Báo cáo Y học: Regulation of peptide-chain elongation in mammalian cells pptx

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:
(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.
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