MINIREVIEW Regulation of peptide-chain elongation in mammalian cells Gareth J. Browne and Christopher G. Proud Division of Molecular Physiology, School of Life Sciences, University of Dundee, MSI/WTB Complex, Dundee, UK The elongation phase of mRNA translation is the stage at which the polypeptide is assembled and requires a substantial amount of metabolic energy. Translation elongation in mammals requires a set of nonribosomal proteins called eukaryotic elongation actors or eEFs. Several of these proteins are subject to phosphorylation in mammalian cells, including the factors eEF1A and eEF1B that are involved in recruitment of amino acyl- tRNAs to the ribosome. eEF2, which mediates ribosomal translocation, is also phosphorylated and this inhibits its activity. The kinase acting on eEF2 is an unusual and specific one, whose activity is dependent on calcium ions and calmodulin. Recent work has shown that the activity of eEF2 kinase is regulated by MAP kinase signalling and by the nutrient-sensitive mTOR signalling pathway, which serve to activate eEF2 in response to mitogenic or hormonal stimuli. Conversely, eEF2 is inactivated by phosphorylation in response to stimuli that increase energy demand or reduce its supply. This likely serves to slow down protein synthesis and thus conserve energy under such circumstances. Keywords: translation; elongation factor; mTOR; rapamy- cin; eEF1; eEF2. INTRODUCTION Recent years have seen major advances in our understand- ing of the control of mRNA translation, both via regulation of proteins that bind to specific mRNAs and modulate their translation and through control of the activities of compo- nents of the core translational machinery. In the latter area, much attention has been directed at understanding the regulation of the initiation process. As described in the accompanying articles [1,2] and elsewhere [3–6], multiple mechanisms operate to modulate translation initiation. Relatively less attention has been devoted to studying the control of elongation. However, there have been important findings in this area too. These cast new light on how elongation, the principal phase of protein synthesis, is regulated. The purpose of this article is to review this recent work in the context of other studies on cell signalling and the control of mRNA translation. The process of translation elongation consumes a great deal of metabolic energy, at least four high energy bonds being consumed for each amino acid added to the nascent chain(twotoformtheaminoacyl-tRNAasATPis hydrolysed to AMP, and two GTP molecules are broken down to GDP during events on the ribosome itself which involve the elongation factors). In the cytoplasm of higher eukaryotes, the process of peptide-chain elongation requires two types of ancillary factor, one to recruit the amino acyl-tRNAs to the A-site of the ribosome and one to mediate the translocation step, in which the ribosome moves relative to the mRNA by the equivalent of one codon (Table 1). In eukaryotes, the factors involved in amino acyl-tRNA recruitment are eEF1A and eEF1B, while translocation requires eEF2. It is not the purpose of this review to describe the mechanism of elongation, which has recently been reviewed in detail by other authors [7]. This article discusses the mechanisms underlying the control of the activity of the elongation factors themselves. WHY REGULATE ELONGATION? As described in a number of recent review articles, including the two that accompany this one [1,2,4,8–10] there are a number of sophisticated mechanisms that regulate transla- tion initiation. Why should the process of elongation also be subject to regulation? Two main points should be made here. When protein synthesis is activated, e.g. by insulin, growth factors or mitogens, translation initiation will be stimulated and the loading of ribosomes onto mRNAs will increase. It seems logical that the rate of elongation by those ribosomes should also be increased to match the increased rate of attachment of ribosomes to the mRNA, and to avoid a limitation in translation rate due to elongation. For example, the increased numbers of ribosomes engaged in translation will require increased activity of the elongation factors that associate with the ribosome during translation. One could argue that cells could just maintain elongation factors at a constitutively high level of activity: however, elongation activity is inversely related to translational fidelity [11], and inap- propriately high levels of elongation activity may lead to missense errors or premature termination. When protein synthesis rates are to be decreased, inhibition of elonga- tion will ensure that polysomes are retained, even if initiation is also inhibited. This will allow translation to be resumed rapidly when required. Correspondence to C. G. Proud, Division of Molecular Physiology, School of Life Sciences, University of Dundee, MSI/WTB Complex, Dow Street, Dundee, DD1 5EH, UK. Fax: + 44 1382 322424, Tel.: + 44 1382 344919, E-mail: c.g.proud@dundee.ac.uk (Received 2 August 2002, revised 23 September 2002, accepted 3 October 2002) Eur. J. Biochem. 269, 5360–5368 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03290.x As noted above, protein synthesis consumes a high proportion of cellular energy, and the vast majority of this is used in elongation. It therefore follows that, under condi- tions of temporarily increased energy demand or decreased energy supply, it would be advantageous for the cell to reduce the rate of protein synthesis, to allow energy to be diverted to other processes, such as maintaining the plasma membrane potential and ion gradients or to support contraction in heart and striated muscle. Again, inhibition of elongation will ensure that polysomes are retained, with advantages for mRNA stability and for the rapid resump- tion of translation once energy availability improves again. eEF1A There have been a number of changes to the nomenclature here, which can be confusing (e.g. for the present authors). The protein formerly known as eEF1a (oralsoasEF-1a)is now termed eEF1A. eEF1A binds guanine nucleotides and in its GTP-liganded form can interact with aminoacyl tRNA to bring it to the A-site of the ribosome [7]. Following hydrolysis of the GTP, eEF1AÆGDP is released from the ribosome. This form cannot bind amino acyl-tRNA and is ÔrecycledÕ to the active GTP-bound form by eEF1B, which consists of three subunits, a, b and c (Table 1). These proteins were formerly designated as subunits of eEF1 (also called EF-1), and were termed eIF1b-d.Thereaderis referred to Table 1 for clarification. eEF1B thus acts as a guanine nucleotide-exchange factor (GEF) for eIF1A. Sequence comparisons reveal similarity between the C-termini of eEF1Ba and b (Fig. 1), and activity measure- ments suggest each can act to stimulate eEF1A [12], presumably by stimulating GDP/GTP exchange. The complete eEF1B complex (abc) stimulates eEF1A more efficiently than eEF1a or eEF1b alone. eEF1Bc likely has a role in the assembly of the eEF1B complex and perhaps in facilitating the effective interactions of eEF1Ba/b with the substrate eEF1A.GDP. Several groups have studied the phosphorylation and regulation of eEF1A/B. For a detailed description the reader is referred to the recent review article by Traugh [13], whose work has contributed very substantially to know- ledge in this area. What follows is a summary of current knowledge. In higher animals, all four polypeptides (eEF1A and eEF1Babc) are phosphoproteins and are targets for a number of protein kinases. These include casein kinase 2 (CK2), a constitutively active protein kinase, which phos- phorylates the a and b subunits of eEF1B from a number of species ([13]; Fig. 1). Phosphorylation of the eEF1B holoprotein by CK2 has essentially no effect on its ability to stimulate eEF1A [14]. Phosphorylation by CK2 does not therefore seem to influence the activity of eEF1B in this assay, although it might affect its interaction with eEF1A or other proteins. Another GEF involved in translation initiation provides a precedent for this – the phosphoryla- tion of two sites in the extreme C-terminus of eIF2Be by CK2 is required for its interaction with its substrate eIF2 [15]. Insulin or phorbol esters increase the phosphorylation of eEF1A, eEF1Ba and eEF1Bb in vivo [16,17]. The available evidence, which includes phosphopeptide mapping data, suggests that the effect of insulin on the phosphorylation of eEF1Ba and eEF1Bb is mediated via a protein kinase termed MS6K which can directly phosphorylate all three polypeptides [13,18]. In the case of eEF1A, MS6K may also Fig. 1. Translation elongation factors in higher eukaryotes. The overall layout and known phosphorylation sites in vertebrate translation elongation factors are depicted. Numbers to the right of each indicate the number of residues in the known mammalian proteins. Phos- phorylation sites are indicated (S ¼ Ser; T ¼ Thr, residues numbers shown) along with kinases known to phosphorylate them. See text for details and for definitions of abbreviations. The GTP-binding and GEF domains are shown where relevant. The figure also shows the histidine residue in eEF2, which is converted to diphthamide and is a target for ADP-ribosylation by diphtheria toxin. Phosphorylation sites for other kinases mentioned in the text have not been identified. Table 1. Mammalian elongation factors. Name Former name(s) Molecular mass (kDa) Function eEF1A EF-1, eEF1a 50.1 Binds GTP and amino acyl-tRNA; recruits amino acyl-tRNA to ribosomal A-site; functionally equivalent to bacterial EF-Tu eEF1B a eEF1d 24.8 Mediates GDP/GTP exchange on eEF1a; eEF1B b eEF1b 31.1 functionally eqeuivalent to bacterial EF-Ts eEF1B c eEF1c 50.0 eEF2 EF-2 95.2 Binds GTP; required for ribosomal translocation during elongation; functionally equivalent to bacterial EF-G Ó FEBS 2002 Control of translation elongation (Eur. J. Biochem. 269) 5361 be involved, since many of the phosphopeptides observed in response to insulin treatment in vivo areseeninmaps generated from eEF1A phosphorylated by MS6K in vitro. However, the in vivo maps also contain additional peptides indicating that further insulin-stimulated kinases also act on eEF1A in vivo. This kinase also phosphorylates other components of the translational machinery such as eIF4B, eIF4G and ribosomal protein S6, at least in vitro [13]. Phosphorylation of eEF1A/B in vitro by MS6K results in modest stimulation of its activity [18]. The degree of stimulation observed is very similar to that seen when the activity of eEF1A/B from insulin-treated cells is compared with that of the proteins from serum-deprived cells, consistent with the idea that phosphorylation by MS6K may be involved in their regulation in response to serum in vivo. eEF1A and eEF1B are also substrates for phosphoryla- tion by the classical protein kinase C (PKC) isoforms in vitro and this may explain the ability of phorbol esters (which activate several PKCs) to increase the phosphorylation of these proteins in vivo [16,17] (Fig. 1). Phorbol esters also increase the phosphorylation of the valyl-tRNA synthetase that associates with eEF1A/B. The available evidence suggests that phosphorylation of eEF1A/B and of valyl- tRNA synthetase by PKC increases their activities in translation elongation and amino acylation, respectively. The increased activity of eEF1A/B appears to result from enhanced GEF activity [19]. Lastly, in Xenopus oocytes, eEF1Bc is phosphorylated during meiotic maturation [13,20] (Fig. 1). It appears to be a direct substrate for the protein kinase activity of the maturation-promoting complex MPF [21] and the major site of phosphorylation was identified as Thr230, which is conserved in mammals. The kinase present in MPF, cdc2, also phosphorylates eEF1Bb, in this case at Thr122 [22]. Although protein synthesis is increased during maturation, there is so far no evidence that these phosphorylation events actually alter the activity of eEF1A/eEF1B. eEF2 eEF2 is a monomeric protein with a mass of about 93 kDa (Fig. 1). It binds guanine nucleotides and is active when bound to GTP. The GTP is hydrolysed late in the translocation process, and the energy released may be coupled to translocation, although there are conflicting data here [7]. eEF2 thus leaves the ribosome as inactive eEF2ÆGDP, but the rate of release of GDP is sufficiently high that no guanine nucleotide-exchange factor is required to produce active eEF2ÆGTP. The GTP-binding motif is located towards the N-terminus of eEF2, in a region that appears to be involved in its binding to the ribosome (Fig. 1). This region also contains the major physiological phosphorylation site in eEF2, at Thr56 [23,24]. Its C-terminus is also thought to contain a region that interacts with the ribosome [25] and a further site of post-transla- tional modification, in this instance the diphthamide residue, which is ADP-ribosylated by diphtheria toxin [25]. ADP-ribosylation inhibits the activity of eEF2. Phosphorylation of eEF2 inhibits its activity, in translo- cation and in poly(U)-directed polyphenylalanine synthesis [26,27], by preventing it from binding to the ribosome [28]. Early data showed that the phosphorylation of eEF2 was increased by very low concentrations of the protein phosphatase inhibitor okadaic acid [29], suggesting that the major phosphatase acting on eEF2 in the cell was protein phosphatase (PP)2A or a closely related enzyme [30]. This may be significant for the control of eEF2 through signalling via the mammalian target of rapamycin (mTOR; see below). In 1987, Ryazanov showed that eEF2 was phosphor- ylated in a Ca 2+ /calmodulin-dependent manner [31] and Palfrey and Nairn identified an abundant substrate for Ca 2+ /calmodulin-dependent kinase III as eEF2 [32]. As eEF2 is the only known substrate for this kinase, it is now known as eEF2 kinase. Nairn and colleagues subsequently showed that agents that affect cytosolic Ca 2+ levels increase the level of phosphorylation of eEF2 [33,34]. eEF2 phosphorylation was also shown to increase during mitosis, when overall rates of protein synthesis decrease [35]. Subsequent work was directed at the purification of eEF2 kinase. This was achieved in 1993 by two groups [36,37] using rabbit reticulocytes and rat pancreas as starting material, and revealed a polypeptide of about 95–103 kDa on denaturing gel electrophoresis. The activity of the purified kinase against eEF2 was strictly dependent upon Ca 2+ ions and calmodulin. In the presence of Ca 2+ ions and calmodulin, eEF2 kinase underwent extensive auto- phosphorylation resulting in it acquiring the ability to phosphorylate eEF2 in the absence of added Ca 2+ ions and calmodulin. In principle, this would prolong its activation in vivo beyond the duration of a Ca 2+ transient and may be important in longer-term inhibition of trans- lation in response to Ca 2+ ion mobilization. The signifi- cance of the fact that eEF2 kinase is regulated by Ca 2+ ions for the physiological control of protein synthesis is still far from clear, although various ideas have been put forward. For example, it has been suggested that the increased phosphorylation of eEF2, and inhibition of protein synthe- sis, observed in neurones in response to excitotoxic activa- tion of glutamate receptors may serve a cytoprotective function [38]. It may also serve to couple activation of muscle contraction to inhibition of protein synthesis, in order to divert the available metabolic energy towards the contractile machinery (see below). Amino acid sequence data generated from the purified protein allowed the isolation of cDNAs for this enzyme, first reported by Redpath et al. [39]. This revealed a sequence that showed little obvious homology to vast majority of other protein kinases. The availability of additional sequence data allowed Ryazanov et al. [40,41] to identify related enzymes in several metazoan species, in particular a myosin heavy chain kinase from Dictyostelium discoideum. Since this enzyme is known to phosphorylate its substrate within an a-helical region, rather than at a b-turn which is often the case for members of main protein kinase superfamily, Ryazanov coined the term Ôa-kinaseÕ for this unusual group of enzymes. Further discussion of this family may be found in a recent review by Ryazanov [42]. No a-kinase homologues are found in Drosophila, Arabidopsis or the known yeast genomes. The catalytic domain of eEF2 kinase lies in the N-terminal half of its primary structure [43,44] (Fig. 2). Immediately N-terminal to this, around residues 77–99, is the calmodulin binding region, and mutation of Trp84 5362 G. J. Browne and C. G. Proud (Eur. J. Biochem. 269) Ó FEBS 2002 within this motif prevents calmodulin binding and thus also activity [43]. Removal of the C-terminus (residues 525–721) prevents phosphorylation of eEF2 but not autophosphory- lation, consistent with the N-terminus containing the catalytic domain. The C-terminus alone can bind eEF2, and Diggle et al. [43] showed that loss of even the last 19 amino acids resulted in an enzyme that could not phos- phorylate eEF2 but did undergo autophosphorylation. Thus, the extreme C-terminus contains a key site for interaction with eEF2 (Fig. 2). The three-dimensional structure of the catalytic domain of one member of the a-kinase family (ChaK, a TRP channel) was recently analysed [45]. This revealed surprising similarities to the overall fold of members of the main kinase superfamily. The structure did not, however, indicate a particular propensity to recognize a-helical substrates. Substrate recognition may involve a glycine-rich motif, which would thus serve quite a different function in these kinases from the role of a similar motif in phosphate binding in other protein kinases. The eEF2 kinase encoded by the known cDNA may not be the only eEF2 kinase. Hait et al. [46] have published immunological evidence for the existence of multiple forms of eEF2 kinase in phaeochromocytoma cells and the regulatory properties of eEF2 kinase in ventricular cardio- myocytes suggest the existence of a distinct form in these cells [47]. Further work will be required to identify possible additional forms of eEF2 kinase. These might potentially be encoded by distinct genes – the mammalian genome does contain additional a-kinases [40,41] – or by alternatively splicing of transcripts derived from the known gene. Ryazanov reports that generation of transgenic mice in which the eEF2 kinase gene was disrupted resulted in loss of eEF2 kinase activity in tissues from these animals [42], which would appear consistent with the second possibility. However, as data are only shown for liver homogenates it is not possible to assess whether this applied, for example, to heart. Such mice showed no apparent abnormalities in growth or reproduction indicating that eEF2 kinase is not essential for life. Similarly, disruption of the putative eEF2 kinase gene in Caenorhabditis elegans yielded viable organisms [42]. REGULATION OF eEF2 KINASE BY PKA The first evidence for an additional mechanism for controlling eEF2 kinase activity besides its activation by Ca 2+ /calmodulin was provided by the observation that cAMP-dependent protein kinase (PKA) can phosphorylate eEF2 kinase [36,48]. This results in eEF2 kinase becoming partially independent of Ca 2+ /calmodulin for activity [48,49], i.e. it activates eEF2 kinase at low basal Ca 2+ levels. This probably explains how agents that activate PKA – including cAMP analogues, forskolin and b-adrenergic agonists – raise the cellular levels of phosphorylation of eEF2 [47,50,51]. Such treatments inhibit protein synthesis and rates of elongation, and the increased phosphorylation of eEF2 may explain both effects. Diggle et al. [49] identified the site phosphorylated by PKA in rat eEF2 kinase as Ser499 (Ser500 is the human sequence), which lies outside the putative catalytic domain (Fig. 2) in a relatively poor consensus for phosphorylation by PKA. Replacement of Ser499 by an acidic residue, Asp, yielded a constitutively active form of eEF2 kinase. In heart cells, as in others studied, elevation of cAMP levels results in phosphorylation of eEF2 [47]. However, under this condition, eEF2 kinase remains entirely depend- ent on Ca 2+ /calmodulin in contrast with findings for eEF2 kinase from other sources. Instead, activation of PKA results in a marked rise in the maximal activity of eEF2 kinase from ventricular myocytes, suggesting that these cells may contain a distinct isoform of eEF2 kinase, as mentioned above. In heart cells, increasing the maximal activity of eEF2 kinase seems a more appropriate way to enhance its intracellular activity than rendering it independ- entofCa 2+ ions. This is because intracellular Ca 2+ ion concentrations fluctuate on a beat-to-beat basis in cardio- myocytes, rather than rising acutely from low basal levels in response to specific stimuli, which is the situation in many other cell-types. What possible physiological significance can be ascribed to the control of eEF2 kinase by PKA? Bearing in mind that PKA is usually activated either under conditions of increased energy demand, e.g. for contraction in striated or cardiac muscle, it may be that it serves to slow down the rate of elongation, and thus conserve energy, which can then be used for more urgent purposes. Two major signalling mechanisms – cAMP and Ca 2+ ions – thus act to activate eEF2 kinase and switch off elongation (Fig. 3). We will return to the issue of energy demand and the control of elongation below. REGULATION OF eEF2 AND eEF2 KINASE BY INSULIN AND OTHER STIMULI Redpath et al. [52] showed that, in Chinese hamster ovary (CHO) cells overexpressing the insulin receptor, insulin causes the rapid dephosphorylation of eEF2 and this effect is inhibited by rapamycin. This indicated a crucial role in this response for the mammalian target of rapamycin, a protein which is discussed in more detail in the accompany- ing review by Proud [1]. Insulin has subsequently been shown to decrease eEF2 phosphorylation in primary cell types such as adipocytes [50] and ventricular myocytes [53]. This is associated with a decrease in the activity of eEF2 Fig. 2. Schematic depiction of the structure of human eEF2 kinase. The known in vivo phosphorylation sites are indicated, together with the kinases known to phosphorylate them. The question mark by Ser359 indicates that it is likely to be a target for a so far unknown kinase that is activated by IGF1 (see text). (NB: numbering of all these sites is shiftedby+1incomparisontotheratorrabbiteEF2kinase sequences). For ease of presentation, this diagram is not drawn to strict scale. Known functional domains are also indicated (see key) as are the tryptophan (Trp85) required for calmodulin binding and the GxGxxG motif that may be involved in substrate binding (see text). Ó FEBS 2002 Control of translation elongation (Eur. J. Biochem. 269) 5363 kinase, which (where tested) is prevented by rapamycin [52,53]. These data suggested that eEF2 kinase was controlled by insulin through events that involved mTOR. Subsequent work revealed that eEF2 kinase is a substrate for p70 S6 kinase (also termed S6K1), a kinase that is activated by insulin in an mTOR-dependent manner. S6K1 phosphory- lates eEF2 kinase at a single site (Ser366 in the human protein; Figs 2,4) and this inactivates it at basal Ca 2+ ion concentrations [54], thus providing a mechanism by which insulin can switch off eEF2 kinase and activate elongation. Ser366 is also phosphorylated by p90 RSK1 , a kinase that lies directly downstream of Erk in the classical MAP kinase pathway (Fig. 4). It is activated by a variety of stimuli including phorbol esters, mitogens, growth factors and certain G-protein coupled receptor agonists. This signalling connection potentially allows such agonists to activate protein synthesis via the MAP kinase pathway. Recent work has revealed that, in cardiomyocytes, angiotensin II [55] and the Gq-coupled agonists phenylephrine and endothelin 1 [56] decrease eEF2 phosphorylation and this requires signalling through the classical MAP kinase pathway. It is likely that these effects involve the phos- phorylation and inactivation of eEF2 kinase by p90 RSK . Ser366 is, however, not the only rapamycin-sensitive phosphorylation site in eEF2 kinase. Knebel et al.[57] identified Ser359 as a substrate for a stress-activated protein kinase 4 (SAPK4)/p38 MAP kinase d, a member of the stress-activated kinase family (Fig. 4). Phosphorylation of this site inactivates eEF2 kinase. This site undergoes increased phosphorylation in response to insulin-like growth factor 1 (IGF1) and this effect is blocked by rapamycin, again revealing a link to mTOR. Given that SAPK4 is not activated by IGF1, and that it is not known to be regulated by mTOR, it seems likely that there is an additional, unknown, kinase that phosphorylates Ser359 in response to insulin (Fig. 4). The mTOR-dependent inputs into the control of eEF2 kinase, and thus elongation itself, also provide mechanisms by which nutrients, especially amino acids (as precursors for protein synthesis), can positively modulate protein synthe- sis. Such regulation makes good physiological sense and is Fig. 3. Inhibition of eEF2 and elongation by energy demand and other stimuli. In response to activation of NMDA receptors or certain G-protein coupled receptors (GPCRs), intracellular Ca 2+ levels rise, activating eEF2 kinase and leading to phosphorylation and inactiva- tion of eEF2. Activation of adenylate cyclase, either by b-adrenergic agonists or by forskolin, increases cAMP levels and activates cyclic AMP-dependent protein kinase (PKA). This phosphorylates eEF2 kinase, activating it (see text for details) and leading to phosphoryla- tion and inactivation of eEF2. Modest depletion of cellular ATP (which causes AMP levels to rise), or the direct activation of the AMP-activated protein kinase by AICA riboside, leads to increased phosphorylation of eEF2, probably through activation of eEF2 kinase although the molecular mechanisms involved here are unclear. NMDAR, N-methyl- D -aspartate receptor; GPCR, G-protein coupled receptor; b-AR, b-adrenergic receptor; AC, adenylate cyclase; IP 3 , inositol trisphosphate. Fig. 4. Activation of eEF2 by insulin, GPCR agonists and other stimuli. Insulin and IGF1 activate p70 S6k (S6K1) via signalling events dependent upon mTOR (which is inhibited by rapamycin, shown). S6K1 phosphorylates eEF2 kinase at Ser366, and this inactivates eEF2 kinase, contributing to the dephosphorylation of eEF2. IGF1 also increases the phosphorylation of Ser359, a site which also inhibits eEF2 kinase activity. The kinase involved here is unknown, but phosphorylation is inhibited by rapamycin. Agents that activate the MEK/ERK pathway lead to activation of p90 RSK1 , which also phos- phorylates Ser366 and inactivates eEF2 kinase. Such stimuli include the indicated GPCR agonists, which have been shown to decrease eEF2 phosphorylation in cardiomyocytes (see text). PD98059 and U0126 inhibit MEK activation, and block the effects of these agents on eEF2 phosphorylation. Anisomycin stimulates several stress-activated protein kinase cascades, as indicated. Use of the p38 MAP kinase (SAPK2a/b) inhibitor SB203580 indicates that this pathway regulates the phosphorylation of Ser359 and Ser377, although SAPK4 is prob- ably also involved in the case of Ser359. IR/IGFR, insulin and/or IGF1 receptors; other abbreviations are defined in the text. 5364 G. J. Browne and C. G. Proud (Eur. J. Biochem. 269) Ó FEBS 2002 discussed in greater detail in the accompanying article by Proud [1]. The fact that Ser359 is phosphorylated by a stress- activated protein kinase raises questions about the control of eEF2 phosphorylation in response to cellular stresses. Recent work shows that the effect depends very much on the nature of the stress, with oxidative stress increasing eEF2 phosphorylation, while osmotic stress decreases it [58]. The mechanisms underlying these effects are unclear. However, it is known that eEF2 kinase can be phosphorylated by stress- regulated protein kinases. For example, stress-activated protein kinase 4 (SAPK4; also termed p38 MAP kinase d) phosphorylates eEF2 kinase at Ser359. Anisomycin and tumour necrosis factor a, which activate SAPK4, increase the phosphorylation at Ser359 of eEF2 kinase in vivo and decrease eEF2 phosphorylation consistent [59] with the observation that phosphorylation of Ser359 inhibits eEF2 kinase activity [57]. However, the ability of low concentra- tions of these agents to increase the phosphorylation of Ser359 is suppressed by the compound SB203580. This suggests an additional role for the SB203580-sensitive p38 MAP kinase a/b pathway in modulating the phosphoryla- tion of this site. This pathway also affects the phosphoryla- tion of eEF2 kinase at Ser377, a site whose phosphorylation increases in response to agents that activate p38 MAP kinase a/b such as anisomycin and TNFa [59]. This may be a consequence of its phosphorylation by MAP kinase-activa- ted protein kinase 2, MK-2. However, phosphorylation of this site does not appear to affect the activity of eEF2 kinase. Regulation of eEF2 by cellular energy status As pointed out above, translation elongation is expensive in terms of metabolic energy, and may be inhibited when energy demands increase. What happens when energy supply is restricted? One important pathological situation where this arises is during cerebral ischaemia (as occurs during a stroke, for example). Recent work has shown that this is accompanied by increased phosphorylation of eEF2 [60], suggesting that translation elongation is inhibited. However, other translational components are also modu- lated under this condition [60–62] and multiple effects probably contribute to the inhibition of protein synthesis observed during ischaemia. Recent work in the authors’ laboratory shows that mild energy depletion, achieved by incubation of cells with 2-deoxyglucose, a metabolic poison, results in a marked increase in the phosphorylation of eEF2 [63]. Low concen- trations of 2-deoxyglucose do not affect the regulation of other targets for mTOR signalling indicating this effect is not connected with the proposed role of mTOR as an ATP- sensor [64]. 2-Deoxyglucose treatment is expected to decrease cellular ATP levels and cause a rise in cellular AMP concentrations. This leads to activation of the AMP- activated protein kinase (AMPK), an important sensor of cellular energy status [65]. AMPK phosphorylates and inactivates proteins involved in energy consuming processes and, conversely, activates proteins that can enhance cellular energy production. In many types of cells, AMPK can be activated by treatment with AICA riboside. To test whether AMPK plays a role in regulating eEF2 phosphorylation, Chinese hamster ovary cells or hepatocytes were treated with AICA riboside. This gave rise to a robust increase in the phosphorylation of eEF2, so that, for example, around 75% of the protein was phosphorylated in AICA riboside- treated hepatocytes (compared with around 10% in controls) [63]. Treatment of hepatocytes with AICA ribo- side or anoxic conditions leads to inhibition of protein synthesis, as well as to increased phosphorylation of eEF2. eEF2 phosphorylation also increases when HEK293 cells are treated with oligomycin (which blocks mitochondrial ATP synthesis). This effect is prevented by expression of a dominant-interfering mutant of AMPK [63]. These data strongly suggest that the AMPK mediates the effects of modest ATP depletion on the phosphorylation of eEF2. AMPK does not directly phosphorylate eEF2 at Thr56, suggesting its effects are mediated through modulation of eEF2 kinase or possibly of the phosphatase acting on eEF2 (Fig. 3). The regulation of eEF2 phosphorylation and thus of elongation represent novel targets for regulation by AMPK, linking a major energy-consuming process to the availability of metabolic energy. The potential physiological significance of these observa- tions is clear: under conditions of modest energy deficit, activation of AMPK leads to increased phosphorylation of eEF2 and thus to inhibition of elongation and of protein synthesis (Fig. 3). This helps conserve valuable metabolic energy for the most essential cellular processes. By inhibiting elongation rather than initiation, polysomes are conserved, so that protein synthesis can quickly resume when energy metabolism recovers. It is less clear how activation of AMPK causes increased phosphorylation of eEF2, and work is underway to address this. REGULATION OF PHOSPHATASE ACTIVITY AGAINST eEF2 As noted above, the main phosphatase(s) acting on eEF2 appear to be those that are highly sensitive to okadaic acid such as PP2A. Interestingly, PP2A and its close relatives are implicated in TOR signalling and the regulation of trans- lation in yeast [66,67]. However, in this case regulation primarily involves the control of translation initiation. This involves the phosphatase-binding protein Tap42 [66], which binds to PP2A and related enzymes. TOR signalling promotes the interaction of Tap42 with these phosphatases and may thereby alter their activity or specificity [68]. Mammalian cells possess a related protein, a4, which interacts with PP2A and also with PP4 and PP6 [69,70]. It is therefore possible that mTOR also regulates protein phos- phatase activity, via a4 [71], and this may also contribute to the control of the phosphorylation of eEF2. Direct evidence suggesting that a4 regulates eEF2 phosphorylation was provided by the finding that transient overexpression of a4 decreased the level of eEF2 phosphorylation [70]. Overex- pression of a4 did not affect S6K1, another target of mTOR signalling, which regulates eEF2 kinase (see above). Thus, as depicted in Fig. 4, mTOR may potentially regulate eEF2 phosphorylation both via eEF2 kinase and via modulation of phosphatase activity. PHOSPHORYLATION OF eEF2 IN NONMAMMALIAN SPECIES In eEF2 from metazoa, the sequence around the equivalent of Thr56 is strongly conserved, indeed almost identical to Ó FEBS 2002 Control of translation elongation (Eur. J. Biochem. 269) 5365 that in mammals. Consistent with this, eEF2 from the insect Spodoptera frugiperda is a substrate for the mammalian eEF2 kinase [72]. However, there is no evidence that insect cells contain a kinase that can phosphorylate eEF2 [72] and, as mentioned above, the published genome data do not reveal a kinase homologous to mammalian eEF2 kinase [73]. The phosphorylation site at Thr56 is conserved in eEF2 from brewer’s yeast, but not in certain other yeast species, and the known yeast genomes do not contain homologues of eEF2 kinase. Thus, the reported phosphorylation of eEF2 from Saccharomyces cerevisiae must involve a differ- ent kinase [74]. The site of phosphorylation is unknown. Regulation of eEF2 by phosphorylation at Thr56 is so far confined to mammalian systems (and perhaps C. elegans). PERSPECTIVES Recent research efforts have shed important new light on the regulation of eEF2 phosphorylation and eEF2 kinase. Important goals for future work must include studies on the interplay between phosphorylation sites in the regulation of eEF2 kinase, and the identification of the kinase(s) acting at the novel mTOR-regulated sites. Indeed, identification of these kinases may well provide important information on the molecular mechanisms by which mTOR signals to other cellular components such as the initiation factor eIF4E- binding proteins and the ribosomal protein S6 kinases [10,75]. In the case of eEF2 kinase, outstanding questions concern its three-dimensional structure – in particular how its catalytic domain compares with those of other kinases including the only a-kinase studied to date, ChaK; the molecular mechanisms by which Ca/calmod- ulin and phosphorylation regulate its activity; and how its extreme C-terminus interacts with and recruits its substrate, eEF2. Further work will also be required to elucidate the physiological role of phosphorylation of subunits of eEF1A/eEF1B in modulating their activity and/or control- ling protein synthesis in vivo. ACKNOWLEDGEMENTS We gratefully acknowledge financial support from the Biotechnology and Biological Sciences Research Council, the Medical Research Council and the British Heart Foundation for our research on the control of elongation. REFERENCES 1. Proud, C.G. (2002) Regulation of mammalian translation factors by nutrients. Eur. J. Biochem. 269, 5338–5349. 2. Scheper, G.C. & Proud, C.G. (2002) Does phosphorylation of the cap-binding protein eIF4E play a role in translation initiation? Eur. J. Biochem. 269, 5350–5359. 3. Gingras, A C., Raught, B. & Sonenberg, N. (2001) Regulation of translation initiation by FRAP/mTOR. Genes Dev. 15, 807–826. 4. Proud, C.G. (2001) Regulation of eukaryotic initiation factor eIF2B. Prog. Mol. Subcell. Biol. 26, 95–114. 5. Macdonald, P. (2001) Diversity in translational regulation. Curr. Opin. Cell. Biol. 13, 326–331. 6. Dever, T.E. (2002) Gene-specific regulation by general translation factors. Cell 108, 545–556. 7. Merrick, W.C. & Nyborg, J. (2000) The protein biosynthesis elongation cycle. In Translational Control of Gene Expression. (Sonenberg, N., Hershey, J.W.B. & Mathews, M.B., eds), pp. 89–125. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 8. Rhoads, R.E. (1999) Signal transduction pathways that regulate eukaryotic protein synthesis. J. Biol. Chem. 274, 30337–30340. 9. Raught, B., Gingras, A C. & Sonenberg, N. (2000) Regulation of ribosome recruitment in eukaryotes. In Translational Control of Gene Expression. (Sonenberg, N., Hershey, J.W.B. & Mathews, M.B., eds), pp. 245–293. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 10. Avruch, J., Belham, C., Weng, Q., Hara, K. & Yonezawa, K. (2001) The p70, S6 kinase integrates nutrient and growth signals to control translational capacity. Prog. Mol. Subcell. Biol. 26, 115–154. 11. Carr-Schmid, A., Valente, L., Loik, V.I., Williams, T., Starita, L.M. & Kinzy, T.G. (1999) Mutations in elongation factor 1b,a guanine nucleotide exchange factor, enhance translational fidelity. Mol. Cell. Biol. 19, 5257–5266. 12. Sheu, G.T. & Traugh, J.A. (1997) Recombinant subunits of mammalianelongationfactor1expressedinEscherichia coli: subunit interactions, elongation activity, and phosphorylation by protein kinase CKII. J. Biol. Chem. 272, 33290–33297. 13. Traugh, J.A. (2001) Insulin, phorbol ester and serum regulate the elongation phase of protein synthesis. Prog.Mol.Subcell.Biol.26, 33–48. 14. Sheu, G.T. & Traugh, J.A. (1999) A structural model for elon- gation factor (EF-1) and phosphorylation by protein kinase CKII. Mol. Cell. Biochem. 191, 181–186. 15. Wang, X., Paulin, F.E.M., Campbell, L.E., Gomez, E., O’Brien, K., Morrice, N. & Proud, C.G. (2001) Eukaryotic initiation factor 2B: identification of multiple phosphorylation sites in the epsilon subunit and their roles in vivo. EMBO J. 20, 4349–4359. 16. Venema, R.C., Peters, H.I. & Traugh, J.A. (1991) Phosphoryla- tion of elongation factor 1 (EF-1) and valyl-tRNA synthetase by protein kinase C and stimulation of EF-1 activity. J. Biol. Chem. 266, 12574–12580. 17. Venema, R.C., Peters, H.I. & Traugh, J.A. (1991) Phosphoryla- tion of valyl-tRNA synthetase and elongation factor 1 in response to phorbol esters is associated with stimulation of both activities. J. Biol. Chem. 266, 11993–11998. 18. Chang, Y.W. & Traugh, J.A. (1997) Phosphorylation of elonga- tion factor-1 and ribosomal protein S6 by multipotential S6 kinase and insulin stimulation of translational elongation. J. Biol. Chem. 272, 28252–28257. 19. Peters, H.I., Chang, Y.W.E. & Traugh, J.A. (1995) Phosphor- ylation of elongation factor-1 (EF-1) by protein kinase C stimu- lates GDP/GTP-exchange activity. Eur. J. Biochem. 234, 550–556. 20. Belle, R., Derancourt, J., Poulhe, R., Capony, J.P., Ozon, R. & Mulner-Lorillon, O. (1989) A purified complex from Xenopus oocytes contains a p47 protein, an in vivo substrate of MPF, and a p30 protein respectively homologous to elongation factors EF-1 gamma and EF-1 beta. FEBS Lett. 255, 101–104. 21. Janssen, G.M., Morales, J., Schipper, A., Labbe, J.C., Mulner- Lorillon, O., Belle, R. & Moller, W. (1991) A major substrate of maturation promoting factor identified as elongation factor 1bcd in Xenopus laevis. J. Biol. Chem. 266, 14885–14888. 22. Mulner-Lorillon, O., Minella, O., Cormier, P., Capony, J.P., Cavadore, J., Morales, J., Poulhe, R. & Belle, R. (1994) Elonga- tion factor EF-1delta, a new target for maturation- promoting factor in xenopus oocytes. J. Biol. Chem. 269, 20201–20207. 23. Ovchinnikov, L.P., Motuz, L.P., Natapov, P.G., Averbuch, L.J., Wettenhall, R.E., Szyszka, R., Kramer, G. & Hardesty, B. (1990) Three phosphorylation sites in elongation factor 2. FEBS Lett. 275, 209–212. 5366 G. J. Browne and C. G. Proud (Eur. J. Biochem. 269) Ó FEBS 2002 24. Price, N.T., Redpath, N.T., Severinov, K.V., Campbell, D.G., Russell, J.M. & Proud, C.G. (1991) Identification of the phos- phorylation sites in elongation factor-2 from rabbit reticulocytes. FEBS Lett. 282, 253–258. 25. Nygard, O. & Nilsson, L. (1990) Translational dynamics. Inter- actions between the translational factors, tRNA and ribo- somes during eukaryotic protein synthesis. Eur. J. Biochem. 191, 1–17. 26. Ryazanov, A.G. & Davydova, E.K. (1989) Mechanism of elon- gation factor 2 (EF-2) inactivation upon phosphorylation. Phos- phorylated EF-2 is unable to catalyze translocation. FEBS Lett. 251, 187–190. 27. Redpath, N.T., Price, N.T., Severinov, K.V. & Proud, C.G. (1993) Regulation of elongation factor-2 by multisite phosphorylation. Eur. J. Biochem. 213, 689–699. 28. Carlberg, U., Nilsson, A. & Nygard, O. (1990) Functional prop- erties of phosphorylated elongation factor 2. Eur. J. Biochem. 191, 639–645. 29. Redpath, N.T. & Proud, C.G. (1989) The tumour promoter okadaic acid inhibits reticulocyte-lysate protein synthesis by increasing the net phosphorylation of elongation factor 2. Biochem. J. 262, 69–75. 30. Redpath, N.T. & Proud, C.G. (1990) Activity of protein phos- phatases against initiation factor-2 and elongation factor-2. Biochem. J. 272, 175–180. 31. Ryazanov, A.G. (1987) Ca 2+ /calmodulin-dependent phosphory- lation of elongation factor 2. FEBS Lett. 214, 331–334. 32. Nairn, A.C. & Palfrey, H.C. (1987) Identification of the major M r 100,000 substrate for calmodulin-dependent protein kinase III in mammalian cells as elongation factor-2. J. Biol. Chem. 262, 17299– 17303. 33. Hincke, M.T. & Nairn, A.C. (1992) Phosphorylation of elonga- tion factor 2 during Ca 2+ -mediated secretion from rat parotid acini. Biochem. J. 282, 877–882. 34. Mackie, K.P., Nairn, A.C., Hampel, G., Lam, G. & Jaffe, E.A. (1989) Thrombin and histamine stimulate the phosphorylation of elongation factor 2 in human umbilical vein endothelial cells. J. Biol. Chem. 264, 1748–1753. 35. Celis, J.E., Madsen, P. & Ryazanov, A.G. (1990) Increased phos- phorylation of elongation factor 2 during mitosis in transformed human amnion cells correlates with a decreased rate of protein synthesis. Proc.Natl.Acad.Sci.USA87, 4231–4235. 36. Mitsui, K., Brady, M., Palfrey, H.C. & Nairn, A.C. (1993) Puri- fication and characterization of calmodulin-dependent protein kinase III from rabbit reticulocytes and rat pancreas. J. Biol. Chem. 268, 13422–13433. 37. Redpath, N.T. & Proud, C.G. (1993) Purification and phosphor- ylation of elongation factor-2 kinase from rabbit reticulocytes. Eur. J. Biochem. 212, 511–520. 38. Marin, P., Nastiuk, K.L., Daniel, N., Girault, J., Czernik, A.J., Glowinski, J., Nairn, A.C. & Premont, J. (1997) Glutamate dependent phosphorylation of elongation factor 2 and inhibition of protein synthesis in neurons. J. Neurosci. 17, 3445–3454. 39. Redpath, N.T., Price, N.T. & Proud, C.G. (1996) Cloning and expression of cDNA encoding protein synthesis elongation factor- 2kinase.J. Biol. Chem. 271, 17547–17554. 40. Ryazanov, A.G., Ward, M.D., Mendola, C.E., Pavur, K.S., Dorovkov, M.V., Wiedmann, M., Erdjument-Bromage, H., Tempst, P., Parmer, T.G., Prostko, C.R., Germino, F.J. & Hait, W.N. (1997) Identification of a new class of protein kinases represented by eukaryotic elongation factor-2 kinase. Proc. Natl. Acad.Sci.USA94, 4884–4889. 41. Ryazanov, A.G., Pavur, K.S. & Dorovkov, M.V. (1999) Alpha kinases: a new class of protein kinases with a novel catalytic domain. Curr. Biol. 9, R43–R45. 42. Ryazanov, A.G. (2002) Elongation factor-2 kinase and its newly discovered relatives. FEBS Lett. 514, 26–29. 43. Diggle, T.A., Seehra, C.K., Hase, S. & Redpath, N.T. (1999) Analysis of the domain structure of elongation factor-2 kinase by mutagenesis. FEBS Lett. 457, 189–192. 44. Pavur, K.S., Petrov, A.N. & Ryazanov, A.G. (2000) Mapping the functional domains of elongation factor-2 kinase. Biochemistry 39, 12216–12224. 45. Yamaguchi, H., Matsushita, M., Nairn, A.C. & Kuriyan, J. (2001) Crystal structure of the atypical protein kinase domain of a TRP channel with phosphotransferase activity. Mol. Cell 7, 1047–1057. 46. Hait, W.N., Ward, M.D., Trakht, I.N. & Ryazanov, A.G. (1996) Elongation factor-2 kinase – immunological evidence for the existence of tissue-specific isoforms. FEBS Lett. 397, 55–60. 47. McLeod, L.E., Wang, L. & Proud, C.G. (2001) b-Adrenergic agonists increase phosphorylation of elongation factor 2 in car- diomyocytes without eliciting calcium-independent eEF2 kinase activity. FEBS Lett. 489, 225–228. 48. Redpath, N.T. & Proud, C.G. (1993) Cyclic AMP-dependent protein kinase phosphorylates rabbit reticulocyte elongation fac- tor-2 kinase and induces calcium-independent activity. Biochem. J. 293, 31–34. 49. Diggle, T.A., Subkhankulova, T., Lilley, K.S., Shikotra, N., Willis, A.E. & Redpath, N.T. (2001) Phosphorylation of elonga- tion factor-2 kinase on serine 499 by cAMP-dependent protein kinase induces Ca 2+ /calmodulin-independent activity. Biochem. J. 353, 621–626. 50.Diggle,T.A.,Redpath,N.T.,Heesom,K.J.&Denton,R.M. (1998) Regulation of protein synthesis elongation factor-2 kinase by cAMP in adipocytes. Biochem. J. 336, 525–529. 51. Hovland, R., Eikhom, T.S., Proud, C.G., Cressey, L.I., Lanotte, M., Doskeland, S.O. & Houge, G. (1999) cAMP inhibits trans- lation by inducing Ca 2+ /calmodulin-independent elongation fac- tor 2 kinase activity in IPC-81 cells. FEBS Lett. 444, 97–101. 52. Redpath, N.T., Foulstone, E.J. & Proud, C.G. (1996) Regulation of translation elongation factor-2 by insulin via a rapamycin- sensitive signalling pathway. EMBO J. 15, 2291–2297. 53. Wang, L., Wang, X. & Proud, C.G. (2000) Activation of mRNA translation by insulin in rat cardiomyocytes involves multiple rapamycin-sensitive steps. Am. J. Physiol. 278, H1056–H1068. 54. Wang, X., Li, W., Williams, M., Terada, N., Alessi, D.R. & Proud, C.G. (2001) Regulation of elongation factor 2 kinase by p90 RSK1 and p70, S6 kinase. EMBO J. 20, 4370–4379. 55. Everett, A.D., Stoops, T.D., Nairn, A.C. & Brautigan, D. (2001) Angiotensin II regulates phosphorylation of translation elonga- tion factor-2 in cardiac myocytes. Am.J.Physiol.281, H161–H167. 56. Wang, L. & Proud, C.G. (2002) Ras/Erk signalling is required for regulation of translation factors linked to mTOR and protein synthesis by hypertrophic agents in cardiomyocytes. Circ. Res. in press. 57. Knebel, A., Morrice, N. & Cohen, P. (2001) A novel method to identify protein kinase substrates: eEF2 kinase is phosphorylated and inhibited by SAPK4/p38delta. EMBO J. 20, 4360–4369. 58. Patel, J., McLeod, L.E., Vries, R.G., Flynn, A., Wang, X. & Proud, C.G. (2002) Cellular stresses profoundly inhibit protein synthesis and modulate the states of phosphorylation of multiple translation factors. Eur. J. Biochem. 269, 3076–3085. 59. Knebel, A., Haydon, C.E., Morrice, N. & Cohen, P. (2002) Stress- induced regulation of eEF2 kinase by SB203580-sensitive and – insensitive pathways. Biochem. J. 367, 525–532. 60. Althausen, S., Mengesdorf, T., Mies, G., Olah, L., Nairn, A.C., Proud, C.G. & Paschen, W. (2001) Changes in the phosphoryla- tion of initiation factor eIF-2a, elongation factor eEF-2 and p70, S6 kinase after transient focal cerebral ischaemia in mice. J. Neurochem. 78, 779–787. 61. DeGracia, D.J., Neumar, R.W., White, B.C. & Krause, G.S. (1996) Global brain ischemia and reperfusion: modifications in eukaryotic initiation factors associated with inhibition of transla- tion initiation. J. Neurochem. 67, 2005–2012. Ó FEBS 2002 Control of translation elongation (Eur. J. Biochem. 269) 5367 62. Martin de la Vega, C., Burda, J., Nemethova, M., Quevedo, C., Alcazar,A.,Martin,M.E.,Danielsova,V.,Fando,J.L.&Salinas, M. (2001) Possible mechanisms involved in the down-regulation of translation during transient global ischaemia in the rat brain. Biochem. J. 357, 819–826. 63. Horman, S., Browne, G.J., Krause, U., Patel, J.V., Vertommen, D., Bertrand, L., Lavoinne, A., Hue, L., Proud, C.G. & Rider, M.H. (2002) Activation of AMP-activated protein kinase leads to the phosphorylation of elongation factor 2 and an inhibition of protein synthesis. Curr. Biol. 12, 1419–1423. 64. Dennis, P.B., Jaeschke, A., Saitoh, M., Fowler, B., Kozma, S.C. & Thomas, G. (2001) Mammalian TOR: a homeostatic ATP sensor. Science 294, 1102–1105. 65. Hardie, D.G., Carling, D. & Carlson, M. (1998) The AMP-acti- vated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell? Annu.Rev.Biochem.67, 821–855. 66. Di Como, C.J. & Arndt, K.T. (1996) Nutrients, via the TOR proteins, stimulate the association of Tap42 with Type phospha- tases. Genes Dev. 10, 1904–1916. 67. Barbet, N.C., Schneider, U., Helliwell, S.B., Stansfield, I. & Tuite, M.F. (1996) TOR controls translation initiation and early G1 progression in yeast. Mol. Biol. Cell 7, 25–42. 68. Jiang, Y. & Broach, J.R. (1999) Tor proteins and protein phos- phatase 2A reciprocally regulate Tap42p in controlling cell growth in yeast. EMBO J. 18, 2782–2792. 69. Chen, J., Peterson, R.T. & Schreiber, S.L. (1998) Alpha-4 associates with protein phosphatases 2A, 4 and 6. Biochem. Biophys. Res. Commun. 247, 827–832. 70. Chung, H., Nairn, A.C., Murata, K. & Brautigan, D.L. (1999) Mutation of Tyr307 and Leu309 in the protein phosphatase 2A catalytic subunit favours association with the alpha4 subunit which promotes dephosphorylation of elongation factor-2. Biochemistry 10, 10371–10376. 71. Peterson, R.T., Desai, B.N., Hardwick, J.S. & Schreiber, S.L. (1999) Protein phosphatase 2A interacts with the 70 kDa S6 kinase and is activated by inhibition of FKBP-12-rapamycin-associated protein. Proc. Natl. Acad. Sci. USA 96, 4438–4442. 72. Oldfield, S. & Proud, C.G. (1993) Phosphorylation of elongation factor-2 from the lepidopteran insect, Spodoptera frugiperda. FEBS Lett. 327, 71–74. 73. Morrison, D.K., Murakami, M.S. & Cleghon, V. (2000) Protein kinases and phosphatases in the Drosophila genome. J. Cell. Biol. 150, F57–F62. 74. Donovan, M.G. & Bodley, J.W. (1991) Saccharomyces cerevisiae elongation factor 2 is phosphorylated by an endogenous kinase. FEBS Lett. 291, 303–306. 75. Gingras, A C., Raught, B., Gygi, S.P., Niedzwieka, A., Miron, M., Burley, S.K., Polakiewicz, R.D., Wyslouch-Cieczyska, A., Aeber- sold, R. & Sonenberg, N. (2001) Hierarchical phosphorylation of the translation inhibitor 4E-BP1. Genes Dev. 15, 2852–2864. 5368 G. J. Browne and C. G. Proud (Eur. J. Biochem. 269) Ó FEBS 2002 . activity of eEF2. Phosphorylation of eEF2 inhibits its activity, in translo- cation and in poly(U)-directed polyphenylalanine synthesis [26,27], by preventing. myocytes, suggesting that these cells may contain a distinct isoform of eEF2 kinase, as mentioned above. In heart cells, increasing the maximal activity of