Glucose Activates TORC2-Gad8 Protein via Positive Regulation of the cAMP/cAMP-dependent Protein Kinase A (PKA) Pathway and Negative Regulation of the Pmk1 Protein-Mitogen-activated Protein Kinase Pathway

The target of rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol 3-kinase-related kinases. TOR proteins are found at the core of two evolutionary conserved complexes, known as TORC1 and TORC2. In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. TORC2 has been implicated in a wide variety of functions; however, the signals that regulate TORC2 activity have so far remained obscure. TORC2 has one known direct substrate, the AGC kinase Gad8, which is related to AKT in human cells. Gad8 is phosphorylated by TORC2 at Ser-546 (equivalent to AKT Ser-473), leading to its activation. Here, we show that glucose is necessary and sufficient to induce Gad8 Ser-546 phosphorylation in vivo and Gad8 kinase activity in vitro. The glucose signal that activates TORC2-Gad8 is mediated via the cAMP/PKA pathway, a major glucose-sensing pathway. By contrast, Pmk1, similar to human extracellular signal-regulated kinases and a major stress-induced mitogen activated protein kinase (MAPK) in fission yeast, inhibits TORC2-dependent Gad8 phosphorylation and activation. Inhibition of TORC2-Gad8 also occurs in response to ionic or osmotic stress, in a manner dependent on the cAMP/PKA and Pmk1-MAPK signaling pathways. Our findings highlight the significance of glucose availability in regulation of TORC2-Gad8 and indicate a novel link between the cAMP/PKA, Pmk1/MAPK, and TORC2-Gad8 signaling.     

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.     

The mammalian target of rapamycin complex 2 controls folding and stability of Akt and protein kinase C

The target of rapamycin (TOR), as part of the rapamycin-sensitive TOR complex 1 (TORC1), regulates various aspects of protein synthesis. Whether TOR functions in this process as part of TORC2 remains to be elucidated. Here, we demonstrate that mTOR, SIN1 and rictor, components of mammalian (m)TORC2, are required for phosphorylation of Akt and conventional protein kinase C (PKC) at the turn motif (TM) site. This TORC2 function is growth factor independent and conserved from yeast to mammals. TM site phosphorylation facilitates carboxyl-terminal folding and stabilizes newly synthesized Akt and PKC by interacting with conserved basic residues in the kinase domain. Without TM site phosphorylation, Akt becomes protected by the molecular chaperone Hsp90 from ubiquitination-mediated proteasome degradation. Finally, we demonstrate that mTORC2 independently controls the Akt TM and HM sites in vivo and can directly phosphorylate both sites in vitro. Our studies uncover a novel function of the TOR pathway in regulating protein folding and stability, processes that are most likely linked to the functions of TOR in protein synthesis.     

Phosphorylation of Wat1, human Lst8 homolog is critical for the regulation of TORC2 –Gad8 dependent pathway in fission yeast Schizosacchromyces pombe

Mammalian Lst8 interacts with the kinase domain of mTOR and stabilizes its interaction with Raptor regulating cell growth through the mTOR-S6K1 signalling pathway. Fission yeast Wat1, an ortholog of mammalian Lst8 is also an essential component of TOR complex 1 (TORC1) and TOR Complex 2 (TORC2) that control protein kinases essential for metabolic pathways. Here, we show that in response to osmotic stress, the Wat1 protein undergoes hyper-phosphorylation at S116 position. Wat1 interacts with the C-terminal region of Tor1 that also contain kinase domain. Co-immunoprecipitation and molecular modelling studies suggest that Wat1-Tor1 interaction is stabilized by FATC domain of Tor1 protein present at the C-terminal region. We have also demonstrated a physical interaction of Wat1 with Gad8, an AGC family protein kinase that is dependent on phosphorylation of Wat1 at S116 residue. Wat1 phosphorylation is required for the maintenance of vacuolar integrity and sexual differentiation. Collectively, our study reveals Wat1 phosphorylation regulates Gad8 function in a manner dependent on Tor1 interaction.     

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.     

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.     

Structure of TOR and its complex with KOG1

The target of rapamycin (TOR) is a large (281 kDa) conserved Ser/Thr protein kinase that functions as a central controller of cell growth. TOR assembles into two distinct multiprotein complexes: TORC1 and TORC2. A defining feature of TORC1 is the interaction of TOR with KOG1 (Raptor in mammals) and its sensitivity to a rapamycin-FKBP12 complex. Here, we have reconstructed in three dimensions the 25 A resolution structures of endogenous budding yeast TOR1 and a TOR-KOG1 complex, using electron microscopy. TOR features distinctive N-terminal HEAT repeats that form a curved tubular-shaped domain that associates with the C-terminal WD40 repeat domain of KOG1. The N terminus of KOG1 is in proximity to the TOR kinase domain, likely functioning to bring substrates into the vicinity of the catalytic region. A model is proposed for the molecular architecture of the TOR-KOG1 complex explaining its sensitivity to rapamycin.     

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.     

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.     

Target of rapamycin complex 2 signals to downstream effector yeast protein kinase 2 (Ypk2) through adheres-voraciously-to-target-of-rapamycin-2 protein 1 (Avo1) in Saccharomyces cerevisiae

The conserved Ser/Thr kinase target of rapamycin (TOR) serves as a central regulator in controlling cell growth-related functions. There exist two distinct TOR complexes, TORC1 and TORC2, each coupling to specific downstream effectors and signaling pathways. In Saccharomyces cerevisiae, TORC2 is involved in regulating actin organization and maintaining cell wall integrity. Ypk2 (yeast protein kinase 2), a member of the cAMP-dependent, cGMP-dependent, and PKC (AGC) kinase family, is a TORC2 substrate known to participate in actin and cell wall regulation. Employing avo3(ts) mutants with defects in TORC2 functions that are suppressible by active Ypk2, we investigated the molecular interactions involved in mediating TORC2 signaling to Ypk2. GST pulldown assays in yeast lysates demonstrated physical interactions between Ypk2 and components of TORC2. In vitro binding assays revealed that Avo1 directly binds to Ypk2. In avo3(ts) mutants, the TORC2-Ypk2 interaction was reduced and could be restored by AVO1 overexpression, highlighting the important role of Avo1 in coupling TORC2 to Ypk2. The interaction was mapped to an internal region (amino acids 600-840) of Avo1 and a C-terminal region of Ypk2. Ypk2(334-677), a truncated form of Ypk2 containing the Avo1-interacting region, was able to interfere with Avo1-Ypk2 interaction in vitro. Overexpressing Ypk2(334-677) in yeast cells resulted in a perturbation of TORC2 functions, causing defective cell wall integrity, aberrant actin organization, and diminished TORC2-dependent Ypk2 phosphorylation evidenced by the loss of an electrophoretic mobility shift. Together, our data support the conclusion that the direct Avo1-Ypk2 interaction is crucial for TORC2 signaling to the downstream Ypk2 pathway.     

Sch9 is a major target of TORC1 in Saccharomyces cerevisiae

The Target of Rapamycin (TOR) protein is a Ser/Thr kinase that functions in two distinct multiprotein complexes: TORC1 and TORC2. These conserved complexes regulate many different aspects of cell growth in response to intracellular and extracellular cues. Here we report that the AGC kinase Sch9 is a substrate of yeast TORC1. Six amino acids in the C terminus of Sch9 are directly phosphorylated by TORC1. Phosphorylation of these residues is lost upon rapamycin treatment as well as carbon or nitrogen starvation and transiently reduced following application of osmotic, oxidative, or thermal stress. TORC1-dependent phosphorylation is required for Sch9 activity, and replacement of residues phosphorylated by TORC1 with Asp/Glu renders Sch9 activity TORC1 independent. Sch9 is required for TORC1 to properly regulate ribosome biogenesis, translation initiation, and entry into G0 phase, but not expression of Gln3-dependent genes. Our results suggest that Sch9 functions analogously to the mammalian TORC1 substrate S6K1 rather than the mTORC2 substrate PKB/Akt.     

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.     

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.     

Growth control via TOR kinase signaling,an intracellular sensor of amino acid and energy availability,with crosstalk potential to proline metabolism

The TOR (Target of Rapamycin) protein kinase pathway plays a central role in sensing and responding to nutrients, stress, and intracellular energy state. TOR complex 1 (TORC1) is comprised of TOR, Raptor, and Lst8 and its activity is sensitive to inhibition by the macrolide antibiotic rapamycin. TORC1 regulates protein synthesis, ribosome biogenesis, autophagy, and ultimately cell growth through the phosphorylation of S6 K, 4E-BP, and other substrates. As TORC1 activity is positively or negatively modulated in response to upstream regulators, cellular growth rate is, respectively, enhanced or suppressed. A separate multiprotein TOR complex, TORC2, is insensitive to direct inhibition by rapamycin and does not regulate growth patterns directly; TORC2 can, however, impact certain aspects of TORC1 signaling and cell survival. TOR signaling is an ancient pathway, conserved among the yeasts, Dictyostelium, C. elegans, Drosophila, mammals, and Arabidopsis. This review will focus on the regulation of TORC1 in mammalian cells in the context of amino acid sensing/regulation and intracellular ATP homeostasis, but will also include comparisons among other organisms.     

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.     

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