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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 C The C 2013 Biochemical Society Authors Journal compilation 923 924 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 C The C 2013 Biochemical Society Authors Journal compilation completely dependent upon Ca2 + ions/calmodulin for 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. References 1 Merrick, W.C. (2010) Eukaryotic protein synthesis: still a mystery. J. Biol. Chem. 285, 21197–21201 2 Thoreen, C.C., Chantranupong, L., Keys, H.R., Wang, T., Gray, N.S. and Sabatini, D.M. (2012) A unifying model for mTORC1-mediated regulation of mRNA translation. Nature 485, 109–113 3 Hsieh, A.C., Liu, Y., Edlind, M.P., Ingolia, N.T., Janes, M.R., Sher, A., Shi, E.Y., Stumpf, C.R., Christensen, C., Bonham, M.J. et al. 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