The FKBP-rapamycin binding domain of human TOR undergoes strong conformational changes in the presence of membrane mimetics with and without the regulator phosphatidic acid

The Ser/Thr kinase target of rapamycin (TOR) is a central controller of cellular growth and metabolism. Misregulation of TOR signaling is involved in metabolic and neurological disorders and tumor formation. TOR can be inhibited by association of a complex of rapamycin and FKBP12 to the FKBP12-rapamycin binding (FRB) domain. This domain was further proposed to interact with phosphatidic acid (PA), a lipid second messenger present in cellular membranes. Because mammalian TOR has been localized at various cellular membranes and in the nucleus, the output of TOR signaling may depend on its localization, which is expected to be influenced by the interaction with complex partners and regulators in response to cellular signals. Here, we present a detailed characterization of the interaction of the FRB domain with PA and how it is influenced by the surrounding membrane environment. On the basis of nuclear magnetic resonance- and circular dichroism-monitored binding studies using different neutral and negatively charged lipids as well as different membrane mimetics (micelles, bicelles, and liposomes), the FRB domain may function as a conditional peripheral membrane protein. However, the data for the isolated domain just indicate an increased affinity for negatively charged lipids and membrane patches but no specific preference for PA or PA-enriched regions. The membrane-mimetic environment induces strong conformational changes that largely maintain the α-helical secondary structure content but presumably disperse the helices in the lipidic environment. Consistent with overlapping binding surfaces for different lipids and the FKBP12-rapamycin complex, binding of the inhibitor complex protects the FRB domain from interactions with membrane mimetics at lower lipid concentrations.     

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.     

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.     

The FRB domain of mTOR: NMR solution structure and inhibitor design

The mammalian target of rapamycin (mTOR) is a protein that is intricately involved in signaling pathways controlling cell growth. Rapamycin is a natural product that binds and inhibits mTOR function by interacting with its FKBP-rapamycin-binding (FRB) domain. Here we report on the NMR solution structure of FRB and on further studies aimed at the identification and characterization of novel ligands that target the rapamycin binding pocket. The biological activity of the ligands, and that of rapamycin in the absence of FKBP12, was investigated by assaying the kinase activity of mTOR. While we found that rapamycin binds the FRB domain and inhibits the kinase activity of mTOR even in the absence of FKBP12 (in the low micromolar range), our most potent ligands bind to FRB with similar binding affinity but inhibit the kinase activity of mTOR at much higher concentrations. However, we have also identified one low-affinity compound that is also capable of inhibiting mTOR. Hence, we have identified compounds that can directly mimic rapamycin or can dissociate the FRB binding from the inhibition of the catalytic activity of mTOR. As such, these ligands could be useful in deciphering the complex regulation of mTOR in the cell and in validating the FRB domain as a possible target for the development of novel therapeutic compounds.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> FKBP12 binds <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> TOR and its expression in plants leads to rapamycin susceptibility

Background  

The eukaryotic TOR pathway controls translation, growth and the cell cycle in response to environmental signals such as nutrients or growth-stimulating factors. The TOR protein kinase can be inactivated by the antibiotic rapamycin following the formation of a ternary complex between TOR, rapamycin and FKBP12 proteins. The TOR protein is also found in higher plants despite the fact that they are rapamycin insensitive. Previous findings using the yeast two hybrid system suggest that the FKBP12 plant homolog is unable to form a complex with rapamycin and TOR, while the FRB domain of plant TOR is still able to bind to heterologous FKBP12 in the presence of rapamycin. The resistance to rapamycin is therefore limiting the molecular dissection of the TOR pathway in higher plants.     

FRB domain of human TOR protein induces compromised proliferation and mitochondrial dysfunction in Leishmaniadonovani promastigotes

Visceral leishmaniasis (VL) or Kala-azar, the second-largest parasitic killer worldwide, is caused by Leishmania donovani. The drugs to treat VL are toxic and expensive. Moreover, their indiscriminate use gave rise to resistant strains. The high rate of parasite proliferation within the host macrophage cells causes pathogenesis. In the proliferative pathway, FRB domain of TOR protein is ubiquitously essential. Although orthologues of mTOR protein are reported in trypanosomatids and Leishmania but therein depth molecular characterization is yet to be done. Considerable protein sequence homology exists between the TOR of kinetoplastidas and mammals. Interestingly, exogenous human FRB domain was shown to block G1 to S transition in mammalian cancer cells. Thus, we hypothesized that expression of human FRB domain would inhibit the proliferation of Leishmaniadonovani. Indeed, promastigotes stably expressing wild type human FRB domain show 4.7 and 1.5 folds less intra- and extra-cellular proliferations than that of untransfected controls. They also manifested 2.65 times lower rate of glucose stimulated oxygen consumption. The activities of all respiratory complexes were compromised in the hFRB expressing promastigotes. In these cells, depolarized mitochondria were 2-fold more than control cells. However, promastigotes expressing its mutant version (Trp²⁰²⁷-Phe) has shown similar characteristics like untransfected cells. Thus, this study reveals greater insights on the conserved role of TOR in the regulation of the respiratory complexes in L. donovani. The slow growing variant of FRB expressing promastigotes will have great potential to be exploited as a prophylactic agent against leishmaniasis.     

The fission yeast TOR homolog, tor1+, is required for the response to starvation and other stresses via a conserved serine

Targets of rapamycin (TORs) are conserved phosphatidylinositol kinase-related kinases that are involved in the coordination between nutritional or mitogenic signals and cell growth. Here we report the initial characterization of two Schizosaccharomyces pombe TOR homologs, tor1(+) and tor2(+). tor2(+) is an essential gene, whereas tor1(+) is required only under starvation and other stress conditions. Specifically, Deltator1 cells fail to enter stationary phase or undergo sexual development and are sensitive to cold, osmotic stress, and oxidative stress. In complex with the prolyl isomerase FKBP12, the drug rapamycin binds a conserved domain in TORs, FRB, thus inhibiting some of the functions of TORs. Mutations at a conserved serine within the FRB domain of Saccharomyces cerevisiae TOR proteins led to rapamycin resistance but did not otherwise affect the functions of the proteins. The S. pombe tor1(+) exhibits different features; substitution of the conserved serine residue, Ser(1834), with arginine compromises its functions and has no effect on the inhibition that rapamycin exerts on sexual development in S. pombe.     

The rapamycin-binding domain of the protein kinase mammalian target of rapamycin is a destabilizing domain

Rapamycin is an immunosuppressive drug that binds simultaneously to the 12-kDa FK506- and rapamycin-binding protein (FKBP12, or FKBP) and the FKBP-rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR) kinase. The resulting ternary complex has been used to conditionally perturb protein function, and one such method involves perturbation of a protein of interest through its mislocalization. We synthesized two rapamycin derivatives that possess large substituents at the C-16 position within the FRB-binding interface, and these derivatives were screened against a library of FRB mutants using a three-hybrid assay in Saccharomyces cerevisiae. Several FRB mutants responded to one of the rapamycin derivatives, and twenty of these mutants were further characterized in mammalian cells. The mutants most responsive to the ligand were fused to yellow fluorescent protein, and fluorescence levels in the presence and absence of the ligand were measured to determine stability of the fusion proteins. Wild-type and mutant FRB domains were expressed at low levels in the absence of the rapamycin derivative, and expression levels rose up to 10-fold upon treatment with ligand. The synthetic rapamycin derivatives were further analyzed using quantitative mass spectrometry, and one of the compounds was found to contain contaminating rapamycin. Furthermore, uncontaminated analogs retained the ability to inhibit mTOR, although with diminished potency relative to rapamycin. The ligand-dependent stability displayed by wild-type FRB and FRB mutants as well as the inhibitory potential and purity of the rapamycin derivatives should be considered as potentially confounding experimental variables when using these systems.     

Pharmacological characterization of purified recombinant mTOR FRB-kinase domain using fluorescence-based assays

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in nutrient sensing and cell growth and is a validated target for oncology and immunosuppression. Two modes of direct small-molecule inhibition of mTOR activity are known: targeting of the kinase active site and a unique mode in which the small molecule rapamycin, in complex with FKBP12 (the 12-kDa FK506 binding protein), binds to the FRB (FKBP12/rapamycin binding) domain of mTOR and inhibits kinase activity through a poorly defined mechanism. To facilitate the study of these processes, the authors have expressed and purified a truncated version of mTOR that contains the FRB and kinase domains and have developed homogeneous fluorescence-based assays to study mTOR activity. They demonstrate the utility of these assays in studies of active site-directed and FRB domain-directed mTOR inhibition. The results suggest that these assays can replace traditional radiometric or Western blot-based assays.     

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     

Molecular Docking studies of FKBP12-mTOR inhibitors using binding predictions

Mammalian target of rapamycin (mTOR) is a key regulator of cell growth, proliferation and angiogenesis. mTOR signaling is frequently hyper activated in a broad spectrum of human cancers thereby making it a potential drug target. The current drugs available have been successful in inhibiting the mTOR signaling, nevertheless, show low oral bioavailability and suboptimal solubility. Considering the narrow therapeutic window of the available inhibitors, through computational approaches, the present study pursues to identify a compound with optimal oral bioavailability and better solubility properties in addition ensuing high affinity between FKBP12 and FRB domain of mTOR. Current mTOR inhibitors; Everolimus, Temsirolimus Deforolimus and Echinomycin served as parent molecules for similarity search with a threshold of 95%. The query molecules and respective similar molecules were docked at the binding cleft of FKBP12 protein. Aided by MolDock algorithm, high affinity compounds against FKBP12 were retrieved. Patch Dock supervised protein-protein interactions were established between FRB domain of mTOR and ligand (query and similar) bound and free states of FKBP12. All the similar compounds thus retrieved showed better solubility properties and enabled better complex formation of mTOR and FKBP12. In particular Everolimus similar compound PubChem ID: 57284959 showed appreciable drugs like properties bestowed with better solubility higher oral bioavailability. In addition this compound brought about enhanced interaction between FKBP12 and FRB domain of mTOR. In the study, we report Everolimus similar compound PubChem ID: 57284959 to be potential inhibitor for mTOR pathway which can overcome the affinity and solubility concerns of current mTOR drugs.

Abbreviations

mTOR - Mammalian Target of Rapamycin, FRB domain - FKBP12-rapamycin associated protein, FKBP12 - FK506-binding protein 12, OPLS - Optimized Potentials for Liquid Simulations, Akt - RAC-alpha serine/threonine-protein kinase, PI3K - phosphatidylinositide 3-kinases.     

Elucidating TOR signaling and rapamycin action: lessons from Saccharomyces cerevisiae.

TOR (target of rapamycin) is a phosphatidylinositol kinase-related protein kinase that controls cell growth in response to nutrients. Rapamycin is an immunosuppressive and anticancer drug that acts by inhibiting TOR. The modes of action of TOR and rapamycin are remarkably conserved from S. cerevisiae to humans. The current understanding of TOR and rapamycin is derived largely from studies with S. cerevisiae. In this review, we discuss the contributions made by S. cerevisiae to understanding rapamycin action and TOR function.     

Raptor, a binding partner of target of rapamycin

The mammalian target of rapamycin (mTOR) controls cell growth in response to amino acids and growth factors, in part by regulating p70 S6 kinase alpha (p70 alpha) and eukaryotic initiation factor 4E binding protein 1 (4EBP1). Raptor (regulatory associated protein of mTOR) is a 150 kDa mTOR binding protein that is essential for TOR signaling in vivo and also binds 4EBP1 and p70alpha through their respective TOS (TOR signaling) motifs, a short conserved segment previously shown to be required for amino acid- and mTOR-dependent regulation of these substrates in vivo. Raptor appears to serve as an mTOR scaffold protein, the binding of which to the TOS motif of mTOR substrates is necessary for effective mTOR-catalyzed phosphorylation. Further understanding of regulation of the mTOR-raptor complex in response to the nutritional environment would require identification of the interplay between the mTOR-raptor complex and its upstream effectors such as the protein products of tumor suppressor gene tuberous sclerosis complexes 1 and 2, and the Ras-related small G protein Rheb.     

Elucidating TOR Signaling and Rapamycin Action: Lessons from Saccharomyces cerevisiae

TOR (target of rapamycin) is a phosphatidylinositol kinase-related protein kinase that controls cell growth in response to nutrients. Rapamycin is an immunosuppressive and anticancer drug that acts by inhibiting TOR. The modes of action of TOR and rapamycin are remarkably conserved from S. cerevisiae to humans. The current understanding of TOR and rapamycin is derived largely from studies with S. cerevisiae. In this review, we discuss the contributions made by S. cerevisiae to understanding rapamycin action and TOR function.     

基于雷帕霉素与FRB结合的蛋白质间相互作用的相关技术

FKBP12-rapamycin复合物的结合位点(FKBP1 2-rapamycin binding,FRB)为雷帕霉素(rapamycin,RAP)与哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)结合的结构城,基于RAP介导FK506结合蛋白12(12 kD FK506-binging protein,FKBP12)与FRB蛋白质相互作用相关研究技术的发展与应用,使人们对小分子介导的蛋白质相互作用有了更多的认识.就研究RAP作用于FRB域及研究FKBP12-RAP-FRB三元复合物形成的相关技术作一综述,为确认新的mTOR抑制剂的作用机制及认识其他蛋白质间相互作用提供参考.     

Inhibition of target of rapamycin signaling by rapamycin in the unicellular green alga Chlamydomonas reinhardtii

The macrolide rapamycin specifically binds the 12-kD FK506-binding protein (FKBP12), and this complex potently inhibits the target of rapamycin (TOR) kinase. The identification of TOR in Arabidopsis (Arabidopsis thaliana) revealed that TOR is conserved in photosynthetic eukaryotes. However, research on TOR signaling in plants has been hampered by the natural resistance of plants to rapamycin. Here, we report TOR inactivation by rapamycin treatment in a photosynthetic organism. We identified and characterized TOR and FKBP12 homologs in the unicellular green alga Chlamydomonas reinhardtii. Whereas growth of wild-type Chlamydomonas cells is sensitive to rapamycin, cells lacking FKBP12 are fully resistant to the drug, indicating that this protein mediates rapamycin action to inhibit cell growth. Unlike its plant homolog, Chlamydomonas FKBP12 exhibits high affinity to rapamycin in vivo, which was increased by mutation of conserved residues in the drug-binding pocket. Furthermore, pull-down assays demonstrated that TOR binds FKBP12 in the presence of rapamycin. Finally, rapamycin treatment resulted in a pronounced increase of vacuole size that resembled autophagic-like processes. Thus, our findings suggest that Chlamydomonas cell growth is positively controlled by a conserved TOR kinase and establish this unicellular alga as a useful model system for studying TOR signaling in photosynthetic eukaryotes.     

Protein Kinase Activity and Identification of a Toxic Effector Domain of the Target of Rapamycin TOR Proteins in Yeast

In complex with FKBP12, the immunosuppressant rapamycin binds to and inhibits the yeast TOR1 and TOR2 proteins and the mammalian homologue mTOR/FRAP/RAFT1. The TOR proteins promote cell cycle progression in yeast and human cells by regulating translation and polarization of the actin cytoskeleton. A C-terminal domain of the TOR proteins shares identity with protein and lipid kinases, but only one substrate (PHAS-I), and no regulators of the TOR-signaling cascade have been identified. We report here that yeast TOR1 has an intrinsic protein kinase activity capable of phosphorylating PHAS-1, and this activity is abolished by an active site mutation and inhibited by FKBP12-rapamycin or wortmannin. We find that an intact TOR1 kinase domain is essential for TOR1 functions in yeast. Overexpression of a TOR1 kinase-inactive mutant, or of a central region of the TOR proteins distinct from the FRB and kinase domains, was toxic in yeast, and overexpression of wild-type TOR1 suppressed this toxic effect. Expression of the TOR-toxic domain leads to a G1 cell cycle arrest, consistent with an inhibition of TOR function in translation. Overexpression of the PLC1 gene, which encodes the yeast phospholipase C homologue, suppressed growth inhibition by the TOR-toxic domains. In conclusion, our findings identify a toxic effector domain of the TOR proteins that may interact with substrates or regulators of the TOR kinase cascade and that shares sequence identity with other PIK family members, including ATR, Rad3, Mei-41, and ATM.     

TOR regulation of AGC kinases in yeast and mammals

The TOR (target of rapamycin), an atypical protein kinase, is evolutionarily conserved from yeast to man. Pharmacological studies using rapamycin to inhibit TOR and yeast genetic studies have provided key insights on the function of TOR in growth regulation. One of the first bona fide cellular targets of TOR was the mammalian protein kinase p70 S6K (p70 S6 kinase), a member of a family of kinases called AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases, which include PKA (cAMP-dependent protein kinase A), PKG (cGMP-dependent kinase) and PKC (protein kinase C). AGC kinases are also highly conserved and play a myriad of roles in cellular growth, proliferation and survival. The AGC kinases are regulated by a common scheme that involves phosphorylation of the kinase activation loop by PDK1 (phosphoinositide-dependent kinase 1), and phosphorylation at one or more sites at the C-terminal tail. The identification of two distinct TOR protein complexes, TORC1 (TOR complex 1) and TORC2, with different sensitivities to rapamycin, revealed that TOR, as part of either complex, can mediate phosphorylation at the C-terminal tail for optimal activation of a number of AGC kinases. Together, these studies elucidated that a fundamental function of TOR conserved throughout evolution may be to balance growth versus survival signals by regulating AGC kinases in response to nutrients and environmental conditions. This present review highlights this emerging function of TOR that is conserved from budding and fission yeast to mammals.     

Rapamycin and glucose-target of rapamycin (TOR) protein signaling in plants

Target of rapamycin (TOR) kinase is an evolutionarily conserved master regulator that integrates energy, nutrients, growth factors, and stress signals to promote survival and growth in all eukaryotes. The reported land plant resistance to rapamycin and the embryo lethality of the Arabidopsis tor mutants have hindered functional dissection of TOR signaling in plants. We developed sensitive cellular and seedling assays to monitor endogenous Arabidopsis TOR activity based on its conserved S6 kinase (S6K) phosphorylation. Surprisingly, rapamycin effectively inhibits Arabidopsis TOR-S6K1 signaling and retards glucose-mediated root and leaf growth, mimicking estradiol-inducible tor mutants. Rapamycin inhibition is relieved in transgenic plants deficient in Arabidopsis FK506-binding protein 12 (FKP12), whereas FKP12 overexpression dramatically enhances rapamycin sensitivity. The role of Arabidopsis FKP12 is highly specific as overexpression of seven closely related FKP proteins fails to increase rapamycin sensitivity. Rapamycin exerts TOR inhibition by inducing direct interaction between the TOR-FRB (FKP-rapamycin binding) domain and FKP12 in plant cells. We suggest that variable endogenous FKP12 protein levels may underlie the molecular explanation for longstanding enigmatic observations on inconsistent rapamycin resistance in plants and in various mammalian cell lines or diverse animal cell types. Integrative analyses with rapamycin and conditional tor and fkp12 mutants also reveal a central role of glucose-TOR signaling in root hair formation. Our studies demonstrate the power of chemical genetic approaches in the discovery of previously unknown and pivotal functions of glucose-TOR signaling in governing the growth of cotyledons, true leaves, petioles, and primary and secondary roots and root hairs.