The mammalian target of rapamycin (mTOR) is a Ser/Thr kinase structurally and functionally conserved from yeast to humans that positively regulates cell growth, proliferation, and survival, while inhibition of mTOR signaling extends lifespan (Harrison et al. 2009). In eukaryotes, mTOR is ubiquitously expressed and whole-organism knockout has demonstrated that it is essential for cell growth and viability. mTOR forms two multiprotein complexes, namely, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). The macrolide rapamycin acutely inhibits mTORC1 but not mTORC2. mTORC1 and mTORC2 regulate functionally distinct, yet partially overlapping, signaling networks that collectively control the spatial and temporal regulation of cell growth.
Nutrients and growth factors activate mTORC1, whereas low cellular energy levels or stress inhibits mTORC1. Under favorable conditions for growth, mTORC1 is activated to promote translation, ribosome biogenesis, and inhibit autophagy. Thus, mTORC1 regulates when a cell grows. mTORC2 is activated by growth factors to promote cell survival and actin cytoskeleton remodeling in yeast, dictyostelium, and mammals. Thus, in broad terms, mTORC2 regulates where a cell grows.
As primary regulators of cell growth, the mTORC1 and mTORC2 signaling networks function as key regulatory nodes whose functions are important for development, aging, and have been linked to cancer and metabolic disorders. These signaling networks will be discussed in detail below:
mTORC2 is composed of mTOR, Rictor, Sin1, mLST8, and PRR5/PRR5L (Protor 1/Protor 2) (reviewed in Cybulski and Hall 2009). PRR5 is not required for the interaction between mTOR, Rictor, Sin1, and mLST8. Deletion of Rictor or Sin1 is embryonic lethal, as mice die after day E10–E10.5. Loss of Sin1 disrupts Rictor’s association with mTORC2. Interestingly, ablation of Rictor or Sin1 results in loss of Akt/PKB hydrophobic motif (HM) phosphorylation (at Ser473), yet the phosphorylation of several Akt/PKB targets is unaffected. This suggests either that phosphorylation of Ser473 may alter substrate specificity, or that in the absence of active Akt/PKB, other AGC kinases such as SGK, S6K, or RSK can phosphorylate a subset of Akt/PKB substrates. Surprisingly, although mLST8 is a component of both mTORC1 and mTORC2, mLST8 knockout mice die at day E10.5, phenotypically resembling Rictor−/− mice. Furthermore, mLST8 is required for Akt/PKB HM phosphorylation, but not S6K signaling. These data suggest that mLST8 has an essential role only in mTORC2.
In mice, tissue-specific knockouts of mTOR or mTORC1/mTORC2 components have demonstrated that mTOR plays a key role in regulating muscle atrophy, glucose metabolism, and lipid metabolism (reviewed in Polak and Hall 2009). mTORC1 and mTORC2 signal via distinct pathways to control a wide variety of cellular processes. These mTOR-regulated processes mediate the accumulation of cellular mass and, thereby, ultimately determine cell size.
The mTORC1 Signaling Network
In contrast to growth factors that inhibit TSC1/2, low cellular energy and stress activate TSC1/2 GTPase-activator protein (GAP) activity. Low energy (high AMP:ATP ratio) activates AMP-activated protein kinase (AMPK) to phosphorylate TSC2. This action inhibits mTORC1 by increasing TSC2 GAP activity toward Rheb, a Ras-like GTPase. In addition to phosphorylating TSC2, AMPK also inhibits mTORC1 by directly phosphorylating Raptor. Mutations of the AMPK upstream activating kinase, LKB1, result in hyperactive mTORC1 signaling, thereby linking LKB1 to the TSC1/2 mTORC1 pathway. Hypoxia inhibits mTORC1 signaling through the HIF1-mediated upregulation of two homologous proteins REDD1 and REDD2 (Regulated in Development and DNA damage response genes 1 and 2) REDD acts to activate TSC1/2, independently of LKB1-AMPK, in order to inhibit mTORC1. The stress and energy signaling pathways are likely to be further associated, as prolonged hypoxia leads to ATP depletion and activation of AMPK.
Inactivation of TSC1/2 then allows GTP-bound Rheb to activate mTORC1. However, amino acids are required for proper mTORC1 localization to a late endosome/lysosome compartment containing active Rheb. In contrast to growth factor stimulation, amino-acid activation of mTORC1 occurs independently of TSC1/2, as amino acid withdrawal also downregulates mTORC1 signaling in TSC2-deficient cells. Activation of mTORC1 by amino acids requires the Ras-like GTPases Rheb and Rag, both of which bind directly to mTORC1. The Rag GTPase heterodimer (RagA or RagB binding to RagC or RagD) mediates the localization of mTORC1 to a late endosomal/lysosomal compartment containing Rheb. Rag heterodimers are recruited to lysosomes via an aptly named protein complex, Ragulator, which is composed of the genes encoded by the MAPKSP1, ROBLD3, and c11orf59 (Sancak et al. 2010). Ragulator-to-Rag-to-mTORC1 binding then facilitates the activation of mTORC1 by GTP-bound Rheb. hVps34 has also been implicated in the amino-acid stimulation of mTORC1 in a TSC1/2-independent fashion. The hVps34 product phosphatidylinositol-3-phosphate (PI(3)P) may help recruit mTORC1 through an unidentified scaffold protein. However, in flies, Vps34 functions downstream of TOR. How amino acids GTP-load, and thereby activate the Rag GTPases to ultimately activate mTORC1 remains to be determined. Intriguingly, MAP4K3 acts upstream of Rag-mediated mTORC1 activation (Yan et al. 2010). Amino acids stimulate MAP4K3 activity to phosphorylate an unknown substrate upstream of the Rag/Ragulator complex, whereas under amino acid–starved conditions, PP2A T61 epsilon inhibits MAP4K3 and subsequent mTORC1 activation. Upon activation, mTORC1 controls protein synthesis, promotes ribosome biogenesis, and inhibits autophagy.
Downstream effectors of mTORC1: Translation: mTORC1 activates cap-dependent translation initiation and elongation by phosphorylating the eukaryotic initiation factor 4E (eIF4E)–binding protein 1 (4E-BP1) and the p70 ribosomal S6 kinase (S6K). The phosphorylation of 4E-BP1 prevents its binding to eIF4E, enabling eIF4E to then associate with eIF4G to stimulate translation initiation, whereas phosphorylation of S6K by mTORC1 and phosphoinositide-dependent kinase 1 (PDK1) is required for complete activation. Activated S6K promotes translation initiation by phosphorylating eIF4B, programmed cell death protein 4 (PDCD4), and eEF2 kinase (eEF2K). Phosphorylation of eIF4B and PDCD4 activates translation initiation, whereas phosphorylation of eEF2K upregulates translation elongation. S6K also promotes the translation efficiency of spliced mRNAs via S6K-Aly/REF-like substrate (SKAR) (Ma et al. 2008). SKAR associates with mRNAs in a splicing-dependent manner, where it then recruits activated S6K and thereby preferentially enhances translation of spliced mRNAs. Additionally, S6K phosphorylates 40S ribosomal protein S6, but the significance of this phosphorylation is unknown. mTORC1 associates with its substrates 4E-BP1 and S6K through Raptor and a TOR signaling (TOS) motif in 4E-BP1 and S6K. The TOS motif is a conserved five-amino-acid sequence (FEMDI and FDIDL in the C terminus of 4E-BP1 and in the N terminus of S6K, respectively) that is necessary for the phosphorylation of these proteins by mTORC1. However, TOS motifs have not been identified in recently discovered substrates, suggesting mTORC1 may bind to different substrates through distinct regions or alternative scaffold proteins.
Ribosome biogenesis: mTORC1 promotes the synthesis of ribosomes and transfer RNAs (tRNAs). Rapamycin blocks the biosynthesis of ribosomes by inhibiting transcription of RNA polymerase I (Pol I)–dependent rRNA genes, Pol II–dependent ribosomal protein genes (RP genes), and Pol III–dependent tRNA genes. mTOR controls Pol I via the essential transcription initiation factor TIF-1A (Transcriptional Intermediary Factor 1A). Rapamycin treatment leads to TIF-1A inactivation and thus impairs formation of the transcription initiation complex. Furthermore, TIF1A translocates from the nucleus to the cytoplasm upon rapamycin-mediated mTORC1 inactivation. In yeast, the forkhead-like transcription factor FHL1 functions as a TOR-dependent regulator of Pol II-dependent RP gene expression. mTORC1 regulates Pol III-mediated gene expression by directly phosphorylating and inhibiting MAF1, a Pol III transcriptional repressor. mTORC1 associates with TFIIIC, is recruited to Pol III-transcribed genes, and relieves MAF1-mediated repression, thus allowing Pol III transcription to occur (Michels et al. 2010).
Autophagy: mTORC1 negatively regulates macroautophagy, a starvation-induced catabolic process where bulk cytoplasm is enclosed in a double membrane structure and delivered to the vacuole for degradation. Rapamycin induces autophagy in yeast and human tissue culture, demonstrating the conserved role of mTORC1 as an inhibitor of autophagy. mTORC1 directly inhibits autophagy by phosphorylating and repressing a conserved protein complex composed of unc-51-like kinase 1 (ULK1), autophagy-related gene 13 (ATG13), and focal adhesion kinase family–interacting protein with a molecular weight of 200 kDa (FIP200) (Jung et al. 2009). mTORC1 also directly phosphorylates and represses DAP1 (Koren et al. 2010). Unlike the ULK1/ATG13/FIP200 complex, DAP1 is a negative regulator of autophagy. Thus via positive and negative regulation, mTORC1 activity tightly controls the absolute level of cellular autophagy. Additionally, dysregulation of mTORC1 may contribute to cancer cell survival, as tumor cells may temporarily activate autophagy to overcome nutrient deprivation under poor growth conditions. Through the mechanisms described above, mTORC1 regulates temporal cell growth.
The mTORC2 Signaling Network
Downstream effectors of mTORC2: Unlike mTORC1, which can be specifically inhibited by rapamycin, mTORC2 is insensitive to acute rapamycin treatment. Thus, many of the downstream effectors and physiological functions of mTORC2 remain unknown.
Cell survival: mTORC2-deficient cells are sensitive to stress-induced apoptosis. mTORC2 promotes cell survival through the activation of Akt/PKB and serum and glucocorticoid-inducible kinase (SGK), two AGC kinases that have both distinct and overlapping substrates. Akt/PKB negatively regulates the pro-apoptotic protein BAD, while SGK regulates the phosphorylation of NDRG1, and both kinases negatively regulate FOXO. Interestingly, mTORC2 activity is required for prostrate tumorigenesis in PTEN null tumors, and not for normal prostate function (Guertin et al. 2009). This suggests that inhibiting mTORC2-mediated cell survival would be attractive cancer therapeutic target with few side effects to healthy tissue.
Cytoskeleton remodeling: mTORC2 regulates actin cytoskeleton remodeling in yeast and is important for chemotaxis and cell migration in dictyostelium and mammals respectively (Charest et al. 2010). Furthermore, loss of mTORC2 alters actin polymerization and perturbs cell morphology. Thus, mTORC2 regulates where cell growth occurs. mTORC2-dependent cytoskeleton remodeling is likely mediated by the mTORC2 effectors PKCα, Paxillin, and Rac1, although the molecular mechanism by which mTORC2 regulates all these processes has not been determined.
AGC kinase activation and stability: mTORC1 and mTORC2 activate many members of the AGC kinase family (reviewed in Jacinto and Lorberg 2008). The AGC kinase activated by mTORC1 includes S6K, while mTORC2 regulates Akt/PKB, SGK, and PKCα. mTOR phosphorylates the hydrophobic motif (HM) in the AGC kinases to stimulate kinase activity. In S6K and SGK, phosphorylation of the HM creates a docking site for PDK1 and subsequent phosphorylation of the AGC kinase activation loop (reviewed in Pearce et al. 2010). mTOR also phosphorylates the turn motif (TM), at least in Akt/PKB and PKCα, to control stability of the kinase. In yeast, TOR phosphorylates and activates the AGC kinases Gad8, Ypk2, and Sch9. Future studies may reveal additional AGC kinases that are regulated by mTOR.
Open Questions and Summary
Expressional regulation of mTORC1: mTOR is classically thought to be ubiquitously expressed in eukaryotes. However, emerging research suggests mTORC1 may also be regulated at the level of expression. mTOR belongs to a subgroup of the atypical protein kinases, called [phosphoinositide 3-kinase]-related kinases (PIKK), which include Ataxia telangiectasia mutated (ATM); ATM and Rad3 related (ATR); DNA-dependent protein kinase (DNA-PK); suppressor with morphological effect on genitalia 1 (SMG1); and transformation/transcription domain–associated protein (TRRAP). All members of the PIKK family, including mTOR, interact with Tel2. These interactions positively regulate PIKK protein stability, but the physiological conditions under which Tel2 might regulate mTOR remain to be defined. Another recently identified regulator of mTOR protein stability is the tumor suppressor FBXW7. FBXW7 physically associates with mTOR and targets it for ubiquitination and degradation. Loss of FBXW7 increases the total amount of mTOR and subsequently the phosphorylation of S6K, indicating that FBXW7 is upstream of mTORC1. Depletion of FBXW7 did not affect the phosphorylation of Akt/PKB, suggesting that FBXW7 is not upstream of mTORC2. Further research is required to identify the physiological conditions when mTORC1 may be expressionally regulated.
Pharmacological inhibition of mTORC1 and mTORC2: Acute treatment with rapamycin, which forms a complex with FKBP12, binds to and allosterically inhibits mTORC1 but not mTORC2. In some cell lines, prolonged treatment with rapamycin (>24 h) indirectly inhibits mTORC2. Recently, several groups have independently developed active-site inhibitors of mTOR, which include PP242, Torin1, Ku-0063794, WAY-354, and AZD8055 (reviewed in Sparks and Guertin 2010). These compounds exhibit IC50 values in the low nanomolar range against mTORC1 and mTORC2. Furthermore, PP242 inhibited cancer cell proliferation to a greater extent than rapamycin (Janes et al. 2010). The increased antiproliferative affects of PP242 over rapamycin could be caused by inhibition of mTORC1 and mTORC2, rapamycin-resistant functions of mTORC1, or inhibition of an off-target kinase. mTOR active-site inhibitors will likely play an important role in the identification of novel mTOR functions and possibly as future cancer therapeutics.
mTORC1 and mTORC2 consensus motif: mTOR, as part of mTORC1 or mTORC2, can canonically phosphorylate two distinct target sites, a serine or threonine flanked by bulky hydrophobic (Φ) residues (Φ-pSer/Thr-Φ) and serine or threonine followed by a proline (pSer/Thr-Pro). Recent research in yeast and human cells to define the mTOR-regulated phosphoproteome revealed that mTORC1 can also phosphorylate serine followed by glutamine target sites (pSer-Gln) such as those in MAF1 (Huber et al. 2009 and Shor et al. 2010). Identification of additional mTORC1 and mTORC2 substrates may help to clarify the true mTOR consensus motif. Alternatively, mTOR may function as a promiscuous protein kinase to phosphorylate a wide variety of consensuses motifs. Under the latter model, a specific subunit of mTORC1 or mTORC2 would function as a scaffold protein to regulate substrate specificity.
Closing remarks: The mTORC1 and mTORC2 signaling networks have emerged as central controllers of cell growth that are important for development, aging, and diseases such as cancer and diabetes. The revelation that mTORC1 and mTORC2 regulate functionally distinct, yet partially overlapping signaling networks to collectively control the spatial and temporal cell growth has been a major conceptual advancement. Finally, the synthesis of mTORC1 and mTORC2 active-site inhibitors have the potential to be promising new therapeutic options to target mTOR dysregulation in cancer and metabolic disorders.
I offer my regrets to our colleagues whose excellent work I could not described due to space limitations. I acknowledge the Werner-Siemens Foundation for funding support and M.N. Hall for advice and the general figure template.
- Robitaille AM, Hall MN. mTOR. UCSD-Nature Molecule Pages. 2008. http://www.signaling-gateway.org/molecule/query?afcsid=A000094. Accessed 15 Oct 2008.