF-2K as a hitherto unknown regulator of ER stress-induced apoptosis. Finally, we use both sal and ER stress models to show that eEF-2 phosphorylation can be activated by at least two signaling mechanisms. Our work identifies eEF-2K as a new component of the ER stress response and underlines the utility of novel small molecules in discovering new cell biology.     

Elongation factor-2 kinase: its role in protein synthesis and autophagy

Elongation factor-2 kinase (eEF-2 kinase; Ca(2+)/calmodulin-dependent kinase III) controls the rate of peptide chain elongation. The activity of eEF-2 kinase is increased in many malignancies, yet its precise function in carcinogenesis remains unknown. Autophagy, a well-defined survival pathway in yeast, may also play an important role in oncogenesis. Furthermore, the autophagic response to nutrient deprivation is regulated by the mammalian target of rapamycin (mTOR). eEF-2 kinase lies downstream of mTOR and is regulated by several kinases in this pathway. Therefore, we studied the role of eEF-2 kinase in autophagy. Knockdown of eEF-2 kinase by RNA interference inhibited autophagy in several cell types as measured by light chain 3 (LC3)-II formation, acidic vesicular organelle staining, and electron microscopy. In contrast, overexpression of eEF-2 kinase increased autophagy. Furthermore, inhibition of autophagy markedly decreased the viability of glioblastoma cells grown under conditions of nutrient depletion. These results suggest that eEF-2 kinase plays a regulatory role in the autophagic process in tumor cells and may promote cancer cell survival under conditions of nutrient deprivation. Therefore, eEF-2 kinase activation may be a part of a survival mechanism in glioblastoma, and targeting this kinase may represent a novel approach to cancer treatment.     

Geraniol inhibits prostate cancer growth by targeting cell cycle and apoptosis pathways

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.     

Antibacterial mechanism of soybean isoflavone on Staphylococcus aureus

Effects of different flavonoids on various bacterial strains have been extensively reported; however, the mechanism(s) of their action on bacterial cells remain largely elusive. In this study, the antibacterial mechanism of soybean isoflavone (SI) on Staphylococcus aureus is systematically investigated using 4′6-diamidino-2-phenylindole (DAPI) staining, pBR322DNA decatenation experiment mediated by topoisomerase and agarose gel electrophoresis for direct decatenation. The results of fluorescence microscopy and fluorescence spectrophotometer indicated that DAPI was integrated in Staphylococcus aureus. Additionally, the quantity of both DNA and RNA reduced to 66.47 and 60.18%, respectively, after treated with SI for 28 h. Effects of SI on topoisomerase I and II were also investigated. SI completely inhibited the pBR322DNA unwinding mediated by topoisomerase I and topoisomerase II at the concentration of 6.4 mg/ml and could denature the plasmid DNA at the concentration of 12.8 mg/ml. These results indicate that topoisomerase I and II are the most important targets by SI to restrain bacterial cell division.     

Elongation Factor-2 Kinase: Its Role in Protein Synthesis and Autophagy

Elongation factor-2 kinase (eEF-2 kinase; Ca²⁺/calmodulin-dependent kinase III) controls the rate of peptide chain elongation. The activity of eEF-2 kinase is increased in many malignancies, yet its precise function in carcinogenesis remains unknown. Autophagy, a well-defined survival pathway in yeast, may also play an important role in oncogenesis. Furthermore, the autophagic response to nutrient deprivation is regulated by the mammalian target of rapamycin (mTOR). eEF-2 kinase lies downstream of mTOR and is regulated by several kinases in this pathway. Therefore, we studied the role of eEF-2 kinase in autophagy. Knockdown of eEF-2 kinase by RNA interference inhibited autophagy in several cell types as measured by light chain 3 (LC3)-II formation, acidic vesicular organelle staining, and electron microscopy. In contrast, overexpression of eEF-2 kinase increased autophagy. Furthermore, inhibition of autophagy markedly decreased the viability of glioblastoma cells grown under conditions of nutrient depletion, which increased eEF-2 kinase activity and decreased the activity of S6 kinase, suggesting an involvement of mTOR pathway in the eEF-2 kinase-mediated regulation of autophagy. These results suggest that eEF-2 kinase plays a regulatory role in the autophagic process in tumor cells and may promote cancer cell survival under conditions of nutrient deprivation. Therefore, eEF-2 kinase activation may be a part of a survival mechanism in glioblastoma, and targeting this kinase may represent a novel approach to cancer treatment.

Addendum to:

Elongation Factor-2 Kinase Regulates Autophagy in Human Glioblastoma Cells

H. Wu, J.-M. Yang, S. Jin, H. Zhang and W.N. Hait

Cancer Res 2006; 66:3015-23     

Adenylyl Cyclases 1 and 8 Initiate a Presynaptic Homeostatic Response to Ethanol Treatment

Background

Although ethanol exerts widespread action in the brain, only recently has progress been made in understanding the specific events occurring at the synapse during ethanol exposure. Mice deficient in the calcium-stimulated adenylyl cyclases, AC1 and AC8 (DKO), demonstrate increased sedation duration and impaired phosphorylation by protein kinase A (PKA) following acute ethanol treatment. While not direct targets for ethanol, we hypothesize that these cyclases initiate a homeostatic presynaptic response by PKA to reactivate neurons from ethanol-mediated inhibition.

Methodology/Principal Findings

Here, we have used phosphoproteomic techniques and identified several presynaptic proteins that are phosphorylated in the brains of wild type mice (WT) after ethanol exposure, including synapsin, a known PKA target. Phosphorylation of synapsins I and II, as well as phosphorylation of non-PKA targets, such as, eukaryotic elongation factor-2 (eEF-2) and dynamin is significantly impaired in the brains of DKO mice. This deficit is primarily driven by AC1, as AC1-deficient, but not AC8-deficient mice also demonstrate significant reductions in phosphorylation of synapsin and eEF-2 in cortical and hippocampal tissues. DKO mice have a reduced pool of functional recycling vesicles and fewer active terminals as measured by FM1-43 uptake compared to WT controls, which may be a contributing factor to the impaired presynaptic response to ethanol treatment.

Conclusions/Significance

These data demonstrate that calcium-stimulated AC-dependent PKA activation in the presynaptic terminal, primarily driven by AC1, is a critical event in the reactivation of neurons following ethanol-induced activity blockade.     

Investigating the kinetic mechanism of inhibition of elongation factor 2 kinase by NH125: evidence of a common in vitro artifact

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.     

Targeting elongation factor-2 kinase (eEF-2K) induces apoptosis in human pancreatic cancer cells

Pancreatic cancer (PaCa) is one of the most aggressive, apoptosis-resistant and currently incurable cancers with a poor survival rate. Eukaryotic elongation factor-2 kinase (eEF-2K) is an atypical kinase, whose role in PaCa survival is not yet known. Here, we show that eEF-2K is overexpressed in PaCa cells and its down-regulation induces apoptotic cell death. Rottlerin (ROT), a polyphenolic compound initially identified as a PKC-δ inhibitor, induces apoptosis and autophagy in a variety of cancer cells including PaCa cells. We demonstrated that ROT induces intrinsic apoptosis, with dissipation of mitochondrial membrane potential (ΔΨm), and stimulates extrinsic apoptosis with concomitant induction of TNF-related apoptosis inducing ligand (TRAIL) receptors, DR4 and DR5, with caspase-8 activation, in PANC-1 and MIAPaCa-2 cells. Notably, while none of these effects were dependent on PKC-δ inhibition, ROT down-regulates eEF-2K at mRNA level, and induce eEF-2K protein degradation through ubiquitin–proteasome pathway. Down-regulation of eEF-2K recapitulates the events observed after ROT treatment, while its over-expression suppressed the ROT-induced apoptosis. Furthermore, eEF-2K regulates the expression of tissue transglutaminase (TG2), an enzyme previously implicated in proliferation, drug resistance and survival of cancer cells. Inhibition of eEF-2K/TG2 axis leads to caspase-independent apoptosis which is associated with induction of apoptosis-inducing factor (AIF). Collectively, these results indicate, for the first time, that the down-regulation of eEF-2K leads to induction of intrinsic, extrinsic as well as AIF-dependent apoptosis in PaCa cells, suggesting that eEF-2K may represent an attractive therapeutic target for the future anticancer agents in PaCa.     

An enzyme-linked immunosorbent assay for the association of the catalytic domain of diphthamide-specific ribosyltransferases to eukaryotic elongation factor-2.

The X-ray structure of the catalytic domain of Pseudomonas aeruginosa exotoxin A (PE24) has recently been solved to high resolution, facilitating studies on the interaction of PE24 with its target substrate, eukaryotic elongation factor-2 (eEF-2). PE24 exhibits mono-ADP-ribosyltransferase (ADPRT) activity in a mechanism that has been proposed to feature a nucleophilic attack by the diphthamide residue (nucleophile) of eEF-2 on the C-1 of the nicotinamide ribose of NAD(+). The interaction of wheat germ eEF-2 with PE24 was studied by employing an enzyme-linked immunosorbent assay (ELISA), devised to assess protein-protein interactions. It was shown that the proteins associate with each other only in the presence of the enzyme's nucleotide substrate, NAD(+), and exhibit a dose-dependent association that is saturable. The apparent dissociation constant (K(d)) for this protein-protein interaction is 50 nM and is salt-dependent. The association is maximal at low ionic strength and is progressively weaker at higher salt concentrations, which corroborates previous findings on the salt dependence of ADPRT activity for this toxin. This finding suggests that the sensitivity of ADPRT activity toward high salt resides in the interaction between the catalytic domain of the toxin and eEF-2. A major product of the glycohydrolase activity of PE24, nicotinamide, inhibits the binding between PE24 and eEF-2 with an ID(50) of 20 microM. The naturally occurring, noncatalytic mutant of PE24, H426Y, did not bind eEF-2 in the ELISA, verifying that His 426 is located at the center of the eEF-2 binding site within ETA.     

Downregulation of the translation elongation factor 2 kinase in Xenopus laevis oocytes at the final stages of oogenesis

Phosphorylation of translation elongation factor 2(eEF-2) by a specific Ca2+/calmodulin-dependent eEF-2 kinase plays an important role in the regulation of protein synthesis in mammalian cells. We show here that an eEF-2 kinase similar to the mammalian enzyme is present in tissues of the amphibian Xenopus laevis. We investigated changes in the activity of eEF-2 kinase in extracts of Xenopus oocytes at different stages of oogenesis. The eEF-2 kinase activity was constant from stage I to stage IV of oogenesis, but dramatically decreased after stage IV. Extracts of fully grown stage-VI oocytes showed no eEF-2 kinase activity. However, when extracts were analyzed by two-dimensional gel electrophoresis, eEF-2 was found to be present mostly, if not exclusively, in the dephosphorylated form throughout oogenesis. It is suggested that eEF-2 kinase disappears late in oogenesis to make protein synthesis insensitive to changes in intracellular Ca2+ concentration. This may be important for the induction of meiotic maturation.     

Mechanisms for increased levels of phosphorylation of elongation factor-2 during hibernation in ground squirrels

Previously, eEF-2 phosphorylation has been identified as a reversible mechanism involved in the inhibition of the elongation phase of translation. In this study, an increased level of phosphorylation of eukaryotic elongation factor-2 (eEF-2) was observed in the brains and livers of hibernating ground squirrels. In brain and liver from hibernators, eEF-2 kinase activity was increased relative to that of active animals. The activity of protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates eEF-2, was also decreased in brain and liver from hibernators. This was associated with an increase in the level of inhibitor 2 of PP2A (I(2)(PP2A)), although there was an increase in the level of the catalytic subunit of PP2A (PP2A/C) in hibernating brains and livers. These results indicate that eEF-2 phosphorylation represents a specific and previously uncharacterized mechanism for inhibition of the elongation phase of protein synthesis during hibernation. Increased levels of eEF-2 phosphorylation in hibernators appear to be a component of the regulated shutdown of cellular functions that permits hibernating animals to tolerate severe reductions in cerebral blood flow and oxygen delivery capacity.     

Molecular dissection of the silkworm ribosomal stalk complex: the role of multiple copies of the stalk proteins

In animal ribosomes, two stalk proteins P1 and P2 form a heterodimer, and the two dimers, with the anchor protein P0, constitute a pentameric complex crucial for recruitment of translational GTPase factors to the ribosome. To investigate the functional contribution of each copy of the stalk proteins, we constructed P0 mutants, in which one of the two C-terminal helices, namely helix I (N-terminal side) or helix II (C-terminal side) were unable to bind the P1–P2 dimer. We also constructed ‘one-C-terminal domain (CTD) stalk dimers’, P1–P2_ΔC and P1_ΔC–P2, composed of intact P1/P2 monomer and a CTD-truncated partner. Through combinations of P0 and P1–P2 variants, various complexes were reconstituted and their function tested in eEF-2-dependent GTPase and eEF-1α/eEF-2-dependent polyphenylalanine synthesis assays in vitro. Double/single-CTD dimers bound to helix I showed higher activity than that bound to helix II. Despite low polypeptide synthetic activity by a single one-CTD dimer, its binding to both helices considerably increased activity, suggesting that two stalk dimers cooperate, particularly in polypeptide synthesis. This promotion of activity by two stalk dimers was lost upon mutation of the conserved YPT sequence connecting the two helices of P0, suggesting a role for this sequence in cooperativity of two stalk dimers.     

Detection of anti-elongation factor 2 kinase (calmodulin-dependent protein kinase III) antibodies in patients with systemic lupus erythematosus

Elongation factor 2 kinase (eEF-2K), also known as calmodulin-dependent protein kinase III, is a member of the calmodulin-mediated signaling pathway that links activation of cell surface receptors to cell division. The activity of eEF-2K is increased in many human cancers and may be a valid target for anti-cancer treatment. It is one of the unconventional eukaryotic protein kinases with respect to its structural domains in comparison to other members of the serine/threonine protein kinase superfamily. eEF-2K is highly conserved in nature. For example, the amino acid sequence of human eEF-2K is 90% identical to mouse and rat eEF-2Ks and 40% identical to that of the C. elegans enzyme. Therefore it has been difficult to generate high-titer and high-specificity antibodies to the human enzyme by traditional techniques. Patients with systemic lupus erythematosus (SLE) produce auto-antibodies to a variety of cellular proteins, including members of the protein translation apparatus. Hence, we developed an ELISA assay that could detect anti-eEF2K antibodies from sera of SLE patients using purified eEF-2K as an antigen. We screened 117 sera from SLE patients. High-titer anti-eEF-2K antibodies were detected in 72 subjects. One of the high-titer sera was used for further characterization. The auto-antibody recognized eEF-2K on immunoblots and immunoprecipitated the kinase with intact enzyme activity. In conclusion, anti-eEF-2K antibodies are found in sera of SLE patients and are useful tools to study the role of this highly conserved enzyme.     

High-resolution one-dimensional polyacrylamide gel isoelectric focusing of various forms of elongation factor-2.

A system for analyzing covalent modifications of elongation factor-2 (eEF-2) by one-dimensional isoelectric focusing in slab polyacrylamide gels is described. Depending on the degree of phosphorylation, four species of eEF-2 could be resolved corresponding to the un-, mono-, bis-, and trisphosphorylated factor. Furthermore, the degree of ADP-ribosylation of the protein could also be assessed by this method. It was also shown that an acidic isoform of eEF-2 exists which appears not to be artifactual and that the relative level of this isoform appeared to vary between different cell types. By Western blotting the gels and using an antibody against eEF-2 it is possible to assess the state of phosphorylation of the factor in cells.     

Heterogeneity in the actions of drugs that bind in the DNA minor groove.

Distamycin and Hoechst 33258 have long served as the model compounds for biochemical, biophysical, and clinical studies of the drugs that bind in the DNA minor groove. However, the results presented in this investigation clearly show that 4,6-diamidino-2 phenylindole (DAPI) is superior to both of these drugs at negating the effects of intrinsic DNA curvature and anisotropic bendability as measured by electrophoretic and ligation analysis. In addition, DAPI was more effective than distamycin and Hoechst 33258 at inhibiting the assembly of nucleosomes onto synthetic and natural sequences that have multiple closely spaced oligo-AT sequences that serve as drug binding sites. Since these effects may be related to the biological action of the drugs, it was of interest to determine the mechanism that was responsible for the enhanced action of DAPI. The possibility that the differential drug potencies resulted from differential overall affinities of the ligands for A-tract molecules was considered, but drug binding studies suggested that this was not the case. It is also unlikely that the differential drug effects resulted from the binding of the drugs to different DNA sites since the oligo A/T binding sites for DAPI and Hoechst were centered on the same nucleotide positions as revealed by footprinting studies using exonuclease III, DNase I, and hydroxyl radical. However, the footprinting studies with DNase I did uncover a potentially important difference between the drugs. DAPI protected only the AT bp in the binding sites, while distamycin and Hoechst protected these bp as well as flanking Gs and Cs. These results permitted us to advance a preliminary model for the enhanced action DAPI. According to the model, the short length of DAPI and its absolute specificity for A/T bps with narrow minor grooves ensures that only particularly minor grooves that give rise to curvature and anisotropic bendability are occupied by the drug. Consequently, each helical deflection induced by an A-tract in the absence of the drug is countered by an opposite deflection induced by DAPI binding, thus effectively neutralizing intrinsic curvature and bending into the minor groove.