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REVIEWS TOR SIGNALLING IN BUGS, BRAIN AND BRAWN Estela Jacinto and Michael N. Hall TOR — a highly conserved atypical protein kinase and the ‘target of rapamycin’, an immunosuppressant and anti-cancer drug — controls cell growth. TOR controls the growth of proliferating yeast, fly and mammalian cells in response to nutrients. Recent findings, however, indicate that TOR also controls the growth of non-proliferating cells, such as neurons and muscle cells. Furthermore, TOR, by associating with regulatory proteins and inhibiting phosphatases, controls the activity of multiphosphorylated effectors. THE PHOSPHATIDYLINOSITOL KINASE (PIK)-RELATED PROTEIN KINASE FAMILY (PIKK). A family of kinases that share sequence homology with lipid kinases but have a protein kinase activity. They are distinguished by the presence of a unique carboxy-terminal region (FATC) that is not present in the PIK family. Division of Biochemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. Correspondence to M.N.H. e-mail [email protected] doi:10.1038/nrm1018 The processes of cell growth and cell division are usually coupled to give rise to cells of a specific size. There are certain exceptions, however, in which one process occurs independently of the other. For example, division without growth occurs during early development when embryonic cells divide before commencing growth. Growth without division occurs in predifferentiated cells, such as immature thymocytes, and in differentiated cells, such as neurons and muscle cells. Why regulate cell growth? Organs and organisms have characteristic sizes that are determined by the size of their constituent cells. The regulation of cell growth therefore ensures that there is an overall body plan with properly proportioned organs. Defects in the regulation of cell growth can result in cancer or a pathological increase or decrease in organ size1–3. What determines cell size? Environmental stimuli, such as the presence of nutrients and growth factors, and physical stimuli induce cell growth4. On reaching a defined size, however, the exposure of cells to a saturating amount of any of these stimuli does not lead to further growth. A genetic programme, therefore, must also govern cell growth and so ultimately control cell, organ and organism size. An increasing body of evidence points to the target of rapamycin (TOR) signalling pathway as a central controller of cell growth5. Early studies in yeast and mammalian cells showed that TOR regulates growth in dividing cells by controlling several processes — including transcription, translation and ribosome biogenesis — NATURE REVIEWS | MOLECUL AR CELL BIOLOGY that collectively determine the mass of the cell. Studies in Drosophila melanogaster have shown recently that the TOR signalling pathway, by controlling cell size, also controls the overall body size. Finally, recent findings in neurons and muscle cells indicate that TOR controls growth of non-dividing cells, which provides further evidence for a conserved and widespread role of TOR in the regulation of cell growth. TOR and its interactors The discovery of the target of the antifungal activity of rapamycin led to the identification of TOR. Mutations in yeast that confer rapamycin resistance resulted in the identification of three genes — FPR1, TOR1 and TOR2 (REF. 6). Rapamycin forms a complex with the FPR1 gene product (FPR1), a peptidyl-prolyl isomerase that is known as FK506-binding protein of 12 kDa (FKBP12) in mammals, and this complex then binds and inhibits TOR (BOX 1). This mode of action of rapamycin is conserved from yeast to humans. TOR — which is found in yeast (TOR1 and TOR2 in Saccharomyces cerevisiae and Schizosaccharomyces pombe)7–9, fungi (TOR1 in Cryptococcus neoformans)10, plants (AtTOR in Arabidopsis thaliana)11, worms (CeTOR)12, flies (dTOR)13,14 and mammals (mTOR; also known as FKBP12-rapamycin associated protein (FRAP), rapamycin and FKBP12 target (RAFT) or rapamycin target (RAPT)15–17) — is a member of the PHOSPHATIDYLINOSITOL KINASE-RELATED PROTEIN KINASE (PIKK) 18 FAMILY . The carboxy-terminal region of TOR shows high VOLUME 4 | FEBRUARY 2003 | 1 1 7 © 2003 Nature Publishing Group REVIEWS Box 1 | Immunosuppressants and their targets The IMMUNOSUPPRESSANTS rapamycin, FK506 and cyclosporin A (CsA) are natural bacterial or fungal secondary metabolites. Rapamycin (also known as sirolimus or RAPA) is a lipophilic MACROLIDE produced by a bacterial strain, Streptomyces hygroscopicus, that was originally isolated from a soil sample collected in Rapa-Nui (Easter Island) — hence the name rapamycin. FK506 (tacrolimus), also a macrolide, is produced by the soil bacterium Streptomyces tsukubaensis. CsA is a cyclic UNDECAPEPTIDE that is produced by Tolypocladium inflatum. All three immunosuppressants function by forming a complex with an IMMUNOPHILIN. Rapamycin and FK506 form a complex with FK506-binding protein protein 12 (FKBP12), and CsA forms a complex with cyclophilin. The rapamycin–FKBP12, FK506–FKBP12 and CsA–cyclophilin complexes elicit an immunosuppressive effect by acting on targets in T-cell signalling pathways. Rapamycin–FKBP12 binds and inhibits the target of rapamycin (TOR), which is a component of the T-cell interleukin-2 (IL-2) signalling pathway. FK506–FKBP12 and CsA–cyclophilin bind and inhibit calcineurin, a type 2B phosphatase in the T-cell receptor pathway. Rapamycin also has several other clinical applications; it is showing promise in clinical trials as an anti-cancer drug110, and has recently been used to coat cardiac STENTS as a means to prevent proliferation of smooth muscle cells during RESTENOSIS111. Finally, rapamycin has a potent antifungal activity. IMMUNOSUPPRESSANT A drug or compound that inhibits an immune response through inhibition of T-cell growth and/or proliferation. It is used mainly to prevent rejection of organ grafts. MACROLIDE Any of several antibiotics that contain a lactone ring and are produced by Streptomyces sp. UNDECAPEPTIDE A peptide that is composed of a chain of 11 amino-acid residues. IMMUNOPHILIN An intracellular protein that binds immunosuppressive drugs. STENT A small, mesh-like tube made from stainless steel that is placed permanently inside an artery to hold it open to improve the flow of blood. RESTENOSIS A re-narrowing or blockage of an artery at the same site at which treatment, such as an angioplasty or stent procedure, has already taken place. 118 homology to lipid kinases, but evidence supports a role for TOR as a serine/threonine protein kinase19–21. TOR has several known or putative protein–protein interaction domains (FIG.1). The amino-terminal region contains ~20 tandem HEAT REPEATS22. An internal region of TOR contains a so-called FAT DOMAIN23. The carboxy-terminal region of TOR contains the FRB domain, which is the FKBP12/FPR1–rapamycin binding site. This is followed by the catalytic domain that shows homology to phosphatidylinositol kinases, and finally the FATC sequence. Consistent with the presence of many protein–protein interaction domains in TOR, TOR is part of multiprotein complexes. TOR complexes in yeast. TOR is a high-molecularweight protein (~280 kDa) that forms a complex with several proteins. In yeast, two functionally distinct TOR complexes have been identified 24. TOR complex 1 (TORC1) contains TOR1 or TOR2 and the evolutionarily conserved proteins kontroller of growth 1 (KOG1) and LST8. KOG1, which contains four internal HEAT motifs and seven WD40 REPEATS, might function as a scaffold protein to couple TOR to its targets24 (FIG.1). LST8 has seven WD40 repeats, and functions in nutrient-sensitive permease sorting and regulation of the retrograde signalling protein (RTG)-1–RTG3 transcription factor (see below)25,26. TOR complex 2 (TORC2) contains TOR2, adheres voraciously to TOR2 (AVO)-1, AVO2, AVO3 and LST8. AVO1 has a domain that is weakly similar to the Ras-binding domain (RBD) of Ras target proteins. AVO3 contains a RasGEFN (guaninenucleotide exchange factor) domain that is found in exchange factors and activating proteins for Ras-like small GTPases. AVO2 contains five ankyrin repeats and is the only non-essential protein among the TORC1 and TORC2 subunits24. | FEBRUARY 2003 | VOLUME 4 TOR complexes in mammalian cells. In mammalian cells, mTOR forms a complex with raptor and mLST8, which are the mammalian orthologues of KOG1 and LST8 (REFS 24,27,28), respectively. This complex, which is the mammalian equivalent of TORC1, has been named the ‘nutrient-sensitive complex’ 28. Recently, tuberous sclerosis 1 (TSC1; also known as hamartin) and TSC2 (tuberin), the products of two tumour suppressor genes, have also been shown to bind mTOR29. TSC1 and TSC2 have a negative effect on TOR signalling, whereas all of the other TOR-binding proteins that have been described above seem to have a positive effect (see below)29–31. Regulators of TOR in yeast In yeast, nutrients such as carbon and nitrogen stimulate cell growth (BOX 2;TABLE 1). Studies in yeast initially showed that the TOR signalling pathway responds to nutrients32. Mutants that are defective in TOR function (that is, they lack both TOR1 and TOR2), or cells that have been treated with rapamycin, phenotypically resemble cells that have been starved or are in G0. This phenotype includes a downregulation of protein synthesis and ribosome biogenesis, an upregulation of autophagy and ubiquitindependent protein degradation, specific changes in transcription and an increase in messenger RNA turnover. As disruption of TORC1 mimics rapamycin treatment, TORC1 seems to mediate the signalling pathway or pathways that control these rapamycin-sensitive, growth-related processes in response to nutrients 24. So, TORC1 mediates the temporal control of cell growth (FIG.2). The disruption of TOR2 alone causes a defect in the cell-cycledependent polarization of the actin cytoskeleton33. A polarized actin cytoskeleton orientates the secretory pathway and, consequently, the delivery of newly made proteins and lipids to the growth site, or bud, on the yeast cell surface. This function, which is specific to TOR2, is rapamycin-insensitive. The disruption of TORC2 mimics TOR2 depletion, and TORC2, unlike TORC1, is rapamycin-insensitive24. So, TORC2 mediates the signalling pathway that controls polarization of the actin cytoskeleton, thereby mediating the spatial control of cell growth. Furthermore, the two structurally and functionally distinct TOR complexes provide a molecular basis for the diversity, specificity and selective rapamycin sensitivity of TOR signalling in yeast. The effects of TOR in yeast Protein synthesis. Yeast TOR1 and TOR2 (as part of TORC1) control protein synthesis probably by activating the translation initiation factor eIF4E. Downregulation of cap-dependent TRANSLATION INITIATION is one of the earliest effects that is observed after rapamycin treatment32. TOR also controls the transcription of ribosomal proteins and the synthesis and processing of ribosomal RNA and transfer RNA, which emphasizes the role of TOR in controlling translation34–37. www.nature.com/reviews/molcellbio © 2003 Nature Publishing Group REVIEWS HEAT FAT HEAT FRB Kinase FATC TOR RNC HEAT WD40 KOG1 (raptor) WD40 LST8 (mLST8) RBD AVO1 Ankyrin AVO2 RasGEFN AVO3 Figure 1 | Structural domains of TOR and its binding partners. The structural domains of the target of rapamycin (TOR) are evolutionarily conserved. The KOG1/raptor domain structure is also conserved. The RNC (raptor N-terminal conserved) domain consists of highly conserved residues that are found in all KOG1/raptor orthologues. Regions in AVO1 and AVO3 have homology to the Ras-binding domain (RBD) and RasGEFN domain, respectively. The structural domain of LST8 is also highly conserved, consisting of seven WD40 repeats. AVO2 contains five ankyrin repeats. AVO, adheres voraciously to TOR2; FAT; a domain that was found in the FRAP (mTOR), ATM and TRRAP proteins; FRB, the binding site for FKBP12–rapamycin; HEAT, an amino-acid sequence motif that was first identified in huntingtin, elongation factor 3, regulatory A subunit of PP2A and TOR; KOG1, kontroller of growth 1. HEAT REPEATS An amino-acid sequence motif that was first identified in huntingtin, elongation factor 3, regulatory A subunit of PP2A and TOR. Each repeat varies in length between 37 and 43 amino acids,occurs as anti-parallel α-helices,and is repeated tandemly at least three times in every protein. Most of the proteins that contain this motif are large,are known to be part of a complex and function in transport processes. FAT DOMAIN (FRAP, ATM, TRRAP). A domain spanning ~500 amino acids that is found in the PIKK and TRRAP protein families . This domain is found aminoterminal to the kinase domain, and in combination with the FATC domain, which is found at the extreme carboxyl terminus. FAT and FATC domains are speculated to function in protein–protein interactions. WD40 REPEAT A repeat of ~40 amino acids with a characteristic central Trp–Asp motif. Protein stability. In the presence of nutrients, TOR controls protein stability by inhibiting both autophagy and ubiquitin-dependent protein degradation. Autophagy, which is the vacuolar targeting and degradation of bulk cytoplasm, is triggered by the dephosphorylation and activation of the APG1–APG13 (autophagy 1; 13) kinase complex in response to starvation. TOR inhibits autophagy by maintaining APG1 in a phosphorylated state38,39. Similarly, TOR prevents ubiquitylation, vacuolar targeting and degradation of the tryptophan transporter TAT2 by maintaining the serine/threonine kinase NPR1 in a phosphorylated — and thereby inactive — state40,41. NPR1 might phosphorylate TAT2 directly to trigger its ubiquitylation and internalization. Interestingly, mTOR also inhibits autophagy and nutrient transporter turnover in mammalian cells, but the mechanism of this inhibition is unknown42–44. Transcription. In yeast, TOR negatively controls the transcription of starvation-specific genes, by regulating the nuclear localization of several nutrient-responsive transcription factors34,35,45. TOR maintains the GATA-TYPE TRANSCRIPTION FACTOR GLN3 in a phosphorylated state and thereby tethered to the cytoplasmic protein URE2 (REF. 45). When TOR is inactivated — by nitrogen starvation or rapamycin treatment — GLN3 is dephosphorylated, released from URE2 and translocated into the nucleus to induce the transcription of genes that are required for the use of secondary nitrogen sources. URE2 is also phosphorylated in a TOR-dependent manner34,35. TOR negatively controls the transcription of stressresponsive genes by sequestering the general stress transcription factors MSN2 and MSN4 in the cytoplasm45. TOR sequesters the MSNs possibly by promoting the association of MSN2 and MSN4 with the cytoplasmic 14-3-3 proteins BMH1 and BMH2 (REF. 45). Finally, TOR negatively regulates the heterodimeric transcription factor that is composed of RTG1 and RTG3 (REFS 46,47). The mechanism by which TOR inhibits RTG1–RTG3 is unknown, but epistasis analysis indicates that this inhibition might occur through RTG2 and MKS1 (REFS 48–50). TOR seems to antagonize RTG2, which is a negative regulator of MKS1 (REF. 49), and MKS1 inhibits RTG1–RTG3 (REFS 49,50). So, a model is that TOR inhibits RTG1–RTG3 through a cascade of three negative regulatory steps. In the absence of glutamine, RTG1–RTG3 moves into the nucleus and induces the transcription of genes that encode enzymes Box 2 | Nutrient signalling in yeast Yeast cells grow and proliferate in response to ambient nutrients such as nitrogen and carbon sources. The quantity and quality of nutrient sources are detected by a diverse set of sensing and regulatory systems. In the presence of good nitrogen sources, such as ammonium or glutamine, the transcription of genes that are involved in the use of a poorer nitrogen source is repressed by the TOR signalling pathway, a phenomenon that is known as ‘nitrogen catabolite repression’112. All nitrogen sources are eventually converted to ammonia and glutamate, which are then converted to glutamine — the preferred nitrogen source and a key intermediate in yeast nitrogen metabolism. In the presence of a poor nitrogen source, such as proline or urea, genes that are required to scavenge and metabolize these secondary nitrogen sources are upregulated as a result of inactivation of the TOR pathway. The TOR pathway, which also responds to nutrients other than the nitrogen source, controls several growth-related processes, including ribosome biogenesis and protein synthesis. SSY1, an AMINO-ACID PERMEASE-like protein at the plasma membrane, is required for sensing extracellular amino acids and inducing genes that encode other amino-acid permeases113. The GCN system (general control non-derepressible) senses intracellular amino acids and induces synthesis of amino-acid biosynthetic genes in response to amino-acid deprivation. Glucose, which is the preferred carbon source of yeast, is detected by RGT2 (restores glucose transport 2) and SNF3 (sucrose non-fermenting 3), two plasma-membrane proteins that resemble glucose transporters, and by the G-protein-coupled receptor GPR1 (REF. 114). A high ambient concentration of glucose causes the repression of genes that are involved in respiration, GLUCONEOGENESIS and metabolism of alternative sugar sources. RGT2 and SNF3 signal through the AMP-activated protein kinase SNF1. GPR1 signals through the cyclic AMP–protein kinase A pathway. NATURE REVIEWS | MOLECUL AR CELL BIOLOGY VOLUME 4 | FEBRUARY 2003 | 1 1 9 © 2003 Nature Publishing Group REVIEWS In mammalian cells, growth is stimulated by a combination of nutrients and growth factors (TABLE 1). Accumulating evidence indicates that mTOR might mediate signalling in response to both stimuli (FIG.3). of mTOR blocks signalling through the PI3K pathway51,52. Reduction of neoplastic proliferation and tumour size by inhibition of mTOR in PTEN-deficient cells is due largely to the inhibition of RIBOSOMAL S6 PROTEIN KINASE (S6K, also known as p70 S6 kinase). The targets of mTOR, S6K and eukaryotic INITIATION FACTOR 4E-BINDING PROTEIN (4E-BP; also known as phosphorylated heat and acid-stable protein regulated by insulin, PHAS-I), are also components of the insulin–PI3K–PDK1 –Akt/PKB (protein kinase B) pathway53. What is the nature of the link between mTOR and the insulin signalling pathway in the control of S6K and 4E-BP? mTOR and the insulin signalling pathway converge on S6K and 4E-BP to mediate the complex, hierarchical phosphorylation of these common downstream targets54–56. However, recent evidence indicates that the insulin pathway might also impinge on mTOR signalling upstream of mTOR. Genetic evidence in flies and biochemical evidence in mammalian cells indicate that the TSC1–TSC2 complex binds and inhibits TOR29,30,57. The TSC1–TSC2 complex is inactivated in response to insulin by Akt-mediated phosphorylation of three sites in TSC2 (REF. 31). Akt also phosphorylates mTOR directly, on Ser2448 (where Ser is serine), but the significance of this phosphorylation is uncertain, as a substitution of Ser2448 with alanine does not affect mTOR signalling58,59. Furthermore, mTOR seems to respond positively to phosphatidic acid and ATP, both of which are produced in response to growth factors60,61. So, mTOR signalling is controlled by growth-factor inputs upstream and downstream of mTOR. Several studies have placed the TSC1–TSC2 complex upstream of TOR29,30,57. However, there is one conflicting report that suggests that the TSC complex regulates S6K1 independently of mTOR62*. It is difficult to reconcile this discrepancy as the conflicting reports30,57,62 describe essentially the same experiment but with opposite results. It is also worth noting that the binding of TSC1 and TSC2 to TOR was shown using overexpressed, recombinant TOR29. As TOR controls cell growth in yeast and plants, which lack a PI3K signalling pathway, the PI3K pathway seems to have been grafted onto the TOR pathway late in evolution. The joint control by the mTOR and PI3K pathways meets the need of multicellular animals to coordinate cell-autonomous growth with overall body growth. Multicellularity evolved independently in plants and animals, and plants must use another strategy to coordinate growth. Interestingly, worms have both a TOR and a PI3K pathway, but, in this case, the PI3K pathway does not seem to be involved in controlling cell growth12. Growth factors. The mTOR pathway mediates growth factor signalling through the phosphatidylinositol 3kinase (PI3K) pathway. Studies using phosphatase and tensin homolog (PTEN)-deficient cancer cells, in which the PI3K pathway is activated owing to the upregulation of the lipid second messenger phosphatidylinositol-3,4,5trisphosphate (PtdIns(3,4,5)P3), indicate that inhibition Nutrients. Similar to yeast TOR, mTOR also responds to the availability of nutrients. Branched-chain amino acids, particularly leucine, activate the mTOR signalling pathway63–66. A high level of ambient amino acids, even in the absence of insulin, promotes phosphorylation and activation of S6K, without activating Akt. Withdrawal of amino acids, which mimics rapamycin Table 1 | Nutrient and/or growth-factor signalling proteins Yeast Human Function* TOR1/2 mTOR Protein kinase, nutrient signalling KOG1 raptor Scaffold protein LST8 mLST8 Permease sorting, negatively regulates RTGs AVO1 hSIN1(?) Actin cytoskeleton, Ras signalling AVO2 – Actin cytoskeleton AVO3 – Actin cytoskeleton, sphingolipid metabolism TAP42 α4 Phosphatase interactor/regulator TIP41 mTIP41 Binds and regulates TAP42 in yeast PPH21/22 PP2A ctalytic Phosphatase catalytic subunit TPD3/CDC55 A/B regulatory PP2A regulatory subunit SIT4 PP2A catalytic Phosphatase catalytic subunit SAPs – SIT4 associated proteins NPR1 – Ser/Thr kinase, permease regulation GLN3 – GATA transcription factor, nitrogen regulation RTG1/3 – Transcription factor, TCA cycle – PI3K PI3 kinase, mitogen signalling – Akt/PKB Protein kinase, binds PtdInsP3 – PDK1 Protein kinase, binds PtdInsP3 – PTEN Phospholipid phosphatase – TSC1 (hamartin) Negatively regulates mTOR – TSC2 (tuberin) Negatively regulates mTOR – S6K Protein kinase, translation activator – 4E-BP eIF4E binding protein, translation inhibitor *Characterized or putative functions are listed. 4E-BP, eukaryotic initiation factor 4E-binding protein; α4, mammalian homologue of TAP42; AVO, adheres voraciously to TOR2; CDC55, cell-divisioncycle mutant 55; eIF4E, eukaryotic initiation factor 4E; GLN3, glutamine; KOG1, kontroller of growth 1: LST8, lethal with sec-thirteen 8 ; NPR1, nitrogen permease reactivator 1; PDK1, 3-phosphoinositide dependent protein kinase-1; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; PP2A, protein phosphatase 2A; PPH, protein phosphatase; PTEN, phosphatase and tensin homologue; RTG, retrograde signalling protein; S6K, ribosomal S6 protein kinase; SAP, SIT4associated protein; SIT4, suppressors of initiation of transcription; SIN1, sty1 interactor; TAP42, type 2A-phosphatase associated protein, 42 kDa; TCA, tricarboxylic acid; TIP41, TAP42-interacting protein, 41 kDa; TOR, target of rapamycin; TPD3, tRNA processing deficient 3; TSC, tuberous sclerosis complex. TRANSLATION INITIATION The first step in protein synthesis, wherein the initiating ribosome scans along the messenger RNA and identifies the initiator codon to begin translation in the proper reading frame. GATA-TYPE TRANSCRIPTION FACTORS A family of transcription factors that contain a zinc-finger motif that was first identified in the vertebrate GATA-1 protein. These transcription factors bind the consensus sequence GATA in the 5′ non-coding regions of constitutive and inducible genes. 120 of the tricarboxylic acid (TCA) cycle. As GLN3 and RTG1–RTG3 respond to intracellular levels of glutamine, the TOR pathway seems to sense glutamine, among other unknown nutrient compounds47. So, TOR broadly controls growth-related metabolism by sequestering several transcription factors in the cytoplasm. Regulators of mammalian TOR | FEBRUARY 2003 | VOLUME 4 www.nature.com/reviews/molcellbio © 2003 Nature Publishing Group REVIEWS Nutrients Cell membrane ? KOG1 LST8 TOR1/2 LST8 Rapamycin AVO1 TOR2 AVO2 AVO3 TAP42 P TIP41 RTG2 TIP41 MKS1 SIT4 PPH GLN3 ? Actin organization Translation TOR as a protein-phosphatase regulator NPR1 MSN2/4 RTG1/3 Permease turnover Transcription Figure 2 | Two functionally distinct TOR complexes in yeast. Target of rapamycin (TOR) complex 1 (TORC1) contains either TOR1 or TOR2, together with KOG1 and LST8. TORC1 mediates the rapamycin-sensitive function of TOR, which leads to the activation of translation, inhibition of protein turnover and inhibition of transcription of starvation-specific genes. TOR controls several of these processes by inhibiting type 2A phosphatases. TOR complex 2 (TORC2) consists of TOR2, LST8, AVO1, AVO2, and AVO3. The TORC2 complex mediates actin cytoskeleton organization and is insensitive to rapamycin. AVO, adheres voraciously to TOR2; GLN3, glutamine; KOG1, kontroller of growth 1; MSN, multicopy suppressors of snf1 mutation; NPR1, nitrogen permease reactivator 1; PPH, protein phosphatase; RTG, retrograde signalling protein; SIT4, suppressors of initiation of transcription 4; TAP42, type 2A-phosphatase associated protein 42 kDa; TIP41, TAP42-interacting protein 41 kDa. AMINO-ACID PERMEASE A protein that transports amino acids from the outside to the inside of the cell. In yeast, these proteins contain 12 membranespanning segments, and are either broadly specific for a group of structurally related amino acids or highly specific for individual amino acids. GLUCONEOGENESIS The metabolic formation of carbohydrates from noncarbohydrate organic precursors. RIBOSOMAL S6 PROTEIN KINASE (S6K). A protein kinase that phosphorylates the ribosomal protein S6. S6 is involved in the translation of messenger RNA transcripts that contain a polypyrimidine tract at their transcriptional start site. through raptor28, although the effect of nutrients on the mTOR–raptor association is debated24,27,67. The precise roles of raptor, TSC1–TSC2 and possibly mLST8 in controlling mTOR activity in response to amino acids remain to be determined. However, as S6K and 4E-BP phosphorylation in response to both insulin and amino acids is rapamycin sensitive, mTOR integrates nutrient and insulin signals to control cell growth. Interestingly, as the production of insulin as well as the response to insulin is sensitive to nutrients, mTOR as a nutrient sensor might also be involved in regulating insulin production68. treatment, leads to rapid dephosphorylation of S6K and 4E-BP. Furthermore, rapamycin treatment renders the phosphorylation of S6K refractory to amino-acid stimulation63. Although amino acids seem to regulate the mTOR pathway, the effect of amino acids on mTOR kinase activity is unclear. Dennis et al. reported that amino acids have no effect on mTOR kinase activity towards S6K, as assayed with mTOR that was immunopurified from amino acid stimulated cells60. However, Kim et al. have carried out similar experiments, but with conditions that favour the inclusion of raptor, the newly identified mTOR interactor protein. In the presence of raptor, mTOR kinase activity is increased when cells are stimulated with leucine28. Consistent with the above observations, raptor has been proposed to function as a scaffold protein that links mTOR to S6K and 4E-BP27. Finally, mTOR is also thought to respond to a mitochondrial signal, but this signal could simply be amino acids that are synthesized in the mitochondria47. How might mTOR kinase activity be regulated by amino acids? The recent observations that loss of the TSC1–TSC2 complex results in an increase in S6K activity and renders cells resistant to amino-acid starvation imply that amino acids, as well as growth factors, signal to TOR through inhibition of the TSC COMPLEX30. The finding that nutrient deprivation stabilizes the mTOR–raptor association and inhibits mTOR kinase activity indicates that nutrients might regulate mTOR NATURE REVIEWS | MOLECUL AR CELL BIOLOGY Regulation of type 2A protein phosphatases (PP2As) is an important mechanism of TOR signalling. Several downstream effectors of TOR, such as NPR1 and GLN3 in yeast, and S6K and 4E-BP in mammals, are multiphosphorylated proteins that are dephosphorylated rapidly after rapamycin treatment. The regulation of phosphatases, in addition to kinases, by TOR ensures a rapid and coordinated response to nutrient deprivation. PP2A. In yeast and mammals, the PP2A catalytic (C) subunits are broadly active; target specificity is determined by the associated regulatory (A or B) subunits69. The regulatory subunits of phosphatases are diverse and, as well as controlling the substrate specificity, they also determine the subcellular localization of the phosphatase complex. The PPH21 or PPH22 catalytic subunit of yeast PP2A associates with the regulatory A subunit, TPD3, and one of two B regulatory subunits, CDC55 and RTS1. Mammalian PP2A consists of a core dimer of a catalytic subunit that is associated with a scaffolding A subunit. This core dimer associates with one of various regulatory B subunit isoforms or splice variants. SIT4. SIT4 is a yeast PP2A-related catalytic subunit that associates with one of four regulatory proteins that are known as SAPs (SIT4-associated proteins) — SAP4, SAP155, SAP185, and SAP190 (REFS 70,71). Deletion of three of the SAP genes (SAP155, SAP185, and SAP190) phenocopies a SIT4 deletion, which indicates that the SAPs regulate SIT4 positively. SIT4, PP2A and TAP42. In yeast, SIT4 and PP2A also interact with the essential protein TAP42 (type 2A associated protein-42kDa)72. TAP42 binds the catalytic subunits of PP2A and SIT4 in a nutrient-dependent, rapamycin-sensitive manner, and a TAP42 mutation confers rapamycin resistance, which indicates that TAP42 is a component of the TOR pathway. TAP42 seems to compete with other regulatory subunits for binding to the phosphatase catalytic subunit73. TOR promotes the binding of TAP42 to the phosphatase catalytic subunit and thereby prevents the binding of other regulatory subunits. What is the consequence of TOR-stimulated binding of TAP42 to SIT4 and PP2A? NPR1 and GLN3 are dephosphorylated rapidly in a SIT4-dependent manner after rapamycin treatment, VOLUME 4 | FEBRUARY 2003 | 1 2 1 © 2003 Nature Publishing Group REVIEWS Growth factors Nutrients Cell membrane ? Raptor mLST8 mTOR Rapamycin TSC1 TSC2 PtdInsP3 PDK1 PtdInsP2 Akt PI3K PTEN PP2A 4E-BP S6K elF4E S6 Translation Transcription Transcription Figure 3 | mTOR integrates signals from nutrients and growth factors leading to cell growth. Growth-factor stimulation activates phosphatidylinositol 3-kinase (PI3K) which phosphorylates phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) at position 3 to generate PtdIns(3,4,5)P3. The protein kinases Akt and 3-phosphoinositide-dependent protein kinase-1 (PDK1) are recruited to the membrane and bind PtdIns(3,4,5)P3through their pleckstrin homology (PH) domains. Membrane-bound Akt is activated, leading to the phosphorylation of TSC2/tuberin. This renders the TSC complex unstable and inactive, and thereby relieves the inhibitory constraint on the mammalian target of rapamycin (mTOR). Nutrients, by an unknown mechanism, activate the mTOR complex, which consists of mTOR bound to raptor and mLST8. mTOR promotes the hierarchical phosphorylation of the translation activator S6K and the translation inhibitor 4E-BP either directly or indirectly by the inhibition of protein phosphatase 2A (PP2A). PDK1 and an unidentified growth factor-responsive kinase also phosphorylate S6K and 4E-BP, respectively. Phosphorylation of S6K and 4E-BP promotes translation, via the ribosomal protein S6 and the eukaryotic initiation factor 4E (eIF4E), respectively. mTOR also controls gene transcription in response to nutrients and in cooperation with growth factor stimulation5. 4E-BP, eukaryotic initiation factor 4E-binding protein; S6K, ribosomal S6 protein kinase; TSC2, tuberous sclerosis complex 2. INITIATION FACTOR 4E-BINDING PROTEIN (4E-BP; PHAS-I). When dephosphorylated, 4E-BP negatively regulates capdependent translation by binding and inhibiting the eukaryotic initiation factor 4E (eIF4E). TSC COMPLEX This consists of TSC1, a protein that is predicted to form coiledcoil structures and contains a putative transmembrane domain, and TSC2, a protein that contains a coiled-coil domain and a Rap GTPaseactivating protein (GAP) domain. Mutations in either TSC1 or TSC2 are responsible for tuberous sclerosis, a genetic disorder that is characterized by hamartomas in various organs. 122 which implies that TAP42 inhibits SIT4 and possibly PP2A40,45,74. Interestingly, TAP42 binds only a small fraction of the more abundant catalytic subunits, which indicates that TAP42 might only inhibit a specific pool of PP2A or SIT4 (REF. 72). TAP42 might also activate PP2A or SIT4 towards specific targets, but such targets are unknown. TOR regulation of TAP42. TOR might regulate TAP42 both directly and indirectly. TOR phosphorylates TAP42 in vitro, and TAP42 is dephosphorylated in a PP2A-dependent manner in rapamycin-treated cells73. This implies that TOR phosphorylates TAP42 and thereby directly controls the binding of TAP42 to PP2A. By contrast, TOR seems to control the binding of TAP42 to SIT4 indirectly, as indicated by the observation that TAP42 dephosphorylation after rapamycin treatment is significantly slower than SIT4–TAP42 dissociation and SIT4 activation40,45,72–74. TOR controls the binding of TAP42 to SIT4 through the TAP42-interacting protein TIP41 (REF. 74). TOR, by maintaining TIP41 phosphorylation, promotes the | FEBRUARY 2003 | VOLUME 4 binding of TAP42 to SIT4. After TOR inactivation, TIP41 is dephosphorylated, binds TAP42 and thereby induces the release and activation of SIT4. SIT4, in turn, dephosphorylates more TIP41. So, TIP41 is part of a feedback loop, the purpose of which is to rapidly amplify phosphatase activity in response to TOR-inactivating conditions. Mammalian TOR and phosphatases. TOR also controls phosphatase activity in mammalian cells. mTOR phosphorylates PP2A in vitro, and rapamycin activates phosphatase activity in vivo75. mTOR might therefore regulate PP2A through direct phosphorylation. Furthermore, PP2A binds and inactivates S6K75. Finally, a mammalian orthologue of TAP42, α4, associates with mammalian PP2A catalytic subunits76,77. Although the rapamycin sensitivity of this association is controversial, S6K activation is defective in α4-disrupted cells, which suggests that α4 might function in the mTOR pathway77. α4 also associates with the microtubuleassociated and OPITZ SYNDROME protein Midline-1 (MID1). MID1, a UBIQUITIN LIGASE that is involved in normal midline development, targets PP2A for degradation78. Interestingly, overexpression of α4 promotes the dephosphorylation of MID1, which indicates that MID1 might be a target of α4–PP2A79. In support of a phosphatase activity for this complex, elongation factor-2 is also dephosphorylated when α4 is overexpressed80. The role of α4 in mTOR signalling remains to be determined. TOR in Drosophila growth control Similar to the situation in mammals, the TOR and insulin signalling pathways in Drosophila converge to coordinately regulate cell size81. Loss-of-function mutations in the genes that encode the positive elements of these pathways, such as dTOR, dIRS (insulin receptor substrate; also known as chico), dPI3K, dPDK1, dAkt and dS6K result in smaller cells and, in some cases, smaller but correctly proportioned flies (FIG. 4)13,14,82–86. Conversely, loss-offunction mutations in the genes that encode the negative elements of these pathways, such as dPTEN, dTSC1, and dTSC2, lead to an increased cell size87–89. In addition, expression of a highly active d4E-BP1, also a negative regulator, decreases cell size90. dTOR regulates dS6K and probably also d4E-BP, which indicates that dTOR might control cell size through protein synthesis. Regulation of dTOR. How is dTOR regulated? A reduction in the dosage of the dTOR gene suppresses the lethality and the cell-size defect that is caused by a dTsc mutation29. Furthermore, in cells with decreased dTSC expression, dS6K phosphorylation is resistant to amino-acid starvation, but remains rapamycin sensitive. Finally, loss of both the dTSC complex and dS6K, like the loss of dS6K alone, results in decreased cell growth (that is, ds6k is epistatic to dTsc29). These results indicate that dTOR is regulated negatively by dTSC, and this inhibition of dTOR by dTSC is relieved by amino acids. But how is dTSC regulated? Similar to mammalian TSC, dTSC2 is phosphorylated and inactivated by dAkt. www.nature.com/reviews/molcellbio © 2003 Nature Publishing Group REVIEWS Growth in non-proliferating cells +/+ I-1/I-1 Figure 4 | A TOR-signalling defect in Drosophila causes smaller cells and a smaller animal. Shown are wild type (+/+) and ds6k (l-1/l-1) mutant flies. The ds6k allele is a partial loss-offunction mutation. dTOR and ds6k-null mutations are lethal. S6K, ribosomal S6 protein kinase; TOR; target of rapamycin. Reproduced with permission from REF. 86 © (1999) American Association for the Advancement of Science. Indeed, most dAkt-mediated growth signalling is transduced by phosphorylation of two highly conserved sites in dTSC231. Mutation of these two sites in dTSC2, such that they can no longer be phosphorylated, stabilizes the dTSC complex and inhibits cell growth. Furthermore, destabilization of the complex by mutation of dTsc1 promotes cell growth that is not further enhanced by dAkt overexpression. Although these observations indicate that dTSC is controlled by dAkt, they do not account for the amino-acid-mediated inactivation of dTSC, as dAkt is not controlled by amino acids. So, in both flies and mammals, it remains to be determined how TSC responds to amino acids. Nevertheless, the above studies indicate that in higher eukaryotes, both the nutrient-induced TOR pathway and the insulin pathway determine cell size. TOR in muscle cells. An increased workload on a muscle causes an increase in muscle mass. This increase in mass, or hypertrophy, is due to an increase in the size of individual muscle cells (BOX 3). A reduced workload, ageing or myopathies lead to atrophy, which is a loss of skeletal-muscle mass due to shrinkage of individual muscle cells. How a mechanical stimulus is propagated as a chemical signal, and the molecular mechanisms that underlie skeletal-muscle hypertrophy are poorly understood. However, muscle hypertrophy requires an increase in the rate of protein synthesis and signalling by the mTOR and the insulin-like growth factor (IGF1) pathways91–94. Treatment with rapamycin prevents IGF1-induced hypertrophy, whereas expression of activated Akt promotes hypertrophy91,92,95.Also, increased phosphorylation of mTOR at Ser2448, a site that is phosphorylated by Akt in vitro, is associated with muscle hypertrophy96. The mTOR effector S6K shows a rapamycin-sensitive increase in phosphorylation in response to muscle load92,97. The role of mTOR and the Akt pathways in musclecell growth is supported by the finding that transgenic mice that overexpress activated Akt specifically in the heart have enlarged cardiomyocytes, which results in a larger heart 98. The Akt-induced size increase is rapamycin-sensitive, indicating that the effect is mediated by mTOR. The observed increase in myocyte size is independent of cell division, as an increase in heart size occurs when myocytes are in a postmitotic state98. Taken together, the above studies on muscle cells show how both cell and organ size are controlled by mTOR, and provide an example of how mTOR can control growth in non-proliferating cells. Box 3 | Skeletal-muscle hypertrophy OPITZ SYNDROME Opitz G/BBB syndrome is a congenital disorder that arises from defects in ventral midline development. Manifestations of this disorder include, among others, mental retardation, cleft lip and palate, and genitourinary defects. UBIQUITIN LIGASE An enzyme that couples the small protein ubiquitin to lysine residues on a target protein; it marks the target protein for destruction by the proteasome. SATELLITE CELLS Myogenic stem cells that are able to proliferate and form new myofibres. In response to an increased workload, skeletal muscles IGF1 Mechanical load Nutrients undergo hypertrophic growth. Load-induced hypertrophy occurs as a result of protein synthesis that is induced by Akt the insulin-growth factor 1 (IGF1) and Ca2+/calmodulin calcineurin–nuclear factor of activated T cells (NFAT) Rapamycin mTOR Calcineurin signalling pathways. However, recent studies challenge the prominent role given to the calcineurin–NFAT pathway, and argue for an important role for Akt and mTOR in NFAT 4E-BP S6K muscle hypertrophy91,92. Activation of Akt after IGF1 stimulation promotes translation by the phosphorylation Transcription of translational regulators and mTOR targets S6K and 4ETranslation BP. Calcium could couple a mechanical stimulus to the IGF1 pathway and lead to increased gene transcription by Nucleus activating the calcium-dependent phosphatase calcineurin, which dephosphorylates and activates transcription factors such as NFAT115. Skeletal-muscle hypertrophy also occurs during muscle regeneration. As adult skeletal muscles are terminally differentiated, muscle regeneration involves activation of SATELLITE CELLS that are located at the periphery of myofibres116. Activated satellite cells, which are also known as myoblasts, proliferate and express myogenic markers. The myoblasts then migrate and fuse to existing muscle fibres (hypertrophy) or fuse together to form new myofibres (hyperplasia). Myoblast proliferation and fusion are stimulated by growth factors such as IGF1. NATURE REVIEWS | MOLECUL AR CELL BIOLOGY VOLUME 4 | FEBRUARY 2003 | 1 2 3 © 2003 Nature Publishing Group REVIEWS Box 4 | Synaptic plasticity in neurons Synaptic plasticity refers to Presynapse the ability of synapses to undergo modification in response to a stimulus. The Nutrients morphological and ? ? biochemical changes that Postsynapse Ca2+/ Rapamycin accompany changes in mTOR calmodulin synaptic strength are the Nucleus Translation PKA basis for learning and 117 ERK/MAPK memory . In the model Transcription organism Aplysia, shortterm memory entails the transient stimulation of synapses by neurotransmitters, which leads to the presynaptic production of the second messenger cyclic AMP and the subsequent activation of protein kinase A (PKA). PKA phosphorylates substrates such as ion channels and proteins of the exocytosis machinery in the pre-synapse and consequently enhances the availability and release of transmitters. Short-term synaptic changes therefore involve modification of pre-existing proteins, which leads to modification of pre-existing synaptic connections. The conversion of short-term memory to long-term memory requires new protein synthesis immediately after re-stimulation of a synapse. In the hippocampus of mammalian brain, both presynaptic and postsynaptic changes have been proposed to mediate longterm potentiation (LTP). Ca2+-dependent signalling pathways mediate synaptic changes, for example by enhancing neurotransmitter release in the presynapse and increasing responsiveness of glutamate receptors in the postsynapse. As a neuron makes several synaptic connections, an important issue is whether LTP is cell-wide or synapse-specific. Although nuclear events are required, long-term changes are specific to the stimulated synapse and require local protein synthesis. messenger RNAs that are stored in dendrites are activated after synaptic stimulation. Translation of these mRNAs, which is mediated by the mTOR pathway, facilitates LTP. The mechanism by which mTOR is activated in neurons, possibly by neurotransmitters, neurotrophic factors, or nutrients, is unknown. ERK/MAPK, extracellular-signal-regulated kinase/mitogen-activated protein kinase. LONG-TERM POTENTIATION (LTP). LTP is a specific example of coincidence detection, whereby the high-frequency stimulation of a neuron increases the magnitude of subsequent responses, an effect that can last for days. LTP is believed to underlie some kinds of learning and memory. SYNAPSE The point of contact and transfer of information from one neuron to another. RNA INTERFERENCE (RNAi). The process by which an introduced double-stranded RNA specifically silences the expression of genes through degradation of their cognate messenger RNAs. 124 TOR in neurons. Memory formation is achieved by changes in synaptic strength or plasticity, a process which involves LONG-TERM POTENTIATION (LTP) or, in the snail Aplysia, long-term facilitation (LTF) (BOX 4). Studies in Aplysia have shown that, despite nuclear events, long-term changes in synaptic function and structure are confined to a stimulated SYNAPSE and require local protein synthesis from pre-existing mRNAs. So proteins are synthesized and deposited specifically at a stimulated synapse, which in turn leads to the synapse-specific growth that is necessary to encode memory99,100. As the control of protein synthesis is an important aspect of LTP/LTF, it is perhaps not surprising that mTOR is involved in LTP/LTF100,101. The induction of LTF in invertebrates and LTP in hippocampal slices by electrical stimulation or by brain-derived neurotrophicfactor stimulation is inhibited by rapamycin101,102. Postsynaptic protein synthesis and S6K activity in response to a stimulus are also inhibited by rapamycin103,104. Finally, components of the mTOR and insulin pathways are enriched at postsynaptic sites101,105. These findings show that mTOR controls synaptic protein synthesis and so also memory formation in response to a stimulus. Furthermore, this is an interesting example of mTOR controlling cell growth in a localized manner and, again, in non-proliferating cells. mTOR might also control overall neuronal growth, as the deletion of PTEN in the brain results in enlargement of neuronal cells106. | FEBRUARY 2003 | VOLUME 4 Does mTOR control the size of proliferating cells? Cultured mammalian cells treated with rapamycin have a reduced proliferative rate and are smaller at all stages of the cell cycle107. RNA INTERFERENCE (RNAi)-mediated inhibition of mTOR or raptor also leads to decreased size in proliferating cells28. Finally, S6K1deficient mice have smaller pancreatic β-cells as a result of a growth defect during embryogenesis108. So, mTOR also seems to control cell size in proliferating cells, which is consistent with previous findings for dTOR and yeast TOR. Conclusion and perspective Studies over the past seven years have shown that TOR controls the growth of proliferating yeast, Drosophila and mammalian cells. In addition, TOR also controls the growth of non-proliferating neurons and muscle cells. An important mechanism of TOR signalling is the phosphorylation of multi-phosphorylated effectors, by activating protein kinases and inhibiting protein phosphatases. An unanswered question is how TOR senses and is activated by the presence of nutrients. The further characterization of several recently identified regulatory proteins that associate with TOR should provide clues as to how TOR carries out its many functions in controlling cell growth. The importance of TOR in regulating cell growth is underscored by the finding that rapamycin is not only an immunosuppressant but also an effective anti-cancer drug. The effect of rapamycin on tumour growth seems www.nature.com/reviews/molcellbio © 2003 Nature Publishing Group REVIEWS to be twofold. 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Hall’s laboratory: http://www.biozentrum.unibas.ch/Research/Biochemistry/Hall/ hall.html Access to this interactive links box is free online. *Please note that this sentence has been corrected and differs from the print version. On page 120, right column of the print version, the sentence reads ‘However, there is one conflicting report which suggests that mTOR regulates S6K1and 4E-BP independently of mTOR62.’ 126 | FEBRUARY 2003 | VOLUME 4 www.nature.com/reviews/molcellbio © 2003 Nature Publishing Group