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mTOR and the control of whole body metabolism
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mTOR and the control of whole body metabolism Pazit Polak and Michael N Hall Mammalian Target of Rapamycin (mTOR) is a highly conserved protein kinase that functions as part of two distinct multiprotein complexes to regulate growth and metabolism. This review describes the most important recent advances in the mTOR signaling field. In addition, we provide an overview on the functions of mTOR in different organs, with a special focus on the role of mTOR in whole body energy metabolism. Address Biozentrum, University of Basel, Basel CH-4056, Switzerland Corresponding author: Hall, Michael N ([email protected])
activates the TSC complex. mTORC2 is activated by growth factors via an unknown mechanism. mTORC1 has two well characterized substrates, the translation inhibitor 4E-BP and the AGC kinase and translation activator S6K. It regulates cellular processes such as translation, transcription, ribosome biogenesis, and autophagy. mTORC1, via S6K, also negatively feeds back on insulin receptor substrate 1 (IRS1) in the PI3K-Akt pathway upstream of mTORC1 to decrease insulin sensitivity (Figure 1). mTORC2 phosphorylates the AGC kinase Akt. The reader is referred to other reviews for more detailed descriptions of earlier accounts of mTOR signaling and TOR signaling in model organisms [1–5].
Current Opinion in Cell Biology 2009, 21:209–218 This review comes from a themed issue on Cell regulation Edited by Brian Hemmings and Nikolas Tonks Available online 2nd March 2009 0955-0674/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2009.01.024
Introduction Target of Rapamycin (TOR) is a highly conserved Ser/Thr kinase that is found in two structurally and functionally distinct complexes to regulate growth and metabolism. In mammals, mTOR complex 1 (mTORC1) contains mTOR, mLST8, and raptor, and is sensitive to the immunosuppressive and anti-cancer drug rapamycin. Rapamycin, in complex with the immunophilin FKBP12 (FKBP12-rapamycin), binds directly to mTORC1. mTORC2 contains mTOR, mLST8, rictor, mSIN1, and PRR5, and is insensitive to FKBP12-rapamycin. The two complexes signal via different effector pathways to control distinct cellular processes. This review summarizes the major findings in the mTOR field within the past two years. But, first we briefly describe the status of the mTOR signaling network as it was two years ago. Two years ago, it was known that mTORC1 is positively regulated by amino acids, via an unknown mechanism, and by growth factors via the PI3KAkt signaling pathway (Figure 1). Akt (also known as PKB) inactivates the Tuberous Sclerosis Complex (TSC, composed of the proteins TSC1 and TSC2), which acts as a GAP to inactivate signaling by the small GTPase ras homolog enriched in brain (Rheb). Rheb is a direct activator of mTORC1. mTORC1 is negatively regulated by low cellular energy via AMPK that phosphorylates and www.sciencedirect.com
Upstream and downstream of mTOR In the past couple of years, many important advances have been made in identifying new components of the mTOR network and in further elucidating connections between known components. As throughout the history of mTOR signaling, most breakthroughs have been with regard to mTORC1 rather than mTORC2, due to the availability of the specific mTORC1 inhibitor rapamycin. Some of the new mTOR interactors are found both upstream and downstream of the mTORCs, revealing a complex signaling network with many feedback loops and cell type-specific interactions. Upstream regulators of mTORC1
Amino acids activate mTORC1. Independent studies from the Guan and Neufeld [6] and Sabatini [7] groups provided important insight on the molecular mechanism by which amino acids activate mTORC1. Using complementary approaches, they identified the Rag GTPases as regulators of mTORC1 in response to amino acids. Amino acids induce GTP loading of the Rag proteins that then bind raptor and transport mTORC1 to an ill-defined endomembrane. At this new site, mTORC1 interacts with its activator Rheb [7] that is independently activated by growth factors. The amino acids–Rag GTPases–mTORC1 axis, at least in flies, participates in regulation of cellular processes such as autophagy and cell growth [6]. This model is appealing because it addresses the long-standing question of why growth factors cannot stimulate mTORC1 activity in the absence of amino acids. Full activation of mTORC1 is achieved only via a combination of amino acids localizing mTORC1 to a Rheb-containing compartment and growth factors activating Rheb. But, how does Rheb activate mTORC1? FKBP38 is a protein with structural similarity to FKBP12. FKBP38 binds and inhibits mTORC1 via a mechanism presumably Current Opinion in Cell Biology 2009, 21:209–218
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Figure 1
The mTOR signaling network. mTOR is found in two structurally and functionally distinct complexes that together regulate growth and metabolism. Amino acids (purple) positively regulate the rapamycin-sensitive mTORC1 via the Rag GTPases. Growth factors (green) positively regulate mTORC1 via the Akt-PI3K pathway. Growth factors also positively regulate mTORC2 via an unknown pathway that involves the TSC complex. Low energy status (yellow) negatively regulates mTORC1 via AMPK. Substrates of the mTORCs are depicted in gray. For simplicity, not all known members of the signaling pathway are illustrated. Further members are discussed in the text.
similar to that of the FKBP12-rapamycin complex [8,9]. Bai et al. [9] reported that GTP-Rheb binds FKBP38 and releases it from mTORC1. However, the Proud group showed that while FKBP38 indeed binds Rheb and mTORC1, this binding is not altered by amino acids or insulin, and FKBP38 does not affect mTORC1 activity [10]. The mechanism by which Rheb activates mTORC1 remains to be clarified. Current Opinion in Cell Biology 2009, 21:209–218
Other possible mediators of amino acids signaling to mTOR are the Ste20-related kinase MAP4K3 [11], and the class III PI3K mVps34 [12,13]. While the mechanism by which MAP4K3 regulates mTORC1 is unknown, a mechanism for mVPS34 was recently proposed. According to this proposed mechanism, amino acids induce an extracellular calcium influx that activates calmodulin, which in turn binds and activates mVps34 [14]. mVps34 www.sciencedirect.com
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then produces phosphatidylinositol-3-phosphate that somehow activates mTORC1. The mechanism also involves the formation of a calmodulin-mVps34mTORC1 supercomplex. However, the regulation of mTORC1 by mVps34 may be unique to mammals because in flies Vps34 does not regulate TORC1 [15]. This is surprising because regulation of TORC1 by amino acids is conserved from yeast to human, and the mechanism might also be expected to be conserved, as is the involvement of the Rag GTPases. Also interesting is the fact that while TORC1 is a negative regulator of autophagy, Vps34 positively regulates autophagy [16–18]. Furthermore, in mammalian C2C12 cells, amino acids appear to inhibit rather than activate mVps34 [17]. mVps34 may also control mTORC1 in response to energy status, as mVps34 is inhibited by the AMPK activator AICAR [12]. Another link between mTORC1 and mVps34 is that both mVps34 [19,20] and mTORC1 [21] are activated by resistance exercise, and amino acids have a much stronger effect on mTORC1 activation following exercise [22]. These findings suggest that mVps34 might be involved in the activation of mTORC1 in response to exercise. However, mTORC1 activation following exercise occurs only in fast-twitch muscles [21], whereas mVps34 is mainly expressed in slow-twitch muscles [20]. In addition, activation of mTORC1 is maintained for up to 18 h after exercise, whereas mVps34 is active only for up to 6 h and Akt for only 30 min [20], suggesting that other mechanisms are involved in activation of mTORC1 following exercise. Thus, mVPS34 may have diverse and context-specific roles in the regulation of mTORC1 that require further elucidation. In summary, many interesting advances have been made toward elucidating the mechanism by which amino acids activate mTORC1. The Rag GTPases, FKBP38, MAPK4K3, and mVPS34 have been implicated in this process, but further studies are needed to confirm many observations and to elucidate possible interactions between these proteins.
from phosphorylating and activating TSC2. Thus, Wnt is an upstream activator of mTORC1. The crosstalk between the Wnt and mTORC1 pathways may be bidirectional as suggested by the observation that the TSC complex negatively regulates the stability of b-catenin, the main transcription factor activated by Wnt signaling [25]. Another newly discovered regulator of TSC is the IkB kinase b (IKKb) [26], a key activator of the proinflammatory NF-kB signaling pathway. IKKb phosphorylates and destabilizes TSC1, thus activating mTORC1 in response to inflammatory cytokines (but not growth factors) to stimulate angiogenesis in tumors. In what might be a positive feedback loop, mTORC1 can activate IKK toward NF-kB [27], although this effect is stronger for IKKa than IKKb. This is another mechanism by which mTORC1 promotes cancer development [2]. Growth factors also control mTORC1 independently of the TSC complex. The proline-rich Akt substrate 40 kDa (PRAS40) binds raptor and thereby inactivates mTORC1 [28,29–31,32,33]. Thus, PRAS40 is a direct inhibitor of mTORC1. In response to growth factors, Akt phosphorylates and inhibits PRAS40. Akt phosphorylates Thr246 in PRAS40 that promotes binding of inhibitory 14-3-3 proteins. mTORC1 also phosphorylates PRAS40, on Ser183, but the role of this phosphorylation is unclear [29–31]. Cellular energy status was also shown recently to control mTORC1 independently of the TSC complex. In addition to inhibiting mTORC1 indirectly by phosphorylating and activating the TSC complex, AMPK phosphorylates and inhibits mTORC1 directly [34]. AMPK phosphorylates raptor in mTORC1 thereby promoting binding of 14-3-3 proteins. These findings underscore the importance of downregulating mTORC1 in response to energy stress.
The TSC complex, a GAP for Rheb and thus a negative regulator of mTORC1, integrates many mTORC1 inputs. Most notably, Akt, ERK, and RSK phosphorylate and inhibit TSC2 in response to growth factors. TSC2 phosphorylated by these kinases is inhibited via the direct binding of 14-3-3 to phospho-serines in TSC2. Conversely, AMPK phosphorylates different sites in TSC2 and thereby activates the TSC complex in response to energy depletion. The homologous REDD1 and 2 proteins also activate TSC, in response to hypoxia, by releasing 14-3-3 proteins from TSC2 [23].
p53 is also a negative regulator of the mTORC1 pathway in response to stress [35]. p53 inhibits the mTOR pathway by at least two different mechanisms, in response to DNA damage. In insulin-sensitive tissues, p53 directly activates transcription of AMPKb1, TSC2, IGF-BP3, and PTEN, each of which negatively regulate mTORC1 signaling [36]. In mouse liver and fibroblasts and in several human cancer cell lines, p53 stimulates expression of sestrin1 and sestrin2 that directly activate AMPK toward TSC2 [37]. It should be mentioned that p53 can also be negatively [38] or positively [39] regulated downstream of mTORC1, thus possibly forming another feedback loop.
Wnt signaling regulates development in embryos and cell growth and proliferation in adults. Inoki et al. [24] showed that the canonical Wnt pathway prevents GSK3
Hsu et al. [40] presented genetic and biochemical evidence that the translationally controlled tumor protein (TCTP) is a GEF for Rheb and thereby an activator of
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mTORC1. However, immediately following the report by Hsu et al. [40], three studies appeared suggesting that TCTP is not a GEF for Rheb. Chen et al. [41] showed that S6K phosphorylation is unaffected in TCTP knockout mice. Wang et al. [10] showed that TCTP cannot be co-immunoprecipitated with Rheb, mTOR, or raptor, and that knockdown or overexpression of TCTP has no effect on the phosphorylation of 4E-BP or S6. Rehmann et al. [42] showed that TCTP cannot function as a Rheb GEF in vitro. Unpublished results suggest that the TCTP homolog in S. cerevisiae is unrelated to the TOR pathway (unpublished observation). The reason for the contradictory findings reported by Hsu et al. [40] and the other groups is not clear. Rehmann et al. [42] suggest that TSC and TCTP may control similar targets, such as cyclin E, but via unrelated mechanisms. TOR is the founding member of a conserved family of Ser/Thr kinases known as phosphatidylinositol kinaserelated protein kinases (PIKKs). Tel2, a conserved protein that in yeast controls telomere length, but in mammals is of unknown function, interacts with the non-catalytic region of all six mammalian PIKKs including mTOR [43]. This interaction stabilizes the PIKKs. Knockout of Tel2 in mouse embryonic fibroblasts results in a reduction of mTOR half-life from 18 h to 2 h and reduces mTORC1 signaling. It remains to be determined whether Tel2 is upstream of mTORC2. Another newly discovered regulator of mTOR stability is the tumor suppressor FBXW7 [44]. FBXW7 binds mTOR and targets it for ubiquitination and degradation. Depletion of FBXW7 increases the amount of mTOR and enhances phosphorylation of the mTORC1 substrate S6K, indicating that FBXW7 is upstream of mTORC1. Depletion of FBXW7 has no effect on phosphorylation of the mTORC2 substrate Akt, suggesting that FBXW7 is not upstream of mTORC2. The physiological significance of these observations remains to be determined, but they might be related to tumor suppression. Downstream of mTORC1
Increased mTORC1 signaling leads to increased translation, including the synthesis of secreted proteins. This causes ER stress and activation of the unfolded protein response (UPR) [45]. UPR involves stimulation of JNK that phosphorylates and inhibits the IRS1 in the PI3K pathway upstream of mTORC1, thereby creating another negative feedback loop and insulin resistance. The ER stress induced by mTORC1 hyperactivation also leads to increased apoptosis. The presence of introns within transcripts enhances their translation efficiency. A recent report by Ma et al. [46] demonstrates that mTORC1 plays a role in this phenomenon. S6K1-Aly/REF-like substrate (SKAR) is a cell growth regulator that associates with mRNAs in a spliCurrent Opinion in Cell Biology 2009, 21:209–218
cing-dependent manner. SKAR recruits activated S6K1, and thereby preferentially enhances translation of spliced mRNAs. Upstream regulators of mTORC2
Growth factors (but not amino acids) activate mTORC2. Do growth factors signal to mTORC2 via the TSC complex? This is a difficult question to answer owing to the negative feedback loop from mTORC1 to IRS1 (Figure 1). The best-characterized substrate of mTORC2 is Akt that is also subject to the negative feedback loop. Loss of TSC can lead to loss of Akt phosphorylation because of the negative feedback loop preventing PIP3 synthesis and PIP3-dependent recruitment of Akt to the plasma membrane, or because loss of TSC affects intrinsic mTORC2 activity [47]. Surprisingly, Huang et al. [48] have proposed that TSC binds and activates mTORC2 directly, and this effect of TSC is independent of its GAP activity toward Rheb. How growth factors might activate TSC toward mTORC2 yet inhibit TSC toward mTORC1 remains to be determined. Proline rich protein 5 (PRR5) [49,50] and its close homolog PRR5-like (PRR5L) [31] (also known as protor1 and 2) are two newly discovered mTORC2 interactors of unknown function. These proteins bind mTORC2 via rictor or mSIN1, but do not affect the assembly of the complex nor its in vitro kinase activity. In cell culture, knockdown of PRR5 reduces Akt phosphorylation, as well as S6K and 4E-BP phosphorylation, and results in attenuation of proliferation [50]. Knockdown of PRR5L does not affect Akt or S6K phosphorylation but results in increased apoptosis [31]. Downstream of mTORC2
AGC kinases are a large family of conserved kinases represented by cAMP-dependent protein kinase (PKA), protein kinase G (PKG), and protein kinase C (PKC), but this family also includes Akt, S6K, SGK and other kinases. AGC kinases are activated by phosphorylation of their activation loop by PDK1 and, in some cases, of their hydrophobic motif by a kinase historically termed ‘PDK2’. mTORC1 functions as PDK2 toward S6K (Thr389) and mTORC2 is PDK2 for Akt (Ser473) [51,52]. mTORC2 was recently shown to be PDK2 also for all conventional PKCs and novel PKCe [53,54]. mTOR is the hydrophobic motif kinase for SGK1 (Ser422). However, Hong et al. [55] showed that mTORC1 is the PDK2 for SGK1 whereas Garcia-Martinez et al. [56] demonstrated that the PDK2 for SGK1 is mTORC2 rather than mTORC1. In fission and budding yeast, the SGK1 homologs Gad8 and Ypk2, respectively, are phosphorylated by TORC2, supporting mTORC2 as the PDK2 for SGK1. In summary, mTOR (usually mTORC2) is PDK2 for most, if not all, AGC kinases [52]. www.sciencedirect.com
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Figure 2
Metabolic phenotypes of mice with tissue-specific loss of mTORC1 signaling. Phenotypes include both whole body and tissue-specific phenotypes. See text for details.
The so-called turn motif in Akt (Thr450) and PKC are also phosphorylated in an mTORC2-dependent manner [53,54]. Turn motif phosphorylation stabilizes Akt and PKC. In the absence of growth factor stimulation of mTORC2, Akt is stabilized by Hsp90. The turn motif of PKC is constitutively phosphorylated, suggesting that another kinase can phosphorylate PKC in the absence of growth factors. Conflicting results have been presented on whether mTORC2 phosphorylates the Akt and PKC turn motifs directly [53,54].
Roles of mTOR in whole animal metabolism A full body knockout of any component of mTORC1 or mTORC2 is embryonic lethal [57–62]. Thus, rapamycin treatment or tissue-specific knockout of mTORC components has been used to study mTOR function in animals. To date, these studies have focused on metabolic organs such as adipose tissue, muscle, pancreas, and liver (Figure 2). Mice with adipose-specific knockout of raptor are lean and resistant to diet-induced obesity, owing to an increase in mitochondrial uncoupling in white adipose tissue [63]. Furthermore, these mice have better metabolic parameters, including improved glucose tolerance and www.sciencedirect.com
insulin sensitivity and resistance to diet-induced hypercholesterolemia. The higher insulin sensitivity is attributable to the leanness and to loss of the S6K to IRS1 negative feedback loop in adipose tissue. This phenotype is similar to that of mice lacking the mTORC1 positive effector S6K1 [64], and opposite to that of mice lacking the mTORC1 negative effectors 4E-BP1 and 2 [65]. Thus, mTORC1 signaling in adipose tissue controls whole body energy metabolism. Interestingly, similar to the adipose-specific raptor knockout mice, rapamycin treatment causes weight loss in gerbils [66] and prevents weight gain in rats and humans [67]. Ciliary neurotrophic factor (CNTF) and the adiposesecreted hormone leptin exert their anorexic effect through upregulation of the mTORC1 pathway in the hypothalamus [68,69]. Furthermore, hypothalamusspecific expression of dominant-negative S6K results in increased food intake, whereas expression of constitutively active S6K results in decreased food intake [70]. Thus, hypothalamic mTORC1 signaling negatively regulates appetite. In the central nervous system, mTORC1 also positively regulates axon formation, growth, and polarization [71]. Hyperactivation of mTORC1 via knockout of the TSC complex increases Current Opinion in Cell Biology 2009, 21:209–218
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the synthesis of SAD-A and SAD-B, kinases that regulate axon polarity. In addition, mTORC1 induces axon regeneration after CNS injury [72]. Thus, mTORC1 has several roles in the nervous system. Adiponectin, another important adipose-secreted hormone, enhances peripheral insulin sensitivity. This effect of adiponectin has been attributed to activation of AMPK that in turn inhibits mTORC1 signaling and the inhibitory phosphorylation of IRS1 by S6K, thus enhancing insulin sensitivity in tissue culture models [73]. Metformin is a widely prescribed anti-diabetic drug that enhances insulin sensitivity through a mechanism similar to that of adiponectin. It activates AMPK that inhibits mTORC1and the negative feedback loop, at least in liver [34,74–77]. In agreement with this mode of action for metformin, overexpression of dominant-negative raptor in liver reduces the negative feedback loop and improves insulin sensitivity [78]. Inhibition of TORC1 signaling also extends lifespan in yeast, flies, and worms. As TORC1 is a nutrient sensor, this effect on lifespan is probably equivalent to dietary restriction and its panoply of metabolic effects. It is still under investigation whether mTORC1 controls lifespan also in mammals, but expectations are that it does. In contrast to the improved glucose tolerance in adiposespecific mTORC1 knockout mice, rapamycin-treated mice [79,80] or gerbils [66] are diabetic and hyperlipidemic. This effect is due to smaller pancreatic islets and a decrease in glucose-stimulated insulin synthesis and secretion, resulting in a 90% reduction in serum insulin levels [66]. In agreement with these results, rapamycin impairs the proliferation of murine b-cells in vivo [81], of isolated porcine islets [80], and of pancreatic cancer cell lines [82]. Mice deficient for S6K1 [83], or for its direct substrate S6 [84], also show reduced islet size and low serum insulin levels. By contrast, b-cell-specific knockout of TSC2 results in increased b-cell number and size and hyperinsulinemia, which are reversible by rapamycin treatment [85,86]. The hypoinsulinemia in rapamycintreated animals is accompanied by slight peripheral insulin resistance, possibly due to a so far ill-defined hormonal effect that cannot be offset by inhibition of the negative feedback loop in the peripheral tissues. Thus, pancreatic mTORC1 regulates insulin production and glucose homeostasis. Mice with a muscle-specific knockout of raptor display downregulation of proteins involved in mitochondrial biogenesis (such as PGC1a) and hyperactivation of Akt (due to loss of negative feedback) in muscle. This results in lower oxidative capacity and increased glycogen storage accompanied by muscle dystrophy [87]. Surprisingly, although the negative feedback loop from S6K to IRS1 in muscle of the raptor knockout mice is absent, Current Opinion in Cell Biology 2009, 21:209–218
glucose tolerance is slightly reduced. The observation that absence of the negative feedback loop does not increase glucose tolerance may be an indirect effect of the reduced oxidative capacity and/or increased glycogen accumulation in the muscle, or an unknown hormonal effect on other tissues. A major effect of both the muscle-specific and the adipose-specific raptor knockout is on mitochondrial activity. However, in adipose tissue mTORC1 knockout enhances respiration whereas in muscle the knockout inhibits mitochondrial biogenesis. While adipose-specific raptor knockout mice are similar to S6K1 knockout mice, muscle-specific raptor knockout mice are not. Muscle of S6K1 knockout mice has more mitochondria and increased expression of PGC1a [64,88]. Another recent study found that rapamycin treatment or knockdown of mTOR or raptor in muscle cells in vitro decreases mitochondrial gene expression and oxygen consumption [79]. This effect is via downregulation of PGC1a and the transcription factor YY1, and does not involve S6K1 or Akt [79,89]. Furthermore, many aspects of the phenotype of mice with a muscle-specific raptor knockout are similar to mice with a muscle-specific PGC1a knockout [90]. These findings suggest that mTORC1 controls mitochondrial respiration either negatively via S6K (adipose), or positively via PGC1a (muscle), depending on the mTORC1 downstream effectors that might be found in a particular tissue. PGC1a is only very weakly expressed in white adipose tissue. Muscle-specific rictor (mTORC2) knockout confers littleto-no phenotype [87,91]. The only reported phenotype is reduced Akt Ser473 phosphorylation and slight glucose intolerance. The slight glucose intolerance is probably due to decreased insulin signaling via the Akt pathway. Interestingly, double knockout of raptor and rictor in muscle results in a phenotype identical to knockout of raptor alone, including increased Akt phosphorylation at Ser473 and stable expression and phosphorylation of PKC [87]. This suggests that a kinase other than mTORC2 is able to phosphorylate Akt and PKC under conditions where mTORC1 is also inactive. Further support for this notion comes from mTOR knockout MEFs that show only a very slight reduction in Akt Ser473 phosphorylation [92]. A similar phenomenon is observed when kinase dead mTOR is overexpressed in cardiac muscle [93]. What might this other kinase be? Akt can be phosphorylated by DNA-PK in response to DNA damage [94]. Other candidates have been proposed, such as MAPKAP kinase-2, PKC, integrin-linked kinase, ATM, PDK1, and Akt itself [52].
Conclusions The recent findings in the mTOR field include new upstream regulators (e.g. PRAS40, Rag GTPases, the Wnt and NF-kB pathways, p53 and Tel2) and downwww.sciencedirect.com
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stream targets (e.g., UPR, AGC kinases, and SKAR) of the mTORCs. Notably, mechanisms for how amino acids regulate mTORC1 have been proposed. An important new area in mTOR research has been the study of tissuespecific mTOR functions and how these impinge on whole body growth and metabolism.
Acknowledgements We apologize to colleagues whose work we could not describe owing to space limitations. MNH acknowledges support from the Swiss National Science Foundation and the Canton of Basel.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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41. Chen SH, Wu PS, Chou CH, Yan YT, Liu H, Weng SY, YangYen HF: A knockout mouse approach reveals that TCTP functions as an essential factor for cell proliferation and survival in a tissue- or cell type-specific manner. Mol Biol Cell 2007, 18:2525-2532.
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57. Gangloff YG, Mueller M, Dann SG, Svoboda P, Sticker M, Spetz JF, Um SH, Brown EJ, Cereghini S, Thomas G et al.: Disruption of the mouse mTOR gene leads to early postimplantation lethality and prohibits embryonic stem cell development. Mol Cell Biol 2004, 24:9508-9516.
43. Takai H, Wang RC, Takai KK, Yang H, de Lange T: Tel2 regulates the stability of PI3K-related protein kinases. Cell 2007, 131:1248-1259. This study, together with reference [44], revealed that mTOR is regulated at the level of protein stability. Takai et al. showed that Tel2 binds mTOR and substantially stabilizes it. Tel2 regulates the stability of all mammalian PIKK family members.
58. Murakami M, Ichisaka T, Maeda M, Oshiro N, Hara K, Edenhofer F, Kiyama H, Yonezawa K, Yamanaka S: mTOR is essential for growth and proliferation in early mouse embryos and embryonic stem cells. Mol Cell Biol 2004, 24:6710-6718.
44. Mao J-H, Kim I-J, Wu D, Climent J, Kang HC, DelRosario R, Balmain A: FBXW7 targets mTOR for degradation and Current Opinion in Cell Biology 2009, 21:209–218
59. Guertin DA, Stevens DM, Thoreen CC, Burds AA, Kalaany NY, Moffat J, Brown M, Fitzgerald KJ, Sabatini DM: Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKC[alpha], but not S6K1. Dev Cell 2006, 11:859-871. www.sciencedirect.com
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60. Jacinto E, Facchinetti V, Liu D, Soto N, Wei S, Jung SY, Huang Q, Qin J, Su B: SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. Cell 2006, 127:125-137.
77. Tzatsos A, Kandror KV: Nutrients suppress phosphatidylinositol 3-kinase/Akt signaling via raptor-dependent mTOR-mediated insulin receptor substrate 1 phosphorylation. Mol Cell Biol 2006, 26:63-76.
61. Shiota C, Woo J-T, Lindner J, Shelton KD, Magnuson MA: Multiallelic disruption of the rictor gene in mice reveals that mTOR complex 2 is essential for fetal growth and viability. Dev Cell 2006, 11:583-589.
78. Koketsu Y, Sakoda H, Fujishiro M, Kushiyama A, Fukushima Y, Ono H, Anai M, Kikuchi T, Fukuda T, Kamata H et al.: Hepatic overexpression of a dominant negative form of raptor enhances Akt phosphorylation and restores insulin sensitivity in K/KAy mice. Am J Endocrinol Metab 2008, 294:E719-E725.
62. Yang Q, Inoki K, Ikenoue T, Guan K-L: Identification of Sin1 as an essential TORC2 component required for complex formation and kinase activity. Genes Dev 2006, 20:2820-2832. 63. Polak P, Cybulski N, Feige JN, Auwerx J, Ruegg MA, Hall MN: Adipose-specific knockout of raptor results in lean mice with enhanced mitochondrial respiration. Cell Metab 2008, 8:399-410. This study, together with reference [87], reported the first tissue-specific knockout of an mTORC1 component in mice. Polak et al. demonstrated that adipose-specific knockout of raptor results in lean and healthy mice. The phenotype was attributed to partial transdifferentiation of white to brown adipose tissue, resulting in enhanced mitochondrial uncoupling. 64. Um SH, Frigerio F, Watanabe M, Picard F, Joaquin M, Sticker M, Fumagalli S, Allegrini PR, Kozma SC, Auwerx J et al.: Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity. Nature 2004, 431:200-205. 65. Le Bacquer O, Petroulakis E, Paglialunga S, Poulin F, Richard D, Cianflone K, Sonenberg N: Elevated sensitivity to diet-induced obesity and insulin resistance in mice lacking 4E-BP1 and 4EBP2. J Clin Invest 2007, 117:387-396. 66. Fraenkel M, Ketzinel-Gilad M, Ariav Y, Pappo O, Karaca M, Castel J, Berthault M-F, Magnan C, Cerasi E, Kaiser N et al.: mTOR inhibition by rapamycin prevents {beta}-cell adaptation to hyperglycemia and exacerbates the metabolic state in type 2 diabetes. Diabetes 2008, 57:945-957.
79. Cunningham JT, Rodgers JT, Arlow DH, Vazquez F, Mootha VK, Puigserver P: mTOR controls mitochondrial oxidative function through a YY1-PGC-1[alpha] transcriptional complex. Nature 2007, 450:736-740. This study reported a novel function of mTORC1, as a transcription coactivator. mTORC1 directly interacts with the transcription factor YY1 and the coactivator PGC1 to positively regulate transcription of mitochondrial genes. 80. Bussiere CT, Lakey JR, Shapiro AM, Korbutt GS: The impact of the mTOR inhibitor sirolimus on the proliferation and function of pancreatic islets and ductal cells. Diabetologia 2006, 49:2341-2349. 81. Zahr E, Molano RD, Pileggi A, Ichii H, San Jose S, Bocca N, An W, Gonzalez-Quintana J, Fraker C, Ricordi C et al.: Rapamycin impairs [beta]-cell proliferation in vivo. Transplant Proc 2008, 40:436-437. 82. Azzariti A, Porcelli L, Gatti G, Nicolin A, Paradiso A: Synergic antiproliferative and antiangiogenic effects of EGFR and mTor inhibitors on pancreatic cancer cells. Biochem Pharmacol 2008, 75:1035-1044. 83. Pende M, Kozma SC, Jaquet M, Oorschot V, Burcelin R, Le Marchand-Brustel Y, Klumperman J, Thorens B, Thomas G: Hypoinsulinaemia, glucose intolerance and diminished betacell size in S6K1-deficient mice. Nature 2000, 408:994-997.
67. Rovira J, Arellano EM, Burke JT, Brault Y, Moya-Rull D, BanonManeus E, Ramirez-Bajo MJ, Gutierrez-Dalmau A, Revuelta I, Quintana LF et al.: Effect of mTOR inhibitor on body weight: from an experimental rat model to human transplant patients. Transpl Int 2008, 21:992-998.
84. Ruvinsky I, Sharon N, Lerer T, Cohen H, Stolovich-Rain M, Nir T, Dor Y, Zisman P, Meyuhas O: Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis. Genes Dev 2005, 19:2199-2211.
68. Cota D, Proulx K, Smith KAB, Kozma SC, Thomas G, Woods SC, Seeley RJ: Hypothalamic mTOR signaling regulates food intake. Science 2006, 312:927-930.
85. Rachdi L, Balcazar N, Osorio-Duque F, Elghazi L, Weiss A, Gould A, Chang-Chen KJ, Gambello MJ, Bernal-Mizrachi E: Disruption of Tsc2 in pancreatic beta cells induces beta cell mass expansion and improved glucose tolerance in a TORC1-dependent manner. Proc Natl Acad Sci U S A 2008, 105:9250-9255.
69. Cota D, Matter EK, Woods SC, Seeley RJ: The role of hypothalamic mammalian target of rapamycin complex 1 signaling in diet-induced obesity. J Neurosci 2008, 28:7202-7208. 70. Blouet C, Ono H, Schwartz GJ: Mediobasal hypothalamic p70 S6 kinase 1 modulates the control of energy homeostasis. Cell Metab 2008, 8:459-467. 71. Choi Y-J, Di Nardo A, Kramvis I, Meikle L, Kwiatkowski DJ, Sahin M, He X: Tuberous sclerosis complex proteins control axon formation. Genes Dev 2008, 22:2485-2495. 72. Park KK, Liu K, Hu Y, Smith PD, Wang C, Cai B, Xu B, Connolly L, Kramvis I, Sahin M et al.: Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway. Science 2008, 322:963-966. 73. Wang C, Mao X, Wang L, Liu M, Wetzel MD, Guan K-L, Dong LQ, Liu F: Adiponectin sensitizes insulin signaling by reducing p70 S6 kinase-mediated serine phosphorylation of IRS-1. J Biol Chem 2007, 282:7991-7996. 74. Dowling RJO, Zakikhani M, Fantus IG, Pollak M, Sonenberg N: Metformin inhibits mammalian target of rapamycin dependent translation initiation in breast cancer cells. Cancer Res 2007, 67:10804-10812. 75. Mordier S, Iynedjian PB: Activation of mammalian target of rapamycin complex 1 and insulin resistance induced by palmitate in hepatocytes. Biochem Biophys Res Commun 2007, 362:206-211. 76. Shaw RJ, Lamia KA, Vasquez D, Koo S-H, Bardeesy N, DePinho RA, Montminy M, Cantley LC: The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin. Science 2005, 310:1642-1646. www.sciencedirect.com
86. Shigeyama Y, Kobayashi T, Kido Y, Hashimoto N, Asahara S, Matsuda T, Takeda A, Inoue T, Shibutani Y, Koyanagi M et al.: Biphasic response of pancreatic beta-cell mass to ablation of tuberous sclerosis complex 2 in mice. Mol Cell Biol 2008, 28:2971-2979. 87. Bentzinger CF, Romanino K, Cloetta D, Lin S, Mascarenhas JB, Oliveri F, Xia J, Casanova E, Costa CF, Brink M et al.: Skeletal muscle-specific ablation of raptor, but not of rictor, causes metabolic changes and results in muscle dystrophy. Cell Metab 2008, 8:411-424. This study, together with reference [63], reported the first tissue-specific knockout of an mTORC1 component in mice. Bentzinger et al. showed that muscles of mice with muscle-specific knockout of raptor have impaired oxidative capacity and elevated glycogen levels, and become progressively dystrophic. 88. Aguilar V, Alliouachene S, Sotiropoulos A, Sobering A, Athea Y, Djouadi F, Miraux S, Thiaudiere E, Foretz M, Viollet B et al.: S6 kinase deletion suppresses muscle growth adaptations to nutrient availability by activating AMP kinase. Cell Metab 2007, 5:476-487. 89. Schieke SM, Phillips D, McCoy JP Jr, Aponte AM, Shen R-F, Balaban RS, Finkel T: The mammalian target of rapamycin (mTOR) pathway regulates mitochondrial oxygen consumption and oxidative capacity. J Biol Chem 2006, 281:27643-27652. 90. Handschin C, Chin S, Li P, Liu F, Maratos-Flier E, LeBrasseur NK, Yan Z, Spiegelman BM: Skeletal muscle fiber-type switching, exercise intolerance, and myopathy in PGC-1{alpha} muscle-specific knock-out animals. J Biol Chem 2007, 282:30014-30021. Current Opinion in Cell Biology 2009, 21:209–218
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91. Kumar A, Harris TE, Keller SR, Choi KM, Magnuson MA, Lawrence JC Jr: Muscle-specific deletion of rictor impairs insulinstimulated glucose transport and enhances Basal glycogen synthase activity. Mol Cell Biol 2008, 28:61-70. 92. Carracedo A, Ma L, Teruya-Feldstein J, Rojo F, Salmena L, Alimonti A, Egia A, Sasaki AT, Thomas G, Kozma SC et al.: Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer. J Clin Invest 2008, 118(9):3065-3074.
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mTOR and the control of whole body metabolism Pazit Polak and Michael N Hall Mammalian Target of Rapamycin (mTOR) is a highly conserved protein kinase that functions as part of two distinct multiprotein complexes to regulate growth and metabolism. This review describes the most important recent advances in the mTOR signaling field. In addition, we provide an overview on the functions of mTOR in different organs, with a special focus on the role of mTOR in whole body energy metabolism. Address Biozentrum, University of Basel, Basel CH-4056, Switzerland Corresponding author: Hall, Michael N ([email protected])
activates the TSC complex. mTORC2 is activated by growth factors via an unknown mechanism. mTORC1 has two well characterized substrates, the translation inhibitor 4E-BP and the AGC kinase and translation activator S6K. It regulates cellular processes such as translation, transcription, ribosome biogenesis, and autophagy. mTORC1, via S6K, also negatively feeds back on insulin receptor substrate 1 (IRS1) in the PI3K-Akt pathway upstream of mTORC1 to decrease insulin sensitivity (Figure 1). mTORC2 phosphorylates the AGC kinase Akt. The reader is referred to other reviews for more detailed descriptions of earlier accounts of mTOR signaling and TOR signaling in model organisms [1–5].
Current Opinion in Cell Biology 2009, 21:209–218 This review comes from a themed issue on Cell regulation Edited by Brian Hemmings and Nikolas Tonks Available online 2nd March 2009 0955-0674/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2009.01.024
Introduction Target of Rapamycin (TOR) is a highly conserved Ser/Thr kinase that is found in two structurally and functionally distinct complexes to regulate growth and metabolism. In mammals, mTOR complex 1 (mTORC1) contains mTOR, mLST8, and raptor, and is sensitive to the immunosuppressive and anti-cancer drug rapamycin. Rapamycin, in complex with the immunophilin FKBP12 (FKBP12-rapamycin), binds directly to mTORC1. mTORC2 contains mTOR, mLST8, rictor, mSIN1, and PRR5, and is insensitive to FKBP12-rapamycin. The two complexes signal via different effector pathways to control distinct cellular processes. This review summarizes the major findings in the mTOR field within the past two years. But, first we briefly describe the status of the mTOR signaling network as it was two years ago. Two years ago, it was known that mTORC1 is positively regulated by amino acids, via an unknown mechanism, and by growth factors via the PI3KAkt signaling pathway (Figure 1). Akt (also known as PKB) inactivates the Tuberous Sclerosis Complex (TSC, composed of the proteins TSC1 and TSC2), which acts as a GAP to inactivate signaling by the small GTPase ras homolog enriched in brain (Rheb). Rheb is a direct activator of mTORC1. mTORC1 is negatively regulated by low cellular energy via AMPK that phosphorylates and www.sciencedirect.com
Upstream and downstream of mTOR In the past couple of years, many important advances have been made in identifying new components of the mTOR network and in further elucidating connections between known components. As throughout the history of mTOR signaling, most breakthroughs have been with regard to mTORC1 rather than mTORC2, due to the availability of the specific mTORC1 inhibitor rapamycin. Some of the new mTOR interactors are found both upstream and downstream of the mTORCs, revealing a complex signaling network with many feedback loops and cell type-specific interactions. Upstream regulators of mTORC1
Amino acids activate mTORC1. Independent studies from the Guan and Neufeld [6] and Sabatini [7] groups provided important insight on the molecular mechanism by which amino acids activate mTORC1. Using complementary approaches, they identified the Rag GTPases as regulators of mTORC1 in response to amino acids. Amino acids induce GTP loading of the Rag proteins that then bind raptor and transport mTORC1 to an ill-defined endomembrane. At this new site, mTORC1 interacts with its activator Rheb [7] that is independently activated by growth factors. The amino acids–Rag GTPases–mTORC1 axis, at least in flies, participates in regulation of cellular processes such as autophagy and cell growth [6]. This model is appealing because it addresses the long-standing question of why growth factors cannot stimulate mTORC1 activity in the absence of amino acids. Full activation of mTORC1 is achieved only via a combination of amino acids localizing mTORC1 to a Rheb-containing compartment and growth factors activating Rheb. But, how does Rheb activate mTORC1? FKBP38 is a protein with structural similarity to FKBP12. FKBP38 binds and inhibits mTORC1 via a mechanism presumably Current Opinion in Cell Biology 2009, 21:209–218
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Figure 1
The mTOR signaling network. mTOR is found in two structurally and functionally distinct complexes that together regulate growth and metabolism. Amino acids (purple) positively regulate the rapamycin-sensitive mTORC1 via the Rag GTPases. Growth factors (green) positively regulate mTORC1 via the Akt-PI3K pathway. Growth factors also positively regulate mTORC2 via an unknown pathway that involves the TSC complex. Low energy status (yellow) negatively regulates mTORC1 via AMPK. Substrates of the mTORCs are depicted in gray. For simplicity, not all known members of the signaling pathway are illustrated. Further members are discussed in the text.
similar to that of the FKBP12-rapamycin complex [8,9]. Bai et al. [9] reported that GTP-Rheb binds FKBP38 and releases it from mTORC1. However, the Proud group showed that while FKBP38 indeed binds Rheb and mTORC1, this binding is not altered by amino acids or insulin, and FKBP38 does not affect mTORC1 activity [10]. The mechanism by which Rheb activates mTORC1 remains to be clarified. Current Opinion in Cell Biology 2009, 21:209–218
Other possible mediators of amino acids signaling to mTOR are the Ste20-related kinase MAP4K3 [11], and the class III PI3K mVps34 [12,13]. While the mechanism by which MAP4K3 regulates mTORC1 is unknown, a mechanism for mVPS34 was recently proposed. According to this proposed mechanism, amino acids induce an extracellular calcium influx that activates calmodulin, which in turn binds and activates mVps34 [14]. mVps34 www.sciencedirect.com
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then produces phosphatidylinositol-3-phosphate that somehow activates mTORC1. The mechanism also involves the formation of a calmodulin-mVps34mTORC1 supercomplex. However, the regulation of mTORC1 by mVps34 may be unique to mammals because in flies Vps34 does not regulate TORC1 [15]. This is surprising because regulation of TORC1 by amino acids is conserved from yeast to human, and the mechanism might also be expected to be conserved, as is the involvement of the Rag GTPases. Also interesting is the fact that while TORC1 is a negative regulator of autophagy, Vps34 positively regulates autophagy [16–18]. Furthermore, in mammalian C2C12 cells, amino acids appear to inhibit rather than activate mVps34 [17]. mVps34 may also control mTORC1 in response to energy status, as mVps34 is inhibited by the AMPK activator AICAR [12]. Another link between mTORC1 and mVps34 is that both mVps34 [19,20] and mTORC1 [21] are activated by resistance exercise, and amino acids have a much stronger effect on mTORC1 activation following exercise [22]. These findings suggest that mVps34 might be involved in the activation of mTORC1 in response to exercise. However, mTORC1 activation following exercise occurs only in fast-twitch muscles [21], whereas mVps34 is mainly expressed in slow-twitch muscles [20]. In addition, activation of mTORC1 is maintained for up to 18 h after exercise, whereas mVps34 is active only for up to 6 h and Akt for only 30 min [20], suggesting that other mechanisms are involved in activation of mTORC1 following exercise. Thus, mVPS34 may have diverse and context-specific roles in the regulation of mTORC1 that require further elucidation. In summary, many interesting advances have been made toward elucidating the mechanism by which amino acids activate mTORC1. The Rag GTPases, FKBP38, MAPK4K3, and mVPS34 have been implicated in this process, but further studies are needed to confirm many observations and to elucidate possible interactions between these proteins.
from phosphorylating and activating TSC2. Thus, Wnt is an upstream activator of mTORC1. The crosstalk between the Wnt and mTORC1 pathways may be bidirectional as suggested by the observation that the TSC complex negatively regulates the stability of b-catenin, the main transcription factor activated by Wnt signaling [25]. Another newly discovered regulator of TSC is the IkB kinase b (IKKb) [26], a key activator of the proinflammatory NF-kB signaling pathway. IKKb phosphorylates and destabilizes TSC1, thus activating mTORC1 in response to inflammatory cytokines (but not growth factors) to stimulate angiogenesis in tumors. In what might be a positive feedback loop, mTORC1 can activate IKK toward NF-kB [27], although this effect is stronger for IKKa than IKKb. This is another mechanism by which mTORC1 promotes cancer development [2]. Growth factors also control mTORC1 independently of the TSC complex. The proline-rich Akt substrate 40 kDa (PRAS40) binds raptor and thereby inactivates mTORC1 [28,29–31,32,33]. Thus, PRAS40 is a direct inhibitor of mTORC1. In response to growth factors, Akt phosphorylates and inhibits PRAS40. Akt phosphorylates Thr246 in PRAS40 that promotes binding of inhibitory 14-3-3 proteins. mTORC1 also phosphorylates PRAS40, on Ser183, but the role of this phosphorylation is unclear [29–31]. Cellular energy status was also shown recently to control mTORC1 independently of the TSC complex. In addition to inhibiting mTORC1 indirectly by phosphorylating and activating the TSC complex, AMPK phosphorylates and inhibits mTORC1 directly [34]. AMPK phosphorylates raptor in mTORC1 thereby promoting binding of 14-3-3 proteins. These findings underscore the importance of downregulating mTORC1 in response to energy stress.
The TSC complex, a GAP for Rheb and thus a negative regulator of mTORC1, integrates many mTORC1 inputs. Most notably, Akt, ERK, and RSK phosphorylate and inhibit TSC2 in response to growth factors. TSC2 phosphorylated by these kinases is inhibited via the direct binding of 14-3-3 to phospho-serines in TSC2. Conversely, AMPK phosphorylates different sites in TSC2 and thereby activates the TSC complex in response to energy depletion. The homologous REDD1 and 2 proteins also activate TSC, in response to hypoxia, by releasing 14-3-3 proteins from TSC2 [23].
p53 is also a negative regulator of the mTORC1 pathway in response to stress [35]. p53 inhibits the mTOR pathway by at least two different mechanisms, in response to DNA damage. In insulin-sensitive tissues, p53 directly activates transcription of AMPKb1, TSC2, IGF-BP3, and PTEN, each of which negatively regulate mTORC1 signaling [36]. In mouse liver and fibroblasts and in several human cancer cell lines, p53 stimulates expression of sestrin1 and sestrin2 that directly activate AMPK toward TSC2 [37]. It should be mentioned that p53 can also be negatively [38] or positively [39] regulated downstream of mTORC1, thus possibly forming another feedback loop.
Wnt signaling regulates development in embryos and cell growth and proliferation in adults. Inoki et al. [24] showed that the canonical Wnt pathway prevents GSK3
Hsu et al. [40] presented genetic and biochemical evidence that the translationally controlled tumor protein (TCTP) is a GEF for Rheb and thereby an activator of
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mTORC1. However, immediately following the report by Hsu et al. [40], three studies appeared suggesting that TCTP is not a GEF for Rheb. Chen et al. [41] showed that S6K phosphorylation is unaffected in TCTP knockout mice. Wang et al. [10] showed that TCTP cannot be co-immunoprecipitated with Rheb, mTOR, or raptor, and that knockdown or overexpression of TCTP has no effect on the phosphorylation of 4E-BP or S6. Rehmann et al. [42] showed that TCTP cannot function as a Rheb GEF in vitro. Unpublished results suggest that the TCTP homolog in S. cerevisiae is unrelated to the TOR pathway (unpublished observation). The reason for the contradictory findings reported by Hsu et al. [40] and the other groups is not clear. Rehmann et al. [42] suggest that TSC and TCTP may control similar targets, such as cyclin E, but via unrelated mechanisms. TOR is the founding member of a conserved family of Ser/Thr kinases known as phosphatidylinositol kinaserelated protein kinases (PIKKs). Tel2, a conserved protein that in yeast controls telomere length, but in mammals is of unknown function, interacts with the non-catalytic region of all six mammalian PIKKs including mTOR [43]. This interaction stabilizes the PIKKs. Knockout of Tel2 in mouse embryonic fibroblasts results in a reduction of mTOR half-life from 18 h to 2 h and reduces mTORC1 signaling. It remains to be determined whether Tel2 is upstream of mTORC2. Another newly discovered regulator of mTOR stability is the tumor suppressor FBXW7 [44]. FBXW7 binds mTOR and targets it for ubiquitination and degradation. Depletion of FBXW7 increases the amount of mTOR and enhances phosphorylation of the mTORC1 substrate S6K, indicating that FBXW7 is upstream of mTORC1. Depletion of FBXW7 has no effect on phosphorylation of the mTORC2 substrate Akt, suggesting that FBXW7 is not upstream of mTORC2. The physiological significance of these observations remains to be determined, but they might be related to tumor suppression. Downstream of mTORC1
Increased mTORC1 signaling leads to increased translation, including the synthesis of secreted proteins. This causes ER stress and activation of the unfolded protein response (UPR) [45]. UPR involves stimulation of JNK that phosphorylates and inhibits the IRS1 in the PI3K pathway upstream of mTORC1, thereby creating another negative feedback loop and insulin resistance. The ER stress induced by mTORC1 hyperactivation also leads to increased apoptosis. The presence of introns within transcripts enhances their translation efficiency. A recent report by Ma et al. [46] demonstrates that mTORC1 plays a role in this phenomenon. S6K1-Aly/REF-like substrate (SKAR) is a cell growth regulator that associates with mRNAs in a spliCurrent Opinion in Cell Biology 2009, 21:209–218
cing-dependent manner. SKAR recruits activated S6K1, and thereby preferentially enhances translation of spliced mRNAs. Upstream regulators of mTORC2
Growth factors (but not amino acids) activate mTORC2. Do growth factors signal to mTORC2 via the TSC complex? This is a difficult question to answer owing to the negative feedback loop from mTORC1 to IRS1 (Figure 1). The best-characterized substrate of mTORC2 is Akt that is also subject to the negative feedback loop. Loss of TSC can lead to loss of Akt phosphorylation because of the negative feedback loop preventing PIP3 synthesis and PIP3-dependent recruitment of Akt to the plasma membrane, or because loss of TSC affects intrinsic mTORC2 activity [47]. Surprisingly, Huang et al. [48] have proposed that TSC binds and activates mTORC2 directly, and this effect of TSC is independent of its GAP activity toward Rheb. How growth factors might activate TSC toward mTORC2 yet inhibit TSC toward mTORC1 remains to be determined. Proline rich protein 5 (PRR5) [49,50] and its close homolog PRR5-like (PRR5L) [31] (also known as protor1 and 2) are two newly discovered mTORC2 interactors of unknown function. These proteins bind mTORC2 via rictor or mSIN1, but do not affect the assembly of the complex nor its in vitro kinase activity. In cell culture, knockdown of PRR5 reduces Akt phosphorylation, as well as S6K and 4E-BP phosphorylation, and results in attenuation of proliferation [50]. Knockdown of PRR5L does not affect Akt or S6K phosphorylation but results in increased apoptosis [31]. Downstream of mTORC2
AGC kinases are a large family of conserved kinases represented by cAMP-dependent protein kinase (PKA), protein kinase G (PKG), and protein kinase C (PKC), but this family also includes Akt, S6K, SGK and other kinases. AGC kinases are activated by phosphorylation of their activation loop by PDK1 and, in some cases, of their hydrophobic motif by a kinase historically termed ‘PDK2’. mTORC1 functions as PDK2 toward S6K (Thr389) and mTORC2 is PDK2 for Akt (Ser473) [51,52]. mTORC2 was recently shown to be PDK2 also for all conventional PKCs and novel PKCe [53,54]. mTOR is the hydrophobic motif kinase for SGK1 (Ser422). However, Hong et al. [55] showed that mTORC1 is the PDK2 for SGK1 whereas Garcia-Martinez et al. [56] demonstrated that the PDK2 for SGK1 is mTORC2 rather than mTORC1. In fission and budding yeast, the SGK1 homologs Gad8 and Ypk2, respectively, are phosphorylated by TORC2, supporting mTORC2 as the PDK2 for SGK1. In summary, mTOR (usually mTORC2) is PDK2 for most, if not all, AGC kinases [52]. www.sciencedirect.com
mTOR and the control of whole body metabolism Polak and Hall 213
Figure 2
Metabolic phenotypes of mice with tissue-specific loss of mTORC1 signaling. Phenotypes include both whole body and tissue-specific phenotypes. See text for details.
The so-called turn motif in Akt (Thr450) and PKC are also phosphorylated in an mTORC2-dependent manner [53,54]. Turn motif phosphorylation stabilizes Akt and PKC. In the absence of growth factor stimulation of mTORC2, Akt is stabilized by Hsp90. The turn motif of PKC is constitutively phosphorylated, suggesting that another kinase can phosphorylate PKC in the absence of growth factors. Conflicting results have been presented on whether mTORC2 phosphorylates the Akt and PKC turn motifs directly [53,54].
Roles of mTOR in whole animal metabolism A full body knockout of any component of mTORC1 or mTORC2 is embryonic lethal [57–62]. Thus, rapamycin treatment or tissue-specific knockout of mTORC components has been used to study mTOR function in animals. To date, these studies have focused on metabolic organs such as adipose tissue, muscle, pancreas, and liver (Figure 2). Mice with adipose-specific knockout of raptor are lean and resistant to diet-induced obesity, owing to an increase in mitochondrial uncoupling in white adipose tissue [63]. Furthermore, these mice have better metabolic parameters, including improved glucose tolerance and www.sciencedirect.com
insulin sensitivity and resistance to diet-induced hypercholesterolemia. The higher insulin sensitivity is attributable to the leanness and to loss of the S6K to IRS1 negative feedback loop in adipose tissue. This phenotype is similar to that of mice lacking the mTORC1 positive effector S6K1 [64], and opposite to that of mice lacking the mTORC1 negative effectors 4E-BP1 and 2 [65]. Thus, mTORC1 signaling in adipose tissue controls whole body energy metabolism. Interestingly, similar to the adipose-specific raptor knockout mice, rapamycin treatment causes weight loss in gerbils [66] and prevents weight gain in rats and humans [67]. Ciliary neurotrophic factor (CNTF) and the adiposesecreted hormone leptin exert their anorexic effect through upregulation of the mTORC1 pathway in the hypothalamus [68,69]. Furthermore, hypothalamusspecific expression of dominant-negative S6K results in increased food intake, whereas expression of constitutively active S6K results in decreased food intake [70]. Thus, hypothalamic mTORC1 signaling negatively regulates appetite. In the central nervous system, mTORC1 also positively regulates axon formation, growth, and polarization [71]. Hyperactivation of mTORC1 via knockout of the TSC complex increases Current Opinion in Cell Biology 2009, 21:209–218
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the synthesis of SAD-A and SAD-B, kinases that regulate axon polarity. In addition, mTORC1 induces axon regeneration after CNS injury [72]. Thus, mTORC1 has several roles in the nervous system. Adiponectin, another important adipose-secreted hormone, enhances peripheral insulin sensitivity. This effect of adiponectin has been attributed to activation of AMPK that in turn inhibits mTORC1 signaling and the inhibitory phosphorylation of IRS1 by S6K, thus enhancing insulin sensitivity in tissue culture models [73]. Metformin is a widely prescribed anti-diabetic drug that enhances insulin sensitivity through a mechanism similar to that of adiponectin. It activates AMPK that inhibits mTORC1and the negative feedback loop, at least in liver [34,74–77]. In agreement with this mode of action for metformin, overexpression of dominant-negative raptor in liver reduces the negative feedback loop and improves insulin sensitivity [78]. Inhibition of TORC1 signaling also extends lifespan in yeast, flies, and worms. As TORC1 is a nutrient sensor, this effect on lifespan is probably equivalent to dietary restriction and its panoply of metabolic effects. It is still under investigation whether mTORC1 controls lifespan also in mammals, but expectations are that it does. In contrast to the improved glucose tolerance in adiposespecific mTORC1 knockout mice, rapamycin-treated mice [79,80] or gerbils [66] are diabetic and hyperlipidemic. This effect is due to smaller pancreatic islets and a decrease in glucose-stimulated insulin synthesis and secretion, resulting in a 90% reduction in serum insulin levels [66]. In agreement with these results, rapamycin impairs the proliferation of murine b-cells in vivo [81], of isolated porcine islets [80], and of pancreatic cancer cell lines [82]. Mice deficient for S6K1 [83], or for its direct substrate S6 [84], also show reduced islet size and low serum insulin levels. By contrast, b-cell-specific knockout of TSC2 results in increased b-cell number and size and hyperinsulinemia, which are reversible by rapamycin treatment [85,86]. The hypoinsulinemia in rapamycintreated animals is accompanied by slight peripheral insulin resistance, possibly due to a so far ill-defined hormonal effect that cannot be offset by inhibition of the negative feedback loop in the peripheral tissues. Thus, pancreatic mTORC1 regulates insulin production and glucose homeostasis. Mice with a muscle-specific knockout of raptor display downregulation of proteins involved in mitochondrial biogenesis (such as PGC1a) and hyperactivation of Akt (due to loss of negative feedback) in muscle. This results in lower oxidative capacity and increased glycogen storage accompanied by muscle dystrophy [87]. Surprisingly, although the negative feedback loop from S6K to IRS1 in muscle of the raptor knockout mice is absent, Current Opinion in Cell Biology 2009, 21:209–218
glucose tolerance is slightly reduced. The observation that absence of the negative feedback loop does not increase glucose tolerance may be an indirect effect of the reduced oxidative capacity and/or increased glycogen accumulation in the muscle, or an unknown hormonal effect on other tissues. A major effect of both the muscle-specific and the adipose-specific raptor knockout is on mitochondrial activity. However, in adipose tissue mTORC1 knockout enhances respiration whereas in muscle the knockout inhibits mitochondrial biogenesis. While adipose-specific raptor knockout mice are similar to S6K1 knockout mice, muscle-specific raptor knockout mice are not. Muscle of S6K1 knockout mice has more mitochondria and increased expression of PGC1a [64,88]. Another recent study found that rapamycin treatment or knockdown of mTOR or raptor in muscle cells in vitro decreases mitochondrial gene expression and oxygen consumption [79]. This effect is via downregulation of PGC1a and the transcription factor YY1, and does not involve S6K1 or Akt [79,89]. Furthermore, many aspects of the phenotype of mice with a muscle-specific raptor knockout are similar to mice with a muscle-specific PGC1a knockout [90]. These findings suggest that mTORC1 controls mitochondrial respiration either negatively via S6K (adipose), or positively via PGC1a (muscle), depending on the mTORC1 downstream effectors that might be found in a particular tissue. PGC1a is only very weakly expressed in white adipose tissue. Muscle-specific rictor (mTORC2) knockout confers littleto-no phenotype [87,91]. The only reported phenotype is reduced Akt Ser473 phosphorylation and slight glucose intolerance. The slight glucose intolerance is probably due to decreased insulin signaling via the Akt pathway. Interestingly, double knockout of raptor and rictor in muscle results in a phenotype identical to knockout of raptor alone, including increased Akt phosphorylation at Ser473 and stable expression and phosphorylation of PKC [87]. This suggests that a kinase other than mTORC2 is able to phosphorylate Akt and PKC under conditions where mTORC1 is also inactive. Further support for this notion comes from mTOR knockout MEFs that show only a very slight reduction in Akt Ser473 phosphorylation [92]. A similar phenomenon is observed when kinase dead mTOR is overexpressed in cardiac muscle [93]. What might this other kinase be? Akt can be phosphorylated by DNA-PK in response to DNA damage [94]. Other candidates have been proposed, such as MAPKAP kinase-2, PKC, integrin-linked kinase, ATM, PDK1, and Akt itself [52].
Conclusions The recent findings in the mTOR field include new upstream regulators (e.g. PRAS40, Rag GTPases, the Wnt and NF-kB pathways, p53 and Tel2) and downwww.sciencedirect.com
mTOR and the control of whole body metabolism Polak and Hall 215
stream targets (e.g., UPR, AGC kinases, and SKAR) of the mTORCs. Notably, mechanisms for how amino acids regulate mTORC1 have been proposed. An important new area in mTOR research has been the study of tissuespecific mTOR functions and how these impinge on whole body growth and metabolism.
Acknowledgements We apologize to colleagues whose work we could not describe owing to space limitations. MNH acknowledges support from the Swiss National Science Foundation and the Canton of Basel.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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