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7/28/2019 Mammalian Target of Rapamycin- A Signaling Kinase for Every Aspect of Celllular Life
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Thomas Weichhart (ed.), mTOR: Methods and Protocols, Methods in Molecular Biology, vol. 821,DOI 10.1007/978-1-61779-430-8_1, © Springer Science+Business Media, LLC 2012
Chapter 1
Mammalian Target of Rapamycin: A Signaling Kinasefor Every Aspect of Cellular Life
Thomas Weichhart
Abstract
The mammalian (or mechanistic) target o rapamycin (mTOR) is an evolutionarily conserved serine-threonine kinase that is known to sense the environmental and cellular nutrition and energy status. Diversemitogens, growth actors, and nutrients stimulate the activation o the two mTOR complexes mTORC1and mTORC2 to regulate diverse unctions, such as cell growth, prolieration, development, memory,longevity, angiogenesis, autophagy, and innate as well as adaptive immune responses. Dysregulation o themTOR pathway is requently observed in various cancers and in genetic disorders, such as tuberous scle-rosis complex or cystic kidney disease. In this review, I will give an overview o the current understandingo mTOR signaling and its role in diverse tissues and cells. Genetic deletion o specic mTOR pathway proteins in distinct tissues and cells broadened our understanding o the cell-specic roles o mTORC1and mTORC2. Inhibition o mTOR is an established therapeutic principle in transplantation medicine andin cancers, such as renal cell carcinoma. Pharmacological targeting o both mTOR complexes by novel
drugs potentially expand the clinical applicability and ecacy o mTOR inhibition in various diseasesettings.
Key words: mTOR, Rapamycin, Immunity, Cancer, Kinase
When the antiungal drug rapamycin was isolated rom the soilbacterium Streptomyces hygroscopicus on Easter Island (Rapa Nui)in the 1970s, nobody could have imagined back in those days thatthis drug would be undamental or the identication o a signalingnetwork that regulates so many dierent aspects o cellular lie (1).Initially, rapamycin was developed as antiungal agent, but soonaterward it was ound that rapamycin possesses immunosuppressiveand antiprolierative properties (2). Yeast genetic screens discoveredthat rapamycin inhibits two genes called target o rapamycin 1 and 2(TOR1 and TOR2) and later the mammalian homolog mammalian
1. Identicationo mTOR
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(or mechanistic) TOR (mTOR; also known as FRAP1 or RAFT1) was identied (1). Rapamycin does not directly inhibit mTOR, butinstead binds the FK506-binding protein 12 (FKBP12), and it isthis complex, which inhibits mTOR (Fig. 1) (1). mTOR is a sig-naling kinase that aects broad aspects o cellular unctions, includ-ing metabolism, growth, survival, aging, synaptic plasticity,immunity, and memory (3). It is an atypical serine-threonine pro-
tein kinase belonging to the phosphatidylinositol kinase-relatedkinase (PIKK) amily with a predicted molecular weight o 290 kDa.mTOR is the catalytic subunit o two distinct complexes calledmTOR complex 1 (mTORC1) and mTORC2.
The mTOR signaling pathway is highly conserved rom yeast to
humans and is activated by a variety o divergent stimuli. mTOR senses cellular energy levels by monitoring cellular ATP:AMP levels
via the AMP-activated protein kinase (AMPK), growth actors such
2. The mTORSignaling Pathway
Ribosome
mTORC1mTORRaptormLST8PRAS40
mTORC2mTORRictorSIN1mLST8
TSC1TSC2P
P
P
Rheb
Rheb
GTP
GDP
TCTPRapamycin
4E-BP1 p70S6KP P
protein synthesis & cell growth
FKBP12
Insulin/ IGF-1
TLR ligands
PI3K
IRS-1
P
PIP2
PIP3
PTEN
AMP
Cellular stress
AMPK
LKB1
P
PDK1
GSK3AktP
P
++
Wnt
Rag GTPases
amino acids
Fig. 1. The current understanding of mTOR signaling. All pathway members, which are discussed in the text, are shown.
Please note that additional proteins in the mTOR pathway have been described that are comprehensively reviewed by
Zoncu et al. (3) or Yang and Guan (1).
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as insulin and insulin-like growth actor 1 (IGF-1) via the insulinreceptor and the IGF-1 receptor respectively, amino acids via RagGTPases, and signals rom the Wnt amily via glycogen synthasekinase 3 (GSK3) (1, 4). In the immune system, stimulation o antigen receptors (T and B cell receptors), cytokine receptors (e.g.,
Interleukin [IL]-2 receptor), or toll-like receptors (TLRs) (1, 5–8)all lead to the activation o mTOR (Fig. 1). As the archetypical andbest-documented example, triggering o the insulin receptoractivates tyrosine kinase adaptor molecules at the cell membraneleading to the recruitment o the class I amily o phosphati-dylinositol-3 kinases (PI3K) to the receptor complex (9). Followingreceptor engagement, PI3K phosphorylates phosphatidylinositol4,5-bisphosphate (PIP2) to generate phosphatidylinositol-3,4,5-trisphosphate (PIP3) as a second messenger to recruit and activatedownstream targets, including Akt (Fig. 1). The serine-threonine
kinase Akt, also termed protein kinase B (PKB), connects PI3K and mTOR (10). There are three highly homologous isoorms o
Akt encoded in the genome (Akt1, Akt2, and Akt3) representingsome o the most important survival kinases involved in regulat-ing a similarly wide array o cellular processes as mTOR, includingmetabolism, growth, prolieration, and apoptosis (11). The tumorsuppressor phosphatase and tensin homolog deleted on chromosome10 (PTEN) is a lipid phosphatase and dephosphorylates PIP3 tonegatively regulate PI3K signaling (12). A main eector o Akt isthe tuberous sclerosis complex (TSC) protein 2 (TSC2) (1). TSC2
is a tumor suppressor that orms a heterodimeric complex withTSC1. Mutations in TSC1 or TSC2 give rise to the hamartomasyndrome TSC and the prolierative lung disorder lymphangioleio-myomatosis (13). TSC2 is phosphorylated and inactivated by Aktleading to a loss o suppression o mTOR by the TSC1–TSC2complex (1). Moreover, downstream o TSC1–TSC2, the GTPaseRas homolog enriched in brain (Rheb) is essential or mTOR activation (14). Amino acid-induced activation o the Rag GTPasespromotes the translocation o mTORC1 to lyosomal compartmentsthat contain Rheb (4, 15–17). mTOR controls protein synthesis
through the direct phosphorylation and inactivation o a repressoro mRNA translation, eukaryotic initiation actor 4E-bindingprotein 1 (4E-BP1), and through phosphorylation and activationo S6 kinase (S6K1 or p70S6K), which in turn phosphorylates theribosomal protein S6 (18). Cytokines, growth actors, amino acids,insulin, or TLR ligands activate mTOR and increase the phospho-rylation status o 4E-BP1 and S6K1 in a rapamycin-sensitive manner(18). Importantly, a negative eedback loop has been described inthe insulin receptor pathway involving the insulin receptor substrate-1(IRS-1). Activation o mTORC1 promotes an inhibitory phospho-
rylation o IRS-1 via S6K1 that leads to inactivation o PI3K and Akt (Fig. 1). Conversely, inhibition o mTORC1 should lead tohyperactivation o Akt, which indeed has been documented in
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some rapamycin-treated cancer patients (3). Loss o mTOR unction leads to an arrest in the G1 phase o the cell-cycle along
with a severe reduction in protein synthesis in many cells.mTOR exists in two distinct complexes in the cell. mTORC1
consists o the regulatory-associated protein o mTOR (Raptor),
a conserved 150 kDa protein, which recruits S6K1 and 4-E-BP1,and the adaptor protein mLST8 (19). Rapamycin inhibits mTORC1activity by blocking its interaction with Raptor (20). mTORC2 iscomposed o mTOR, mLST8 and the adaptor proteins Rictor(rapamycin-insensitive companion o mTOR) and Sin1 (21).mTORC2 is usually rapamycin-insensitive and is thought to regu-late actin cytoskeleton dynamics. mTORC2 directly phosphorylates
Akt at serine 473 via cotranslational phosphorylation and througha direct interaction with the ribosome (22–24). The unctional roleo mTORC2, which is upstream o Akt and mTORC1, is not well
understood within this signaling circuit (25). Interestingly, long-term treatment with rapamycin (>18 h) alters the mTORC1:mTORC2 equilibrium resulting in reduced mTORC2 levels and,hence, impaired Akt signaling in some cells (26). Collectively, recep-tor engagement leads to a coordinated activation o PI3K, Akt,TSC1–TSC2 and Rheb, which are integrated at the level o mTORC1 (1, 27). Interestingly, mTORC1 seems to be predomi-nantly cytoplasmic, whereas mTORC2 is abundant in the cytoplasmand the nucleus in human primary broblasts (28). The detailedunctions o the mTOR complexes in these compartments are cur-
rently unknown. Further aspects o mTOR signaling are compre-hensively reviewed, e.g., by Zoncu et al. (3), Yang and Guan (1).
mTORC1 controls growth and prolieration by modulating mRNA translation through phosphorylation o the 4E-BP1, 2, and 3 andthe S6K1 and 2 (29). More specically, the three 4E-BPs do not
regulate cell size, but they block cell prolieration by inhibiting thetranslation o messenger RNAs that encode proteins involved inprolieration and cell cycle progression (29). In T lymphocytes,mTOR controls cell cycle progression rom the G1 into S phase inIL-2-stimulated cells (30). The cyclin-dependent kinase (Cdk)enzymes, when associated with the G1 cyclins D and E, arerate-limiting or the entry into the S phase o the cell cycle. IL-2activates Cdk by causing the elimination o the Cdk inhibitorprotein p27Kip1, a process that is prevented by rapamycin in Tcells (31). Moreover, mTORC2 activates the serum- and
glucocorticoid-induced protein kinase-1 (SGK1), which in turnphosphorylates p27Kip1. Once phosphorylated, p27Kip1 is
3. Cell Growthand Prolieration
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retained in the cytoplasm rendering it incapable o blockingCdk1 or Cdk2 activation and thereore allowing entry into the cellcycle (32, 33).
Autophagy is a starvation-induced degradation o cytosoliccomponents ranging rom individual proteins (microautophagy)to entire organelles (macroautophagy). Autophagy is critical inproviding substrates or energy production under conditions o limited nutrient supply (3). mTORC1 actively suppresses auto-phagy and, conversely, inhibition o mTORC1 strongly inducesautophagy (34). In S. cerevisiae , TOR-dependent phosphorylationo autophagy-related 13 (Atg13) disrupts the Atg1–Atg13–Atg17complex that triggers the ormation o the autophagosome (3).The mammalian homologs o yeast Atg13 and Atg1, ATG13 andULK1, bind to the 200 kDa FAK amily kinase-interacting protein(FIP200; a putative ortholog o Atg17) and the mammalian-specic component ATG101 (35). mTOR phosphorylates ATG13and ULK1 to block autophagosome initiation (35).
As the mTOR pathway is critical or many basic aspects o cell biology,it is not surprising that mTOR also plays a prominent role in devel-opment. For example, embryonic homozygous deletion o mTOR leads to a developmental arrest at E5.5 (36). Moreover, mTOR−/−embryos show a deect in inner cell mass prolieration consistent
with an inability to establish embryonic stem cells rom mTOR-decient embryos (36). The catalytic unction o mTOR is criticalor the embryonic development as knock-in mice carrying a muta-
tion in the catalytic domain o mTOR die beore embryonic day 6.5 (37). Rheb, the essential upstream regulator o mTORC1, islikewise important or embryonic development (38, 39). Interes-tingly, in contrast to mTOR or Raptor mutants, the inner cellmass o Rheb−/− embryos dierentiate normally (38). Moreover,embryonic deletion o Rheb in neural progenitor cells abolishesmTORC1 signaling in the developing brain and increases mTORC2signaling. While embryonic and early postnatal brain developmentappears grossly normal in these mice, there are deects in myelina-tion (39). These results suggest that mTORC1 signaling plays a
role in selective cellular adaptations but is not decisive or generalcellular viability (39).
4. Autophagy
5. DevelopmentalAspects o mTORSignaling
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TSC is an autosomal dominant disease caused by mutations ineither TSC1 or TSC2. TSC is characterized by the presence o benign tumors called hamartomas, which within the brain areknown as cortical tubers. Neurological maniestations in TSCpatients include epilepsy, mental retardation, and autistic eatures(40). In mice, Tsc2 haploin suciency causes aberrant retinogenic-ulate projections and the TSC2–Rheb–mTOR pathway controlsaxon guidance in the visual system (41). In addition, dorsal rootganglial neurons (DRGs) in the peripheral nervous system activatemTOR ollowing damage to enhance axonal growth capacity (42).Hence, the mTOR pathway has a central role in axon guidance,regeneration, and growth (43).
Human embryonic stem cells (hESCs), derived rom blastocyst-stageembryos, can undergo long-term sel-renewal and have the remark-able ability to dierentiate into multiple cell types in the humanbody (44). A role or mTOR in these processes has recently beenappreciated (45). mTOR integrates signals rom extrinsic pluripo-
tency-supporting actors and represses the transcriptional activitieso a subset o developmental and growth inhibitory genes in hESCs.Repression o the developmental genes by mTOR is necessary orthe maintenance o hESC pluripotency (46). A similar mechanismis operative in human amniotic fuid stem cells (47). On the otherhand, it has been proposed that mTOR-mediated activation o S6K1 induces dierentiation o pluripotent hESCs (48). In thatline, mTORC1 activation is detrimental to stem cell maintenancein spermatogonial progenitor cells (SPCs) (49, 50).
mTOR regulates the metabolism, growth and survival o b-cells,the cardinal cells in the pancreas that produce insulin, a hormonethat controls the level o glucose in the blood to regulate oodintake (51). Studies in S6K1 knockout mice demonstrate a centralpositive role o mTOR/S6K1 signaling in b-cell growth and unc-tion (51). Indeed, these mice develop glucose intolerance despite
6. Nerve Functionand Epilepsy
7. mTORand Pluripotencyin Stem Cells
8. Regulationo b-Cell Functionand Obesityby mTOR Signaling
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increased insulin sensitivity. This is associated with depletion o thepancreatic insulin content, hypoinsulinemia and reduced b-cellmass suggesting that lack o S6K1 activity impairs b-cell growthand unction (51). In addition, it has been shown that obesity develops in older hypothalamic Tsc1 knockout animals; however,
young animals display a prominent gain-o-unction b-cell pheno-type prior to the onset o obesity (52). Young hypothalamic Tsc1knockout animals display improved glycemic control due tomTOR-mediated enhancement o b-cell size and insulin production.Thus, mTOR disseminates a dominant signal to promote b cell/islet size and insulin production, and this pathway is crucial orb-cell unction and glycemic control (52).
The immune system is a complex network o cells that protectagainst disease by identiying and killing pathogens and tumorcells, but it is also implicated in homeostatic mechanisms like tissueremodeling and wound healing (53).
A growing body o evidence indicates that in myeloid phago-cytes (monocytes, macrophages, and myeloid DC; mDC) mTOR is crucially implicated in TLR signaling and might serve as a decisionmaker to control the cellular response to pathogens by modulating
cytokines, chemokines, and type I intereron responses (54, 55).Inhibition o mTOR by rapamycin in these cells promotes IL-12and IL-23 production via the transcription actor NF-k B but blocksthe release o IL-10 via Stat3 (7, 8, 56, 57). These results havebeen conrmed in kidney transplant patients in vivo. The mostprominent transcriptional alterations in peripheral blood romrapamycin-treated kidney transplant recipients aect the innateimmune cell compartment and hyperactivation o NF-k B-mediatedproinfammatory pathways (58). Moreover, kidney transplantpatients on rapamycin display an increased infammatory and
immunostimulatory potential o myeloid monocytes and dendriticcells in vivo compared with patients on calcineurin inhibitors(59, 60). Moreover, rapamycin can augment infammation andpulmonary injury by enhancing NF-k B activity in the lung o tobacco-exposed mice (61). In dendritic cells, autophagy acilitatesthe presentation o endogenous proteins on MHC class I and classII molecules. This leads to the activation o CD4+ T cells andconnects autophagy in innate immune cells with enhanced adaptiveimmune responses. For example, in Mycobacterium tuberculosis-inected DCs, rapamycin-induced autophagy enhances the presen-
tation o mycobacterial antigens (62).
9. MyeloidPhagocytesActivate mTORto LimitProinfammatoryResponses
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Plasmacytoid DCs (pDCs) constitute a specialized cell populationthat produce large amounts o type I intererons (IFN) in responseto viral inection via the activation o cytoplasmic receptors orTLRs (63). The mTOR pathway is important or the regulationo type I IFN production in murine pDCs (6, 64). Inhibition o mTOR or its downstream mediators S6K1 and S6K2 during pDCactivation block the phosphorylation and nuclear translocation o the transcription actor IRF-7, which results in impaired IFN-a and b production (6). In addition, translation o IRF-7 in pDCs isnegatively regulated by the 4E-BP pathway downstream o mTOR (65). Hence, mTOR via its two downstream eectors, 4E-BP andS6K, controls translation and activation o IRF-7.
Peripheral CD4+ T helper (Th) lymphocytes are critical in regulatingimmune responses as well as autoimmune and infammatory diseases.Upon activation, naïve CD4+ Th cells dierentiate into distincteector subsets depending on the cytokine milieu (66). Recentdata show that mTOR-decient naïve CD4+ T cells are unable to
dierentiate into Th1, Th2, and Th17 cells, but preerentially develop into induced regulatory T (Treg) cells, which can potently suppress adaptive immune responses (67). In line, rapamycin isable to enrich Treg cells in vitro (68). mTORC2 is important inthese processes as Rictor-decient CD4+ T cells are unable todierentiate into Th2 cells demonstrating that mTORC2 is criticalor Th2 dierentiation (69, 70). On the other hand, Rheb-decientT cells ail to generate Th17 responses in vitro and in vivo (67).The role o mTORC1 and mTORC2 or Th1 dierentiation iscurrently under debate.
Another insight how mTOR regulates adaptive immunity in vivocan be deduced rom recent experiments showing that mTOR infuences the migratory properties o murine CD8+ T lymphocytesand the dierentiation o CD8+ memory T cells (71, 72). Themigratory properties o naïve CD8+ T lymphocytes into the lymph
nodes crucially depends on the constitutive expression o thechemokine receptor 7 (CCR7) and L-selectin (CD62L), which iscontrolled by the transcription actor Krüppel-like actor 2 (KLF2).
10. mTORMediates Type IIntereronProduction in
PlasmacytoidDendritic Cells
11. CD4+ T HelperDierentiation andthe Role o mTOR
12. mTOR ControlsCD8+ T LymphocyteMigrationand Memory
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10 T. Weichhart
with TSC or sporadic lymphangioleiomyomatosis oten developbenign renal neoplasms called angiomyolipomas, which impair renalunction. In initial clinical trials, inhibition o mTOR showed somepromise in leading to a partial angiomyolipoma regression (13, 81).
Rapamycin was initially seen as a holy grail or cancer therapy; how-ever, the potency o rapamycin as an anticancer drug in clinical trials
was limited to the tumor types described above (3). The constrictedsuccess o rapamycin and the appreciation that mTORC1 has bothrapamycin-sensitive and rapamycin-insensitive substrates (82, 83) as
well as the nding that mTORC2 activation is largely unaected or
even increased by acute cellular exposure to rapamycin promotedthe development o novel active-site mTOR inhibitors that ully block both mTOR complexes. These novel ATP-competitive inhib-itors have improved anticancer activity compared to rapamycin in a
variety o solid tumor models in vitro and in vivo and at least threecandidate compounds have entered clinical trials (84–88). They show a consistently potent eect against tumors that are driven by PI3K–Akt; however, the clinical eectiveness o these novel mTOR inhibitors remain to be determined.
In the last years, there has been a tremendous amount o novel dataestablishing how the mTOR pathway is regulated by various envi-ronmental and intracellular molecules and how mTOR controlsmany dierent processes implicated in health and disease. Inhibitiono mTOR is currently established in allogeneic transplantation andin certain orms o cancer. Novel applications or mTOR inhibitors,
such as the generation o high number o Treg cells ex vivo orimmunotherapy or the improvement o vaccines by the promotiono memory CD8+ T-cell responses, are currently evaluated. Theidentication o novel signaling pathways important or the controlo mTOR in dierent tissues o the body may open urther clinicalapplications o inhibiting mTOR in human disease.
Acknowledgments
TW is supported by the Else-Kröner Fresenius Stitung. I apologizeto those authors whose primary work I did not reerence directly in the text.
15. NovelInhibitorsTargeting mTORC1and mTORC2
16. Conclusion
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