mtorc1 signalling and mrna translation

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1 mtor Signalling, Nutrients and Disease 227 mtorc1 signalling and mrna translation Christopher G. Proud 1 School of Biological Sciences, University of Southampton, Southampton SO16 7PX, U.K. Abstract Signalling through mtorc1 (mammalian target of rapamycin complex 1) is important in controlling many cell functions, including protein synthesis, which it activates. mtorc1 signalling is activated by stimuli which promote protein accumulation such as anabolic hormones, growth factors and hypertrophic stimuli. mtorc1 signalling regulates several components of the protein synthetic machinery, including initiation and elongation factors, protein kinases which phosphorylate the ribosome and/or translation factors, and the translation of specific mrnas. However, there are still important gaps in our understanding of the actions of mtorc1 and the relative contributions that different targets of mtorc1 make to the activation of protein synthesis remain to be established. Introduction Signalling through mtor (mammalian target of rapamycin) plays a key role in several cellular functions. mtor actually forms two types of complex, of which only one [mtor (mtor complex) 1] is actually acutely sensitive to inhibition by rapamycin [1]. However, even in the case of mtorc1, not all of its effects appear to be blocked by this drug, as discussed in greater detail below. mtor possesses a kinase domain, which resembles lipid kinases, but actually phosphorylates proteins on serine or threonine residues. Several proteins have been identified as substrates for phosphorylation by mtorc1. The first ones were proteins implicated in the control of mrna translation, and this process remains the one whose control by mtorc1 is best understood [2]. As described in the present review, mtorc1 functions to activate several steps in mrna translation, and it is therefore highly appropriate that mtorc1 signalling requires amino acids (the precursors for protein synthesis) and is stimulated by anabolic, mitogenic and hypertrophic stimuli which enhance protein accumulation. Leucine is the most effective single amino acid in terms of stimulation of mtorc1 signalling. Furthermore, mtorc1 signalling also acts to repress autophagy [3], a form of protein breakdown, which opposes protein synthesis. One of the features that distinguishes mtorc1 from mtorc2 is the presence in only the former complex of the protein raptor (regulatory associated protein of mtor) [1] (Figure 1). Raptor interacts with short motifs in substrates for mtorc1 presumably to recruit them to the complex for phosphorylation: these are termed TOS [TOR (target Key words: elongation factor, initiation factor, mammalian target of rapamycin (mtor), mrna, protein kinase, protein synthesis. Abbreviations used: CaM, Ca 2+ /calmodulin; CH, cardiac hypertrophy; eef2, eukaryotic elongation factor 2; eef2k, eef2 kinase; eif, eukaryotic initiation factor; 4E-BP, eif4e-binding protein; mtor, mammalian target of rapamycin; mtorc, mtor complex; p90 RSK, p90 ribosomal S6 kinase; PABP, poly(a)-binding protein; PE, phenylephrine; raptor, regulatory associated protein of mtor; S6K, S6 kinase; TOS, TOR (target of rapamycin) signalling; UTR, untranslated region; 5 -TOP, 5 -UTR containing a tract of pyrimidines. 1 C.G.Proud@soton.ac.uk of rapamycin) signalling] motifs. mtorc1 contains GβL, which is also found in mtorc2. mtorc1 and protein synthesis Although the best-known targets for control by mtorc1 are involved in the control of mrna translation, in many cell lines, rapamycin has only modest effects on the rate of protein synthesis in the short term (see, e.g., [4]). However, it does have much greater effects on overall protein synthesis rates, e.g. in certain types of primary cell. In primary adult rat ventricular myocytes, agents such as insulin and α 1 -adrenergic agonists [such as PE (phenylephrine)] markedly and rapidly activate the rate of protein synthesis, and this activation is largely blocked by rapamycin, indicting that it involves signalling through mtorc1 [5,6]. mtorc1 also promotes ribosome biogenesis [7], but this effect will not contribute significantly to increased rates of protein synthesis over such short times. Thus rapid activation of the translational machinery by mtorc1 plays an important role in controlling protein synthesis in such cells. The control by α 1 -adrenergic agonists is of particular interest as they play a key role in driving CH (cardiac hypertrophy), a life-threatening disease involving overgrowth of cardiac muscle, which predisposes to cardiac failure. The main factor driving CH is elevated rates of protein synthesis. The fact that mtorc1 is important in the activation of protein synthesis by α 1 -adrenergic agonists is consistent with the findings that rapamycin can prevent or even reverse CH in animal models [8,9]. mtorc1 signalling also regulates the translation of specific mrnas. Among these are a subset of mrnas that contain tracts of pyrimidines in their 5 -UTRs (untranslated regions), hence they are termed 5 -TOP mrnas (Figure 1). The 5 -TOP acts to impair the translation of such mrnas, which, conversely, is enhanced by amino acids and by, for example, serum, and this appears to require signalling through mtorc1 as indicated by the fact that rapamycin Biochem. Soc. Trans. (2009) 37, ; doi: /bst

2 228 Biochemical Society Transactions (2009) Volume 37, part 1 Figure 1 Links between mtorc1 and the control of mrna translation. mtorc1 is activated by amino acids and by hormones and growth factors mtorc1 phosphorylates several sites in 4E-BP1 (via at least two distinct mechanisms as indicated by the two arrows). Release of 4E-BP1 from eif4e activates eif4e by allowing it to bind eif4g, which interacts with other components (see the text for further details). Certain mrnas contain a 5 -TOP which inhibits their translation; this is overcome by signalling through mtorc1, but the mechanism is unknown. mtorc1 also activates the S6Ks, which phosphorylate S6, eif4b and eef2k (see the text for further details). eef2k phosphorylates and inhibits eef2. Phosphorylation of eef2k at each of several inhibitory sites is regulated by mtorc1; cdc2/cyclin B activity is promoted by mtorc1 signalling, by unknown means. An unidentified kinase phosphorylates eef2k at Ser 78. To aid the clarity of presentation, many components have been omitted and the components are not drawn to scale. The green line indicates the mrna, and the blue star denotes the 5 -cap. Broken arrows indicate connections that are so far not understood.?? indicates phosphorylation events whose function remains unclear. Circled P s indicate phosphorylation sites: red or green backgrounds denote sites that respectively inhibit or activate the target protein. Grey indicates sites of either neutral or unknown function. impairs, for example, the serum-induced enhancement of their translation [10]. It is so far unknown how mtorc1 controls the translation of 5 -TOP mrnas; it was suggested previously that this involved the S6Ks (S6 kinases), but it is now clear that this is not the case [11,12]. The 5 -TOP mrnas include those encoding ribosomal proteins and other components of the translational machinery such as elongation factors. This control mechanism provides a way in which mtorc1 signalling can increase the levels of such proteins, thereby providing an increased cellular capacity for protein synthesis. In this context, it is interesting that the expression of the catalytic ε-subunit of the translation initiation factor eif (eukaryotic initiation factor) 2B has recently been reported to be promoted by mtorc1 at the level of translation [13], although the mechanisms involved are unclear (this is not a 5 -TOP mrna). It is very likely that there are additional mrnas whose translation is controlled through mtorc1, e.g. those that contain inhibitory secondary structure in their 5 -UTRs [14,15] (see below). Two classes of proteins that contain TOS motifs are the S6Ks [16] and the 4E-BPs (eif4e-binding proteins) [17]. There are two S6K genes in mammals (S6K1 and S6K2), which give rise, via alternative splicing, to four polypeptides. There are three 4E-BPs, numbered 4E-BP1, 4E-BP2 and 4E-BP3, of which 4E-BP1 is by far the best understood. The ribosomal protein S6Ks The S6Ks phosphorylate several proteins that are associated with mrna translation or its control. These include: ribosomal protein S6, a component of the small ribosomal subunit; eef2 (eukaryotic elongation factor 2) kinase (see below); and eif4b, a protein involved in unravelling inhibitory secondary structure in the 5 -UTRs of certain mrnas (Figure 1). Inactive S6K1 associates with translation initiation complexes containing eif3 (a multisubunit protein which binds several components of the translational machinery). S6K1 dissociates from eif3 upon activation [18]; mtor and raptor show the converse behaviour, as their association with eif3 increases upon stimulation with agents that activate this pathway. S6Ks can also phosphorylate eif4b, an ancillary factor for the initiation factor eif4a, an RNA helicase (see below) [19]. Phosphorylation of eif4b promotes its association with eif3. In addition, S6Ks phosphorylate and thereby inhibit the protein kinase that phosphorylates and inactivates eef2 (the kinase is thus eef2 kinase or eef2k) (Figure 1) [20]. eef2k and eef2 are regulated via mtorc1, as discussed below. However, the significance of the S6Ks in regulating protein synthesis remains unclear; little or no effect on protein synthesis has been reported for cells and tissues in which either the S6Ks have been knocked out or the five phosphorylation sites in ribosomal protein S6 have been mutated to alanines [11]. Cells lacking the phosphorylation sites in S6 actually show higher rates of protein synthesis and proliferation. Although mtorc1 signalling plays a critical role in CH [8,9], S6K-double-knockout mice develop physiological or pathological CH to a similar extent to that of control animals [21]. These data suggest that none of the S6K targets above are critical for the control of protein synthesis (i.e. eif4b and eef2k, as well as S6 itself). However, it must be noted, first, that all three proteins are also substrates for p90 RSK (p90 ribosomal S6 kinase) [which is activated by ERK (extracellular-signalregulated protein kinase) rather than by mtorc1] which targets the same (eif4b and eef2k) or a subset of the same (S6) sites as the S6Ks. Secondly, mtorc1 also regulates the activity of eef2k via additional phosphorylation sites, involving neither S6Ks nor p90 RSK, which inhibit eef2k activity more strongly.

3 mtor Signalling, Nutrients and Disease 229 The 4E-BPs control initiation complex assembly The 4E-BPs are small phosphoproteins which bind to eif4e at a site that overlaps its interaction site for eif4g. 4E-BPs undergo phosphorylation at multiple sites leading to their release from eif4e, allowing eif4e to bind the scaffold protein eif4g (Figure 1). eif4g also interacts with a number of other components of the translational machinery, including the RNA helicase eif4a, its ancillary factor eif4b, eif3 and PABP [poly(a)-binding protein]. The fact that eif4g binds both eif4e and PABP, which interact with opposite ends of the mrna, means that the mrna is, in effect, circularized, a feature that seems to be important for efficient translation (Figure 1). Other major roles of eif4g and its interaction partners are recruitment of the 40S subunit (via eif3) and unwinding inhibitory secondary structure in the 5 -UTR (by eif4a/eif4b). mrnas whose 5 -UTR contains such a structure are generally weakly translated, although this can be enhanced by agents that promote formation of eif4e eif4g eif4a complexes (termed eif4f ) [22]. Translation of mrnas (which include those encoding a number of proteins involved in events linked to cell transformation [14,15]) is expected to be enhanced by mtorc1 signalling via increased levels of eif4f complexes. The phosphorylation of several sites in 4E-BP1 depends upon its TOS motif, particularly those closest to the eif4ebinding site (Ser 65 /Thr 70 in human 4E-BP1). Mutation of the TOS motif almost abolishes the interaction of 4E-BP1 with raptor [23 25]. However, the mtorc1-dependent phosphorylation of the N-terminal sites, Thr 37 /Thr 46, requires a further motif which contains the sequence Arg- Ala-Ile-Pro (hence RAIP motif) [26]. This motif is also involved in binding to raptor: mutating this region greatly decreases, but does not abolish, raptor binding [27]. Thus 4E- BP1 apparently interacts with raptor through both its TOS and RAIP motifs: to date, no other targets for mtorc1 have been shown to contain features analogous to the RAIP motif. Interestingly, in many cell types, Thr 37 /Thr 46 are already heavily phosphorylated under serum-starved conditions, whereas phosphorylation of Thr 70 and especially Ser 65 requires an additional stimulus such as insulin or serum (see, e.g., [23,28]). Starving the cells of amino acids, or of leucine in particular, results in the dephosphorylation of Thr 37 /Thr 46 : thus it appears that the RAIP motif mediates an amino-acid-dependent input from mtorc1 to 4E-BP1, while the TOS motif is involved in an input that requires additional signalling events. Despite our extensive knowledge of eif4g/eif4f and of the control of 4E-BP1, and the fact that this system is expected to function to regulate the assembly of translation initiation, its importance for the overall control of protein synthesis has remained unclear. Given the importance of mtorc1-dependent mechanisms for the control of protein synthesis in cardiomyocytes and for CH, we have sought to explore the role of regulated eif4f formation in these processes. One approach to this would be to make use of the small-molecule eif4e/eif4gbinding inhibitor 4EGI-1 described by Moerke et al. [29]. However, this compound rapidly (<1 h) causes cardiomyocytes to lose viability, rather similarly to the effects of a peptide inhibitor of this interaction in other cell types [30]. We therefore chose to express 4E-BP1 using an adenoviral vector, using a mutant (LM/AA; which cannot bind to eif4e) as a negative control. Overexpressing wild-type 4E-BP1 in individuals with ARVC (arrhythmogenic right ventricular cardiomyopathy) diminishes the basal level of association of eif4g with eif4e (i.e. of eif4f complexes) and reduces the basal rate of protein synthesis (B.P.-H. Huang, Y. Wang, X. Wang and C.G. Proud, unpublished work). This is consistent with eif4f playing a role in the basal protein synthesis rates. PE normally increases the rate of protein synthesis 2-fold or more, and promotes the phosphorylation of 4E-BP1 and its dissociation from eif4e, allowing eif4g to bind to eif4e. In cells that overexpress 4E-BP1, PE fails to promote eif4g binding; this is not because it interferes with mtorc1 signalling, because the PE-induced effects on the phosphorylation of endogenous 4E-BP1, S6 and eef2 are not affected. Strikingly, the ability of PE to activate protein synthesis is completely unaffected by overexpression of 4E-BP1: PE activated protein synthesis to a proportionally greater extent than in control cells, albeit from a lower basal level. Such increases remain highly dependent on mtorc1. Blocking eif4f formation in this way also fails to inhibit the PE-induced growth of cardiomyocytes, consistent with its lack of effect on PE-stimulated protein synthesis rates, but does block the translation of structured mrnas, as predicted by the model outlined above. These data imply that increased formation of eif4f, an mtorc1-regulated process, is not required for the activation of protein synthesis and imply that other mtorc1-dependent events are responsible for this. Studies on mice in which the genes for 4E-BP1 and/or 4E-BP2 have been knocked out have revealed effects on the development or maintenance of adipose tissue [31], on obesity and insulin resistance [32], and on hippocampal memory [33] and animal behaviour [34]. mtorc1 positively regulates translation elongation eef2 mediates the translocation step of peptide-chain elongation where the ribosome migrates by one codon along the mrna and the trnas move between the different sites on the ribosome. Phosphorylation of eef2 (at Thr 56 ) inhibits its activity by impairing its binding to the ribosome (reviewed in [35,36]) (Figure 1). As mentioned above, phosphorylation of eef2 is catalysed by a specific kinase, eef2k. eef2k belongs to the small group of α-kinases which are distinct from the main serine/threonine/tyrosine kinase superfamily. Its activity is normally dependent on CaM (Ca 2+ /calmodulin), which binds to eef2k through a region

4 230 Biochemical Society Transactions (2009) Volume 37, part 1 near the N-terminus, immediately N-terminal of its kinase domain. Many agents that activate protein synthesis, including insulin [5,37] and PE [6], rapidly (<2 15 min) induce the dephosphorylation of eef2 and the inactivation of eef2k, both of which effects are inhibited by rapamycin. How does mtorc1 inactivate eef2k, which does not contain a TOS motif, does not bind raptor and is not a substrate for phosphorylation by mtorc1? This implies that the link between mtorc1 and eef2k is indirect. mtorc1 promotes the phosphorylation of at least three sites in eef2k, each of which results in its decreased activity, although in different ways (Figure 1). Ser 366 is phosphorylated directly by S6Ks (and also by p90 RSK [38]). Phosphorylation of Ser 78, adjacent to the CaM-binding site, inhibits the binding of CaM to eef2k and thereby inhibits eef2k activity [39]. It is not known which kinase phosphorylates Ser 78. Phosphorylation at Ser 359 also inhibits the activity of eef2k and is modulated by mtorc1 [40]. Recent work has shown that cdc2/cyclin B phosphorylates Ser 359 in eef2k (Figure 1) and that its activity appears to be promoted by mt- ORC1 [41]: it declines in response to starving cells of amino acids (or leucine alone) and increases in cells lacking TSC2 (tuberous sclerosis complex 2), a negative regulator of mt- ORC1. The control of eef2k by cdc2/cyclin B may serve to keep eef2 phosphorylation low in mitotic cells [41], in which certain mrnas need to be translated. It is clearly possible that mtorc1 also stimulates the activity of cdc2/cyclin B against other substrates, which could provide a mechanism by which mtorc1 signalling can promote G 2 M-phase progression. It is not yet known how mtorc1 controls cdc2/cyclin B. Overview and perspective We have seen that mtorc1 regulates the availability of eif4e through its control of the 4E-BPs. This is probably mainly a mechanism for controlling the translation of specific mrnas, rather than global protein synthesis. mtorc1 also promotes the translation of the 5 -TOP mrnas which encode ribosomal proteins and other proteins involved in translation, allowing mtorc1 to modulate the protein synthetic capacity of the cell. The mechanisms involved in this are, however, so far unclear. Although the S6Ks actually associate with the initiation machinery and phosphorylate several proteins involved in mrna translation or its control, their importance for the control of protein synthesis remains to be established. One substrate for the S6Ks is eef2k. eef2k is inactivated by signalling through mtorc1, which allows mtorc1 to activate the elongation machinery that is required for the translation of all mrnas. It will be important to define the contribution that the control of elongation makes to overall rates of protein synthesis. 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