Báo cáo Y học: Regulation of mammalian translation factors by nutrients pot

MINIREVIEW
Regulation of mammalian translation factors by nutrients
Christopher G. Proud
Division of Molecular Physiology, School of Life Sciences, University of Dundee, MSI/WTB Complex, Dow Street, UK
Protein synthesis requires both amino acids, as precursors,
and a substantial amount of metabolic energy. It is well
established that starvation or lack of nutrients impairs pro-
tein synthesis in mammalian cells and tissues. Branched
chain amino acids are particularly effective in promoting
protein synthesis. Recent work has revealed important new
information about the mechanisms involved in these effects.
A number of components of the translational machinery are
regulated through signalling events that require the mam-
malian target of rapamycin, mTOR. These include transla-
tional repressor proteins (eukaryotic initiation factor
4E-binding proteins, 4E-BPs) and protein kinases that act
upon the small ribosomal subunit (S6 kinases). Amino acids,
especially leucine, positively regulate mTOR signalling
thereby relieving inhibition of translation by 4E-BPs and
activating the S6 kinases, which can also regulate translation
elongation. However, the molecular mechanisms by which
amino acids modulate mTOR signalling remain unclear.
Protein synthesis requires a high proportion of the cell’s
metabolic energy, and recent work has revealed that meta-
bolic energy, or fuels such as glucose, also regulate targets of
the mTOR pathway. Amino acids and glucose modulate a
further important regulatory step in translation initiation,
the activity of the guanine nucleotide-exchange factor
eIF2B. eIF2B controls the recruitment of the initiator
methionyl-tRNA to the ribosome and is activated by insulin.
However, in the absence of glucose or amino acids, insulin

no longer activates eIF2B. Since control of eIF2B is inde-
pendent of mTOR, these data indicate the operation of
additional, and so far unknown, regulatory mechanisms that
control eIF2B activity.
Keywords: translation; elongation factor; mTOR; amino
acid; glucose; initiation factor.
INTRODUCTION
It has long been known that starvation or lack of nutrients
influence protein synthesis rates in mammalian tissues and
cells. This is not unexpected given that protein synthesis
requires both amino acids, as precursors, and metabolic
energy. Indeed, protein synthesis is one of the major energy
consuming processes of the cell. Recent advances in
understanding of the mechanism of translation and its
control have facilitated studies at the molecular level into
the regulation of protein synthesis by nutrients, and the
interplay between nutrients and hormonal signals. An
important finding in the last few years is that a number of
components of the translational machinery in mammalian
cells are subject to acute regulation by the nutrient status of
the cell. Regulation of most of these components is linked to
the rapamycin-sensitive mTOR (mammalian target of
rapamycin) signalling pathway. These targets for mTOR
signalling include regulators of translation initiation and
elongation, and protein kinases acting on the small
ribosomal subunit. This knowledge has allowed investiga-
tors to return to the key issue of placing this improved
knowledge in a physiological context, and studying the
regulation of protein synthesis by nutrients in physiologi-
cally important tissues such as skeletal muscle. This article

reviews our current understanding of the regulation of
translation factors by nutrients and recent studies applying
this information to tissues such as pancreatic b-cells, skeletal
muscle and heart. Early data suggested that, in muscle
in vivo, the rate of elongation may limit protein synthesis
under fed conditions [1] while initiation may be limiting in
starved animals [2]. Recent work has improved our
understanding of the molecular mechanisms involved in
regulating both translation initiation and elongation.
Early studies focused on the control of protein synthesis
in skeletal muscle, as it is a tissue of particular importance
for whole body protein metabolism. Overnight fasting led to
the disaggregation of polyribosomes in rat skeletal muscle
[1] indicating an impairment of translation initiation.
Fasting of animals for longer periods involved an additional
reduction in the levels of ribosomes in the tissue, manifested
as a fall in its RNA content (the bulk of cellular RNA is
ribosomal RNA) [3]. In this article I shall discuss mecha-
nisms by which nutrients regulate both translation initiation
and ribosome biogenesis.
REGULATION OF eIF4E
BY eIF4E-BINDING PROTEINS
The eukaryotic initiation factor (eIF) 4E binds to the 5¢-cap
structure of eukaryotic mRNAs and likely provides the first
contact between the translational machinery and the
mRNA in de novo translation initiation. eIF4E also interacts
with several types of protein binding partners. One class
comprises the scaffold proteins of the eIF4G group (eIF4G
I
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, 5338–5349 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03292.x
and eIF4G
II
). eIF4G interacts with a number of other
proteins. These include eIF4A, an RNA helicase; the
poly(A)-binding protein, PABP; the multisubunit initiation
factor, eIF3, which provides a link to the 40S subunit; and
the eIF4E kinases, Mnk1 and Mnk2 [4,5] (Fig. 1A). The
eIF4E/4G/4A complex is often referred to as the eIF4F
complex, although the other components are also likely to
be associated with such complexes under physiological
conditions. Such complexes are thought to be of key
importance in mediating normal, cap-dependent, transla-
tion initiation. The second group of eIF4E-interacting
proteins comprises low molecular mass proteins that bind to
the same (or an overlapping) site on eIF4E and block its
interaction with eIF4G (Fig. 1A). In mammals, three
eIF4E-binding proteins are known (4E-BP1/2/3) and they
will be discussed below. A third type of partner for eIF4E is
the nucleocytoplasmic shuttling protein, 4E-T [6], which is
considered to be important in conveying eIF4E from the
cytoplasm into the nucleus, where a significant proportion
of the cellular eIF4E is found [7]. These proteins (eIF4Gs,
4E-BPs, 4E-T) share a common motif through which they

interact with the same, or overlapping, sites in eIF4E [6,8].
Binding is therefore mutually exclusive and, for example,
eIF4E bound to 4E-BP1 cannot interact with eIF4G to
form initiation complexes. 4E-BP1 thus acts as a repressor
of cap-dependent translation [9,10].
Of the three 4E-BPs, 4E-BP1 is easily the most intensively
studied and best understood. It undergoes phosphorylation
at multiple sites in vivo.AsindicatedinFig.1B,thesesites
are located almost throughout its short sequence of around
118 amino acids, only the N-terminus being devoid of sites
of phosphorylation. Phosphorylation of 4E-BP1 shows a
marked hierarchy in vivo [11,12](X.Wang,W.Li,J.L.Parra,
A. Beugent & C.G. Proud, unpublished observations).
Phosphorylation of the threonines near the N-terminus is
required for modification of Thr70, while phosphorylation
at Thr70 is required for phosphorylation of Ser65. Earlier
data suggested that Ser65 and Thr70 were the most
important sites for modulating the binding of 4E-BP1 to
eIF4E – phosphorylation at Thr70 promotes its release and
phosphorylation at Ser65 may prevent rebinding (Fig. 1B).
Phosphorylation of Ser112, at the extreme C-terminus, also
appears to be required for release of 4E-BP1 from eIF4E
([14]; C. G. Proud, unpublished data).
Phosphorylation of several sites in 4E-BP1 is increased by
agents that activate protein synthesis, such as insulin.
Phosphorylation of Ser65 and Thr70, and to a lesser extent,
Thr37/46, is blocked by rapamycin, indicating an essential
role for mTOR in signalling from, e.g., the insulin-receptor
to 4E-BP1 (reviewed in [15]). The complex nature of the
hierarchy of phosphorylation of the other sites in 4E-BP1

suggests that they are targets for a range of proline-directed
kinases that also await identification. Several kinases have
been found to phosphorylate 4E-BP1 in vitro, including
Erk16, but it remains to be established which kinases
actually act on 4E-BP1 in vivo. Several of these, especially
those acting at Ser65 and Thr70, the sites most profoundly
affected by rapamycin, may be regulated by nutrients
through the mTOR pathway.
REGULATION OF 4E-BP1 BY AMINO
ACIDS
In a number of kinds of mammalian cells, amino acids exert
marked effects on the phosphorylation and regulation of 4E-
BP1 (reviewed in [15,
1
17]). For example, when Chinese
Fig. 1. Regulation of eIF4E by 4E-BPs (A) and schematic structure of
4E-BP1 (B). eIF4E (which binds the 5¢-cap of the mRNA, not shown
but see text) can interact either with 4E-BPs (such as 4E-BP1, shown) or
eIF4G, but not both simultaneously. Phosphorylation of 4E-BP1 at
multiple sites induces its release from eIF4E, allowing it to bind to
eIF4G and form functional initiation factor complexes that also contain
the poly(A)-binding protein PABP, the RNA helicase eIF4A and the
eIF4E kinases (such as Mnk1). eIF4G also interacts with eIF3 and
thereby recruits the 40S subunit to the 5¢-end of the mRNA. eIF4E can
also interact with the shuttling protein 4E-T which is involved in
transferring eIF4E to the nucleus: this again involves a binding site on
eIF4E that is occluded by 4E-BP1. A number of agents increase the
phosphorylation of 4E-BP1 including amino acids and insulin (see text).
Regulation by these stimuli is blocked by rapamycin, indicating an
essential role in this for signalling via mTOR. (B) 4E-BP1 contains a

binding motif for interaction with eIF4E, and two regulatory domains
have also been identified – the RAIP motif towards the N-terminus
[110] and the TOS motif at the extreme C-terminus [111]. Six sites of
phosphorylation have been identified. All except S112 (numbering
based on human sequence) are Ser-Pro or Thr-Pro sites (Ser112 is fol-
lowed by Gln). Inhibition of mTOR or amino acid withdrawal results in
dephosphorylation of a number of sites in 4E-BP1, especially Ser65 and
Thr70 (underlined), although Thr37/46 are also affected. Insulin
stimulates phosphorylation of Ser65, Thr70 and Ser112, while Ser83
appear to be basally phosphorylated. Thin arrows indicate interplay
between sites of phosphorylation that underlies the complex hierarchy
of phosphorylation events, while thick arrows indicate the roles of
specific sites in regulating the function of 4E-BP1. For example, phos-
phorylation at Thr37/46 is required for phosphorylation at Thr70, and
phosphorylation at Thr70 is required for phosphorylation at Ser65.
Ó FEBS 2002 Control of translation factors by nutrients (Eur. J. Biochem. 269) 5339
hamster ovary cells are transferred to a medium lacking
amino acids, 4E-BP1 undergoes dephosphorylation [18–20],
which occurs within 15–30 min after amino acid withdrawal.
In control cells, in medium containing amino acids, little or
no 4E-BP1 is bound to eIF4E and high levels of eIF4F
complexes are present. Removal of amino acids quickly
causes a marked increase in the amount of 4E-BP1 associated
with eIF4E and loss of eIF4F complexes [18,19,21]. These
effects are similar to those of adding rapamycin suggesting
that the effects of amino acids are mediated via the mTOR
pathway. They are reversed, within minutes, by the readdi-
tion of amino acids. The most effective single amino acid is
leucine, others having very little or no effect in most cells. This
role for leucine is a feature that

2
will be discussed later.
However, even at concentrations well above those present in
the cells’ normal medium, leucine cannot induce the level of
4E-BP1 phosphorylation seen in amino acid replete cells,
suggesting that the other amino acids present in growth
medium are also important in this effect. These data show
that amino acids themselves, in the absence of hormones such
as insulin, have marked effects on the phosphorylation of 4E-
BP1 and on the formation of translation initiation factor
complexes. They imply that mammalian cells have the ability
to sense the prevailing availability of amino acids and to relay
this information to the translational machinery.
The situation is subtly different in certain other types of
cells: in human embryonic kidney cells, maintained in
medium without serum, basal 4E-BP1 phosphorylation is
much lower, so that much of the eIF4E is bound to 4E-BP1
and levels of eIF4F complexes are accordingly low [22,23].
Upon addition of insulin or the phorbol ester, TPA, 4E-BP1
undergoes phosphorylation leading to its release from eIF4E
and formation of eIF4F complexes. This does not happen in
cells maintained in medium lacking amino acids (E. Hajduch
& C. G. Proud, unpublished). In contrast, in adult cardio-
myocytes, there is no such requirement for external amino
acids, as insulin can induce phosphorylation of 4E-BP1 in
cells kept in amino acid-free medium (L. Wang
4
&C.G.
Proud, unpublished data). Similarly, in primary adult rat
adipocytes insulin can bring about the phosphorylation of

4E-BP1 in the absence of any external amino acids [24,25].
Insulin elicits an increase in 4E-BP1 phosphorylation in
amino acid-replete cells, and it can still do so to some extent
in CHO cells deprived of amino acids, provided that a
metabolizable glucose analogue (or other metabolizable
hexose such as
D
-mannose) is also present [19]. Glucose
increases the basal level of phosphorylation of Thr70, but
has little effect on basal phosphorylation at Ser65 or Thr37/
46. However, the presence of glucose does allow insulin to
elicit phosphorylation at these sites [19]. Glucose thus exerts
a permissive effect with respect to the action of insulin and
promotes the release of 4E-BP1 from eIF4E to allow
formation of eIF4F complexes. This may reflect an input
from metabolic energy to the control of 4E-BP1, perhaps
via modulation of the activity of mTOR [26]. This will be
discussed in more detail below.
Overall, these data indicate a requirement both for amino
acids (especially leucine) and an energy source for activation
of this key step in translation initiation. This clearly makes
excellent sense – amino acids are the precursor for protein
synthesis, leucine being an essential amino acid, and protein
synthesis consumes a large proportion (perhaps 20–25%
[27]) of total cellular energy.
REGULATION BY AMINO ACIDS IN
PRIMARY TISSUES AND CELL TYPES
While many groups have studied the control of translation
factors in established cell lines, Kimball, Jefferson and
colleagues have extensively investigated the effects of amino

acids on 4E-BP1 in primary cells and in tissues. These
include isolated adipocytes, perfused liver and skeletal
muscle, studied both in vitro and in vivo (reviewed in [28–
30]). Their data again indicate that amino acids exert a
positive effect on the phosphorylation of 4E-BP1, with
leucine being effective when given alone [31–34]. In muscle,
amino acids were required for insulin to enhance the
formation of complexes between eIF4E and eIF4G and the
rate of protein synthesis. These authors have argued that
insulin may serve a permissive function here: for example,
giving leucine alone, orally, to rats activates protein
synthesis and translation initiation as effectively as a
complete meal, but without a rise in plasma insulin
concentration [35]. This again underlines the primary role
of leucine in regulating protein synthesis. In mice with
defective insulin signalling (with similarities to type II
diabetes) feeding still stimulates protein synthesis in a
similar way to the effects observed in control animals [36].
However, marked reduction in circulating insulin levels
(achieved using anti-insulin Ig) does impair the response to
feeding [37–39]. Taken together these data indicate that
insulin is required for the feeding-induced activation of
translation, but that increases in insulin levels may not be,
thus suggesting insulin plays a permissive role here.
REGULATION OF THE S6 KINASES
The protein kinases that phosphorylate ribosomal protein
S6 are a second set of proteins that are regulated via mTOR
and implicated in the control of mRNA translation [40,41]
(Fig. 2). Through alternative splicing, the S6 kinase 1 (also
termed a)and2(b) genes each give rise to two distinct

proteins, yielding a total of four S6 kinases. The activation
of S6K1 and S6K2 by all stimuli so far tested (e.g. insulin,
growth factors and phorbol esters) is blocked by rapamycin
[40,42,43].
Activation of the S6Ks involves their phosphorylation at
multiple sites, some of which lie in the catalytic domain or its
Fig. 2. The S6 kinases. The structures of S6K1 and S6K2 are depicted
schematically, including their splice variants (forms I and II is each
case). The domains within each sequence are indicated, as are major
sites of phosphorylation that are associated with activation of these
enzymes, and the nuclear localization signals (NLS) in S6K1 I and the
S6K2 isoforms. See text for further information.
5340 C. G. Proud (Eur. J. Biochem. 269) Ó FEBS 2002
so-called ÔextensionÕ or ÔlinkerÕ while the majority are located
in the C-terminal regulatory domain [40,41] (Fig. 2). Thr229
in the T-loop of the catalytic domain has been shown to be
phosphorylated by phosphoinositide-dependent kinase 1
(PDK1) in vitro [44,45]. The protein kinases responsible for
phosphorylating the other sites await conclusive identifica-
tion. While mTOR can phosphorylate T389 in vitro, it is not
clear that it is the physiological T389 kinase (discussed in
[40]). The sensitivity of S6K regulation to rapamycin
nevertheless shows that mTOR makes an essential input
to the control of the S6Ks. The interplay between the
phosphorylation sites in S6K1 is complex and for a more
detailed discussion the reader is directed to a recent
comprehensive review [40]. For the present purposes, this
can be summarized to say (a) that phosphorylation of the
sites in the C-terminus of S6K1 is believed to facilitate access
(by the relevant kinases) to Thr229 and Thr389, phos-

phorylation of which is critical for activity; (b) phosphory-
lation is again ordered, with modification of Thr229 and
especially Thr389 occurring later or even last in the
hierarchy; and (c) phosphorylation of multiple sites is
sensitive to rapamycin and therefore to signalling via
mTOR (reviewed in [40,46]).
AMINO ACIDS ARE POSITIVE
REGULATORS OF S6K1
In common with the 4E-BPs, the activity and regulation of
S6K1 is also sensitive to the nutrient status of the cell [40],
although differences are again seen between cell types.
Amino-acid-replete CHO cells show a substantial basal
activity of S6K1 that is further enhanced by addition of
insulin [18,21]. When cells are transferred to amino-acid-free
medium, basal activity falls sharply and S6K1 is refractory
to stimulation by insulin. Removal of even a single amino
acid (especially of arginine or leucine) leads to a marked fall
in S6K1 activity [20]. Addition of amino acids partially
restores both basal activity and insulin-responsiveness,
although the addition of both amino acids and glucose is
required for substantial recovery of both effects [18,20,21].
This situation shows similarities and differences with respect
to that described above for 4E-BP1/eIF4F in these cells. In
both cases, amino acids exert very marked effects in CHO
cells: however, whereas amino acids/glucose suffice for
complete formation of eIF4F complexes and to maintain a
high level of phosphorylation of phosphorylation of
4E-BP1, full activation of S6K1 requires inputs from both
amino acids/glucose and insulin. It is currently unclear
whether the requirement, in CHO cells, for both amino

acids and an additional input (e.g. from insulin) reflects
effects of these agents on different (subsets of) phosphory-
lation sites in S6K1. In this context it is notable that Hara
et al. [20] reported that addition of high levels of amino
acids to CHO cells overexpressing the insulin receptor
resulted in as high a degree of activation of S6K1 as was
observed with normal levels of amino acids plus insulin.
This suggests that amino acids can elicit the full response if
present at sufficiently high levels. It seems likely that in
CHO cells, and probably in other cell types too, amino-acid-
replete cells contain only enough amino acids to give partial
activation of S6K1 and insulin provides a further input to its
activation. Some cell types appear to contain enough amino
acids for regulation of S6K1 even when starved for external
amino acids (e.g. hepatoma cells [47]). The fact that such
cells become dependent upon external amino acids when
treated with a compound that inhibits autophagy suggests
that this intracellular supply of amino acids is derived from
this form of protein breakdown. Autophagy is especially
active in hepatocytes and related cell-types, and this is
perhaps why some other cell types are more dependent on
external amino acids.
The effects on S6K1 of nutrient stimuli and agents such as
insulin are blocked by rapamycin [30]. In fact, removal of
amino acids from CHO cells leads to effects on 4E-BP1 and
S6K1, which are qualitatively similar to those of rapamycin
treatment. Similar data have been reported for a number of
other cell types including adipocytes and HEK 293 cells
[20,48], giving rise to the notion that the effects of amino
acids are transmitted via mTOR, although there is no

formal evidence for this. Evidence in favour of this idea was
provided by Hara et al. [20] who showed that amino acid
deprivation led to complete dephosphorylation of S6K1 at
T389 (T412 in the numbering system used by these authors),
this being a major rapamycin-sensitive and thus mTOR-
controlled phosphorylation site in S6K1 [49]. Furthermore,
and more importantly, they found that a mutant of S6K1
that is resistant to inhibition by rapamycin was also resistant
to the effects of amino acid withdrawal. These data suggest
that amino acids may signal to S6K1 via mTOR. There is as
yet no data on the regulation of S6K2 by amino acids.
However, since, like S6K1, S6K2 is regulated via mTOR, it
is likely that amino acids also modulate the activity of this
enzyme.
Protein synthesis itself consumes amino acids. It has long
been known that cycloheximide (an inhibitor of protein
synthesis) activates S6K1 [50]. Our recent data show that it
facilitates the activation of both S6K1 and 4E-BP1 by
insulin in amino-acid-deprived CHO cells (A. Beugnet et al.
5
,
unpublished data). Consistent with its effect on 4E-BP1
phosphorylation, treatment of cells with cycloheximide also
allows insulin to bring about the release of 4E-BP1 from
eIF4E and the formation of complexes between eIF4E and
eIF4G. Furthermore, three other inhibitors of protein
synthesis (anisomycin, emetine or puromycin) each exert
very similar effects. Their effects are not due to release of
amino acids into the medium. Taken together the data are
consistent with the idea (Fig. 3) that regulation of S6K1 and

other targets for mTOR signalling is influenced by the size
of an intracellular pool of amino acids whose size is
determined by the rates of protein degradation and
synthesis, and by the availability of extracellular amino
acids (which presumably enter this pool following their
transport into the cell [51]).
Studies using ÔrealÕ cells, adipocytes and skeletal muscle,
generally reflect the data obtained in other, transformed, cell
lines. For example, amino acids have been shown to
stimulate S6K1 in rat adipocytes, and this effect is blocked
by rapamycin [48]. However, insulin can activate S6K1 in
isolated rat adipocytes in the absence of added amino acids
[52]. Orally administered leucine elicits the phosphorylation
of S6K1 in skeletal muscle, and this requires insulin, but not
an increase in insulin concentration [53]. In human forearm
muscle, branched-chain amino acids elicit phosphorylation
of S6K1 [54]. These data are largely similar to those
discussed above for the regulation of 4E-BP1 by amino
acids.
Ó FEBS 2002 Control of translation factors by nutrients (Eur. J. Biochem. 269) 5341
A ROLE FOR S6 PHOSPHORYLATION
IN RIBOSOME BIOGENESIS?
Work, in particular from the laboratory of George Thomas,
has suggested that the S6 kinases may play a role in
regulating the translation of a set of mRNAs termed the
5¢-TOP (tract of oligopyrimidine) mRNAs (Fig. 3). This
group of mRNAs includes those for each of the ribosomal
proteins in mammals, and those for certain other proteins
involved in mRNA translation such as the elongation
factors eEF1A and eEF2 [55] and the poly(A)-binding

protein PABP [56]. These mRNAs are characterized by the
presence at their extreme 5¢-ends of a short sequence of
pyrimidines (the 5¢-TOP), which represses their translation
in serum-deprived cells. Following stimulation of the cells
by serum, 5¢-TOP mRNAs such as that for eEF1A shift into
polyribosomes (i.e. initiation onto them is presumably
enhanced [57,58]). Amino acids themselves also promote
increased synthesis of proteins encoded by 5¢-TOP mRNAs
such as eEF1A [59] and ribosomal proteins [60]. This
amino-acid- and hormone-regulated translational control
mechanism provides a way in which synthesis of compo-
nents of the translational machinery can be quickly switched
on following treatment of mammalian cells by an anabolic/
proliferative stimulus, to increase the cellular capacity for
protein synthesis. Defects in this may therefore underlie the
small size phenotype of cells or animals in which S6K genes
have been knocked out [61,62].
The studies of Jefferies and Thomas [57,58,63] indicated
that the translational activation of the 5¢-TOP mRNAs was
inhibited by rapamycin – the drug prevented or reversed
their increased incorporation into polysomes [58,63]. The
same group subsequently reported that expression of a
mutant of S6K1 that is relatively insensitive to rapamycin
resulted in decreased sensitivity of 5¢-TOP mRNA transla-
tion to this drug [63]. This seemed to indicate that S6K1,
and perhaps phosphorylation of S6, was involved in
activating 5¢-TOP mRNA translation. However, more
recent work has challenged this view. Tang et al.[64]
demonstrated that 5¢-TOP mRNA translation is enhanced
by amino acids (which would be consistent with a role for

S6K1) but concluded that, by various criteria, S6 phos-
phorylation did not appear to be sufficient for increased
5¢-TOP mRNA translation, at least in response to amino
acids. For example, amino acid regulation of 5¢-TOP
mRNA translation is still observed in cells in which both
alleles of the S6K1 gene are knocked out and in which no
phosphorylation of S6 is observed in response to amino
acids. This also casts doubt on the role of S6Ks and thus S6
phosphorylation in the control of 5¢-TOP mRNA transla-
tion at least in response to amino acids.
The above findings underline the need for further work to
elucidate the mechanisms by which 5¢-TOP mRNA trans-
lation is controlled and to define the cellular functions of the
S6Ks, which clearly do include roles in events linked to the
control of cell and organism size. One such function that has
recently been reported is in the control of the elongation
factor eEF2.
Elongation factor 2 (eEF2) is regulated through the
mTOR pathway and by cellular energy status eEF2
mediates the translocation step of elongation. Phosphory-
lation of eEF2 at Thr56 inhibits its activity by preventing it
from binding to the ribosome. Phosphorylation of eEF2 is
catalysed by eEF2 kinase, an unusual and highly specific
enzyme. A more detailed discussion of eEF2 and eEF2
kinase can found in the accompanying article by Browne
and Proud [65].
In CHO cells overexpressing the insulin receptor, insulin
brings about the rapid dephosphorylation of eEF2, con-
comitantly with accelerating the rate of elongation. Both
effects were blocked by rapamycin [66]. Insulin also elicits

the dephosphorylation of eEF2 in other types of cells [67–
70], and decreases the activity of eEF2 kinase, an effect
blocked by rapamycin [66,70]. These data suggested a link
between mTOR and the control of eEF2 kinase although
the nature of the links between mTOR and eEF2 kinase
remained unclear for several years. It is now clear that one
link between mTOR and the control of eEF2 kinase
involves the phosphorylation of eEF2 kinase at Ser366 by
Fig. 3. mTOR positively regulates the phosphorylation and function of
the ribosomal protein (rp) S6 kinases and of the eIF4E-binding protein,
4E-BP1. Phosphorylation of S6Ks leads to their activation. S6Ks
phosphorylate rpS6, which is considered to play a role in the regulation
of the translation of the subset of mRNAs containing a 5¢-terminal
tractofoligopyrimidines(5¢-TOP mRNAs). S6Ks also phosphorylate
elongation factor 2 (eEF2) kinase leading to a decrease in its activity at
basal calcium concentrations. Inactivation of eEF2 kinase facilitates
the dephosphorylation of eEF2 and the activation of elongation. There
appear to be additional mTOR-dependent inputs into the phos-
phorylation and control of eEF2 kinase (see accompanying article by
Browne and Proud [65]). Phosphorylation of 4E-BP1 at certain sites
(see Fig. 1B) leads to its release from eIF4E, which can then interact
with eIF4G to form initiation complexes than can recruit the 40S
ribosomal subunit to the 5¢-end of the mRNA. Amino acids and cel-
lular energy act as positive modulators of mTOR. The mechanisms by
which amino acids exert this effect are unclear, but recent evidence
suggests this may involve sensing of intracellular amino acid levels.
Insulin and a range of other stimuli increase the phosphorylation of
S6Ks and 4E-BP1. However, it is not clear (??) whether they do so by
modulating the activity/function of mTOR, or whether they provide
separate inputs that nonetheless require the mTOR-dependent input.

The question mark by the role of S6 phosphorylation in the translation
of 5¢-TOP mRNAs denotes the fact that Tang et al. [64] have recently
challenged the prevailing concept that these mRNAs are regulated via
S6 kinases/phosphorylation of rpS6, at least in response to amino
acids. The question mark by the role of ATP in regulating mTOR
activity [26] is to indicate that recent data also suggest a role for the
AMP-activated kinase in regulating mTOR signalling in skeletal
muscle (see text [88]).
5342 C. G. Proud (Eur. J. Biochem. 269) Ó FEBS 2002
S6K1, which results in decreased activity of eEF2 kinase
[69]. Given these inputs from mTOR into the control of
eEF2 kinase, one might anticipate that the phosphorylation
of eEF2 would be modulated by nutrients. Indeed, insulin
cannot fully elicit the dephosphorylation of eEF2 in CHO
cells lacking amino acids or glucose [21].
A further input from the nutritional status of the cells to
the control of eEF2 phosphorylation appears to be related
to cellular ATP levels, and may underlie the requirement for
glucose referred to above. It clearly makes physiological
sense for protein synthesis to be matched to energy
availability: protein synthesis is a major energy-consuming
process, using up around 25–30% of total cellular energy
[27]. This is discussed further in the article by Browne and
Proud [65] that accompanies this one.
mTOR
Frequent mention of mTOR in the regulation of translation
has been made in this article without any discussion, so far,
of this protein itself. mTOR is large (around 290 kDa) and
its primary sequence indicates the presence of a number of
potential functional domains [71] (Fig. 4). These include a

series of so-called HEAT domains towards its N-terminus,
which are likely to be involved in protein–protein interac-
tions and a domain with similarity to lipid kinases towards
its C-terminus (Fig. 4). This region shows similarity to the
phosphoinositide (PI) kinases although mTOR has not been
shown to phosphorylate any lipid substrates. Similar
domains are also found in several protein kinases such as
the ataxia talengiectasia (mutated) kinase ATM and the
related kinase ATR. As noted above, mTOR can also
phosphorylate certain proteins (e.g. 4E-BP1 and S6K1, see,
e.g [72–74]), at least in vitro and in the presence of
unphysiologically high concentrations of Mn
2+
-ions. It
remains to be established that its in vivo function is as a
protein kinase and that it does indeed act on proteins such
as 4E-BP1 and S6K1 in vivo. Nonetheless, an intact kinase
domain is essential for the function of yeast TOR (see [71])
or of mTOR [73,75] and rapamycin inhibits the kinase
activity of mTOR.
In addition to possessing in vitro kinase activity itself,
mTOR has been shown to be associated with other protein
kinase activities, which can be separated from mTOR, e.g.
by treatment of mTOR immunoprecipitates with detergent
[76,77]. This illustrates that these kinase activities are not
intrinsic to mTOR but, presumably, arise from proteins
noncovalently bound to mTOR, perhaps via its HEAT
domains. The role of these extrinsic kinases in the functions
of mTOR and in downstream signalling to proteins such as
4E-BP1, the S6Ks and eEF2 kinase is a potentially

important area for future study. One further profitable area
for investigation is likely to be the identification of proteins
that interact with mTOR; this may shed important light on
the regulation of mTOR and on downstream signalling
from mTOR to the translational machinery. Indeed, two
recent studies identified a novel 150 kDa protein termed
Raptor (regulatory associated protein of mTOR) that
interacts with mTOR [78,79]. Although raptor has a
positive role in nutrient stimulated signalling, its association
with mTOR negatively regulates mTOR kinase activity.
Nutrient withdrawal results in an increased association of
raptor with mTOR [79]. Biochemical studies show that
raptor is required for phosphorylation of 4E-BP1 by mTOR
and that raptor also enhances phosphorylation of S6k1 by
mTOR [78]. Raptor seems to have a positive role in the
regulation of cell size. It was shown to interact with 4E-BP1,
especially hypophosphorylated forms of the latter and thus
appears to act as a scaffold protein by forming ternary
complexes with mTOR and 4E-BP1 (and perhaps also
S6k1). Further work will clearly be required to determine
how nutrients modulate the mTOR–raptor interaction, and
whether raptor promotes phosphorylation of all sites in
S6k1 and 4E-BP1, or only specific residues.
Other recent work has provided insights into the
upstream regulation of mTOR. The proteins hamartin
and tuberin (also termed TSC1/2) form a complex that
suppresses signalling via mTOR (reviewed in [80]). The
genes for TSC1/2 are mutated in people suffering from
certain types of benign tumours. TSC2 is phosphorylated by
protein kinase B, this providing a potential link between

phosphatidylinositide 3-kinase signalling and regulation of
mTOR, and there is some evidence that this may result in
dissociation of complexes between TSC1 and TSC2, thus
relieving inhibition of mTOR. However, it is not clear
whether TSC1/2 are also involved in the regulation of
mTOR by nutrients.
Rapamycin binds as a complex with FKBP12 to a region
close to the kinase domain in the primary sequence of
mTOR (Fig. 4). Recent work suggests that this region may
also bind phosphatidic acid and that such binding may be
important for the physiological function of mTOR [81]. The
authors suggest that the rapamycin-FKBP12 complex may
displace PA from mTOR thereby inhibiting its activity [82].
Although proteins that are regulated by mTOR are
controlled by a range of stimuli (amino acids, glucose,
insulin, growth factors, G-protein-coupled receptor agon-
ists), the control of mTOR itself remains very poorly
understood. It has proved hard to observe robust changes in
mTOR activity (measured in vitro against 4E-BP1, for
example) in response to the various cellular treatments
tested. This could be for several reasons – for example, the
nature of the antibody used to immunoprecipitated mTOR
seems to have marked effects upon the (changes in) activity
seen in vitro [83].
One way to assess whether amino acids regulate mTOR
itself would be to measure the activity of mTOR extracted
(immunopurified) from amino acid-replete or -starved cells.
Awidelyusedin vitro assay for mTOR relies on its ability to
phosphorylate 4E-BP1 or S6K1. However, using this assay
(with S6K1 as substrate), Dennis et al. [26] were unable to

observe any effect of amino acid withdrawal on the kinase
activity of mTOR. This and several considerations lead to
doubts whether mTOR is indeed the physiological T389
Fig. 4. mTOR. The principal features of mTOR are indicated: these
include domains termed ÔtoxicÕ or ÔrepressorÕ basedonexperiments
performed with the yeast homologue TOR. FRB, FKBP12/rapamycin
binding domain. Further information is given in the text.
Ó FEBS 2002 Control of translation factors by nutrients (Eur. J. Biochem. 269) 5343
kinase [40]. Furthermore, while Thr389 appears to be the
final site in the ordered phosphorylation of S6K1, the
phosphorylation of other, ÔearlierÕ, sites is sensitive to
rapamycin (see [40] and above) strongly implying that
mTOR makes further inputs to the regulation of S6K1
additional to any effect it has on Thr389. Such inputs may
involve regulation of the (unknown) kinases acting at the
C-terminal proline-directed sites or effects on the protein
phosphatases acting on S6K1. A potential regulator of the
phosphatases acting on S6K1 is a4, which interacts with the
catalytic subunit of protein phosphatase (PP)2A [84] and is
the mammalian homologue of the yeast phosphatase
partner Tap42p [85,86], which in turn is implicated in
signalling from yeast TOR to translation. The observations
that PP2A interacts with S6K1 and is activated by
rapamycin treatment of cells might provide a mechanism
by which rapamycin causes dephosphorylation of S6K1
[40,87].
mTOR has a high K
m
for ATP for its phosphorylation of
4E-BP1 or S6K1 in vitro (around 1 m

M
[26]). Thus mTOR
may act as an energy sensor, its activity decreasing as ATP
levels fall. However, this K
m
value is still considerably lower
than normal cellular ATP concentrations and thus ATP
levels would have to fall drastically to have a substantial
effect on the activity of mTOR (especially given that the
relationship between [ATP] and the reaction rate is a
hyperbolic rather than a linear one). Indeed, these authors
used 2-deoxyglucose at very high concentrations (200 m
M
)
to deplete ATP. As noted above, effects on another energy
sensing system, AMP-activated protein kinase (AMPK), are
observed under much milder conditions (1–5 m
M
2-deoxy-
glucose), where increases in eEF2 phosphorylation have
also been seen [67]. Severe energy depletion (such as that
obtained with very high 2-deoxyglucose) will interfere with
many cellular processes, and it is likely that the Ôenergy-
sensingÕ function of mTOR described by Dennis et al.[26]
would only come into play under dire cellular conditions! A
different mechanism may serve to inhibit translation under
conditions of milder energy depletion: this involves activa-
tion of
6
the AMPK

7
and inhibition of eEF2, and is discussed
in the article by Browne and Proud [65]. Bolster et al.[88]
have recently reported that injection of a drug that activates
AMPK causes inhibition of mTOR signalling in skeletal
muscle. However, activation of AMPK may also interfere
with the synthesis and/or release of insulin [89,90], making it
hard to interpret these data. The effects on muscle mTOR
signalling may, for example, reflect changes in circulating
insulin levels.
CONTRIBUTION OF mTOR SIGNALLING
TO THE REGULATION OF PROTEIN
SYNTHESIS
IN VIVO
An important question is, to what extent do the above
regulatory events, linked to mTOR signalling, contribute to
the activation of protein synthesis in cells and tissues? This
question can be addressed by exploring the effect of
rapamycin on the control of overall rates of protein
synthesis. In cell lines, rapamycin generally exerts only a
small inhibitory effect on the rate of protein synthesis [10].
In skeletal muscle, the activation of protein synthesis
elicited by leucine is inhibited by rapamycin, but only
partially [91]. This suggests that leucine may operate to
stimulate protein synthesis both via mTOR-dependent and
-independent pathways. Indeed, leucine is still able to
activate muscle protein synthesis in alloxan-diabetic rats
where there is no S6K1 phosphorylation or eIF4E/eIF4G
binding in response to oral leucine [53]. This points to the
operation of perhaps two amino acid regulated responses:

firstly, regulation of proteins, such as S6K1 and 4E-BP1,
which are linked to mTOR and requires insulin, and,
secondly, an insulin-independent pathway which does not
involve these two proteins and is therefore distinct from
events linked to mTOR (and therefore insensitive to
rapamycin). Additional, leucine-sensitive, regulatory inputs
must therefore also operate to control protein synthesis in
skeletal muscle. Indeed, in L6 myoblasts, regulation of
eIF2B (a translation factor not linked to mTOR signalling,
see below) appears to be of more importance than the
control of eIF4E (by 4E-BP1) for the activation of protein
synthesis by amino acids [92]. In isolated rat heart cells
(ventricular myocytes), protein synthesis is acutely activated
by insulin [70] and this is blocked by around 50% by
rapamycin, indicating that mTOR signalling does play an
important role in the activation of protein synthesis here.
mTOR homologues exist in other eukaryotes, indeed
probably in all of them. In yeast there are two homologues,
TOR1 and TOR2. Recent studies have revealed roles for
these proteins in controlling the cell cycle and cell growth, in
transcription and translation, and in the stability of both
mRNA and proteins [71,93–97]. It is likely that mTOR also
regulates such processes (in addition to the effects on
translation described here).
OTHER TARGETS FOR THE CONTROL
OF TRANSLATION BY NUTRIENTS
Cellular nutrition also affects the control of translation
factors that are not linked to mTOR signalling, in particular
eIF2B. eIF2B is a multisubunit protein that mediates
nucleotide-exchange on eIF2, the translation initiation

factor that recruits the initiator methionyl-tRNA to the
40S subunit to recognize the start codon during translation
initiation [98] (Fig. 5). Binding of eIF2ÆGTPÆMet-tRNAi
complexes to the 40S subunit is therefore required for every
initiation event. The activity of eIF2B plays a role in
regulating overall and transcript specific translational con-
trol in eukaryotes from yeast to mammals, and is regulated
by a variety of inputs [98]. It can be regulated by amino
acids, apparently via several distinct mechanisms, although
these do not appear to involve signalling via mTOR. For
example, rapamycin does not affect the ability of insulin to
activate eIF2B in CHO.T cells [99]. Activation of eIF2B in
these cells requires the presence of amino acids and glucose
in the medium [21]. These effects do not appear to be
connected with defects in the ability of insulin to promote
the dephosphorylation of a regulatory (inhibitory) phos-
phorylation site at Ser535 in the e-subunit of eIF2B (which
still occurs in the absence of glucose or amino acids [21]).
In yeast, amino acids regulate the phosphorylation of the
a-subunit of eIF2, via the eIF2a kinase GCN2 (Fig. 5).
During amino acid starvation, uncharged tRNA accumu-
lates and activates Gcn2, leading to phosphorylation of
eIF2, to yield eIF2(aP), a potent inhibitor of eIF2B and
hence of translation initiation [98]. Inhibition of eIF2B leads
to increased translation of the mRNA for GCN4, an
5344 C. G. Proud (Eur. J. Biochem. 269) Ó FEBS 2002
activator of genes required for amino acid biosynthesis,
allowing yeast cells to make the necessary amino acids.
Although orthologues of Gcn2 exist in mammals, such a
mechanism does not seem to be involved in the effects of

amino acid withdrawal in CHO cells [21], as this manipu-
lation did not affect levels of eIF2a phosphorylation [19,21].
The effects of nutrients on the control of eIF2B therefore
appear to be mediated via alternative regulatory mecha-
nisms, which may include changes in the phosphorylation of
eIF2B. eIF2B undergoes phosphorylation at multiple sites
in vivo, and it is quite possible that one or more sites are
modulated by amino acids/glucose contributing to the
regulation of eIF2B activity. Indeed, our recent data show
that dephosphorylation of Ser535 alone does not result in
activation of eIF2B, implying the operation of additional
regulatory mechanisms [100].
Changes in the activity of eIF2B are implicated in the
overall control of protein synthesis in the liver in response to
amino acid imbalance (elevated levels of leucine, glutamine
and tyrosine), on the basis of correlations between eIF2B
activity (but not levels of eIF4F, for example) and overall
protein synthetic rates [31]. In L6 myoblasts, histidine and
leucine were each able to activate eIF2B and total protein
synthesis, while only leucine was able to modulate 4E-BP1,
via the mTOR pathway [59,92]. These data also imply that
the regulation of the activity of eIF2B, rather than the
control of 4E-BP1 (or by implication, S6K1), is important
for the overall regulation of protein synthesis in these cells.
Control of 4E-BP1 and S6K1, via mTOR, in response to
leucine, appears rather to control the translation of specific
mRNAs [59]. The molecular mechanisms involved here are
unclear, but may involve changes in the activity of a protein
kinase that phosphorylates the catalytic e-subunit of eIF2B
[31]. This kinase showed decreased activity in response to

amino acid imbalance and was probably not one of the
kinases previously shown to phosphorylate eIF2Be.This
finding further underlines the possibility that nutrient
regulation of eIF2B involves changes in its state of
phosphorylation. The activity of this unidentified kinase
was not affected by rapamycin pretreatment of the cells,
again indicating that mTOR is not involved here.
Why should mammalian cells have an additional mech-
anism to regulate eIF2B in response to amino acids given
that they also possess orthologues of GCN2? As discussed
above, in yeast, a rise in uncharged tRNA ultimately
switches on amino acid biosynthetic pathways. In contrast,
mammalian cells are unable to make many of these amino
acids, to provide substrates for tRNA charging. An
uncontrolled accumulation of uncharged tRNA could have
serious consequences for the cell by leading to misincorpo-
ration or premature termination during elongation. It may
therefore be important for mammalian cells to react to
amino acid deficiency before significant accumulation of
uncharged tRNAs occurs.
HOW DO MAMMALIAN CELLS SENSE
AMINO ACIDS?
Leucine appears to be the only amino acid capable of
eliciting an effect on 4E-BP1 and S6K1 in skeletal muscle
[35,91,101], while other branched-chain amino acids (iso-
leucine, valine) are also effective in liver [28,60]. In CHO
cells, leucine was the only one of the amino acids tested
which, when added alone, stimulated 4E-BP1 phosphory-
lation [18]. Similarly, it was omission of leucine that had the
most profound effect on the activity of S6K1 [20]. Omission

of arginine also resulted in a marked fall in S6K1 activity. It
seems likely that cell types differ in their sensitivities to the
omission or addition of specific amino acids. Whether this
reflects the operation of different sensing mechanisms in
different cell types awaits further information on the sensing
process itself.
This raises the key issue of the mechanism by which
amino acids are sensed by mammalian cells. Little infor-
mation is currently available on this. Iiboshi et al. [102]
published information suggesting that levels of tRNA
charging may underlie the control of the mTOR pathway.
Such effects could conceivably be mediated via mGcn2,
which is probably activated by uncharged tRNA. However,
the importance of this mechanism on the short term effects
of leucine and other amino acids on signalling through the
mTOR pathway in questionable. We have consistently been
unable to see any change in the state of phosphorylation of
eIF2a in response to amino acid addition or withdrawal in
CHO cells [19,21]. Only very small changes in eIF2a
phosphorylation were seen upon leucine deprivation in L6
myoblasts [92] and Dennis et al. [26] saw no effect of amino
acid withdrawal on the level of charging of tRNA. Amino
acid alcohols can inhibit amino acyl-tRNA synthetases and
thus block tRNA charging. Iiboshi et al. [102] reported that
treatment of T-lymphoblastoid (Jurkat) cells with amino
Fig. 5. Nutrient inputs into the control of eIF2B in mammalian cells.
The role of eIF2B as the guanine nucleotide exchange factor for eIF2 is
depicted. In yeast, amino acid starvation leads, via the accumulation of
uncharged tRNA, to activation of the eIF2a kinase GCN2 and
phosphorylation of eIF2. eIF2(aP) acts a potent inhibitor of eIF2B

and thus also of overall translation. The importance of this mechanism
in the control of translation by amino acids in mammalian cells is so far
less clear. Recent work suggests that also that eIF2(aP) may also
positively regulate autophagy [112]. Amino acids do, however,
modulate the activity of eIF2B in mammalian cells. Insulin can activate
eIF2B by inducing dephosphorylation of an inhibitory site in eIF2B
(see text), via a pathway involving PKB and the inactivation of GSK3.
Ó FEBS 2002 Control of translation factors by nutrients (Eur. J. Biochem. 269) 5345
acid alcohols led to decreased S6K1 activity. In contrast,
neither we ([19]; A. Beugnet & C.G. Proud, unpublished
data)
8
nor others [34] have observed effects of amino acid
alcohols on targets of mTOR signalling. Inhibition of
hepatic protein synthesis by low amino acid levels seems
independent of uncharged tRNA [103].
How then are amino acids sensed? Amino acids also
regulate (repress) autophagy, e.g. in the liver, and this
prompted a number of studies into the mechanisms involved
in this effect. Byproducts of leucine metabolism seem
unlikely to be involved here (see discussion of [31])
suggesting a role for leucine itself. Mortimore et al.
[104,105] were able to show that a non-cell-permeant leucine
ÔanalogueÕ could still inhibit autophagy, implying that the
effect was mediated by extracellular leucine; they went on to
use photoaffinity labelling to show that this reagent could
label a membrane-associated protein, suggesting the exist-
ence of a plasma membrane leucine ÔsensorÕ [105]. This idea
is attractive in view of the discovery of a plasma membrane
amino acid sensor (Ssy1p) in yeast [106], but the molecular

identity of this protein in mammalian cells has not been
established. As mentioned above, other evidence suggests
that mTOR may be regulated by intracellular amino acid
levels (A. Beugnet, A.R. Tee, P.M. Taylor & C.G. Proud
9
,
unpublished data).
Various structural homologues of leucine can also
mediate the effect [31,107], but the evidence suggests that
leucine, rather than its immediate transamination product
a-ketoisocaproate, is the mediator of the effect [108]. The
specificity for leucine may vary between cell types: while it is
the primary regulator in adipocytes, this is not universally
the case [109]. Deprivation of amino acids caused a marked
depletion of branched chain amino acids [26], consistent
with the alternative possibility that vertebrate cells respond
to intracellular amino acid levels. Other data consistent with
this idea are the observations (a) that injection of leucine
into Xenopus oocytes activated TOR signalling (specifically,
the phosphorylation and activity of S6K [51] and (b) the
finding that manipulation designed to alter intracellular
amino acid levels affect mTOR signalling (see above). As
mentioned above, blocking autophagy renders hepatoma
cells dependent upon added, external amino acids, perhaps
by decreasing the pool of intracellular amino acids [47]. A
further example of the regulation of translation factors by
nutrients is the ability of glucose to modulate the
phosphorylation of eIF2a in the pancreas. This appears to
involve the unfolded protein response and modulation of
the activity of the eIF2a kinase PERK, and may be an

important role in regulating insulin synthesis in this tissue
(see [113]).
FUTURE DIRECTIONS
The last four years or so have seen several important
advances in our understanding of the control of translation
factors by nutrients. However, as is so often the case, the
recent data raise even more questions. Particularly import-
ant issues concerning the role of mTOR signalling in the
control of translation factors include: the nature of the
machinery by which amino acids are sensed in mammalian
cells, and how this information is relayed to mTOR and the
links between mTOR and the control of the S6Ks and
4E-BPs. For example, it is important to identify the protein
kinases that act on 4E-BP1 and the S6Ks. The complex
hierarchy of phosphorylation of 4E-BP1, in particular,
suggests that multiple kinases are involved, some of which
may be basally active (due to an input from mTOR) while
others may be turned on by insulin. The role of protein
phosphatases in the control of 4E-BPs and S6Ks also needs
to be explored. In a wider context, it is crucial to identify the
other regulatory mechanisms, independent of mTOR, that
function to activate protein synthesis in muscle, for example,
in response to feeding.
ACKNOWLEDGEMENTS
Work in the author’s laboratory is supported by the Biotechnology and
Biological Sciences Research Council, the British Heart Foundation,
The European Union, The Medical Research Council and the
Wellcome Trust.
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