Download mTORC1 regulates the efficiency and cellular capacity for protein

Talks About TORCs: Recent Advances in Target of Rapamycin Signalling
mTORC1 regulates the efficiency and cellular
capacity for protein synthesis
Christopher G. Proud1
Centre for Biological Sciences, University of Southampton, Life Sciences Building (B85), Southampton, SO17 1BJ, U.K.
Abstract
mTORC1 (mammalian target of rapamycin complex 1) is activated by nutrients, growth factors and certain
hormones. Signalling downstream of mTORC1 promotes protein synthesis by both activating the processes of
translation initiation and elongation, in the short term, and the production of new ribosomes, in the longer
term. mTORC1 signalling stimulates the translation of the mRNAs encoding the ribosomal proteins, activates
RNA polymerases I and III, which make the rRNAs, and promotes the processing of the precursor for the
main rRNAs. Taken together, these effects allow mTORC1 signalling to drive cell growth and proliferation.
Introduction
Signalling through mTORC1 [mTOR (mammalian target
of rapamycin) complex 1], plays key roles in controlling
a variety of aspects of cellular function. Among these, the
best understood in terms of its control by mTORC1 is
protein synthesis. Since changes in protein synthesis affect
cell growth, cellular metabolism and, via changes in the
levels of specific proteins, a broad range of other cellular
processes, control of this process has a broad impact on the
cell. mTORC1 signalling is activated by certain hormones
and by growth factors, as well as by nutrients such as amino
acids which are required for protein synthesis. It can thus, on
one hand, co-ordinate a range of inputs, and, on the other,
regulate diverse cellular processes, especially ones involved
in cell growth and proliferation.
Control of the synthesis of specific proteins
by mTORC1
The process of mRNA translation is conventionally divided
into four main stages, the first of which is initiation [1].
During this process, ribosomes are recruited to the 5 end of the mRNA, indirectly, via an interaction between
eIF (eukaryotic initiation factor) 4E and the 5 -cap (which
contains a 7-methyl-GTP moiety). eIF4E binds the scaffold
protein eIF4G, which interacts with other proteins, including
eIF3, resulting in recruitment of the 40S ribosomal subunit,
together with other factors, to the mRNA. The resulting
complex then ‘scans’ along the 5 -UTR (5 -untranslated
region) of the mRNA to find the correct start codon, which
Key words: eukaryotic elongation factor 2 (eEF2), mammalian target of rapamycin complex 1
(mTORC1), mRNA translation, ribosome biogenesis, rRNA, translation initiation.
Abbreviations used: eEF2, eukaryotic elongation factor; eEF2K, eEF2 kinase; eIF, eukaryotic
initiation factor; 4E-BP, eIF4E-binding protein; ERK, extracellular-signal-regulated kinase; mTOR,
mammalian target of rapamycin; mTORC1, mTOR complex 1; Pol, RNA polymerase; S6K, ribosomal
protein S6 kinase; 5 -TOP, 5 -terminal oligopyrimidine tract; 5 -UTR, 5 -untranslated region.
1
email [email protected]
Biochem. Soc. Trans. (2013) 41, 923–926; doi:10.1042/BST20130036
is recognized by the anticodon of the initiator methionyltRNA, a component of the complex.
Features in the 5 -UTR of the mRNA can strongly
affect the efficiency with which it undergoes initiation.
For example, regions of secondary structure are inhibitory,
probably because they impede movement of the initiation
complex along the 5 -UTR. This complex does contain a
helicase (eIF4A), which can unwind secondary structure, and
a further protein, eIF4B, which promotes eIF4A activity.
mTORC1 signalling affects this in two main ways: first,
eIF4B is phosphorylated by the S6Ks (ribosomal protein
S6 kinases) which are activated by mTORC1, and this is
considered to promote eIF4B function. Secondly, eIF4A
is brought to the mRNA through an interaction with
eIF4G, and this depends on eIF4E binding to eIF4G;
that interaction is prevented when eIF4E binds to small
heat-stable phosphoproteins called 4E-BPs (eIF4E-binding
proteins). In their hypophosphorylated states, 4E-BPs bind
to a site on eIF4E similar to that occupied by eIF4G,
thus sequestering eIF4E. 4E-BPs are phosphorylated by
mTORC1, leading to the release of eIF4E which can now
bind eIF4G (Figure 1).
Since mTORC1 can regulate the recruitment of eIF4A
and the activity of eIF4B, it has been thought that
mTORC1 signalling will promote translation of mRNAs
with structured 5 -UTRs, although recent high-throughput
studies do not appear to support this [2,3]. Instead, those
studies showed that inhibiting mTOR or mTORC1 most
strongly inhibits the translation of mRNAs with a 5 terminal oligopyrimidine tract (so-called 5 -TOP mRNAs)
[2–4]. Their translation has long been known to be positively
regulated by signalling downstream of mTORC1 (see, e.g.,
[5]), although the mechanisms underlying this remained
unclear. The recent studies [2,3] suggest that the 4E-BPs
play a key role in this, although the details of the mechanism
remain to be fully elucidated. It is important to note that the
set of 5 -TOP mRNAs includes those encoding ribosomal
proteins (and several translation factors) [6], this allows
mTORC1 signalling to rapidly up-regulate ribosome protein
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Biochemical Society Transactions (2013) Volume 41, part 4
Figure 1 mTORC1 regulates translation initiation and ribosome
Figure 2 mTORC1 signalling regulates eEF2K
biogenesis
mTORC1 directly phosphorylates 4E-BP1 and relieves its inhibition of
mTORC1 phosphorylates and activates S6Ks, which in turn phosphorylate
eEF2K at Ser366 , an event which inhibits its activity. In addition, mTORC1
eIF4E, thereby promoting translation initiation. This may explain the
ability of mTORC1 signalling to promote translation of the 5 -TOP mRNAs
which include those for all ribosomal proteins. mTORC1 also activates
signalling also regulates additional sites in eEF2K, including Ser78 and
Ser359 . For example, phosphorylation of Ser78 interferes with the binding
of eEF2K to calmodulin (CaM). The red circles marked ‘P’ denote these
the S6Ks, which appear to contribute to activation of Pol I and thus
to enhanced synthesis of the major rRNAs. mTORC1 also promotes the
processing of the precursor for these rRNAs and the synthesis, by Pol III,
inhibitory phosphorylation sites in eEF2K. mTORC1-mediated inhibition
of eEF2K leads to the dephosphorylation and activation of eEF2.
of the 5S rRNA. Newly made ribosomes are assembled in the nucleolus,
where Pol I and the processing of the main rRNAs also takes place.
Question marks indicate some of the points that remain to be clarified.
production, as well as the synthesis of rRNAs, as part of a
concerted programme of ribosome biogenesis (Figure 1).
Control of translation elongation by
mTORC1
During the elongation stage of mRNA translation, the
ribosome moves along the mRNA deciding it, and the new
polypeptide is assembled. Elongation consumes almost all
of the energy (as ATP and GTP) and amino acids that are
used in mRNA translation (>99 %). The rate of elongation
can be regulated through the phosphorylation of eukaryotic
elongation factor 2 at Thr56 (Figure 2). Phosphorylation of
this site inhibits the ability of eEF2 (eukaryotic elongation
factor 2) to interact with the ribosome and thus carry out
its function in translation, i.e. to allow the translocation of
the ribosome along the mRNA from one codon to the next
[7].
eEF2 is phosphorylated by a highly specific, and unusual,
protein kinase, eEF2K (eEF2 kinase). eEF2K is not a member
of the main protein kinase superfamily, but instead belongs
to a small group of enzymes termed ‘α-kinases’, which
comprise six genes in human cells. eEF2K is normally
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activity. Many agents which activate protein synthesis,
such as growth hormone, insulin (in many cell types) or
hypertrophic agents in cardiomyocytes, induce the rapid
(15–30 min) dephosphorylation of eEF2 [8–10]. They also
cause the inactivation of eEF2K and, in almost all cases, this
effect, and the dephosphorylation of eEF2, are blocked by
rapamycin, showing that the effects are mediated through
mTORC1.
The finding that eEF2K is phosphorylated (on Ser366 )
and inactivated by S6K [11] seemed to provide the
link between mTORC1 and the control of eEF2K
(Figure 2). It also provided a connection to the oncogenic Ras/Raf/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase)
kinase]/ERK pathway, since the same site is also phosphorylated by p90RSK s downstream of that pathway. However, it is now clear that the control of eEF2K is more complex
than this (Figure 2). First, there are additional rapamycinsensitive phosphorylation sites in eEF2K; secondly, in
some cases, the S6K inhibitor PF-4708671 [12] does not
block the mTORC1-dependent dephosphorylation of eEF2,
and, thirdly, rapamycin, or even mTOR kinase inhibitors
such as AZD8055 (which, unlike rapamycin, block all
functions of mTORC1 and mTORC2) fail to inhibit the
dephosphorylation of eEF2 in some settings.
We are now studying which regulatory events account
for the mTORC1-dependent and -independent control of
Talks About TORCs: Recent Advances in Target of Rapamycin Signalling
eEF2K. Two sites additional in eEF2K that are regulated
in an mTORC1-dependent manner have already been
identified, Ser78 [10] and Ser359 [13]; phosphorylation of each
inhibits eEF2K activity (Figure 2). We have now identified
further mTORC1-controlled sites in eEF2K. The additional
rapamycin-sensitive sites in eEF2K are not targets for S6Ks
[11]. It is therefore an exciting possibility that they are instead
substrates for additional novel mTORC1-regulated kinases.
Importantly, these enzymes may link mTORC1to the control
of other processes.
components involved in ribosome biogenesis might provide
potential targets for cancer therapy [20].
Acknowledgements
I am grateful to Dr Xuemin Wang for her careful reading of the paper
and helpful comments before submission.
Funding
Control of ribosome biogenesis by mTORC1
The regulation of translation initiation (through eIF4E/4EBPs) and elongation (via eEF2/eEF2K) provide ways in which
mTORC1 signalling can rapidly activate protein synthesis by
increasing the efficiency of the translational machinery. In
the longer term, mTORC1 signalling also enhances ribosome
production to augment the cellular capacity for protein
synthesis [14]. This involves the enhanced translation of the
5 -TOP mRNAs, many of which encode ribosomal proteins,
as mentioned above. mTORC1 also activates the two RNA
polymerases involved in making rRNAs, i.e. Pol I, which
synthesizes the major rRNAs, and Pol III, which makes the
5S rRNA and tRNAs. Pol I transcribes a large precursor
47S pre-rRNA which must then be processed to create the
mature 5.8S, 18S and 28S rRNAs (Figure 1). This occurs in
regions of the nucleus termed nucleoli, where ribosomes are
also assembled.
Recent work has shown that mTORC1 signalling
promotes pre-rRNA processing [15]. This may involve some
of the ribosomal proteins whose synthesis is controlled by
mTORC1 and which participate in pre-rRNA processing.
Alternatively, mTORC1 may regulate one or more of
the other, very numerous, components involved in rRNA
maturation.
The S6Ks appear to play a role in linking mTORC1
signalling and the control of Pol I (reviewed in [16]), although
additional mechanisms may also be involved. mTORC1
activates Pol III by alleviating its inhibition by Maf1
[17,18]. mTORC1 co-localizes with Maf1 and promotes its
phosphorylation.
Given the complexity of ribosome biogenesis, with the
necessity to generate sufficient quantities of almost 80
proteins and four different rRNAs, the role of mTORC1 in
co-ordinating their production seems logical. One may expect
that additional regulatory mechanisms exist to help sense
and compensate for imbalances in the production of these
components, to ensure efficient ribosome biogenesis and its
co-ordination with the ongoing rate of protein synthesis.
Rapidly dividing cells, such as some tumour cells, require
a high rate of ribosome biogenesis so that each daughter
cell can receive enough ribosomes. In fact, mTORC1
signalling lies downstream of many proto-oncogenes and
tumour suppressors and is activated in a high percentage of
tumour cells. Significantly, an early investigator noted altered
nucleolar morphology in cancer cells [19]. It is possible that
Work on mTORC1 signalling in my laboratory is supported by funding
from the Biotechnology and Biological Sciences Research Council, the
British Heart Foundation, Cancer Research UK, the Royal Society and
the Wellcome Trust.
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Received 20 March 2013
doi:10.1042/BST20130036