Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive

The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of cell growth. In budding yeast, TOR is found in structurally and functionally distinct protein complexes: TORC1 and TORC2. A mammalian counterpart of TORC1 (mTORC1) has been described, but it is not known whether TORC2 is conserved in mammals. Here, we report that a mammalian counterpart of TORC2 (mTORC2) also exists. mTORC2 contains mTOR, mLST8 and mAVO3, but not raptor. Like yeast TORC2, mTORC2 is rapamycin insensitive and seems to function upstream of Rho GTPases to regulate the actin cytoskeleton. mTORC2 is not upstream of the mTORC1 effector S6K. Thus, two distinct TOR complexes constitute a primordial signalling network conserved in eukaryotic evolution to control the fundamental process of cell growth.     

mTORC2 targets AGC kinases through Sin1-dependent recruitment

The protein kinase TOR (target of rapamycin) is a key regulator of cell growth and metabolism with significant clinical relevance. In mammals, TOR signals through two distinct multi-protein complexes, mTORC1 and mTORC2 (mammalian TOR complex 1 and 2 respectively), the subunits of which appear to define the operational pathways. Rapamycin selectively targets mTORC1 function, and the emergence of specific ATP-competitive kinase inhibitors has enabled assessment of dual mTORC1 and mTORC2 blockade. Little is known, however, of the molecular action of mTORC2 components or the relative importance of targeting this pathway. In the present study, we have identified the mTORC2 subunit Sin1 as a direct binding partner of the PKC (protein kinase C) ε kinase domain and map the interaction to the central highly conserved region of Sin1. Exploiting the conformational dependence for PKC phosphorylation, we demonstrate that mTORC2 is essential for acute priming of PKC. Inducible expression of Sin1 mutants, lacking the PKC-interaction domain, displaces endogenous Sin1 from mTORC2 and disrupts PKC phosphorylation. PKB (protein kinase B)/Akt phosphorylation is also suppressed by these Sin1 mutants, but not the mTORC1 substrate p70(S6K) (S6 kinase), providing evidence that Sin1 serves as a selectivity adaptor for the recruitment of mTORC2 targets. This inducible selective mTORC2 intervention is used to demonstrate a key role for mTORC2 in cell proliferation in three-dimensional culture.     

Nutrient-dependent multimerization of the mammalian target of rapamycin through the N-terminal HEAT repeat region

The mammalian target of rapamycin (mTOR) plays a pivotal role in the regulation of cell growth in response to a variety of signals such as nutrients and growth factors. mTOR forms two distinct complexes in vivo. mTORC1 (mTOR complex 1) is rapamycin-sensitive and regulates the rate of protein synthesis in part by phosphorylating two well established effectors, S6K1 (p70 ribosomal S6 kinase 1) and 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1). mTORC2 is rapamycin-insensitive and likely regulates actin organization and activates Akt/protein kinase B. Here, we show that mTOR forms a multimer via its N-terminal HEAT repeat region in mammalian cells. mTOR multimerization is promoted by amino acid sufficiency, although the state of multimerization does not directly correlate with the phosphorylation state of S6K1. mTOR multimerization was insensitive to rapamycin treatment but hindered by butanol treatment, which inhibits phosphatidic acid production by phospholipase D. We also found that mTOR forms a multimer in both mTORC1 and mTORC2. In addition, Saccharomyces cerevisiae TOR proteins Tor1p and Tor2p also exist as homomultimers. These results suggest that TOR multimerization is a conserved mechanism for TOR functioning.     

Tsc tumour suppressor proteins antagonize amino-acid-TOR signalling

Target of Rapamycin (TOR) mediates a signalling pathway that couples amino acid availability to S6 kinase (S6K) activation, translational initiation and cell growth. Here, we show that tuberous sclerosis 1 (Tsc1) and Tsc2, tumour suppressors that are responsible for the tuberous sclerosis syndrome, antagonize this amino acid-TOR signalling pathway. We show that Tsc1 and Tsc2 can physically associate with TOR and function upstream of TOR genetically. In Drosophila melanogaster and mammalian cells, loss of Tsc1 and Tsc2 results in a TOR-dependent increase of S6K activity. Furthermore, although S6K is normally inactivated in animal cells in response to amino acid starvation, loss of Tsc1-Tsc2 renders cells resistant to amino acid starvation. We propose that the Tsc1-Tsc2 complex antagonizes the TOR-mediated response to amino acid availability. Our studies identify Tsc1 and Tsc2 as regulators of the amino acid-TOR pathway and provide a new paradigm for how proteins involved in nutrient sensing function as tumour suppressors.     

A MAP4 kinase related to Ste20 is a nutrient-sensitive regulator of mTOR signalling

The mTOR (mammalian target of rapamycin) signalling pathway is a key regulator of cell growth and is controlled by growth factors and nutrients such as amino acids. Although signalling pathways from growth factor receptors to mTOR have been elucidated, the pathways mediating signalling by nutrients are poorly characterized. Through a screen for protein kinases active in the mTOR signalling pathway in Drosophila we have identified a Ste20 family member (MAP4K3) that is required for maximal S6K (S6 kinase)/4E-BP1 [eIF4E (eukaryotic initiation factor 4E)-binding protein 1] phosphorylation and regulates cell growth. Importantly, MAP4K3 activity is regulated by amino acids, but not the growth factor insulin and is not regulated by the mTORC1 inhibitor rapamycin. Our results therefore suggest a model whereby nutrients signal to mTORC1 via activation of MAP4K3.     

mTOR Signalling in Health and Disease

The TOR (target of rapamycin) proteins are found in all eukaryotes. TOR has a protein kinase domain, as well as other domains through which it interacts with partner proteins to form at least two types of multiprotein complex, TORC1 and TORC2 (TOR complexes 1 and 2). Rapamycin, an antibiotic and immunosuppressant, inhibits functions of TORC1. Use of this drug has revealed roles for TORC1 and its mammalian counterpart, mTORC1, in promoting many anabolic processes. mTORC1 signalling is activated by growth factors and nutrients. It is highly active in many cancers and plays a role in tumorigenesis and in other diseases. Much less is known so far about the functions and regulation of (m)TORC2. The goal of this meeting was to bring together researchers studying the roles of mTORC1/2 in normal cell and animal physiology in diverse systems, as well as scientists exploring the therapeutic value of inhibiting mTOR (mammalian TOR) signalling.     

Signal integration in the (m)TORC1 growth pathway

Background

The protein kinase Target Of Rapamycin (TOR) is a nexus for the regulation of eukaryotic cell growth. TOR assembles into one of two distinct signalling complexes, TOR complex 1 (TORC1) and TORC2 (mTORC1/2 in mammals), with a set of largely non-overlapping protein partners. (m)TORC1 activation occurs in response to a series of stimuli relevant to cell growth, including nutrient availability, growth factor signals and stress, and regulates much of the cell’s biosynthetic activity, from proteins to lipids, and recycling through autophagy. mTORC1 regulation is of great therapeutic significance, since in humans many of these signalling complexes, alongside subunits of mTORC1 itself, are implicated in a wide variety of pathophysiologies, including multiple types of cancer, neurological disorders, neurodegenerative diseases and metabolic disorders including diabetes.

Methodology

Recent years have seen numerous structures determined of (m)TOR, which have provided mechanistic insight into (m)TORC1 activation in particular, however the integration of cellular signals occurs upstream of the kinase and remains incompletely understood. Here we have collected and analysed in detail as many as possible of the molecular and structural studies which have shed light on (m)TORC1 repression, activation and signal integration.

Conclusions

A molecular understanding of this signal integration pathway is required to understand how (m)TORC1 activation is reconciled with the many diverse and contradictory stimuli affecting cell growth. We discuss the current level of molecular understanding of the upstream components of the (m)TORC1 signalling pathway, recent progress on this key biochemical frontier, and the future studies necessary to establish a mechanistic understanding of this master-switch for eukaryotic cell growth.

     

PRAS40 and PRR5-like protein are new mTOR interactors that regulate apoptosis

TOR (Target of Rapamycin) is a highly conserved protein kinase and a central controller of cell growth. TOR is found in two functionally and structurally distinct multiprotein complexes termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). In the present study, we developed a two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) based proteomic strategy to identify new mammalian TOR (mTOR) binding proteins. We report the identification of Proline-rich Akt substrate (PRAS40) and the hypothetical protein Q6MZQ0/FLJ14213/CAE45978 as new mTOR binding proteins. PRAS40 binds mTORC1 via Raptor, and is an mTOR phosphorylation substrate. PRAS40 inhibits mTORC1 autophosphorylation and mTORC1 kinase activity toward eIF-4E binding protein (4E-BP) and PRAS40 itself. HeLa cells in which PRAS40 was knocked down were protected against induction of apoptosis by TNFalpha and cycloheximide. Rapamycin failed to mimic the pro-apoptotic effect of PRAS40, suggesting that PRAS40 mediates apoptosis independently of its inhibitory effect on mTORC1. Q6MZQ0 is structurally similar to proline rich protein 5 (PRR5) and was therefore named PRR5-Like (PRR5L). PRR5L binds specifically to mTORC2, via Rictor and/or SIN1. Unlike other mTORC2 members, PRR5L is not required for mTORC2 integrity or kinase activity, but dissociates from mTORC2 upon knock down of tuberous sclerosis complex 1 (TSC1) and TSC2. Hyperactivation of mTOR by TSC1/2 knock down enhanced apoptosis whereas PRR5L knock down reduced apoptosis. PRR5L knock down reduced apoptosis also in mTORC2 deficient cells. The above suggests that mTORC2-dissociated PRR5L may promote apoptosis when mTOR is hyperactive. Thus, PRAS40 and PRR5L are novel mTOR-associated proteins that control the balance between cell growth and cell death.     

Regulation of neurogenesis and epidermal growth factor receptor signaling by the insulin receptor/target of rapamycin pathway in Drosophila

Determining how growth and differentiation are coordinated is key to understanding normal development, as well as disease states such as cancer, where that control is lost. We have previously shown that growth and neuronal differentiation are coordinated by the insulin receptor/target of rapamycin (TOR) kinase (InR/TOR) pathway. Here we show that the control of growth and differentiation diverge downstream of TOR. TOR regulates growth by controlling the activity of S6 kinase (S6K) and eIF4E. Loss of s6k delays differentiation, and is epistatic to the loss of tsc2, indicating that S6K acts downstream or in parallel to TOR in differentiation as in growth. However, loss of eIF4E inhibits growth but does not affect the timing of differentiation. We also show, for the first time in Drosophila, that there is crosstalk between the InR/TOR pathway and epidermal growth factor receptor (EGFR) signaling. InR/TOR signaling regulates the expression of several EGFR pathway components including pointedP2 (pntP2). In addition, reduction of EGFR signaling levels phenocopies inhibition of the InR/TOR pathway in the regulation of differentiation. Together these data suggest that InR/TOR signaling regulates the timing of differentiation through modulation of EGFR target genes in developing photoreceptors.     

The TOR nutrient signalling pathway phosphorylates NPR1 and inhibits turnover of the tryptophan permease.

The Saccharomyces cerevisiae targets of rapamycin, TOR1 and TOR2, signal activation of cell growth in response to nutrient availability. Loss of TOR or rapamycin treatment causes yeast cells to arrest growth in early G1 and to express several other physiological properties of starved (G0) cells. As part of this starvation response, high affinity amino acid permeases such as the tryptophan permease TAT2 are targeted to the vacuole and degraded. Here we show that the TOR signalling pathway phosphorylates the Ser/Thr kinase NPR1 and thereby inhibits the starvation-induced turnover of TAT2. Overexpression of NPR1 inhibits growth and induces the degradation of TAT2, whereas loss of NPR1 confers resistance to rapamycin and to FK506, an inhibitor of amino acid import. NPR1 is controlled by TOR and the type 2A phosphatase-associated protein TAP42. First, overexpression of NPR1 is toxic only when TOR function is reduced. Secondly, NPR1 is rapidly dephosphorylated in the absence of TOR. Thirdly, NPR1 dephosphorylation does not occur in a rapamycin-resistant tap42 mutant. Thus, the TOR nutrient signalling pathway also controls growth by inhibiting a stationary phase (G0) programme. The control of NPR1 by TOR is analogous to the control of p70 s6 kinase and 4E-BP1 by mTOR in mammalian cells.     

Rheb is an essential regulator of S6K in controlling cell growth in Drosophila

Understanding the mechanisms through which multicellular organisms regulate cell, organ and body growth is of relevance to developmental biology and to research on growth-related diseases such as cancer. Here we describe a new effector in growth control, the small GTPase Rheb (Ras homologue enriched in brain). Mutations in the Drosophila melanogaster Rheb gene were isolated as growth-inhibitors, whereas overexpression of Rheb promoted cell growth. Our genetic and biochemical analyses suggest that Rheb functions downstream of the tumour suppressors Tsc1 (tuberous sclerosis 1)-Tsc2 in the TOR (target of rapamycin) signalling pathway to control growth, and that a major effector of Rheb function is ribosomal S6 kinase (S6K).     

TOR signalling regulates mitotic commitment through the stress MAP kinase pathway and the Polo and Cdc2 kinases

The coupling of growth to cell cycle progression allows eukaryotic cells to divide at particular sizes depending on nutrient availability. In fission yeast, this coupling involves the Spc1/Sty1 mitogen-activated protein kinase (MAPK) pathway working through Polo kinase recruitment to the spindle pole bodies (SPBs). Here we report that changes in nutrients influence TOR signalling, which modulates Spc1/Sty1 activity. Rapamycin-induced inhibition of TOR signalling advanced mitotic onset, mimicking the reduction in cell size at division seen after shifts to poor nitrogen sources. Gcn2, an effector of TOR signalling and modulator of translation, regulates the Pyp2 phosphatase that in turn modulates Spc1/Sty1 activity. Rapamycin- or nutrient-induced stimulation of Spc1/Sty1 activity promotes Polo kinase SPB recruitment and Cdc2 activation to advance mitotic onset. This advanced mitotic onset is abolished in cells depleted of Gcn2, Pyp2, or Spc1/Sty1 or on blockage of Spc1/Sty1-dependent Polo SPB recruitment. Therefore, TOR signalling modulates mitotic onset through the stress MAPK pathway via the Pyp2 phosphatase.     

Arabidopsis TARGET OF RAPAMYCIN interacts with RAPTOR, which regulates the activity of S6 kinase in response to osmotic stress signals

TARGET OF RAPAMYCIN (TOR) kinase controls many cellular functions in eukaryotic cells in response to stress and nutrient availability and was shown to be essential for embryonic development in Arabidopsis thaliana. We demonstrated that Arabidopsis RAPTOR1 (a TOR regulatory protein) interacts with the HEAT repeats of TOR and that RAPTOR1 regulates the activity of S6 kinase (S6K) in response to osmotic stress. RAPTOR1 also interacts in vivo with Arabidopsis S6K1, a putative substrate for TOR. S6K1 fused to green fluorescent protein and immunoprecipitated from tobacco (Nicotiana tabacum) leaves after transient expression was active in phosphorylating the Arabidopsis ribosomal S6 protein. The catalytic domain of S6K1 could be phosphorylated by Arabidopsis 3-phosphoinositide-dependent protein kinase-1 (PDK1), indicating the involvement of PDK1 in the regulation of S6K. The S6K1 activity was sensitive to osmotic stress, while PDK1 activity was not affected. However, S6K1 sensitivity to osmotic stress was relieved by co-overexpression of RAPTOR1. Overall, these observations demonstrated the existence of a functional TOR kinase pathway in plants. However, Arabidopsis seedlings do not respond to normal physiological levels of rapamycin, which appears to be due its inability to bind to the Arabidopsis homolog of FKBP12, a protein that is essential for the binding of rapamycin with TOR. Replacement of the Arabidopsis FKBP12 with the human FKBP12 allowed rapamycin-dependent interaction with TOR. Since homozygous mutation in TOR is lethal, it suggests that this pathway is essential for integrating the stress signals into the growth regulation.     

TOR and S6K1 promote translation reinitiation of uORF‐containing mRNAs via phosphorylation of eIF3h

Mammalian target‐of‐rapamycin (mTOR) triggers S6 kinase (S6K) activation to phosphorylate targets linked to translation in response to energy, nutrients, and hormones. Pathways of TOR activation in plants remain unknown. Here, we uncover the role of the phytohormone auxin in TOR signalling activation and reinitiation after upstream open reading frame (uORF) translation, which in plants is dependent on translation initiation factor eIF3h. We show that auxin triggers TOR activation followed by S6K1 phosphorylation at T449 and efficient loading of uORF‐mRNAs onto polysomes in a manner sensitive to the TOR inhibitor Torin‐1. Torin‐1 mediates recruitment of inactive S6K1 to polysomes, while auxin triggers S6K1 dissociation and recruitment of activated TOR instead. A putative target of TOR/S6K1—eIF3h—is phosphorylated and detected in polysomes in response to auxin. In TOR‐deficient plants, polysomes were prebound by inactive S6K1, and loading of uORF‐mRNAs and eIF3h was impaired. Transient expression of eIF3h‐S178D in plant protoplasts specifically upregulates uORF‐mRNA translation. We propose that TOR functions in polysomes to maintain the active S6K1 (and thus eIF3h) phosphorylation status that is critical for translation reinitiation.     

PAT-related amino acid transporters regulate growth via a novel mechanism that does not require bulk transport of amino acids

Growth in normal and tumour cells is regulated by evolutionarily conserved extracellular inputs from the endocrine insulin receptor (InR) signalling pathway and by local nutrients. Both signals modulate activity of the intracellular TOR kinase, with nutrients at least partly acting through changes in intracellular amino acid levels mediated by amino acid transporters. We show that in Drosophila, two molecules related to mammalian proton-assisted SLC36 amino acid transporters (PATs), CG3424 and CG1139, are potent mediators of growth. These transporters genetically interact with TOR and other InR signalling components, indicating that they control growth by directly or indirectly modulating the effects of TOR signalling. A mutation in the CG3424 gene, which we have named pathetic (path), reduces growth in the fly. In a heterologous Xenopus oocyte system, PATH also activates the TOR target S6 kinase in an amino acid-dependent way. However, functional analysis reveals that PATH has an extremely low capacity and an exceptionally high affinity compared with characterised human PATs and the CG1139 transporter. PATH and potentially other PAT-related transporters must therefore control growth via a mechanism that does not require bulk transport of amino acids into the cell. As PATH is likely to be saturated in vivo, we propose that one specialised function of high-affinity PAT-related molecules is to maintain growth as local nutrient levels fluctuate during development.     

PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase

The heterotrimeric mTORC1 protein kinase nucleates a signaling network that promotes cell growth in response to insulin and becomes constitutively active in cells missing the TSC1 or TSC2 tumor suppressors. Insulin stimulates the phosphorylation of S6K1, an mTORC1 substrate, but it is not known how mTORC1 kinase activity is regulated. We identify PRAS40 as a raptor-interacting protein that binds to mTORC1 in insulin-deprived cells and whose in vitro interaction with mTORC1 is disrupted by high salt concentrations. PRAS40 inhibits cell growth, S6K1 phosphorylation, and rheb-induced activation of the mTORC1 pathway, and in vitro it prevents the great increase in mTORC1 kinase activity induced by rheb1-GTP. Insulin stimulates Akt/PKB-mediated phosphorylation of PRAS40, which prevents its inhibition of mTORC1 in cells and in vitro. We propose that the relative strengths of the rheb- and PRAS40-mediated inputs to mTORC1 set overall pathway activity and that insulin activates mTORC1 through the coordinated regulation of both.     

Signaling by target of rapamycin proteins in cell growth control.

Target of rapamycin (TOR) proteins are members of the phosphatidylinositol kinase-related kinase (PIKK) family and are highly conserved from yeast to mammals. TOR proteins integrate signals from growth factors, nutrients, stress, and cellular energy levels to control cell growth. The ribosomal S6 kinase 1 (S6K) and eukaryotic initiation factor 4E binding protein 1(4EBP1) are two cellular targets of TOR kinase activity and are known to mediate TOR function in translational control in mammalian cells. However, the precise molecular mechanism of TOR regulation is not completely understood. One of the recent breakthrough studies in TOR signaling resulted in the identification of the tuberous sclerosis complex gene products, TSC1 and TSC2, as negative regulators for TOR signaling. Furthermore, the discovery that the small GTPase Rheb is a direct downstream target of TSC1-TSC2 and a positive regulator of the TOR function has significantly advanced our understanding of the molecular mechanism of TOR activation. Here we review the current understanding of the regulation of TOR signaling and discuss its function as a signaling nexus to control cell growth during normal development and tumorigenesis.     

Simultaneous inhibition of mTORC1 and mTORC2 by mTOR kinase inhibitor AZD8055 induces autophagy and cell death in cancer cells

mTOR is a major biological switch, coordinating an adequate response to changes in energy uptake (amino acids, glucose), growth signals (hormones, growth factors) and environmental stress. mTOR kinase is highly conserved through evolution from yeast to man and in both cases, controls autophagy and cellular translation in response to nutrient stress. mTOR kinase is the catalytic component of two distinct multiprotein complexes called mTORC1 and mTORC2. In addition to mTOR, mTORC1 contains Raptor, mLST8 and PRAS40. mTORC2 contains mTOR, Rictor, mSIN1 and Protor-1. mTORC1 activates p70S6K, which in turn phosphorylates the ribosomal protein S6 and 4E-BP1, both involved in protein translation. mTORC2 activates AKT directly by phosphorylating Serine 473. pAKT(S473) phosphorylates TSC2 (tuberin) and inactivates it, preventing its association with TSC1 (hamartin) and the inhibition of Rheb, an activator of mTOR. pAKT also phosphorylates PRAS40, releasing it from the mTORC1 complex, increasing its kinase activity. Finally, AKT regulates FOXO3 phosphorylation, sequestering it in the cytosol in an inactive state.