Regulation of protein synthesis at the elongation stage. New insights into the control of gene expression in eukaryotes.

There are many reports which demonstrate that the rate of protein biosynthesis at the elongation stage is actively regulated in eukaryotic cells. Possible physiological roles for this type of regulation are: the coordination of translation of mRNA with different initiation rate constants; regulation of transition between different physiological states of a cell, such as transition between stages of the cell cycle; and in general, any situation where the maintenance of a particular physiological state is dependent on continuous protein synthesis. A number of covalent modifications of elongation factors offer potential mechanisms for such regulation. Among the various modifications of elongation factors, phosphorylation of eEF-2 by the specific Ca2+calmodulin-dependent eEF-2 kinase is the best studied and perhaps the most important mechanism of regulation of elongation rate. Since this phosphorylation is strictly Ca(2+)-dependent, and makes eEF-2 inactive in translation, this mechanism could explain how changes in the intracellular free Ca2+ concentration may regulate elongation rate. We also discuss some recent findings concerning elongation factors, such as the discovery of developmental stage-specific elongation factors and the regulated binding of eEF-1 alpha to cytoskeletal elements. Together, these observations underline the importance of the elongation stage of translation in the regulation of the cellular processes essential for normal cell life.     

2-Deoxyglucose and NMDA inhibit protein synthesis in neurons and regulate phosphorylation of elongation factor-2 by distinct mechanisms

Cerebral ischaemia is associated with brain damage and inhibition of neuronal protein synthesis. A deficit in neuronal metabolism and altered excitatory amino acid release may both contribute to those phenomena. In the present study, we demonstrate that both NMDA and metabolic impairment by 2-deoxyglucose or inhibitors of mitochondrial respiration inhibit protein synthesis in cortical neurons through the phosphorylation of eukaryotic elongation factor (eEF-2), without any change in phosphorylation of initiation factor eIF-2alpha. eEF-2 kinase may be activated both by Ca(2+)-independent AMP kinase or by an increase in cytosolic Ca2+. Although NMDA decreases ATP levels in neurons, only the effects of 2-deoxyglucose on protein synthesis and phosphorylation of elongation factor eEF-2 were reversed by Na(+) pyruvate. Protein synthesis inhibition by 2-deoxyglucose was not as a result of a secondary release of glutamate from cortical neurons as it was not prevented by the NMDA receptor antagonist 5-methyl-10,11-dihydro-5H-dibenzo-(a,d)-cyclohepten-5,10-imine hydrogen maleate (MK 801), nor to an increase in cytosolic-free Ca2+. Conversely, 2-deoxyglucose likely activates eEF-2 kinase through a process involving phosphorylation by AMP kinase. In conclusion, we provide evidence that protein synthesis can be inhibited by NMDA and metabolic deprivation by two distinct mechanisms involving, respectively, Ca(2+)-dependent and Ca(2+)-independent eEF-2 phosphorylation.     

High hydrostatic pressure inhibits the biosynthesis of eukaryotic elongation factor-2

High continuous hydrostatic pressure is known to inhibit the total cellular protein synthesis. In this study, our goal was to identify pressure-regulated proteins by using two dimensional gel electrophoresis and mass spectrometry. This analysis showed that under 30 MPa continuous hydrostatic pressure the biosynthesis of eukaryotic elongation factor-2 (eEF-2) was inhibited both in HeLa carcinoma and T/C28a4 chondrocytic cell lines. Western blot analysis of HeLa cells revealed that the cellular protein level of eEF-2 decreased by 40%-50% within 12 h of the pressure treatment. However, the steady-state mRNA level of eEF-2 was not affected by the pressure. Cycloheximide addition after 4 h-pressure treatment suggested that the half-life of eEF-2 protein was shorter in pressurized cells. eEF-2 is responsible for the translocation of ribosome along the specific mRNA during translation, and its phosphorylation prevents the ribosomal translocation. Therefore, increased phosphorylation of eEF-2 was considered as one mechanism that could explain the reduced level of protein synthesis in pressurized HeLa cell cultures. However, Western blot analysis with an antibody recognizing the Thr56-phosphorylated form of eEF-2 showed that phosphorylation of eEF-2 was not elevated in pressurized samples. In conclusion, the inhibition of protein synthesis under high pressure occurs independent of the phosphorylation of eEF-2. However, this inhibition may result from the decrease of cellular eEF-2 protein.     

Inhibition of protein synthesis in cortical neurons during exposure to hydrogen peroxide

Transient cerebral ischemia, which is accompanied by a sustained release of glutamate and zinc, as well as H(2)O(2) formation during the reperfusion period, strongly depresses protein synthesis. We have previously demonstrated that the glutamate-induced increase in cytosolic Ca(2+) is likely responsible for blockade of the elongation step of protein synthesis, whereas Zn(2+) preferentially inhibits the initiation step. In this study, we provide evidence indicating that H(2)O(2) and thapsigargin mobilized a common intracellular Ca(2+) pool. H(2)O(2) treatment stimulated a slow increase in intracellular Ca(2+), and precluded the effect of thapsigargin on Ca(2+) mobilization. H(2)O(2) stimulated the phosphorylation of both eIF-2alpha and eEF-2, in a time- and dose-dependent manner, suggesting that both the blockade of the elongation and of the initiation step are responsible for the H(2)O(2)-induced inhibition of protein synthesis. However, kinetic data indicated that, at least during the first 15 min of H(2)O(2) treatment, the inhibition of protein synthesis resulted mainly from the phosphorylation of eEF-2. In conclusion, H(2)O(2) inhibits protein translation in cortical neurons by a process that involves the phosphorylation of both eIF-2alpha and eEF-2 and the relative contribution of these two events depends on the duration of H(2)O(2) treatment.     

A critical role of eEF-2K in mediating autophagy in response to multiple cellular stresses

The phosphorylation of the subunit α of eukaryotic translation initiation factor 2 (eIF2α), a critical regulatory event in controlling protein translation, has recently been found to mediate the induction of autophagy. However, the mediators of autophagy downstream of eIF2α remain unknown. Here, we provide evidence that eIF2α phosphorylation is required for phosphorylation of eukaryotic elongation factor 2 (eEF-2) during nutrient starvation. In addition, we show that eukaryotic elongation factor 2 kinase (eEF-2K) is also required for autophagy signaling during ER stress, suggesting that phosphorylation

of eEF-2 may serve as an integrator of various cell stresses for autophagy signaling. On the other hand, although the activation of eEF-2K in response to starvation requires the phosphorylation of eIF2α, additional pathways relying partly on Ca²⁺ flux may control eEF-2K activity during ER stress, as eIF2α phosphorylation is dispensable for both eEF-2 phosphorylation and autophagy in this context.     

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.     

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.     

Overview: phosphorylation and translation control

Protein synthesis is controlled by the phosphorylation of proteins comprising the translational apparatus. At least 12 initiation factor polypeptides, 3 elongation factors and a ribosomal protein are implicated. Stimulation of translation correlates with enhanced phosphorylation of eIF-4F, eIF-4B, eIF-2B, eIF-3 and ribosomal protein S6, whereas inhibition correlates with phosphorylation of eEF-2 and the alpha-subunit of eIF-2. Strong evidence for regulatory roles exists for eIF-2, eIF-4F and eEF-2, whereas changes in other factor activities due to phosphorylation remain to be demonstrated. Regulation of the specific activity of the translational apparatus by phosphorylation appears to be a general mechanism for the control of rates of global protein synthesis, and may also play a role in modulating the translation of specific mRNAs.     

Regulation of translation elongation factor-2 by insulin via a rapamycin-sensitive signalling pathway.

It is well established that insulin and serum stimulate gene expression at the level of mRNA translation in animal cells, and previous studies have mainly focused on the initiation process. Here we show that, in Chinese hamster ovary cells expressing the human insulin receptor, insulin causes decreased phosphorylation of elongation factor eEF-2 and that this is associated with stimulation of the rate of peptide-chain elongation. eEF-2 is phosphorylated by a very specific Ca 2+/calmodulin-dependent protein kinase (eEF-2 kinase) causing its complete inactivation. The decrease in eEF-2 phosphorylation induced by insulin reflects a fall in eEF-2 kinase activity. Rapamycin, a macrolide immunosuppressant which blocks the signalling pathway leading to the stimulation of the 70/85 kDa ribosomal protein S6 kinases, substantially blocks the activation of elongation, the fall in eEF-2 phosphorylation and the decrease in eEF-2 kinase activity, suggesting that p7O S6 kinase (p70s6k) and eEF-2 kinase may tie on a common signalling pathway. Wortmannin, an inhibitor of phosphatidylinositide-3-OH kinase, had similar effects. eEF-2 kinase was phosphorylated in vitro by purified p70s6k but this had no significant effect on the in vitro activity of eEF-2 kinase.     

Zinc inhibits protein synthesis in neurons. Potential role of phosphorylation of translation initiation factor-2alpha.

In the central nervous system, Zn(2+) is concentrated in the cerebral cortex and hippocampus and has been found to be toxic to neurons. In this study, we show that exposure of cultured cortical neurons from mouse to increasing concentrations of Zn(2+) (10-300 microM) induces a progressive decrease in global protein synthesis. The potency of Zn(2+) was increased by about 2 orders of magnitude in the presence of Na(+)-pyrithione, a Zn(2+) ionophore. The basal rate of protein synthesis was restored 3 h after Zn(2+) removal. Zn(2+) induced a sustained increase in phosphorylation of the alpha subunit of the translation eukaryotic initiation factor-2 (eIF-2alpha), whereas it triggered a transient increase in phosphorylation of eukaryotic elongation factor-2 (eEF-2). Protein synthesis was still depressed 60 min after the onset of Zn(2+) exposure while the state of eEF-2 phosphorylation had already returned to its basal level. Moreover, Zn(2+) was less effective than glutamate to increase eEF-2 phosphorylation, whereas it induced a more profound inhibition of protein synthesis. These results suggest that Zn(2+)-induced inhibition of protein synthesis mainly correlates with the increase in eIF-2alpha phosphorylation. Supporting further that Zn(2+) acts at the initiation step of protein synthesis, it strongly decreased the amount of polyribosomes.     

Kinetic characterisation of the enzymatic activity of the eEF-2-specific Ca2(+)-and calmodulin-dependent protein kinase III purified from rabbit reticulocytes.

The Ca2(+)-and calmodulin-dependent protein kinase III, which specifically phosphorylates the eukaryotic elongation factor 2 (eEF-2), has been purified to apparent homogeneity from the post-ribosomal fraction of rabbit reticulocytes by an efficient four-step method. The method results in a more than 4000-fold purification of the enzyme. SDS-gel electrophoresis showed that the purified kinase contained only one polypeptide with the apparent molecular mass of 90 kDa. The kinase activity was associated with the 90-kDa protein as shown by analyzing the phosphorylating activity of SDS gel electrophoretically purified protein electroblotted to nitrocellulose membranes. The purified kinase was dependent on Ca2+, Mg2+ and calmodulin for activity. Kinetic analysis of the phosphorylation reaction indicates that the turnover number of the kinase was approximately 1 s-1. The Km for the two substrates ATP and eEF-2 was calculated to be approximately 100 microM and 10 microM, respectively. The activity of the kinase was competitively inhibited by cAMP. The inhibition constant Ki (0.5 mM) was found to be in the same order of magnitude as that calculated for the competitive product inhibition caused by ADP. GTP was ten-times less efficient as competitor, indicating that the kinase had a preference for adenosine nucleotides. Phosphorylation of eEF-2 did not interfere with the diphtheria-toxin-catalysed ADP-ribosylation of the factor nor did ADP-ribosylation inhibit phosphorylation.     

Regulation of elongation factor-2 kinase by pH

Elongation factor-2 kinase (eEF-2K) is a Ca(2+)/calmodulin-dependent protein kinase that phosphorylates and inactivates eEF-2 and that can regulate the rate of protein synthesis at the elongation stage. Here we report that a slight decrease in pH, within the range observed in vivo, leads to a dramatic activation of eEF-2K. The activity of eEF-2K in mouse liver extracts, as well as the activity of purified recombinant human eEF-2K, is low at pH 7.2-7.4 and is increased by severalfold when the pH drops to 6.6-6.8. eEF-2K requires calmodulin for activity at neutral as well as acidic pH. Kinetic studies demonstrate that the pH does not affect the K(M) for ATP or eEF-2 and activation of eEF-2K at acidic pH is due to an increase in V(max). To analyze the potential role of eEF-2K in regulating protein synthesis by pH, we constructed a mouse fibroblast cell line that expresses eEF-2K in a tetracycline-regulated manner. Overexpression of eEF-2K led to a decreased rate of protein synthesis at acidic pH, but not at neutral pH. Our results suggest that pH-dependent activation of eEF-2K may play a role in the global inhibition of protein synthesis during tissue acidosis, which accompanies such processes as hypoxia and ischemia.     

Calcium/calmodulin stimulates the autophosphorylation of elongation factor 2 kinase on Thr-348 and Ser-500 to regulate its activity and calcium dependence

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.     

An Examination of the Role of Increased Cytosolic Free Ca2+Concentrations in the Inhibition of mRNA Translation

Mobilization of Ca²⁺sequestered by the endoplasmic reticulum (ER) produces the phosphorylation of initiation factor (eIF) 2, whereas an increase in cytosolic free Ca²⁺([Ca²⁺]_i) due to plasmalemmal Ca²⁺influx increases the phosphorylation of elongation factor (eEF) 2. In nucleated mammalian cells, depletion of ER Ca²⁺stores has been demonstrated to inhibit translational initiation, but evidence that increased [Ca²⁺]_iper se causes slowing of peptide chain elongation is lacking. L-type Ca²⁺channel activity of GH₃pituitary cells, which are enriched in calmodulin-dependent eEF-2 kinase, was manipulated such that the impact of [Ca²⁺]_ion eEF-2 phosphorylation and translational rate could be examined for up to 10 min without inhibiting initiation. At 1 mM extracellular Ca²⁺, resting [Ca²⁺]_ivalues were high (154–255 nM) and eEF-2 was phosphorylated. The Ca²⁺channel antagonist, nisoldipine, lowered [Ca²⁺]_iand reduced eEF-2 phosphorylation by half but had no effect on amino acid incorporation. The Ca²⁺channel agonist, Bay K 8644, produced sustained elevations of [Ca²⁺]_ithat were associated with 25–50% increases in eEF-2 phosphorylation, but no changes in protein synthetic rates occurred. Larger Ca²⁺influxes were achievable with either 25 mM KCl or KCl plus Bay K 8644. These treatments further increased eEF-2 phosphorylation (50–100% above control) and inhibited leucine incorporation by 20–70% but ATP content was reduced by 25–50% and total cell-associated Ca²⁺contents rose by 3- to 13-fold. eIF-2α was not phosphorylated during these treatments. Addition of low concentrations of ionomycin, which do not lower ATP content, was associated with complex changes in [Ca²⁺]_ithat resembled alterations in eEF-2 phosphorylation. The inhibition of leucine incorporation in response to ionomycin, however, coincided only with the phosphorylation of eIF-2α, not eEF-2. It is concluded that changes in [Ca²⁺]_ioccurring in the absence of ATP depletion alter the phosphorylation state of eEF-2 but are not regulatory for mRNA translation.     

Phosphorylation regulates the activity of the eEF-2-specific Ca(2+)- and calmodulin-dependent protein kinase III

The activity of the eukaryotic elongation factor 2 (eEF-2)-specific Ca(2+)- and calmodulin-dependent protein kinase III (CaM PK III) is regulated by phosphorylation. The kinase can be inactivated by treatment with alkaline phosphatase and subsequently reactivated by endogenous protein kinase. This kinase can be substituted for by the catalytic subunit of cAMP-dependent protein kinase but not by casein kinase II. The purified kinase preparation contains only one protein as judged by gel electrophoresis. This protein has a molecular mass of approximately 90 kDa and an isoelectric point of 5.2. Reactivation of the eEF-2 kinase is associated with the phosphorylation of this protein. The amino acid sequence obtained from the 90-kDa protein reveals substantial homology with that of murine heat shock protein 86 (HSP 86) a member of the HSP 90-family. Conventional preparations of HSP 90 contain an inactive eEF-2 kinase that could be activated after dephosphorylation and phosphorylation by the catalytic subunit of cAMP-dependent protein kinase.     

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.     

Co-expression of calcium-dependent protein kinase with the inward rectified guard cell K+ channel KAT1 alters current parameters in Xenopus laevis oocytes

Increased guard cell cytosolic [Ca2+] is known to be involved in signal transduction pathways leading to stomatal closure, and inhibit the inward rectifying guard cell K+ channel KAT1. Guard cell calcium-dependent protein kinase (CDPK) has been shown to phosphorylate KAT1; such phosphorylation is known to modulate other K+ channels involved in signal transduction cascades. The work reported here focused on demonstrating CDPK-dependent inhibition of KAT1 currents. A cDNA encoding soybean CDPK was generated and it's translation product was shown to be functional; demonstrating Ca2+-dependent autophosphorylation and phosphorylation of a target protein. Ion currents were monitored using voltage clamp techniques upon expression of KAT1 in Xenopus laevis oocytes. Coexpression of recombinant CDPK with KAT1 in oocytes altered the kinetics and magnitude of induced K+ currents; at a given hyperpolarizing command voltage, the magnitude of KAT1 currents was reduced and the half-time for channel activation was increased. This finding supports a model of Ca2+-dependent ABA inhibition of inward K+ currents in guard cells as being mediated by CDPK phosphorylation of KAT1.     

Phosphorylation and the control of calcium fluxes

Cell activation, e.g. stimulus-contraction or stimulus-secretion coupling, is brought about by a 100-fold increase in cytosolic free Ca2+ concentration from 0.1 to 10 microM, upon release of Ca2+ from intrareticular or extracellular stores along the concentration gradient. A return to steady state is achieved by either Na+-Ca2+ exchange or ATP-dependent Ca2+ transport against the concentration gradient. Both processes, Ca2+ influx and Ca2+ efflux, are regulated by sophisticated covalent mechanisms. The positive inotropic effect of adrenalin is mediated by the cyclic-AMP-dependent phosphorylation of cardiac sarcolemmal proteins, among which calciductin is the major phosphate acceptor. Upon cyclic-AMP-dependent phosphorylation, the slow Ca2+ channel is activated 3.5 time above its basal low-conductance state, and retains its characteristics, competition by divalent metals, inhibition by La3+ and Ca2+ entry blockers. The adrenalin-induced abbreviation of systole is also explained in terms of the dual phosphorylation of the cardiac sarcoplasmic reticulum calcium pump activator, phospholamban, by cyclic-AMP-dependent protein kinase on the one hand and Ca2+-calmodulin-dependent phospholamban kinase on the other. Calciductin and phospholamban are closely similar acidic proteolipids. A phospholamban-like protein is also found in platelet Ca2+-accumulating vesicles, where its cyclic-AMP-dependent phosphorylation doubles the rate of Ca2+ efflux. These observations raise the possibility that calcium fluxes are regulated by phosphorylation of membrane-bound proteolipids. More generally, phosphorylation modulates K+, Na+ and Ca2+ fluxes through membranes, i.e. the general excitability properties of the cell.     

Differing effects of the protein phosphatase inhibitors okadaic acid and microcystin on translation in reticulocyte lysates.

The effects of the cyanobacterial toxin and protein phosphatase inhibitor, microcystin, on translation in rabbit reticulocyte lysates have been studied. Microcystin inhibited translation with similar potency to the protein phosphatase inhibitor okadaic acid. Unlike low concentrations of okadaic acid, however, it inhibited both the initiation and elongation stages. This was demonstrated using EGTA to inhibit the phosphorylation and inactivation of elongation factor eEF-2. A method for detecting changes in eEF-2 phosphorylation was developed. eEF-2 was found to exist as three different species: eEF-2 was largely monophosphorylated in reticulocyte lysates under control conditions, the remainder being unphosphorylated. Okadaic acid and microcystin increased the level of the bisphosphorylated species. The implications of multiple phosphorylation of eEF-2 for the control of translation is discussed. Microcystin was also found to increase the phosphorylation of eIF-2 alpha (and therefore to inhibit initiation) at lower concentrations than okadaic acid, suggesting that the major eIF-2 alpha phosphatase in the reticulocyte lysate is phosphatase-1.