Caffeine targets TOR complex I and provides evidence for a regulatory link between the FRB and kinase domains of Tor1p

The target of rapamycin (TOR) kinase is an important regulator of growth in eukaryotic cells. In budding yeast, Tor1p and Tor2p function as part of two distinct protein complexes, TORC1 and TORC2, where TORC1 is specifically inhibited by the antibiotic rapamycin. Significant insight into TORC1 function has been obtained using rapamycin as a specific small molecule inhibitor of TOR activity. Here we show that caffeine acts as a distinct and novel small molecule inhibitor of TORC1: (i) deleting components specific to TORC1 but not TORC2 renders cells hypersensitive to caffeine; (ii) rapamycin and caffeine display remarkably similar effects on global gene expression; and (iii) mutations were isolated in Tor1p, a component specific to TORC1, that confers significant caffeine resistance both in vivo and in vitro. Strongest resistance requires two simultaneous mutations in TOR1, the first at either one of two highly conserved positions within the FRB (rapamycin binding) domain and a second at a highly conserved position within the ATP binding pocket of the kinase domain. Biochemical and genetic analyses of these mutant forms of Tor1p support a model wherein functional interactions between the FRB and kinase domains, as well as between the FRB domain and the TORC1 component Kog1p, regulate TOR activity as well as contribute to the mechanism of caffeine resistance.     

Phosphorylation of the TOR ATP binding domain by AGC kinase constitutes a novel mode of TOR inhibition

TOR (target of rapamycin) signaling coordinates cell growth, metabolism, and cell division through tight control of signaling via two complexes, TORC1 and TORC2. Here, we show that fission yeast TOR kinases and mTOR are phosphorylated on an evolutionarily conserved residue of their ATP-binding domain. The Gad8 kinase (AKT homologue) phosphorylates fission yeast Tor1 at this threonine (T1972) to reduce activity. A T1972A mutation that blocked phosphorylation increased Tor1 activity and stress resistance. Nitrogen starvation of fission yeast inhibited TOR signaling to arrest cell cycle progression in G1 phase and promoted sexual differentiation. Starvation and a Gad8/T1972-dependent decrease in Tor1 (TORC2) activity was essential for efficient cell cycle arrest and differentiation. Experiments in human cell lines recapitulated these yeast observations, as mTOR was phosphorylated on T2173 in an AKT-dependent manner. In addition, a T2173A mutation increased mTOR activity. Thus, TOR kinase activity can be reduced through AGC kinase–controlled phosphorylation to generate physiologically significant changes in TOR signaling.     

Two TOR complexes,only one of which is rapamycin sensitive,have distinct roles in cell growth control

The target of rapamycin (TOR) proteins in Saccharomyces cerevisiae, TOR1 and TOR2, redundantly regulate growth in a rapamycin-sensitive manner. TOR2 additionally regulates polarization of the actin cytoskeleton in a rapamycin-insensitive manner. We describe two functionally distinct TOR complexes. TOR Complex 1 (TORC1) contains TOR1 or TOR2, KOG1 (YHR186c), and LST8. TORC2 contains TOR2, AVO1 (YOL078w), AVO2 (YMR068w), AVO3 (YER093c), and LST8. FKBP-rapamycin binds TORC1, and TORC1 disruption mimics rapamycin treatment, suggesting that TORC1 mediates the rapamycin-sensitive, TOR-shared pathway. FKBP-rapamycin fails to bind TORC2, and TORC2 disruption causes an actin defect, suggesting that TORC2 mediates the rapamycin-insensitive, TOR2-unique pathway. Thus, the distinct TOR complexes account for the diversity, specificity, and selective rapamycin inhibition of TOR signaling. TORC1 and possibly TORC2 are conserved from yeast to man.     

TOR Signaling in Fission Yeast

Fission yeast has two TOR kinases, Tor1 and Tor2. Recent studies have indicated that this microbe has a TSC/Rheb/TOR pathway like higher eukaryotes. Two TOR complexes, namely TORC1 and TORC2, have been identified in this yeast, as in budding yeast and mammals. Fission yeast TORC1, which contains Tor2, and TORC2, which contains Tor1, apparently have opposite functions with regard to the promotion of G1 arrest and sexual development. Rapamycin does not inhibit growth of wild-type fission yeast cells, unlike other eukaryotic cells, but precise analyses have revealed that rapamycin affects certain cellular functions involving TOR in this yeast. It appears that fission yeast has a potential to be an ideal model system to investigate the TOR signaling pathways.     

TOR signaling in fission yeast

Fission yeast has two TOR kinases, Tor1 and Tor2. Recent studies have indicated that this microbe has a TSC/Rheb/TOR pathway like higher eukaryotes. Two TOR complexes, namely TORC1 and TORC2, have been identified in this yeast, as in budding yeast and mammals. Fission yeast TORC1, which contains Tor2, and TORC2, which contains Tor1, apparently have opposite functions with regard to the promotion of G1 arrest and sexual development. Rapamycin does not inhibit growth of wild-type fission yeast cells, unlike other eukaryotic cells, but precise analyses have revealed that rapamycin affects certain cellular functions involving TOR in this yeast. It appears that fission yeast has a potential to be an ideal model system to investigate the TOR signaling pathways.     

TOR signaling in growth and metabolism

The target of rapamycin (TOR) is a conserved Ser/Thr kinase that regulates cell growth and metabolism in response to environmental cues. Here, highlighting contributions from studies in model organisms, we review mammalian TOR complexes and the signaling branches they mediate. TOR is part of two distinct multiprotein complexes, TOR complex 1 (TORC1), which is sensitive to rapamycin, and TORC2, which is not. The physiological consequences of mammalian TORC1 dysregulation suggest that inhibitors of mammalian TOR may be useful in the treatment of cancer, cardiovascular disease, autoimmunity, and metabolic disorders.     

Inhibition of protein synthesis by TOR inactivation revealed a conserved regulatory mechanism of the BiP chaperone in Chlamydomonas

The target of rapamycin (TOR) kinase integrates nutritional and stress signals to coordinately control cell growth in all eukaryotes. TOR associates with highly conserved proteins to constitute two distinct signaling complexes termed TORC1 and TORC2. Inactivation of TORC1 by rapamycin negatively regulates protein synthesis in most eukaryotes. Here, we report that down-regulation of TOR signaling by rapamycin in the model green alga Chlamydomonas reinhardtii resulted in pronounced phosphorylation of the endoplasmic reticulum chaperone BiP. Our results indicated that Chlamydomonas TOR regulates BiP phosphorylation through the control of protein synthesis, since rapamycin and cycloheximide have similar effects on BiP modification and protein synthesis inhibition. Modification of BiP by phosphorylation was suppressed under conditions that require the chaperone activity of BiP, such as heat shock stress or tunicamycin treatment, which inhibits N-linked glycosylation of nascent proteins in the endoplasmic reticulum. A phosphopeptide localized in the substrate-binding domain of BiP was identified in Chlamydomonas cells treated with rapamycin. This peptide contains a highly conserved threonine residue that might regulate BiP function, as demonstrated by yeast functional assays. Thus, our study has revealed a regulatory mechanism of BiP in Chlamydomonas by phosphorylation/dephosphorylation events and assigns a role to the TOR pathway in the control of BiP modification.     

TORC2 Plasma Membrane Localization Is Essential for Cell Viability and Restricted to a Distinct Domain

The conserved target of rapamycin (TOR) kinases regulate many aspects of cellular physiology. They exist in two distinct complexes, termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2), that posses both overlapping and distinct components. TORC1 and TORC2 respond differently to the drug rapamycin and have different cellular functions: whereas the rapamycin-sensitive TORC1 controls many aspects of cell growth and has been characterized in great detail, the TOR complex 2 is less understood and regulates actin polymerization, cell polarity, and ceramide metabolism. How signaling specificity and discrimination between different input signals for the two kinase complexes is achieved is not understood. Here, we show that TORC1 and TORC2 have different localizations in Saccharomyces cerevisiae. TORC1 is localized exclusively to the vacuolar membrane, whereas TORC2 is localized dynamically in a previously unrecognized plasma membrane domain, which we term membrane compartment containing TORC2 (MCT). We find that plasma membrane localization of TORC2 is essential for viability and mediated by lipid binding of the C-terminal domain of the Avo1 subunit. From these data, we suggest that the TOR complexes are spatially separated to determine downstream signaling specificity and their responsiveness to different inputs.     

Complexity of the TOR signaling network

The target of rapamycin (TOR) is a serine/threonine kinase of the phosphatidylinositol kinase-related kinase family and is highly conserved from yeast to mammals. TOR functions as a central regulator of cell growth and is itself regulated by a wide range of signals, including growth factors, nutrients and stress conditions. Recent studies in eukaryotic cells have identified two distinct TOR complexes, TORC1 and TORC2, which phosphorylate different substrates and have distinct physiological functions. Here, we discuss new findings that have extended the complexity of TOR signaling and the different roles of the TORC complexes in yeast, flies and mammals.     

What controls TOR?

The target of rapamycin (TOR) is a protein kinase with numerous functions in cell growth control. Some of these functions can be potently inhibited by rapamycin, an immunosuppressive and potential anticancer drug. TOR exists as part of two functionally distinct protein complexes. The functions of TOR complex 1 (TORC1) are effectively inhibited by rapamycin, but the mechanism for this inhibition remains elusive. The identification of TORC2 and recent reports that rapamycin can inhibit TORC2 functions, in some cases, challenge current models of TOR regulation. This review discusses the latest findings in yeast and mammals on the possible mechanisms that control TOR activity leading to its many cellular functions     

Activation of mTORC2 by association with the ribosome

The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of growth. Mammalian TOR complex 2 (mTORC2) regulates AGC kinase family members and is implicated in various disorders, including cancer and diabetes. Here, we investigated the upstream regulation of mTORC2. A genetic screen in yeast and subsequent studies in mammalian cells revealed that ribosomes, but not protein synthesis, are required for mTORC2 signaling. Active mTORC2 was physically associated with the ribosome, and insulin-stimulated PI3K signaling promoted mTORC2-ribosome binding, suggesting that ribosomes activate mTORC2 directly. Findings with melanoma and colon cancer cells suggest that mTORC2-ribosome association is important in oncogenic PI3K signaling. Thus, TORC2-ribosome interaction is a likely conserved mechanism of TORC2 activation that is physiologically relevant in both normal and cancer cells. As ribosome content determines growth capacity of a cell, this mechanism of TORC2 regulation ensures that TORC2 is active only in growing cells.     

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.     

TORC2 signaling is antagonized by protein phosphatase 2A and the Far complex in Saccharomyces cerevisiae

The target of rapamycin (TOR) kinase, a central regulator of eukaryotic cell growth, exists in two essential, yet distinct, TOR kinase complexes in the budding yeast Saccharomyces cerevisiae: rapamycin-sensitive TORC1 and rapamycin-insensitive TORC2. Lst8, a component of both TOR complexes, is essential for cell viability. However, it is unclear whether the essential function of Lst8 is linked to TORC1, TORC2, or both. To that end, we carried out a genetic screen to isolate lst8 deletion suppressor mutants. Here we report that mutations in SAC7 and FAR11 suppress lethality of lst8Δ and TORC2-deficient (tor2-21) mutations but not TORC1 inactivation, suggesting that the essential function of Lst8 is linked only to TORC2. More importantly, characterization of lst8Δ bypass mutants reveals a role for protein phosphatase 2A (PP2A) in the regulation of TORC2 signaling. We show that Far11, a member of the Far3-7-8-9-10-11 complex involved in pheromone-induced cell cycle arrest, interacts with Tpd3 and Pph21, conserved components of PP2A, and deletions of components of the Far3-7-8-9-10-11 complex and PP2A rescue growth defects in lst8Δ and tor2-21 mutants. In addition, loss of the regulatory B' subunit of PP2A Rts1 or Far11 restores phosphorylation to the TORC2 substrate Slm1 in a tor2-21 mutant. Mammalian Far11 orthologs FAM40A/B exist in a complex with PP2A known as STRIPAK, suggesting a conserved functional association of PP2A and Far11. Antagonism of TORC2 signaling by PP2A-Far11 represents a novel regulatory mechanism for controlling spatial cell growth of yeast.     

TOR signaling is a determinant of cell survival in response to DNA damage

The conserved TOR (target of rapamycin) kinase is part of a TORC1 complex that regulates cellular responses to environmental stress, such as amino acid starvation and hypoxia. Dysregulation of Akt-TOR signaling has also been linked to the genesis of cancer, and thus, this pathway presents potential targets for cancer chemotherapeutics. Here we report that rapamycin-sensitive TORC1 signaling is required for the S-phase progression and viability of yeast cells in response to genotoxic stress. In the presence of the DNA-damaging agent methyl methanesulfonate (MMS), TOR-dependent cell survival required a functional S-phase checkpoint. Rapamycin inhibition of TORC1 signaling suppressed the Rad53 checkpoint-mediated induction of ribonucleotide reductase subunits Rnr1 and Rnr3, thereby abrogating MMS-induced mutagenesis and enhancing cell lethality. Moreover, cells deleted for RNR3 were hypersensitive to rapamycin plus MMS, providing the first demonstration that Rnr3 contributes to the survival of cells exposed to DNA damage. Our findings support a model whereby TORC1 acts as a survival pathway in response to genotoxic stress by maintaining the deoxynucleoside triphosphate pools necessary for error-prone translesion DNA polymerases. Thus, TOR-dependent cell survival in response to DNA-damaging agents coincides with increased mutation rates, which may contribute to the acquisition of chemotherapeutic drug resistance.     

TOR complex 1 includes a novel component, Tco89p (YPL180w), and cooperates with Ssd1p to maintain cellular integrity in Saccharomyces cerevisiae

The Tor1p and Tor2p kinases, targets of the therapeutically important antibiotic rapamycin, function as components of two distinct protein complexes in yeast, termed TOR complex 1 (TORC1) and TORC2. TORC1 is responsible for a wide range of rapamycin-sensitive cellular activities and contains, in addition to Tor1p or Tor2p, two highly conserved proteins, Lst8p and Kog1p. By identifying proteins that co-purify with Tor1p, Tor2p, Lst8p, and Kog1p, we have characterized a comprehensive set of protein-protein interactions that define further the composition of TORC1 as well as TORC2. In particular, we have identified Tco89p (YPL180w) and Bit61p (YJL058c) as novel components of TORC1 and TORC2, respectively. Deletion of TOR1 or TCO89 results in two specific and distinct phenotypes, (i) rapamycin-hypersensitivity and (ii) decreased cellular integrity, both of which correlate with the presence of SSD1-d, an allele of SSD1 previously associated with defects in cellular integrity. Furthermore, we link Ssd1p to Tap42p, a component of the TOR pathway that is believed to act uniquely downstream of TORC1. Together, these results define a novel connection between TORC1 and Ssd1p-mediated maintenance of cellular integrity.     

Ubiquitin regulates TORC1 in yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae the TOR complex 1 (TORC1) controls many growth‐related cellular processes and is essential for cell growth and proliferation. Macrolide antibiotic rapamycin, in complex with a cytosol protein named FKBP12, specifically inhibits TORC1, causing growth arrest. The FKBP12‐rapamycin complex interferes with TORC1 function by binding to the FRB domain of the TOR proteins. In an attempt to understand the role of the FRB domain in TOR function, we identified a single point mutation (Tor2^W2041R) in the FRB domain of Tor2 that renders yeast cells rapamycin resistant and temperature sensitive. At the permissive temperature, the Tor2 mutant protein is partially defective for binding with Kog1 and TORC1 is impaired for membrane association. At the restrictive temperature, Kog1 but not the Tor2 mutant protein, is rapidly degraded. Overexpression of ubiquitin stabilizes Kog1 and suppresses the growth defect associated with the tor2 mutant at the nonpremissive temperature. We find that ubiquitin binds non‐covalently to Kog1, prevents Kog1 from degradation and stabilizes TORC1. Our data reveal a unique role for ubiquitin in regulation of TORC1 and suggest that Kog1 requires association with the Tor proteins for stabilization.