Download Regulation of protein synthesis by insulin

Insulin, calcium and the control of mammalian metabolism
Regulation of protein synthesis by insulin
C.G. Proud1
Department of Biochemistry and Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, Canada V6T 1Z3
Abstract
Insulin rapidly activates protein synthesis by activating components of the translational machinery including
eIFs (eukaryotic initiation factors) and eEFs (eukaryotic elongation factors). In the long term, insulin also
increases the cellular content of ribosomes to augment the capacity for protein synthesis. The rapid activation
of protein synthesis by insulin is mediated primarily through phosphoinositide 3-kinase. This involves the
activation of PKB (protein kinase B). In one case, PKB acts to phosphorylate and inactivate glycogen synthase
kinase 3, which in turn phosphorylates and inhibits eIF2B. Insulin elicits the dephosphorylation and activation
of eIF2B. Since eIF2B is required for recycling of eIF2, a factor required for all cytoplasmic translation initiation events, this will contribute to overall activation of protein synthesis. PKB also phosphorylates the TSC1
(tuberous sclerosis complex 1)–TSC2 complex to relieve its inhibitory action on the mTOR (mammalian target
of rapamycin). Inhibition of mTOR by rapamycin markedly impairs insulin-activated protein synthesis. mTOR
controls translation initiation and elongation. The cap-binding factor eIF4E can be sequestered in inactive
complexes by 4E-BP1 (eIF4E-binding protein 1). Insulin elicits phosphorylation of 4E-BP1 and its release from
eIF4E, allowing eIF4E to form initiation factor complexes. Insulin induces dephosphorylation and activation
of eEF2 to accelerate elongation. Both effects are blocked by rapamycin. Insulin inactivates eEF2 kinase by
increasing its phosphorylation at several mTOR-regulated sites. Insulin also stimulates synthesis of ribosomal
proteins by promoting recruitment of their mRNAs into polyribosomes. This is inhibited by rapamycin. Several
key questions remain about, for example, the mechanisms by which mTOR controls 4E-BP1 and eEF2 kinase
and the control of ribosomal protein translation.
Overview
Insulin can stimulate protein synthesis in many types of cells
and tissues, including human and rat muscles, and many types
of cells in culture [1,2]. This involves two major kinds of effects: the rapid activation of existing components of the translational apparatus and the longer term increase in the capacity
of the cell or tissue for protein synthesis, which includes an
increase in ribosome number. Studies in this laboratory have
focused primarily on the short-term regulation of proteins
involved in controlling mRNA translation. These are termed
translation factors. They are extrinsic to the ribosome but
may bind transiently to it to promote individual steps of the
mRNA translation process.
Protein synthesis (mRNA translation) is conventionally
divided into three stages: initiation, elongation and termination. Both initiation and elongation can be controlled by
insulin. Initiation is the stage where the mRNA and the initiator tRNAm Met (methionine tRNA) are recruited to the
ribosome, initially to the small 40 S subunit. The start codon is
then located by a process known as ‘scanning’ during which it
is recognized by the anticodon of tRNAm Met . This culminates
Key words: eukaryotic initiation factor (eIF), G-protein, insulin, mRNA translation, mammalian
target of rapamycin (mTOR), tuberous sclerosis complex (TSC).
Abbreviations used: CaM, calmodulin; eEF, eukaryotic elongation factor; eIF, eukaryotic
initiation factor; 4E-BP1, eIF4E-binding protein 1; GAP, GTPase-activating protein; GSK3, glycogen
synthase kinase 3; mTOR, mammalian target of rapamycin; tRNAm Met , methionine tRNA;
PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; S6K, S6 kinase; 5 -TOP, 5 -terminal
oligopyrimidine tract; TSC, tuberous sclerosis complex.
1
email [email protected]
in assembly of the complete 80 S ribosome at the start codon,
ready to proceed into elongation where the new polypeptide
is assembled.
Insulin activates eIF2B (eukaryotic
initiation factor 2)
tRNAm Met is brought to the ribosome by eIF2, a heterotrimeric GTP-binding protein. The GTP is hydrolysed upon
successful engagement of tRNAm Met with the start codon.
eIF2–GDP is recycled back to the active eIF2–GTP complex
by eIF2B (Figure 1). This GDP/GTP exchange process is
a key regulatory step for translation initiation under many
conditions: for example, it is inhibited by phosphorylation of
eIF2’s α-subunit by any one of four mammalian eIF2 kinases,
which are activated under specific stress conditions [3]. The
resulting inhibition of eIF2B impairs the recycling of eIF2
and thus inhibits overall translation initiation.
Conversely, eIF2B is activated by insulin [4]. This involves
a change in the phosphorylation of its largest ε-subunit, which
contains the catalytic domain. We discovered that insulin
inactivated a kinase that phosphorylates eIF2Bε, which was
subsequently identified as GSK3 (glycogen synthase kinase 3)
[4,5]. GSK3 was already implicated in the control of another
major anabolic process (glycogen deposition) by insulin,
but little was known about its regulation. Phosphorylation
of eIF2B by GSK3 occurs at a single serine residue, immediately N-terminal to the catalytic region, and this impairs
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Figure 1 Regulation by insulin of eIF2B and recruitment of
tRNAm Met to the 40 S subunit
Insulin activates initiation factor eIF2 and the recruitment of tRNAm Met
to the 40 S ribosomal subunit via signalling through PI3K, PKB and GSK3.
Please see the text for further details. IR, insulin receptor; IRS, IR substrate.
its GDP/GTP exchange activity [5]. The insulin-induced inactivation of GSK3 leads to the dephosphorylation of eIF2B,
contributing to its activation. Subsequent studies in other
laboratories revealed that insulin switches off GSK3 via signalling through PI3K (phosphoinositide 3-kinase), via PKB
(protein kinase B or Akt), which phosphorylates both isoforms of GSK3 directly [6].
These studies delineated a complete signalling pathway
from the insulin receptor via PKB to the activation of eIF2B.
It serves to promote the activation of at least two major anabolic processes, glycogen synthesis and mRNA translation.
Since eIF2B is required for all translation initiation events,
this mechanism promotes overall protein synthesis.
Insulin controls the cap-binding complex
via mTOR (mammalian target of
rapamycin) signalling
Recruitment of the mRNA to the ribosome involves a set
of protein factors. eIF4E binds the 5 -cap structure of the
mRNA and interacts with a large multidomain protein,
eIF4G, which can bind a number of other translation factors
as well as the PABP [poly(A)-binding protein] and the eIF4E
kinases, the Mnks [7]. One of the factors that binds to eIF4G
is eIF3, a multisubunit protein that interacts with the 40 S
subunit.
eIF4G binds to eIF4E through a short motif that is also
found in other binding partners for eIF4E. These include
the 4E-BPs (eIF4E-binding proteins), which are small heatand acid-stable phosphoproteins. The best understood of the
three 4E-BPs in mammals, 4E-BP1, was first described by
Denton and colleagues in the early 1980s as ‘22K’ [8], a protein
whose phosphorylation was enhanced by insulin in a PI3Kdependent manner [9]. As PHAS-I (phosphorylated heatand acid-stable protein regulated by insulin), it was cloned
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by Lawrence and colleagues [10] and was shown to be a
4E-BP by Sonenberg and co-workers [11]. By binding to
eIF4E, 4E-BPs prevent eIF4E from interacting with eIF4G
and forming productive initiation complexes. 4E-BPs thus
impair the recruitment of ribosomes to normal cap-dependent
mRNAs.
4E-BP1 undergoes phosphorylation at several sites, and
there appears to be a complex interplay between them
[12]. Overall, the phosphorylation of 4E-BP1 leads to its
release from eIF4E, allowing eIF4E to bind eIF4G and form
initiation complexes. Among the protein partners for eIF4G
is eIF4A, an RNA helicase, which together with eIF4G and
eIF4E forms the eIF4F [7]. eIF4A is thought to unwind
regions of complementary secondary structure in the 5 -UTR
(5 -untranslated region) which otherwise impede scanning.
Formation of eIF4F complexes may be especially important
for efficient translation of those mRNAs that have substantial
5 -secondary structure.
The phosphorylation of several sites in 4E–BP1 is controlled via the mTOR. mTOR is a protein kinase whose
catalytic domain resembles lipid kinases such as PI3K. Some
functions of mTOR are blocked by rapamycin, which also
impairs the phosphorylation of 4E-BP1 and blocks its release
from eIF4E.
mTOR signalling is activated by hormones such as insulin
and by amino acids, notably leucine (reviewed in [13,14])
(Figure 2). Our recent results show that amino acids and
insulin regulate different sets of phosphorylation sites in
4E-BP1 and that the insulin-modulated ones are sensitive
to rapamycin, but the amino acid-modulated ones are largely
insensitive [12]. This implies that amino acids and insulin
modulate downstream mTOR signalling in distinct ways. It
remains unclear, in particular, how mTOR exerts its rapamycin-insensitive, but amino-acid-dependent, effects on
4E-BP1. Phosphorylation of the N-terminal sites in 4E-BP1
(Thr37 /Thr46 in the human protein) depends on a short Nterminal motif RAIP (Arg-Ala-Ile-Pro) [12,15]. It is these
sites that are regulated by amino acids in a rapamycin-insensitive manner. The sites immediately C-terminal to the
eIF4E-binding site (Ser65 , Thr70 ) are sensitive to rapamycin,
are stimulated by insulin, and depend on prior phosphorylation of the N-terminal sites and on a second motif at the
extreme C-terminus. This TOS (target of rapamycin signalling) motif (but not the RAIP feature) binds the mTOR partner raptor (see e.g. [16,17]), and may recruit mTOR–raptor
complexes to 4E-BP1 to phosphorylate it at certain sites.
Upstream control of mTOR by insulin
Insulin regulates mTOR signalling through the TSC1
(tuberous sclerosis complex 1)–TSC2 protein complex: this
normally acts as a brake on mTOR signalling. Mutations
in TSC1 or TSC2 that impair their function or expression
therefore lead to hyperactivation of mTOR and this causes
excessive cell growth, giving rise to the large cell tumours
that are seen in patients with such mutations. The resulting
condition is termed ‘tuberous sclerosis’. TSC1–TSC2 acts as
Insulin, calcium and the control of mammalian metabolism
Figure 2 mTOR signalling controls several components of the
translational machinery
The Figure depicts the activation of mTOR by insulin, through PKB
and the inactivation of TSC1–TSC2, and downstream signalling events
from mTOR that impinge upon the translational machinery. These include
(i) the inactivation of 4E-BP1 and the consequent formation of eIF4F
complexes, (ii) the stimulation of the translation of 5 -TOP mRNAs,
(iii) the activation of the S6Ks and (iv) the inactivation of eEF2 kinase
and consequent stimulation of eEF2. Question marks denote steps that
remain incompletely understood: see the text for further details. IR, insulin receptor; IRS, IR substrate.
a GAP (GTPase-activating protein) complex for the small
G-protein Rheb, which in turn binds to, and in its GTPliganded form activates, mTOR [18]. TSC2 is phosphorylated
by PKB in response to insulin and this appears to impair its
GAP activity, although it is not known how [19,20]. Amino
acids regulate mTOR by a different mechanism, which is
independent of TSC1–TSC2 [21] (Figure 2). A key question
is therefore how do amino acids turn on mTOR?
In addition to 4E-BP1, other proteins involved in the
translational machinery are also controlled by mTOR. These
include the S6Ks (S6 kinases), which phosphorylate multiple
sites in the C-terminus of the 40 S ribosomal protein S6 [22]
(Figure 2). The S6Ks are activated by insulin and other agents
that turn on protein synthesis, and in many types of cells
require that the cellular medium contains amino acids [13,23].
The S6Ks were thought to regulate the translation of the set
of mRNAs in mammalian cells that encode the ribosomal
proteins: these contain a 5 -TOP (5 -terminal oligopyrimidine
tract) which impairs their translation under serum-starved
conditions. Serum stimulation enhances their translation in a
rapamycin-sensitive manner. However, recent results from
animals in which both S6K genes have been inactivated
reveal that the S6Ks are not required for the normal control
of 5 -TOP mRNA translation. Thus additional and so far
unknown targets for mTOR signalling must be involved in
modulating 5 -TOP mRNA translation (Figure 2). The ability
of agents such as insulin to activate ribosomal protein biosynthesis probably contributes to the long-term effects of
insulin on cellular ribosome content referred to at the
beginning of the present paper. S6Ks actually play a role in
the down-regulation of insulin signalling, by phosphorylating
IRS1 (insulin receptor substrate 1) [24,25].
Translation elongation in mammalian cells requires two
eEFs (eukaryotic elongation factors), eEF1 and eEF2. eEF2
mediates the translocation of the ribosome by three nucleotides along the mRNA after addition of each new amino
acid. Phosphorylation of eEF2 on Thr56 impairs its ability
to bind ribosomes, thus inactivating it [26]. Insulin induces
the rapid dephosphorylation of eEF2, leading to accelerated
rates of elongation as measured by transit-time measurements
[27]. Both effects are blocked by rapamycin, implying a key
role for mTOR. Insulin also causes a decrease in the activity
of eEF2 kinase, the specific Ca2+ /CaM (calmodulin)-dependent enzyme that phosphorylates eEF2. This effect is also
prevented by rapamycin. This raises the issue of how mTOR
regulates eEF2 kinase.
eEF2 kinase is a phosphoprotein and mTOR signalling
affects at least three different sites in eEF2 kinase. We showed
that Ser366 is a target for S6K1 and that phosphorylation here
inhibits the activity of eEF2 kinase, especially at low [Ca2+ ]
[28]. Phosphorylation of Ser359 is also regulated in an mTORdependent manner [29] and phosphorylation of this site also
impairs eEF2 kinase activity. Most recently, we identified
Ser78 as a novel mTOR-dependent phosphorylation site in
eEF2 kinase [30]. It lies immediately adjacent to the CaMbinding domain of eEF2 kinase and its phosphorylation
impairs CaM binding, thus decreasing eEF2 kinase activity.
The identities of the protein kinases acting at the latter two
mTOR-modulated sites are so far unknown. The amino acid
input to mTOR signalling makes excellent sense in terms
of the control of elongation: when amino acid supply is
limited, mTOR signalling is impaired and phosphorylation
of these inhibitory sites in eEF2 kinase will be decreased.
This will result in increased eEF2 kinase activity and eEF2
phosphorylation, thereby slowing the rate of elongation
and the consumption of amino acids. Conversely, when
amino acid supply improves, eEF2 can be re-activated and
elongation can accelerate.
Insulin thus activates multiple steps in mRNA translation,
including (i) tRNAm Met and mRNA recruitment to the
ribosome, (ii) the translation of specific mRNAs (the 5 TOP mRNAs) and (iii) the elongation process. The relative
quantitative contributions that each effect makes to the
overall short- and long-term stimulation of protein synthesis
remain to be established. The effects of insulin and other
stimuli on protein synthesis rates in immortalized cell lines
are often rather modest compared with the much larger effects
seen in primary cells, e.g. cardiomyocytes [31]. Primary
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cells may therefore be a more useful system in which to
address these questions, as well as probably yielding data
that are more physiologically relevant.
Work on insulin control of mRNA translation in my laboratory has
been funded by the Medical Research Council (U.K.), the Biotechnology and Biological Sciences Research Council (U.K.), the
British Heart Foundation and the Wellcome Trust. It is currently
supported by the Heart and Stroke Foundation of Canada and the
University of British Columbia.
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Received 14 September 2005