The Mtor Story

J Averous1 and CG Proud2

1Unite´ de Nutrition Humaine, INRA de Theix, Saint Gene`s Champanelle, France and 2Department of Biochemistry & Molecular Biology, University of British Columbia, Life Sciences Centre, Vancouver, BC, Canada

There is currently a high level of interest in signalling strongly implicated in diverse human cancers. Rapamy- through the mammalian target of rapamycin (mTOR). cin, which inhibits certain mTOR functions, inhibits the This reflects both its key role in many cell functions and proliferation of certain tumour cells and can cooperate its involvement in disease states such as cancers. The best with other agents to induce apoptosis. We will also understood targets for mTOR signalling are proteins highlight related areas that are poorly understood and involved in controlling the translational machinery, require further study. including the ribosomal protein S6 kinases and proteins that regulate the initiation and elongation phases of translation. Indeed, there is compelling evidence that at least one of these targets of mTOR (eukaryotic initiation Control of mTOR factor eIF4E) plays a key role in tumorigenesis. It is regulated through the mTOR-dependent phosphorylation As described in detail in the article by (Wullschleger of inhibitory proteins such as eIF4E-binding protein 1. et al., 2006), mTOR is a multidomain protein that forms Thus, targeting mTOR signalling may be an effective complexes with other proteins (Figure 1a and b). Some anticancer strategy, in at least a significant subset of of these partners appear to regulate the activity of tumours. Not all effects of mTOR are sensitive to the mTOR (e.g., the small G-protein Rheb) while others, classical anti-mTOR drug rapamycin, and this compound such as raptor and rictor, are involved in mediating also interferes with other processes besides eIF4E downstream signalling. The interactions of raptor and function. Developing new approaches to targeting mTOR rictor with mTOR seem to be mutually exclusive. The for cancer therapy requires more detailed knowledge of complexes in which they participate are termed mTOR- signalling downstream of mTOR. Such advances are complex 1 (mTORC1) and mTORC2, respectively likely to come from further work to understand the (Sarbassov et al., 2005) (Figure 1b). Raptor binds to regulation of mTOR targets such as components of the short (five residue) motifs in certain targets of mTOR translational apparatus. signalling whose control is sensitive to rapamycin, such Oncogene (2006) 25, 6423–6435. doi:10.1038/sj.onc.1209887 as the ribosomal protein S6 (p70) kinases and eIF4E- binding proteins (4E-BPs). These motifs are termed Keywords: apoptosis; mRNA translation; protein kinase; TOR-signalling or TOS motifs (Schalm and Blenis, initiation; elongation; rapamycin 2002). mTORC1 mediates effects of mTOR that show the ‘classical’ signature of rapamycin-sensitivity, while the known effects of mTORC2 are insensitive to this drug. Thus, by no means all of the biological functions The signalling pathway involving the mammalian target of effects of mTOR are blocked by rapamycin. of rapamycin (mTOR) is now the centre of substantial The basis of the rapamycin-sensitivity of mTORC1- attention due to its key roles in regulating cell function mediated effects is unclear – rapamycin does not, for and its links with human diseases, including certain example, affect the overall integrity of mTORC1, types of cancer. The best-understood roles of mTOR in although it may alter the stability of the interactions mammalian cells are related to the control of mRNA between some of the partner proteins (Jacinto et al., translation. The purpose of this article is, firstly, to 2004; Sarbassov et al., 2006). The latter paper also review current knowledge about signalling downstream showed that long-term treatment of certain types of cells of mTOR to the translational machinery and, secondly, results in the loss of mTORC2 complexes, in addition to to described the links between mTOR signalling and the short-term inhibitory effects of rapamycin on the control of the eukaryotic translation initiation mTORC1. It is suggested that this effect on mTORC2 factor eIF4E. eIF4E plays important roles in mRNA reflects the ability of the rapamycin-FKBP12 complex to translation and probably in mRNA metabolism, and is prevent the binding of rictor to newly-synthesized mTOR. It is also unclear why short-term control by mTORC2 of protein kinase B (PKB, also termed Akt) Correspondence: Dr CG Proud, Department of Biochemistry & Molecular Biology, University of British Columbia, Life Sciences and the cytoskeleton are insensitive to rapamycin. More Centre, 2350 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3. work is required, for example, to understand how E-mail: [email protected] rapamycin interferes with mTORC1 signalling and The mTOR signalling J Averous and CG Proud 6424 a function of mTORC1 requires a supply of amino acids, HEAT domains FAT kinase FRB FATC with the essential branched-chain amino acid leucine being most effective (Kimball and Jefferson, 2006). It is b Growth factors, etc. Growth factors, insulin, etc. not yet clear how leucine activates mTOR, but it does so by acting within the cell, and can do so by mechanisms Ras/Raf PTEN PI 3-kinase that are independent of the TSC1/2 complex that LKB1 mediates the effects of anabolic and mitogenic signals PDK1 β (Byfield et al., 2005; Nobukuni et al., 2005; Smith et al., Erk/p90 RSK G L 2005). Recent evidence suggests a role for the PI mTOR AMKP PKB/Akt 3-kinase-related kinase vps34 in the control of mTOR

rictor mTORC2 TSC2 TSC1 by amino acids (Byﬁeld et al., 2005; Nobukuni et al., 2005). vps34 functions in the regulation of macroauto- PKCα? GβL phagy, a process that is negatively controlled by mTOR ? Rheb.GTP Amino acids mTOR (Klionsky and Emr, 2000). Since autophagy likely

raptor mTORC1 supplies intracellular amino acids to maintain mTOR signalling, there is some logic in connecting these 4E-BPs S6K’s processes, although the mechanisms by which vps34 Others? may link to mTOR signalling and to the control of Via TOS motif autophagy remain to be established. The role of the Figure 1 Summary of mTOR signalling, showing (a) the basic TSC1/2 complex in controlling mTOR is described in features of mTOR and (b) its upstream control, and its complexes detail elsewhere in this volume by Corradetti and Guan. mTORC1 and mTORC2. In (b), components in red boxes are To understand how inhibition of mTOR by rapamy- tumour suppressors, while those in green boxes are proto- cin may exert antiproliferative effects, it is crucial to oncogenic. The function of GbL is unclear, but it is found in mTORC1 and mTORC2. As indicated, mTORC2 effectively acts consider the target proteins and processes that are upstream of mTORC1, by phosphorylating and activating PKB, a known to be controlled through mTOR in a rapamycin- positive regulator of mTOR signalling. Phosphorylation of TSC2 sensitive manner. There is now substantial evidence by PKB and Erk/p90RSK appears to inhibit its function as a Rheb. that dysregulation of the translational machinery can GTPase-activator protein (GAP) while AMPK-mediated phosphorylation is thought to activate this function. contribute to cell transformation (for a reviews see, e.g., Bjornsti and Houghton, 2004; Clemens, 2004; Mamane et al., 2004; Guertin and Sabatini, 2005).

how the mTORC2 complex functions to regulate its targets. By analogy with mTORC1 and raptor, rictor Signalling downstream of mTOR may also bind specific features of its targets, but so far there is no information on this. Furthermore, there is The S6 kinases at least one effect of mTOR that is not sensitive Ribosomal protein S6 has been known for more than 30 to rapamycin, but appears not to be mediated by years to be a phosphoprotein (Gressner and Wool, mTORC2, that is, the phosphorylation of certain 1974): at least five sites of phosphorylation exist in its regulatory sites in 4E-BP1/2 (Wang et al., 2005). This C-terminus. In the following years, two classes of protein hints at the existence of further complexity in signalling kinases were identified as being able to phosphorylate S6 downstream of mTOR. in vitro, the 70 and 90 kDa S6 kinases, which are now The upstream control of mTOR is described else- known as the p70 S6 kinases (S6Ks) and the p90 where in this issue of Oncogene. mTOR (or at least ribosomal protein S6 kinases, or RSKs (Avruch et al., mTORC1) is regulated by signalling pathways linked to 2001; Roux and Blenis, 2004). The subsequent discovery several oncoproteins or tumour suppressors including that the phosphorylation of S6 is sensitive to, and in phosphatidyl inositol (PI) 3-kinase, the lipid phospha- many cases completely blocked by, rapamycin pointed tase PTEN, the protein kinase LKB1, Ras and Raf. to the former enzymes as the main physiological S6 These effects involve signalling through PKB, the AMP- kinases, as their activation is mediated by mTOR activated protein kinase (AMPK) or the ‘classical’ MAP (Avruch et al., 2001). The RSKs, in contrast, are not kinase ERK (Guertin and Sabatini, 2005; Ma et al., affected by rapamycin as they are activated via the 2005) (see article by Corradetti and Guan, in this classical MAP kinase (ERK) pathway (Roux and Blenis, volume). mTOR signalling is activated in cells with 2004). Recent data from S6 kinase knockout mice have loss-of-function mutations in, for example, TSC1/2 or revealed an input from ERK signalling to S6 phospho- PTEN. As assessed by the phosphorylation of 4E-BP1, rylation, at least in response to certain stimuli (Pende it is also hyperactivated in Src-transformed cells et al., 2004). Thus, RSKs or other kinases downstream (Tuhackova et al., 1999). Several aspects of the links of ERK may contribute to S6 phosphorylation under between mTOR signalling and cancer have recently been specific conditions. reviewed (Guertin and Sabatini, 2005; Wullschleger Two p70S6 kinase genes exist in mouse and man et al., 2006). (S6K1 and S6K2), each of which can give rise to two mTOR provides a link between the availability of distinct proteins due to alternative splicing of the amino acids and the the regulation of translation. The mRNAs (Avruch et al., 2001; Figure 2a). The longer

Oncogene The mTOR signalling J Averous and CG Proud 6425 Upstream which all five of the serines that are phosphorylated by NLS PDK1 mTOR kinases? the S6 kinases were mutated to alanine. This work (only in long indicated a role for S6 phosphorylation in cell growth splice variants) P P P P P P P P Catalytic domain (Ruvinsky et al., 2005). Despite this decrease in cell size, the knock-in cells actually showed faster rates of protein Phosphoregulatory synthesis. This could reflect increased access of the S6 region kinases to other substrates involved in translation, such as eIF4B and eEF2 kinase (Figure 2). The phosphorylation of these proteins by S6K1 is likely to lead to more BAD mTORIRS1 SKAR rpS6 eEF2 eIF4B CREMτ efficient initiation or elongation, respectively (Wang kinase et al., 2001; Raught et al., 2004). An earlier idea that the Figure 2 Summary of the 70 kDa S6 kinases (based on S6K1), role of the S6 kinases, and probably S6 phosphoryla- showing the main features of their structures and control (e.g., tion, was to control the translation of the mRNAs for phosphorylation sites) as well as known substrates. As indicated, ribosomal proteins (so-called 50-TOP mRNAs) was not phosphorylation at the mTOR site (Thr389in the short form of substantiated by later data from the S6K knockout mice S6K1) allows PDK1 to phosphorylate and fully turn on the S6 kinases. NLS ¼ nuclear localisation signal; the black box denotes or from the animals in which the phosphorylation sites the kinase catalytic domain. Other phosphorylation sites are in S6 were converted to alanines (Pende et al., 2004; mentioned in the text. Ruvinsky et al., 2005). In many investigations that focused on the role of version of each contains, in its N-terminal region (which mTOR in transformation, the levels of phosphorylation is not present in the shorter version), a potential nuclear- of S6K1 and S6 have been studied as a read-out for localization signal. S6K2 also possesses a functional mTOR activity. This reflects the fact that phosphoryla- nuclear localization signal in the C-terminus (Koh et al., tion of S6K and S6 is very sensitive to rapamycin 1999). treatment. The antiproliferative/apoptotic effects of The S6 kinases are activated by phosphorylation rapamycin were often attributed to its effect on S6K at multiple sites (Figure 2). Several lie within the and the presumed role of S6K in controlling the C-terminus, while two others lie immediately C-terminal translational machinery, although there is little evidence to the catalytic domain. One of these, Thr389in the that S6 phosphorylation/the S6Ks play a major role in shorter form of S6K1, is directly phosphorylated by promoting protein synthesis. However, it is clear that mTOR as part of the mTORC1 complex (Alessi et al., S6K activity is enhanced in several cancer cell lines or 1998; Pullen et al., 1998). Phosphorylation here is tumours. In numerous cases, this is due to the increase required for the phosphorylation of S6K1 by PDK1 of mTOR activity that occurs when the tumour (phosphoinositide-dependent kinase 1) at a threonine in suppressors LKB1, PTEN or TSC are mutated with the activation loop of the catalytic domain, Thr229. loss of function. It has also been observed that the level Phosphorylation at Thr229allows complete activation of S6K1 could be also increased. It is established that of S6K1 (Avruch et al., 2001). S6K2 is likely regulated in the amplification of chromosomal locus 17q23, which a similar manner. The N-termini of S6K1 and 2 each contains the S6K1 gene, is a feature of numerous contain a TOS motif that interacts with the mTOR cancers such as meningioma (Surace et al., 2004) or partner, raptor, to facilitate phosphorylation of S6Ks by breast cancer (Barlund et al., 2000). This suggests that mTORC1 (Schalm and Blenis, 2002). The phosphoryla- S6K1 may participate in carcinogenesis independently tion of the sites in the C-terminal phosphoregulatory of other targets of mTOR, but this idea requires further region is believed to allow access to the sites (Thr389/ investigation. Ser229) whose phosphorylation leads to full activation Few studies have addressed the role of S6K in the (Avruch et al., 2001). It is not clear how the processes leading to carcinogenesis. Single and double phosphorylation of the C-terminal sites is brought knockout (S6K1À/À, S6K2À/À and S6K1À/À S6K2À/À) about or which kinase catalyses this: mTORC1 prima- mice have been generated. The major observations are rily phosphorylates Thr389, which as discussed is that the S6K1À/À animals exhibit a smaller size and controlled by the C-terminal phosphorylation sites, reduced growth and the S6K1À/À S6K2À/À, which exhibit which are sensitive to rapamycin. This implies that a pronounced perinatal lethality (Pende et al., 2004). mTOR also, probably indirectly, brings about the However, there are no data concerning tumorigenesis in phosphorylation of the C-terminal sites. A motif such cells or animals. Addressing the role of S6Ks in RSPRR present in this region appears to plays a crucial transformation presents a number of technical difficul- role in the inhibitory effect of the C-terminal region. It is ties. For example, (i) there is no specific inhibitor of S6K proposed that a negative regulator of S6K1 binds to this activity; (ii) the elimination of S6K1 could potentially be motif and that mTOR is able to disrupt its binding rescued by S6K2; and (iii) the elimination of both S6K1 (Schalm et al., 2005). The C-terminal region of S6K1 and S6K2 may profoundly alter the physiology of the also governs whether S6K1 can be phosphorylated by cell. The overexpression of a rapamycin-resistant form mTORC2. A deletion mutant lacking this region is a of S6K1 increased cell size (Fingar et al., 2002) and the substrate for mTORC2 (Ali and Sabatini, 2005). proportion of cells in S phase in the presence of serum The physiological role of S6 phosphorylation is still (Fingar et al., 2004). Conversely, depletion of S6K1 quite unclear. Recent studies used ‘knock-in’ mice in by RNAi decreases the percentage of cell in S phase

Oncogene The mTOR signalling J Averous and CG Proud 6426 (Fingar et al., 2004). This effect on cell cycle could mTOR be the consequence of a direct regulation of the activity of one or more cell cycle regulators. This effect kinase? kinase? S6K could also be an indirect consequence of the regulation of cell growth by S6K1 via mechanisms that are so 78 359 366 P P P far obscure. More recently, it has been demonstrated Catalytic domain eEF2-binding? that the overexpression of a rapamycin-resistant S6K1 CaM- mutant in ovarian cancer cell line increased the binding expression of metalloproteinase 9, which favours the invasiveness of the cells (Zhou and Wong, 2006). S6K1 eEF2(P) has recently been shown to phosphorylate eIF4B (inactivation) (Raught et al., 2004) and facilitate its recruitment into Figure 3 Overview of eEF2 and eEF2 kinase, including regulatory initiation complexes (Holz et al., 2005), and this could phosphorylation sites in eEF2 kinase that are linked to mTOR play an important role in the regulation of the signaling. The (mTOR-linked) kinases that act at Ser78 and Ser359 translation by mTOR. remain to be identified. S6K1 has also been shown to be responsible for a negative feedback control of IRS1 and 2. This point eEF2 kinase is a phosphoprotein. At least three should be taken carefully into consideration. For phosphorylation sites are regulated by mTOR, as judged example, in S6K1À/À mice, it appears that insulin- by their sensitivities to rapamycin and/or amino-acid sensitivity is increased and this apparently decreases starvation (which lead to their dephosphorylation) the onset of obesity and diabetes with age (Pende et al., (Browne and Proud, 2002; Browne and Proud, 2004; 2000). So it appears that in cancer cells where S6K1 is Figure 3). However, eEF2 kinase does not possess a specifically inhibited, IRS 1 and 2 could be sensitized to TOS motif, does not bind to raptor, and is not a direct insulin (and other stimuli) or even constitutively substrate for phosphorylation by mTOR in vitro (Smith activated. This effect on the PI3K/PKB axis could and Proud, unpublished). Ser366 (in human eEF2 actually improve the survival of cancer cell lines by kinase) is phosphorylated by S6K1 (and by RSKs), and inhibiting apoptotic pathway components such as phosphorylation here causes the inactivation of eEF2 FOXO. Thus, inhibiting S6K could actually promote kinase at low calcium concentrations (i.e., lowers its sensi- antiapoptotic signalling. Finally, the evidence that tivity to activation by Ca2 þ )(Wanget al., 2001). Phospho- S6K1 is a good target in cancer therapy is not so rylation at Ser359also inactivates eEF2 kinase (Knebel clear (for a review see Manning, 2004). Although et al., 2001), but the kinase acting here has yet to be identi- additional functions have been ascribed to S6K1 (see, fied. Phosphorylation at the third site, Ser78, also causes e.g., Holz et al., 2005; for a review see Ruvinsky and inactivation of eEF2 kinase, in this case by inhibiting Meyuhas, 2006), it is not clear how they could the binding of CaM, which binds immediately C-terminal contribute to transformation. to Ser78 (Browne and Proud, 2004). The mTOR-controlled kinase acting at this site is also unknown. There are data indicating that eEF2 kinase activity is Regulation of eEF2 and eEF2 kinase elevated in certain cancer cells (e.g., breast cancer; Parmer et al., 1999) and that its activity is enhanced in Eukaryotic elongation factor 2 (eEF2) mediates the proliferating cells (Bagaglio et al., 1993; Bagaglio and translocation step of translation elongation in which the Hait, 1994; Cheng et al., 1995; Parmer et al., 1997). This ribosome moves relative to the mRNA by one codon, appears surprising since eEF2 kinase is a negative and the peptidyl-tRNA shifts from the A- to the P-site regulator of protein synthesis, and thus presumably of (the spent tRNA moving to the exit site). eEF2 binds cell growth/proliferation. Recent studies show that a GTP and this nucleotide is hydrolysed during the compound that acts as a selective inhibitor of eEF2 translocation process. eEF2 undergoes phosphorylation kinase (NH125) decreases the viability of a range of at Thr56 within the GTP-binding domain and this cancer cell lines, and this effect is countered by artificial modification interferes with its ability to bind the overexpression of eEF2 kinase consistent with the idea ribosome, thus inhibiting its function (Carlberg et al., that eEF2 kinase plays a role in cell viability (Arora 1990; Browne and Proud, 2002). et al., 2003). Earlier data obtained with another The kinase acting on eEF2 is eEF2 kinase, a calcium/ compound that inhibits eEF2 kinase, rottlerin, indicated calmodulin-dependent enzyme that has a very unusual loss of viability and growth, as well as changes in cell catalytic domain, unrelated in sequence to almost all morphology (Parmer et al., 1997). However, it is now other protein kinases (Ryazanov et al., 1997) (Figure 3). clear that rottlerin can inhibit other kinases in addition Insulin and other agents that activate protein synthesis to eEF2 kinase (Davies et al., 2000) and caution must cause the rapid dephosphorylation of eEF2. The effect therefore be exercised in interpreting data obtained with of insulin is accompanied by accelerated rates of such reagents. elongation, and by decreased activity of eEF2 kinase: Recent data suggest a role for eEF2 kinase in the all three effects are blocked by rapamycin (Redpath activation of autophagy (Wu et al., 2006), which as et al., 1996). mTOR thus negatively regulates eEF2 noted above is negatively regulated by mTOR signalling. kinase and thereby activates eEF2. Analysis of cells, tissues or animals that lack eEF2

Oncogene The mTOR signalling J Averous and CG Proud 6427 kinase will be required to establish the contribution a site in its C-terminus (Ser209; Flynn and Proud, 1995; made by phosphorylation of eEF2 to the control of Joshi et al., 1995) and sequestration by small, heat- protein synthesis and to cell physiology. There is stable phosphoproteins termed 4E-binding proteins, or currently a high level of interest in autophagy based 4E-BPs (Gingras et al., 1999; Figure 4a). Very recent on recent data that autophagy may suppress tumour data also show that its intracellular localization is also development, although the situation is not clearcut (Ng regulated by mTOR. and Huang, 2005; Hait et al., 2006). The effect of phosphorylation on the function of eIF4E has been the subject of mixed reports, and its physiological role remains unclear (Scheper and Control of eIF4E Proud, 2002). Recent data suggest that phosphorylation of eIF4E weakens its binding to 7-methylguanosine Eukaryotic initiation factor 4E (eIF4E) is subject to triphosphate and to capped RNA (Scheper et al., 2002; two main types of rapid regulation: phosphorylation at Slepenkov et al., 2006). Detailed biophysical studies are

a Inhibition of translation initiation

mTOR,…?

P P P P P MAPK (Erk, 4E-BP1 p38 MAPK αβ)

P P P eIF4E MNKs

? eIF4G ? ? P P P P P 4E-T

eIF4E nuclear import Translation mTOR? mRNA degradation (P-bodies) initiation

b GβL

mTOR

2 1 ? 4 3 raptor

T37 T46 S65 T70 S101 S112 P P P P P P RAIP 4E-BP1 TOS

Figure 4 (a) Summary of eIF4E and its binding partners (and their roles, i.e., eIF4G/F, 4E-BPs, 4E-T. Mnks); binding of 4E-BP1 to eIF4E prevents the formation of eIF4F complexes by competing with eIF4G. The phosphorylation of 4E-BP1 prevents its binding to eIF4E. The Mnks are activated by phosphorylation and need to bind to eIF4G in order to phosphorylate eIF4E. The biological role of eIF4E phosphorylation is not yet well-deﬁned (see text). The role of mTOR-dependent phosphorylation of eIF4G is quite unclear. 4E-T is a phosphoprotein but the kinase(s) involved and the effects of the phosphorylation are unknown. (b) Outline of 4E-BPs (based on 4E-BP1), showing motifs and phosphorylation sites. It is not clear how the RAIP motif functions in the mTOR-dependent control of 4E-BP1. It is also not clear whether mTOR directly phosphorylates all four sites (Thr37/46/70 and Ser65) in vivo or recruits another kinase(s) to do so. Other kinases that are able to phosphorylate 4E-BP1 in vivo or in vitro independently of mTOR are not shown as their relevance for the physiological control of 4E-BP1 is unclear. The 101 and 112 sites are constitutively phosphorylated in HEK-293 cells. The numbered arrows indicate the apparent order of the hierarchical phosphorylation of these sites by mTOR signalling. The dashed arrows denote phosphorylation events that depend on mTOR but may not be catalysed directly by mTOR.

Oncogene The mTOR signalling J Averous and CG Proud 6428 consistent with the idea that electrostatic repulsion required orientation (as well as increasing their effective between the phosphoserine and negative charges on the local concentrations) (Pyronnet et al., 1999). capped RNA contribute to weakening cap-eIF4E A second mode of regulation of eIF4E is through its binding (Zuberek et al., 2003, 2004). One may speculate regulated binding to partner proteins, as described next. that this serves to allow additional ribosomal initiation This involves mTOR. complexes to bind the mRNA while the previous one is still engaged in scanning, or that it may permit eIF4E to be released from one mRNA in order to bind others, Binding partners for eIF4E: roles for eIF4E in mRNA whose translation has been stimulated by other mechan- translation, transport and degradation isms (Scheper and Proud, 2002). Both could contribute to the activation of mRNA translation. However, there A surface on the so-called ‘dorsal’ face of eIF4E plays a are so far no data on the function of eIF4E phospho- key role in its binding to several partner proteins, one rylation in mRNA translation in mammalian cells. residue, Trp73, being especially important – mutation of The kinases that phosphorylate eIF4E are the Mnks this residue to alanine abolishes these interactions. (MAP kinase-interacting kinases or MAP kinase signal- These partners include the translation initiation scaffold integrating kinases) (Scheper and Proud, 2002; Ueda protein eIF4G, the eIF4E-binding proteins (4E-BPs) et al., 2004; Figure 4a). There are two Mnk genes in and the protein 4E-T, which was first reported as a rodents and man. In man, each gives rise to two proteins nucleocytoplasmic shuttling protein for eIF4E (Dostie differing at their C-termini. The longer form of Mnk1 et al., 2000). All these proteins contain similar motifs (corresponding to the only Mnk1 species identified in involved in their binding to eIF4E (Figure 4a). There are mice) is tightly regulated by MAP kinase signalling, two eIF4G genes in mammals, eIF4GI and eIF4GII through the ERK and p38 MAP kinase a/b pathways (Gingras et al., 1999). The eIF4GI protein is much (Fukunaga and Hunter, 1997; Waskiewicz et al., 1997) better characterized than eIF4GII. It exists as multiple (these enzymes bind its C-terminus and switch it on). In isoforms that differ at their N-termini (Bradley et al., contrast, murine Mnk2 (or the longer form of the 2002; Byrd et al., 2005). human enzyme) has high basal activity that is affected eIF4G binds a number of other proteins, which little by activation or inhibition of the ERK pathway include the Mnks (through a region in the extreme (Scheper et al., 2001), probably because it can bind to C-terminus of eIF4G), the RNA helicase eIF4A, the activated ERK, allowing Mnk2 to remain active in cells multisubunit factor eIF3 (which brings to together the that have not been stimulated or have even been treated 40S ribosomal subunit and eIF4G and its partners) and with compounds that block ERK activation (Parra the poly(A)-binding protein, PABP. The eIF4E/eIF4G/ et al., 2005). It binds relatively less well to p38 MAP eIF4A subcomplex is often referred to as eIF4F. The kinase a/b than Mnk1 does. Given that the ERK helicase function of eIF4A is thought to be of particular pathway is activated by oncoproteins such as Ras and importance for the translation of mRNAs that have Raf, it is possible that the Mnks and eIF4E phospho- long and structured 50-untranslated regions (50-UTRs), rylation play roles in cell transformation. Indeed, it has which require ‘unwinding’ in order for ribosomes to been shown that overexpression of a mutant of eIF4E in be able to traverse the 50-UTR and reach the start which Ser209has been altered to alanine is much less codon (Gingras et al., 1999). Increased engagement of efficient than wildtype eIF4E in transforming NIH 3T3 eIF4E with eIF4G (formation of ‘eIF4F’) will, accord- cells, as judged by its ability to induce the formation of ing to this idea, favour the translation of such structured foci (Topisirovic et al., 2004). The overexpression of wild mRNAs. There is (limited) experimental evidence in type, but not mutant, eIF4E is also accompanied by favour of this (see, e.g., Koromilas et al., 1992), as an increase in cyclin D1 levels. The fact the Mnk inhibi- reviewed more fully in Mamane et al. (2004). A number tor CGP57380 was able to decrease cyclin D1 expression of transformation-related proteins are encoded by ‘less reinforced the idea that the phosphorylation of eIF4E competitive’ mRNAs, including cyclin D1, c-myc and may participate in the process of transformation. vascular endothelial-growth factor (VEGF; reviewed by The short forms of human Mnk1 and 2 (termed Graff and Zimmer (2003) and Mamane et al. (2004)). Mnk1b/2b) are partially nuclear (Scheper et al., 2003; Their translation is expected to be increased by the O’Loghlen et al., 2004): in the case of Mnk1, this is due ‘activation’ of eIF4E that results from activated to the absence of the nuclear export signal found in mTOR signalling and the release of eIF4E from Mnk1a. The Mnks may thus have substrates that are inhibition by 4E-BP1. nuclear: these would include eIF4E itself, which is The activity of eIF4A is enhanced by eIF4B (an partially nuclear (Strudwick and Borden, 2002) (see RNA-binding protein) and eIF4B is a substrate for below). The N-termini of all Mnk isoforms contains a S6K1 (Raught et al., 2004). Its phosphorylation by polybasic sequence that serves both to recruit the Mnks S6K1 favours its binding to eIF3 (Holz et al., 2005). to eIF4G and to interact with importin a and allow their Indeed, S6K1 is actually recruited to the translational nuclear import (Waskiewicz et al., 1999; Parra-Palau machinery through an interaction with eIF3 (Holz et al., et al., 2003). The association of Mnks with eIF4G is 2005). eIF4G is subject to rapamycin-sensitive phos- important for the efficient phosphorylation of eIF4E phorylation at several sites although the functional in vivo, presumably because, by binding both kinase and significance of these phosphorylations and the kinase(s) substrate, eIF4G brings these proteins together in the responsible for them are so far unknown (Raught et al.,

Oncogene The mTOR signalling J Averous and CG Proud 6429 2000). Nevertheless, it has recently been reported that insufficient to bring about release from eIF4E. insulin stimulates the association of eIF3 with eIF4G Phosphorylation of both Ser65 and Thr70 depends and that this is inhibited by rapamycin (Harris et al., upon the prior phosphorylation of the N-terminal 2006). It has not so far been reported whether cells from threonines, and modification of Thr46 may precede S6K1À/À S6K2À/À mice show any defect in protein and be required for phosphorylation of Thr37 (Mothe- synthesis. Satney et al., 2000b; Gingras et al., 2001; Herbert et al., Interestingly, overexpression of other components 2002). The phosphorylation of Thr70 and Ser65 in of eIF4F is also associated with transformation. human 4E-BP1 depends upon a further site, Ser101 The mRNA for eIF4A is highly expressed in some (Wang et al., 2003). 4E-BP1 is thus subject to a complex tumours or tumour cells (Eberle et al., 1997; Shuda series of hierarchical phosphorylation events (summa- et al., 2000). Artificial overexpression of eIF4GI in rized in Figure 4b). NIH3T3 cells leads to anchorage-independent cell The phosphorylation of the N-terminal threonines in proliferation and tumour formation in nude mice 4E-BP1 and 4E-BP2 depends upon a motif in the (Fukuchi-Shimogori et al., 1997), which is consistent N-terminus of 4E-BP1, which includes the sequence Arg- with a key role for eIF4F in events that lead to Ala-Ile-Pro (hence ‘RAIP’ motif) (Tee and Proud, 2002; transformation. Thus, inhibiting mTOR signalling and Beugnet et al., 2003) (Figure 4b). Their phosphorylation thereby promoting the loss of such complexes may is not greatly affected by inactivation of the TOS motif restrain transformation, in an analogous way to the and is rather insensitive to rapamycin (Wang et al., effect of overexpressing 4E-BPs, which also prevent 2005). This could suggest that it is mediated indepen- eIF4F complex formation (Rousseau et al., 1996a). dently of mTOR. However, several lines of evidence Consistent with this, expression of phosphorylation- suggest that their phosphorylation is mediated by defective mutants of 4E-BP1 suppressed the tumori- mTOR: (i) it is inhibited by starvation of cells for genicity of breast carcinoma cells and the spontaneous amino acids, in common with effects mediated by loss of expression of these 4E-BP1 mutants correlated mTORC1; (ii) it is activated by Rheb, a stimulator of with reversion of the cells to a malignant phenotype mTOR (or least, mTORC1); (iii) it is suppressed by (Avdulov et al., 2004). TSC1/2, the GTPase-activating complex for Rheb; (iv) 4E-T also contains the type of eIF4E-binding its is sensitive to inhibitors of the kinase activity of motif found in eIF4GI/II and the 4E-BPs (Dostie mTOR, such as wortmannin, and (v) it is decreased in et al., 2000). It was initially described as a shuttling cells in which mTOR levels have been knocked down protein for eIF4E, mediating its import into the (Wang et al., 2005). The insensitivity to rapamycin could nucleus. Recent data indicate that it also recruits suggest that mTORC2 mediates the phosphorylation of eIF4E to P (processing)-bodies, which are sites for Thr37/46, but the other data argue against this: for mRNA degradation (Ferraiuolo et al., 2005). 4E-T is a example, amino acids are not known to regulate phosphoprotein, although the role (if any) of phospho- mTORC2. mTORC2 cannot phosphorylate 4E-BP1 on rylation in controlling its function or localization is Thr 37/46 (Fonseca and Proud, unpublished data). unclear. Thus, it seems likely that the phosphorylation of Thr37/ 46 in 4E-BP1 is mediated by mTOR through a mechanism that is independent of both raptor Control of the 4E-BPs by mTOR (mTORC1) and rictor (mTORC2). Identifying how this works would generate important new data on mTOR The regulation of the 4E-BP1 is much better understood signalling as well as perhaps providing a novel and than that of 4E-BP2/3. This protein undergoes phos- specific approach to inhibiting 4E-BP1 phosphorylation phorylation at seven sites, at least four of which are and thus eIF4E function, without inhibiting with S6Ks linked to mTOR signalling (Mothe-Satney et al., and promoting PI 3-kinase signalling. 2000a, b; Gingras et al., 2001; Wang et al., 2005). These Whereas 4E-BP1 is mainly cytoplasmic, 4E-BP3 is are threonines 37, 46 and 70, and serine 65 (numbering also found in the nucleus (Kleijn et al., 2002). It shares based on human 4E-BP1; numbers for the rodent this property with its partner, eIF4E, although the proteins differ by À1) (Figure 4b). Ser65 and Thr70 lie biological significance of this is unclear. 4E-BP2 and close to the eIF4E-binding site and, in many cells, their 4E-BP3 both possess C-terminal TOS motifs that are phosphorylation is stimulated by insulin (etc) in a identical in sequence to that in 4E-BP1. Only 4E-BP2 rapamycin-sensitive manner. In some cells, such as contains a RAIP motif, which apparently contributes to CHO cells, they are constitutively phosphorylated controlling 4E-BP2 phosphorylation by amino acids in a (Wang et al., 2005). In HEK 293 cells, for example, similar way to its role in controlling 4E-BP1 (Wang their phosphorylation is dependent upon the C-terminal et al., 2005). TOS motif (Beugnet et al., 2003). Although there are data that suggest that phosphorylation of this site is required for release of eIF4E from 4E-BP1, the eIF4E: roles in cell transformation and survival role of phosphorylation of Ser65 is still controversial (Ferguson et al., 2003). Molecular dynamics findings eIF4E may be described as a proto-oncogene. There is (Tomoo et al., 2005) and earlier biophysical data compelling evidence that it plays a role in the processes suggest that phosphorylation of Ser65 and Thr70 is that lead to cell transformation. Its expression is

Oncogene The mTOR signalling J Averous and CG Proud 6430 increased in several kinds of tumors (reviewed in leukaemia protein, PML (Cohen et al., 2001; Kentsis Bjornsti and Houghton, 2004; De Benedetti and Graff, et al., 2001), which is also associated with Mnk2b 2004). Moreover, the overexpression of eIF4E can (Scheper et al., 2003). Nuclear eIF4E may be involved in induce cellular transformation in vitro and in vivo the export of certain mRNAs to the cytoplasm (Lazaris-Karatzas et al., 1990; Ruggero et al., 2004; (Rousseau et al., 1996b; Culjkovic et al., 2005): over- Wendel et al., 2004). Using a murine model, eIF4E was expression of eIF4E enhances the expression of cyclin found to recapitulate the effect of PKB overexpression D1 not by increasing the translation of its mRNA in terms of tumorigenesis and drug resistance (Wendel directly, but rather by increasing the level of the cyclin et al., 2004). PKB overexpression did not increase the D1 mRNA in the cytoplasm (Rousseau et al., 1996b). level of eIF4E but led to activation of mTOR and The mechanism by which eIF4E does this is unclear, but hyperphosphorylation of 4E-BP1. This results in release it involves a feature in the 30-UTR of the cyclin D1 (activation) of eIF4E and to increased formation of mRNA (Culjkovic et al., 2005), as well as the cap- eIF4F complexes. While rapamycin was effective in binding activity of eIF4E. Recent kinetic studies suggest restoring the sensitivity of PKB-expressing cells to that eIF4E may interact with additional regions of the proapoptotic stimuli, it did not do so in eIF4E- mRNA, via interactions with positively charged side overexpressing cells. The data suggest that eIF4E may chains in the groove in the 3-D structure of eIF4E where play a key role in the mechanisms by which PKB the mRNA may be located (Slepenkov et al., 2006). transforms cells, and can be interpreted as suggesting However, the increase in cyclin D1 expression caused by that the ‘target’ through which rapamycin acts to enhanced levels of eIF4E does not require the ability of sensitize cells to proapoptotic stimuli is likely to be eIF4E to engage in translation, as illustrated by the 4E-BP1/eIF4E. Thus, control of the translational ability of the Trp73Ala mutant of eIF4E, which cannot machinery may be a key element of the mechanism by bind eIF4G, to increase cyclin D1 levels (Topisirovic which PKB transforms cells. et al., 2002, 2003). 4E-T binds eIF4E through a similar The overexpression of eIF4E does not lead to a global motif to that found in eIF4G, so this residue should be increase in mRNA translation but rather to the needed for binding of eIF4E to 4E-T and for nuclear increased expression of specific proteins such as ODC, import of eIF4E. This prompts the question: how does c-myc or cyclin D1. In several cases, their mRNAs eIF4E(W73A) get into the nucleus to promote export of possess a long and structured 50-UTRs and, as the cyclin D1 message? It would be important also to mentioned earlier, the translation of such mRNAs is define whether the binding of 4E-BP1 to eIF4E could thought to be highly dependent on the eIF4F complex interferes with eIF4E localization and so the transport (for reviews see Clemens, 2004; Mamane et al., 2004). of cyclin D1 mRNA, it will define a new role for mTOR Several of these mRNAs encode proteins that are signalling. Nevertheless, the existing data provoke the involved or implicated in tumorigenicity (see review idea that one way in which PML exerts its tumour- Mamane et al., Oncogene 2004). The c-myc mRNA, in suppressor function is by decreasing the binding of particular, has been studied extensively. In a recent eIF4E to the cyclin D1 mRNA (PML diminishes the study, Ruggero et al. (2004) have shown that over- cap-binding ability of eIF4E), thereby impairing export expression of eIF4E under the control of the b-actin of this mRNA, and inhibiting the G1/S transition of the promoter leads to tumour formation. When these mice cell cycle (Cohen et al., 2001). were crossed with others expressing c-myc under the 4E-BP1 should counter the ability of eIF4E to control of the immunoglobulin heavy-chain enhancer promote cell transformation. It has been shown in cells element, lymphoma formation was greatly accelerated. in culture that overexpression of 4E-BP1 can reduce the It appears that enhanced expression of eIF4E not only tumorigenic effect of eIF4E overexpression (Avdulov upregulates expression of c-myc but also that eIF4E et al., 2004). Moreover, the same authors have cooperates with it in the process of transformation. implanted mammary cancer cell MDA-MB-468 trans- Although c-myc is an oncogene, it can also promote fected with 4E-BP1 or a mutant of 4E-BP1, which apoptosis, and this effect is antagonized by eIF4E constitutively binds to eIF4E, into mice. Expression of overexpression which exerts antiapoptotic effects (Li wildtype 4E-BP1 decreased tumour size and expression et al., 2003). On the other hand, c-myc antagonizes the of the mutant had an even larger effect. Lynch et al. effect of eIF4E overexpression on cellular senescence. In (2004) have also shown that the overexpression of a addition to the reported ability of c-myc to increase the mutant of 4E-BP1 inhibits transformation induces by level of expression of other proteins involved in eIF4E and overexpression. However, this mutant is also translation (reviewed in Schmidt, 2004), it likely also able to block the transformation induces by c-myc cooperates with eIF4E in transformation by virtue of its overexpression in Rat1 cells. They observe a decrease of ability to counter the effect of eIF4E on cellular colony formation in soft agar assay and also of tumour senescence. It is also important to consider that eIF4E size in nude mice implanted with the Rat1 cells. The overexpression may play a different kind of role in authors have shown that this effect of the 4E-BP1 cellular transformation, independently of its effect on mutant involved a slower G1 progression and did not translation. decrease global protein synthesis. This last result A fraction of the total cellular eIF4E is found within reinforced the idea that ‘activation’ of eIF4E does not the nucleus (reviewed by Strudwick and Borden, 2002). increase the level of general protein synthesis but rather In this compartment, it associates with the promyelocytic enhances the translation of specific mRNAs that encode

Oncogene The mTOR signalling J Averous and CG Proud 6431 proteins which play an important role, for example, in activity. In one report (Yokogami et al., 2000), the cell cycle. Conversely, overexpression of 4E-BP1 rapamycin was shown to block the phosphoryla- enhanced the ability of rapamycin to inhibit the tion of Tyr727 in STAT3 that is induced by ciliary proliferation of PC3 (prostate cancer) cells. This effect neurotrophic factor, and STAT3 activity. In response to was not apparent in cells overexpressing a mutant of interferon (IFN)-g, STAT3 undergoes dephosphoryla- 4E-BP1 that cannot bind to or inhibit eIF4E (Beugnet tion at another site, and this effect is also inhibited by and Proud, unpublished data). rapamycin (Fang et al., 2006). In this case, rapamycin actually inhibited IFNg-induced apoptosis. The links 4E-BPs and apoptosis between mTOR signalling and the control of the (de)phosphorylation of STAT3 are unknown. However, Several lines of data point to links between 4E-BP1 since the phosphorylation and function of STAT3 are (and perhaps other 4E-BPs) and programmed cell dysregulated (e.g., through constitutive tyrosine phos- death (reviewed in Proud, 2005). For example, expres- phorylation) in a range of human cancers (Klampfer, sion of 4E-BP1 promoted apoptosis of Ras-transformed 2006), inhibition of mTOR signalling may be of value in ﬁbroblasts (Polunovsky et al., 2000). Treatment of cells the context of such tumours. with cell-permeant peptides that inhibit eIF4E in a As mentioned above, it is well-established that mTOR similar way to the 4E-BPs leads to rapid cell death controls the translation of the subset of mRNAs that (Herbert et al., 2000). Conversely, as noted above, include those encoding ribosomal proteins and some expression of eIF4E exerts antiapoptotic effects (see, elongation factors (Meyuhas and Hornstein, 2000). In e.g., Polunovsky et al., 1996; Li et al., 2003, 2004; response to rapamycin treatment, these mRNAs shift Wendel et al., 2004). out of polyribosomes indicating impairment of their DNA damage, which can cause apoptosis, has two translation (reviewed by Meyuhas and Hornstein, 2000). types of effect on 4E-BP1. Firstly, prior to the onset of This control is conferred by a sequence in their extreme apoptosis (as gauged, e.g., by caspase activation), 5-ends comprising a series of pyrimidines (hence 50-tract mTOR signalling is inhibited, causing increased binding of oligopyrimidines; 50-TOP). However, it is not known of 4E-BP1 to eIF4E and inhibition of eIF4F formation how this motif confers control by mTOR or how mTOR (Tee and Proud, 2000, 2001), which may impair the modulates their translation. translation of antiapoptotic mRNAs and enhance the mTOR is also reported to regulate the transcription translation of mRNAs for proapoptotic proteins such of rDNA genes (genes encoding ribosomal RNA as APAF-1, a component of the apoptosome which (Hannan et al., 2003; Mayer et al., 2004)), as described is encoded by an mRNA that contains an internal elsewhere in this volume. A role for mTOR in ribosome entry segment (IRES) that allows eIF4E- controlling the synthesis or ribosomal proteins and independent translation (Coldwell et al., 2000). p53 may rRNA is clearly logical to link, for example, amino- be involved in mediating this effect, as activation of p53 acid availability and anabolic/mitogenic signalling to leads to inhibition of mTOR signalling (Horton et al., ribosome biogenesis. 2002; Feng et al., 2005) and dephosphorylation and degradation of eIF4GI and 4E-BP1 (Constantinou and Clemens, 2005). However, the data in the last paper Conclusions and perspectives suggest that p53 activation inhibits protein synthesis through mechanisms that are independent of rapamycin- mTOR is one of the main regulators of cell growth sensitive mTOR function. and proliferation. Its functions in controlling the Secondly, following caspase activation, 4E-BP1 is translational machinery are summarized in Figure 5. cleaved, removing the section containing the RAIP mTOR is located at the cross road of several major motif (Tee and Proud, 2002). 4E-BP1 therefore signalling pathways (including PI 3-kinase, Erk and becomes dephosphorylated, causing it to bind to AMPK) and is able to integrate a large panel of stress eIF4E in a manner that is ‘irreversible’, in the sense signal such as nutrient deprivation, energy depletion that it is no longer possible to bring about the and oxidative or hypoxic stress. It can thereby phosphorylation-mediated release of 4E-BP1. This will modulate numerous cellular functions. Inhibition of further serve to shut down protein synthesis in an mTOR by rapamycin and its analogs appears to be an ordered manner and, perhaps, also promote the efﬁcient way, in certain cases, to stop the growth of upregulation of pro-apoptotic proteins encoded by tumour cells and/or to promote apoptosis. The use of IRES-containing mRNAs. rapamycin as an anticancer drug is now being evaluated on patients with solid or haematological tumours. Other targets of mTOR signalling However, it is important to consider that the effectiveness of inhibition of mTOR very likely differs There are no other direct targets for mTOR signalling substantially between different types of tumours. It that can really be described as ‘well-characterized’ in appears logical that one of the main determinants of this terms of their control by mTOR. The transcription sensitivity is the level of activity of mTOR itself. By factor STAT3 has been reported to be regulated by virtue of its location in the signalling network, its mTOR in terms both of its phosphorylation and its activity is regulated by several tumour suppressors such

Oncogene The mTOR signalling J Averous and CG Proud 6432

60S

P 4E-BP1 P P UTR R 60S 3’- ’-UT 60S 5 A 40S 60S A 40S A -cap 40S A 5’ P P 40S A -cap A 5’ eIF4E eEF2 A A eIF4E ?? eEF2 40S P P 4E-BP1 eIF3 P P P eIF4G P eEF2 P eIF4B P kinase ?? ?? ?? Overall mTOR S6K translational capacity ?? RIBOSOME 60S BIOGENESIS 40S

Figure 5 Overview of the links between mTOR and the translational machinery. The thick black line represents an mRNA, with its 50-cap, start codon (gold star) and other features shown. Questions marks denote signaling connections or functional consequences that are not understood. Solid lines indicate signaling connections including phosphorylation events, while dashed straight lines signify association or dissociation events. The ﬁgure is entirely schematic, with no attempt to depict relative sizes of components, or actual numbers or locations of phosphorylation sites.

as PTEN or LKB1. It has been also described that p53 cyclin D1 through its control over eIF4E availability. It can inhibit mTOR activity (Feng et al., 2005). As a will be interesting to determine which other proteins consequence of this, mTOR activity is enhanced in exhibit similar regulation. Notably, one may suggest different kinds of cancers. One may hypothesize that the that HIF is one of them. HIF is a transcription factor inhibition of different targets of mTOR by rapamycin involved in the adaptation to hypoxia. HIF is known will have differing levels of importance according to the to be downregulated by rapamycin treatment. HIF ex- type of cancer. These points raise a question: is it pression increases when the tumour suppressor VHL appropriate to inhibit all the (rapamycin-sensitive) (von Hippel-Lindau), a ubiquitin ligase, is mutated targets of mTOR or just the one(s) which seems to play (Thomas et al., 2006). In that case, mTOR inhibition is an active role in the transformation or in the main- particularly efficient in inhibiting cell proliferation. tenance of the malignant phenotype. The first option is Mutating the 50-UTR of the HIF mRNA abolishes the the one currently being tested. Concerning the second effect of inhibiting mTOR (see comments in Choo and possibility, it will first be necessary to identify the Blenis (2006)). Another interesting point is that, despite relevant other targets of mTOR and then to generate the fact that mTOR is described as being inhibited by specific inhibitors that interfere with the target or its hypoxia, HIF expression is actually enhanced in control by mTOR. hypoxia. In fact, it appears that residual mTOR activity This notion that the inhibition of a specific target and is maintained under hypoxic conditions and allows the not all the targets could be beneficial is illustrated with translation of specific mRNA such as HIF (Hui et al., the example of S6K1 inhibition, which may enhance the 2006). In their work, Hui et al. observed a less profound antiapoptotic effect of the PI3 K pathway due the effect of hypoxia on 4E-BP1 phosphorylation than absence of the negative feed-back control of IRS1 by on S6K1 phosphorylation. One can thus envisage S6K1 (see review Manning). In the case of 4E-BP1, it the use of rapamycin treatment for tumours presenting may be envisaged that its activation may also lead to a low level of vascularization even if the level of the increased translation of antiapoptotic proteins. mTOR activity is low. It would be also interesting to This notion that in some case mTOR inhibition could study the regulation of the translation of proteins lead to resistance to apoptosis despite a growth involved in the control of autophagy. It is known than arrest should be studied with attention. For example, mTOR regulates this process (Meijer and Codogno, although most studies describe rapamycin as a pro- 2004) and there is a growing interest in the role of apoptotic agent, it has been already observed that autophagy in cancer (Ng and Huang, 2005). It has been rapamycin can increase levels of Bcl-2 and thus exert an shown that the oncogene Bcl-2 maintains cell survival antiapoptotic effect (Calastretti et al., 2001a, b). It may by inhibiting autophagy (Pattingre and Levine, be that the so-called mTOR inhibition elicited by 2006). a stress leads to the activation of apoptotic effectors It is important to understand more clearly the and this activation is not reproduced by rapamycin signalling events that operate downstream of mTOR. treatment. One example is the phosphorylation of 4E-BP1, notably Among the best-known targets of mTOR, 4E-BP1 the rapamycin-insensitive regulation which is not presents the most direct link with cancer by regulating dependent on mTORC2. This pathway appears to the level of key proteins such as c-myc, VEGF and be sensitive to amino acids and so could perhaps

Oncogene The mTOR signalling J Averous and CG Proud 6433 involve the class III PI3 K, but this remains to be dephosphorylation of 4E-BP1 at the rapamycin-resis- established. The importance of this rapamycin- tant site(s). The other rapamycin-insensitive pathway insensitive regulation is enforced by the work of that involves mTORC2 is also a new ﬁeld of investiga- Constantinou and Clemens (2005). Using a tempera- tion, which will perhaps allow the development of new ture-sensitive mutant of p53 in erythroleukaemia approaches to inhibit mTOR functions in a more cells, they show that p53 activation can cause the speciﬁc manner.

References

Alessi DR, Kozlowski MT, Weng Q-P, Morrice N, Avruch J. Ferraiuolo MA, Basak S, Dostie J, Murray EL, (1998). Curr Biol 8: 69–81. Schoenberg DR, Sonenberg N. (2005). J Cell Biol 170: Ali SM, Sabatini DM. (2005). J Biol Chem 280: 19445–19448. 913–924. Arora S, Yang JM, Kinzy TG, Utsumi R, Okamoto T, Fingar DC, Richardson CJ, Tee AR, Cheatham L, Tsou C, Kitayama T et al. (2003). Cancer Res 63: 6894–6899. Blenis J. (2004). Mol Cell Biol 24: 200–216. Avdulov S, Li S, Michalek V, Burrichter D, Peterson M, Fingar DC, Salama S, Tsou C, Harlow E, Blenis J. (2002). Perlman DM et al. (2004). Cancer Cell 5: 553–563. Genes Dev 16: 1472–1487. Avruch J, Belham C, Weng Q, Hara K, Yonezawa K. (2001). Flynn A, Proud CG. (1995). J Biol Chem 270: 21684–21688. Prog Mol Subcell Biol 26: 115–154. Fukuchi-Shimogori T, Ishii I, Kashiwagi K, Mashiba H, Bagaglio DM, Cheng EH, Gorelick FS, Mitsui K, Nairn AC, Ekimoto J, Igarashi K. (1997). Cancer Res 57: 5041–5044. Hait WN. (1993). Cancer Res 53: 2260–2264. Fukunaga R, Hunter T. (1997). EMBO J 16: 1921–1933. Bagaglio DM, Hait WN. (1994). Cell Growth Differ 5: Gingras A-C, Raught B, Gygi SP, Niedzwieka A, Miron M, 1403–1408. Burley SK et al. (2001). Genes Dev 15: 2852–2864. Barlund M, Monni O, Kononen J, Cornelison R, Torhorst J, Gingras A-C, Raught B, Sonenberg N. (1999). Annu Rev Sauter G et al. (2000). Cancer Res 60: 5340–5344. Biochem 68: 913–963. Beugnet A, Wang X, Proud CG. (2003). J Biol Chem 278: Graff JR, Zimmer SG. (2003). Clin Exp Metastasis 20: 40722. 265–273. Bjornsti MA, Houghton PJ. (2004). Cancer Cell 5: 519–523. Gressner AM, Wool IG. (1974). Biochem Biophys Res Bradley CA, Padovan JC, Thompson TL, Benoit CA, Commun 60: 1482–1490. Chait BT, Rhoads RE. (2002). JBiolChem277: 12559–12571. Guertin DA, Sabatini DM. (2005). Trends Mol Med 11: Browne GJ, Proud CG. (2002). Eur J Biochem 269: 5360–5368. 353–361. Browne GJ, Proud CG. (2004). Mol Cell Biol 24: 2986–2997. Hait WN, Jin S, Yang JM. (2006). Clin Cancer Res 12: Byﬁeld MP, Murray JT, Backer JM. (2005). J Biol Chem 280: 1961–1965. 33076–33082. Hannan KM, Brandenburger Y, Jenkins A, Sharkey K, Byrd MP, Zamora M, Lloyd RE. (2005). J Biol Chem 280: Cavanaugh A, Rothblum L et al. (2003). Mol Cell Biol 23: 18610–18622. 8862–8877. Calastretti A, Bevilacqua A, Ceriani C, Vigano S, Zancai P, Harris TE, Chi A, Shabanowitz J, Hunt DF, Rhoads RE, Capaccioli S et al. (2001a). Oncogene 20: 6172–6180. Lawrence Jr JC. (2006). EMBO J 25: 1659–1668. Calastretti A, Rancati F, Ceriani MC, Asnaghi L, Canti G, Herbert TP, Fahraeus R, Prescott A, Lane DP, Proud CG. Nicolin A. (2001b). Eur J Cancer 37: 2121–2128. (2000). Curr Biol 10: 793–796. Carlberg U, Nilsson A, Nygard O. (1990). Eur J Biochem 191: Herbert TP, Tee AR, Proud CG. (2002). J Biol Chem 277: 639–645. 11591–11596. Cheng EH, Gorelick FS, Czernik AJ, Bagaglio DM, Hait WN. Holz MK, Ballif BA, Gygi SP, Blenis J. (2005). Cell 123: (1995). Cell Growth Differ 6: 615–621. 569–580. Choo AY, Blenis J. (2006). Cancer Cell 9: 77–79. Horton LE, Bushell M, Barth-Baus D, Tilleray VJ, Clemens MJ. (2004). Oncogene 23: 3180–3188. Clemens MJ, Hensold JO. (2002). Oncogene 21: 5325–5334. Cohen N, Sharma M, Kentsis A, Perez JM, Strudwick S, Houghton PJ, Huang S. (2004). Curr Top Microbiol Immunol Borden KLB. (2001). EMBO J 20: 4547–4559. 279: 339–359. Coldwell MJ, Mitchell SA, Stoneley M, MacFarlane M, Hui AS, Bauer AL, Striet JB, Schnell PO, Czyzyk- Willis AE. (2000). Oncogene 19: 899–905. Krzeska MF. (2006). FASEB J 20: 466–475. Constantinou C, Clemens MJ. (2005). Oncogene 24: Jacinto E, Loewith R, Schmidt A, Lin S, Ruegg MA, Hall A 4839–4850. et al. (2004). Nat Cell Biol 6: 1122–1128. Culjkovic B, Topisirovic I, Skrabanek L, Ruiz-Gutierrez M, Joshi B, Cai AL, Keiper BD, Minich WB, Mendez R, Borden KL. (2005). J Cell Biol 169: 245–256. Beach CM et al. (1995). J Biol Chem 270: 14597–14603. Davies SP, Reddy H, Caivano M, Cohen P. (2000). Biochem Kentsis A, Campbell Dwyer E, Perez JM, Sharma M, Chen A, J 351: 95–105. Pan ZQ et al. (2001). J Mol Biol 312: 609–623. De Benedetti A, Graff JR. (2004). Oncogene 23: 3189–3199. Kimball SR, Jefferson LS. (2006). J Nutr 136: 227S–231S. Dostie J, Ferraiuolo M, Pause A, Adam SA, Sonenberg N. Klampfer L. (2006). Curr Cancer Drug Targets 6: 107–121. (2000). EMBO J 19: 3142–3156. Kleijn M, Scheper GC, Wilson ML, Tee AR, Proud CG. Eberle J, Krasagakis K, Orfanos CE. (1997). Int J Cancer 71: (2002). FEBS Lett 532: 319–323. 396–401. Klionsky DJ, Emr SD. (2000). Science 290: 1717–1721. Fang P, Hwa V, Rosenfeld RG. (2006). Exp Cell Res 312: Knebel A, Morrice N, Cohen P. (2001). EMBO J 20: 1229–1239. 4360–4369. Feng Z, Zhang H, Levine AJ, Jin S. (2005). Proc Natl Acad Sci Koh H, Jee K, Lee B, Kim J, Kim D, Yun YH et al. (1999). USA 102: 8204–8209. Oncogene 18: 5115–5119. Ferguson G, Mothe-Satney I, Lawrence Jr JC. (2003). J Biol Koromilas AE, Lazaris-Karatzas A, Sonenberg N. (1992). Chem 278: 47459–47465. EMBO J 11: 4153–4158.

Oncogene The mTOR signalling J Averous and CG Proud 6434 Lazaris-Karatzas A, Montine KS, Sonenberg N. (1990). Ruvinsky I, Meyuhas O. (2006). Trends Biochem Sci 31: Nature 345: 544–547. 342–348. Li S, Perlman DM, Peterson MS, Burrichter D, Avdulov S, Ruvinsky I, Sharon N, Lerer T, Cohen H, Stolovich-Rain M, Polunovsky VA et al. (2004). J Biol Chem 279: 21312–21317. Nir T et al. (2005). Genes Dev 19: 2199–2211. Li S, Takasu T, Perlman DM, Peterson MS, Burrichter D, Ryazanov AG, Ward MD, Mendola CE, Pavur KS, Avdulov S et al. (2003). J Biol Chem 278: 3015–3022. Dorovkov MV, Wiedmann M et al. (1997). Proc Natl Acad Lynch M, Fitzgerald C, Johnston KA, Wang S, Schmidt EV. Sci USA 94: 4884–4889. (2004). J Biol Chem 279: 3327–3339. Sarbassov dD, Ali SM, Sabatini DM. (2005). Curr Opin Cell Ma L, Chen Z, Erdjument-Bromage H, Tempst P, Pandolﬁ PP. Biol 17: 596–603. (2005). Cell 121: 179–193. Sarbassov dD, Ali SM, Sengupta S, Sheen JH, Hsu PP, Bagley Mamane Y, Petroulakis E, Rong L, Yoshida K, Ler LW, AF et al. (2006). Mol Cell 22: 159–168. Sonenberg N. (2004). Oncogene 23: 3172–3179. Schalm SS, Blenis J. (2002). Curr Biol 12: 632–639. Manning BD. (2004). J Cell Biol 167: 399–403. Schalm SS, Tee AR, Blenis J. (2005). J Biol Chem 280: Mayer C, Zhao J, Yuan X, Grummt I. (2004). Genes Dev 18: 11101–11106. 423–434. Scheper GC, Morrice NA, Kleijn M, Proud CG. (2001). Mol Meijer AJ, Codogno P. (2004). Int J Biochem Cell Biol 36: Cell Biol 21: 743–754. 2445–2462. Scheper GC, Parra J-L, Wilson ML, van Kollenburg B, Meyuhas O, Hornstein E. (2000). In: Sonenberg N, Vertegaal ACO, Han Z-G et al. (2003). Mol Cell Biol 23: Hershey JWB, Mathews MB (eds). Cold Spring Harbor 5692–5705. Laboratory Press: Cold Spring Harbor, NY, pp. 671–693. Scheper GC, Proud CG. (2002). Eur J Biochem 269: Mothe-Satney I, Brunn GJ, McMahon LP, Capaldo CT, 5350–5359. Abraham RT, Lawrence JC. (2000a). J Biol Chem 275: Scheper GC, van Kollenburg B, Hu J, Luo Y, Goss DJ, 33836–33843. Proud CG. (2002). J Biol Chem 277: 3303–3309. Mothe-Satney I, Yang D, Fadden P, Haystead TAJ, Lawrence Schmidt EV. (2004). Oncogene 23: 3217–3221. JC. (2000b). Mol Cell Biol 20: 3558–3567. Shuda M, Kondoh N, Tanaka K, Ryo A, Wakatsuki T, Ng G, Huang J. (2005). Mol Carcinog 43: 183–187. Hada A et al. (2000). Anticancer Res 20: 2489–2494. Nobukuni T, Joaquin M, Roccio M, Dann SG, Kim SY, Slepenkov SV, Darzynkiewicz E, Rhoads RE. (2006). J Biol Gulati P et al. (2005). Proc Natl Acad Sci USA 102: Chem 281: 14927–14938. 14238–14243. Smith EM, Finn SG, Tee AR, Browne GJ, Proud CG. (2005). O’Loghlen A, Gonzalez VM, Pineiro D, Perez-Morgado MI, J Biol Chem 280: 18717–18727. Salinas M, Martin ME. (2004). Exp Cell Res 299: 343–355. Strudwick S, Borden KL. (2002). Differentiation 70: 10–22. Parmer TG, Ward MD, Hait WN. (1997). Cell Growth Differ Surace EI, Lusis E, Haipek CA, Gutmann DH. (2004). Ann 8: 327–334. Neurol 56: 295–298. Parmer TG, Ward MD, Yurkow EJ, Vyas VH, Kearney TJ, Tee AR, Proud CG. (2000). Oncogene 19: 3021–3031. Hait WN. (1999). Br J Cancer 79: 59–64. Tee AR, Proud CG. (2001). Cell Death Differ 8: 841–849. Parra JL, Buxade M, Proud CG. (2005). J Biol Chem 280: Tee AR, Proud CG. (2002). Mol Cell Biol 22: 1674–1683. 37623–37633. Thomas GV, Tran C, Mellinghoff IK, Welsbie DS, Chan E, Parra-Palau JL, Scheper GC, Wilson ML, Proud CG. (2003). Fueger B et al. (2006). Nat Med 12: 122–127. J Biol Chem 278: 44197–44204. Tomoo K, Matsushita Y, Fujisaki H, Abiko F, Shen X, Pattingre S, Levine B. (2006). Cancer Res 66: 2885–2888. Taniguchi T et al. (2005). Biochim Biophys Acta 1753: Pende M, Kozma SC, Jaquet M, Oorschot V, Burcelin R, 191–208. Le Marchand-Brustel Y et al. (2000). Nature 408: 994–997. Topisirovic I, Capili AD, Borden KL. (2002). Mol Cell Biol 22: Pende M, Um SH, Mieulet V, Sticker M, Goss VL, Mestan J 6183–6198. et al. (2004). Mol Cell Biol 24: 3112–3124. Topisirovic I, Culjkovic B, Cohen N, Perez JM, Skrabanek L, Polunovsky VA, Gingras AC, Sonenberg N, Peterson M, Tan A, Borden KL. (2003). EMBO J 22: 689–703. Rubins JB et al. (2000). J Biol Chem 275: 24776–24780. Topisirovic I, Ruiz-Gutierrez M, Borden KL. (2004). Cancer Polunovsky VA, Rosenwald IB, Tan AT, White J, Chiang L, Res 64: 8639–8642. Sonenburg N et al. (1996). Mol and Cell Biol 16: 6573–6581. Tuhackova Z, Sovova V, Sloncova E, Proud CG. (1999). Proud CG. (2005). Cell Death Differ 12: 541–546. Int J Cancer 81: 963–969. Pullen N, Dennis PB, Andjelkovic M, Dufner A, Kozma SC, Ueda T, Watanabe-Fukunaga R, Fukuyama H, Nagata S, Hemmings BA et al. (1998). Science 279: 707–710. Fukunaga R. (2004). Mol Cell Biol 24: 6539–6549. Pyronnet S, Imataka H, Gingras AC, Fukunaga R, Hunter T, Wang X, Beugnet A, Murakami M, Yamanaka S, Proud CG. Sonenberg N. (1999). EMBO J 18: 270–279. (2005). Mol Cell Biol 25: 2558–2572. Raught B, Gingras AC, Gygi SP, Imataka H, Morino S, Wang X, Li W, Parra J-L, Beugnet A, Proud CG. (2003). Gradi A et al. (2000). EMBO J 19: 434–444. Mol Cell Biol 23: 1546–1557. Raught B, Peiretti F, Gingras AC, Livingstone M, Shahbazian D, Wang X, Li W, Williams M, Terada N, Alessi DR, Proud CG. Mayeur GL et al. (2004). EMBO J 23: 1761–1769. (2001). EMBO J 20: 4370–4379. Redpath NT, Foulstone EJ, Proud CG. (1996). EMBO J 15: Waskiewicz AJ, Flynn A, Proud CG, Cooper JA. (1997). 2291–2297. EMBO J 16: 1909–1920. Rousseau D, Gingras A-C, Pause A, Sonenberg N. (1996a). Waskiewicz AJ, Johnson JC, Penn B, Mahalingam M, Oncogene 13: 2415–2420. Kimball SR, Cooper JA. (1999). Mol Cell Biol 19: Rousseau D, Kaspar R, Rosenwald I, Gehrke L, Sonenberg N. 1871–1880. (1996b). Proc Natl Acad Sci USA 93: 1065–1070. Wendel HG, De Stanchina E, Fridman JS, Malina A, Ray S, Roux PP, Blenis J. (2004). Microbiol Mol Biol Rev 68: 320–344. Kogan S et al. (2004). Nature 428: 332–337. Ruggero D, Montanaro L, Ma L, Xu W, Londei P, Cordon- Wu H, Yang JM, Jin S, Zhang H, Hait WN. (2006). Cancer Cardo C et al. (2004). Nat Med 10: 484–486. Res 66: 3015–3023.

Oncogene The mTOR signalling J Averous and CG Proud 6435 Wullschleger S, Loewith R, Hall MN. (2006). Cell 124: Zuberek J, Jemielity J, Jablonowska A, Stepinski J, Dadlez M, 471–484. Stolarski R et al. (2004). Biochemistry 43: 5370–5379. Yokogami K, Wakisaka S, Avruch J, Reeves SA. (2000). Curr Zuberek J, Wyslouch-Cieszynska A, Niedzwiecka A, Biol 10: 47–50. Dadlez M, Stepinski J, Augustyniak W et al. (2003). Zhou HY, Wong AS. (2006). Endocrinology 147: 2557–2566. RNA 9: 52–61.

Oncogene