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

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.

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

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

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

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

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

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

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-

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

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.

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

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