TOR1 and TOR2 are structurally and functionally similar but not identical phosphatidylinositol kinase homologues in yeast.

The Saccharomyces cerevisiae genes TOR1 and TOR2 were originally identified by mutations that confer resistance to the immunosuppressant rapamycin. TOR2 was previously shown to encode an essential 282-kDa phosphatidylinositol kinase (PI kinase) homologue. The TOR1 gene product is also a large (281 kDa) PI kinase homologue, with 67% identity to TOR2. TOR1 is not essential, but a TOR1 TOR2 double disruption uniquely confers a cell cycle (G1) arrest as does exposure to rapamycin; disruption of TOR2 alone is lethal but does not cause a cell cycle arrest. TOR1-TOR2 and TOR2-TOR1 hybrids indicate that carboxy-terminal domains of TOR1 and TOR2 containing a lipid kinase sequence motif are interchangeable and therefore functionally equivalent; the other portions of TOR1 and TOR2 are not interchangeable. The TOR1-1 and TOR2-1 mutations, which confer rapamycin resistance, alter the same potential protein kinase C site in the respective protein's lipid kinase domain. Thus, TOR1 and TOR2 are likely similar but not identical, rapamycin-sensitive PI kinases possibly regulated by phosphorylation. TOR1 and TOR2 may be components of a novel signal transduction pathway controlling progression through G1.     

TOR1 and TOR2 have distinct locations in live cells

TOR is a structurally and functionally conserved Ser/Thr kinase found in two multiprotein complexes that regulate many cellular processes to control cell growth. Although extensively studied, the localization of TOR is still ambiguous, possibly because endogenous TOR in live cells has not been examined. Here, we examined the localization of green fluorescent protein (GFP) tagged, endogenous TOR1 and TOR2 in live S. cerevisiae cells. A DNA cassette encoding three copies of green fluorescent protein (3XGFP) was inserted in the TOR1 gene (at codon D330) or the TOR2 gene (at codon N321). The TORs were tagged internally because TOR1 or TOR2 tagged at the N or C terminus was not functional. The TOR1^D330-3XGFP strain was not hypersensitive to rapamycin, was not cold sensitive, and was not resistant to manganese toxicity caused by the loss of Pmr1, all indications that TOR1-3XGFP was expressed and functional. TOR2-3XGFP was functional, as TOR2 is an essential gene and TOR2^N321-3XGFP haploid cells were viable. Thus, TOR1 and TOR2 retain function after the insertion of 748 amino acids in a variable region of their noncatalytic domain. The localization patterns of TOR1-3XGFP and TOR2-3XGFP were documented by imaging of live cells. TOR1-3XGFP was diffusely cytoplasmic and concentrated near the vacuolar membrane. The TOR2-3XGFP signal was cytoplasmic but predominately in dots at the plasma membrane. Thus, TOR1 and TOR2 have distinct localization patterns, consistent with the regulation of cellular processes as part of two different complexes.     

FKBP12-rapamycin target TOR2 is a vacuolar protein with an associated phosphatidylinositol-4 kinase activity.

In complex with the immunophilin FKBP12, the natural product rapamycin inhibits signal transduction events required for G1 to S phase cell cycle progression in yeast and mammalian cells. Genetic studies in yeast first implicated the TOR1 and TOR2 proteins as targets of the FKBP12-rapamycin complex. We report here that the TOR2 protein is membrane associated and localized to the surface of the yeast vacuole. Immunoprecipitated TOR2 protein contains readily detectable phosphatidylinositol-4 (PI-4) kinase activity attributable to either a TOR2 intrinsic activity or to a PI-4 kinase tightly associated with TOR2. Importantly, we find that rapamycin stimulates FKBP12 binding to wild-type TOR2 but not to a rapamycin-resistant TOR2-1 mutant protein. Surprisingly, FKBP12-rapamycin binding does not markedly inhibit the PI kinase activity associated with TOR2, but does cause a delocalization of TOR2 from the vacuolar surface, which may deprive the TOR2-associated PI-4 kinase activity of its in vivo substrate. Several additional findings indicate that vacuolar localization is important for TOR2 function and, conversely, that TOR2 modulates vacuolar morphology and segregation. These studies demonstrate that TOR2 is an essential, highly conserved component of a signal transduction pathway regulating cell cycle progression conserved from yeast to man.     

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.     

GTPase ROP2 binds and promotes activation of target of rapamycin,TOR, in response to auxin

Target of rapamycin (TOR) promotes reinitiation at upstream ORFs (uORFs) in genes that play important roles in stem cell regulation and organogenesis in plants. Here, we report that the small GTPase ROP2, if activated by the phytohormone auxin, promotes activation of TOR, and thus translation reinitiation of uORF-containing mRNAs. Plants with high levels of active ROP2, including those expressing constitutively active ROP2 (CA-ROP2), contain high levels of active TOR. ROP2 physically interacts with and, when GTP-bound, activates TOR in vitro. TOR activation in response to auxin is abolished in ROP-deficient rop2 rop6 ROP4 RNAi plants. GFP-TOR can associate with endosome-like structures in ROP2-overexpressing plants, indicating that endosomes mediate ROP2 effects on TOR activation. CA-ROP2 is efficient in loading uORF-containing mRNAs onto polysomes and stimulates translation in protoplasts, and both processes are sensitive to TOR inhibitor AZD-8055. TOR inactivation abolishes ROP2 regulation of translation reinitiation, but not its effects on cytoskeleton or intracellular trafficking. These findings imply a mode of translation control whereby, as an upstream effector of TOR, ROP2 coordinates TOR function in translation reinitiation pathways in response to auxin.     

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.     

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.     

TOR2 is part of two related signaling pathways coordinating cell growth in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae genes TOR1 and TOR2 encode phosphatidylinositol kinase homologs. TOR2 has two essential functions. One function overlaps with TOR1 and mediates protein synthesis and cell cycle progression. The second essential function of TOR2 is unique to TOR2 and mediates the cell-cycle-dependent organization of the actin cytoskeleton. We have isolated temperature-sensitive mutants that are defective for either one or both of the two TOR2 functions. The three classes of mutants were as follows. Class A mutants, lacking only the TOR2-unique function, are defective in actin cytoskeleton organization and arrest within two to three generations as small-budded cells in the G2/M phase of the cell cycle. Class B mutants, lacking only the TOR-shared function, and class C mutants, lacking both functions, exhibit a rapid loss of protein synthesis and a G1 arrest within one generation. To define further the two functions of TOR2, we isolated multicopy suppressors that rescue the class A or B mutants. Overexpression of MSS4, PKC1, PLC1, RHO2, ROM2, or SUR1 suppressed the growth defect of a class A mutant. Surprisingly, overexpression of PLC1 and MSS4 also suppressed the growth defect of a class B mutant. These genes encode proteins that are involved in phosphoinositide metabolism and signaling. Thus, the two functions (readouts) of TOR2 appear to involve two related signaling pathways controlling cell growth.     

The expanding TOR signaling network

Cell growth (increase in cell mass or size) is tightly coupled to nutrient availability, growth factors and the energy status of the cell. The target of rapamycin (TOR) integrates all three inputs to control cell growth. The discovery of upstream regulators of TOR (AMPK, the TSC1-TSC2 complex and Rheb) has provided new insights into the mechanism by which TOR integrates its various inputs. A recent finding in flies reveals that TOR controls not only growth of the cell in which it resides (cell-autonomous growth) but also the growth of distant cells, thereby determining organ and organism size in addition to the size of isolated cells. In yeast and mammals, the identification of two structurally and functionally distinct multiprotein TOR complexes (TORC1 and TORC2) has provided a molecular basis for the complexity of TOR signaling. Furthermore, TOR has emerged as a regulator of growth-related processes such as development, aging and the response to hypoxia. Thus, TOR is part of an intra- and inter-cellular signaling network with a remarkably broad role in eukaryotic biology.     

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.     

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     

Drosophila target of rapamycin kinase functions as a multimer

Target of rapamycin (TOR) is a conserved regulator of cell growth and metabolism that integrates energy, growth factor, and nutrient signals. The 280-kDa TOR protein functions as the catalytic component of two large multiprotein complexes and consists of an N-terminal HEAT-repeat domain and a C-terminal Ser/Thr kinase domain. Here we describe an allelic series of mutations in the Drosophila Tor gene and show that combinations of mutations in the HEAT and kinase domains of TOR display the rare genetic phenomenon of intragenic complementation, in which two or more defective proteins assemble to form a functional multimer. We present biochemical evidence that TOR self-associates in vivo and show that this multimerization is unaffected by positive or negative signals upstream of TOR. Consistent with multimerization of TOR, recessive mutations in the HEAT and kinase domains can dominantly interfere with wild-type TOR function in cells lacking TSC1 or TSC2. TOR multimerization thus partially accounts for the high apparent molecular weight of TOR complexes and offers novel therapeutic strategies for pathologies stemming from TOR hyperactivity.     

The PP2A regulatory subunit Tap46, a component of the TOR signaling pathway, modulates growth and metabolism in plants

Tap42/α4, a regulatory subunit of protein phosphatase 2A, is a downstream effector of the target of rapamycin (TOR) protein kinase, which regulates cell growth in coordination with nutrient and environmental conditions in yeast and mammals. In this study, we characterized the functions and phosphatase regulation of plant Tap46. Depletion of Tap46 resulted in growth arrest and acute plant death with morphological markers of programmed cell death. Tap46 interacted with PP2A and PP2A-like phosphatases PP4 and PP6. Tap46 silencing modulated cellular PP2A activities in a time-dependent fashion similar to TOR silencing. Immunoprecipitated full-length and deletion forms of Arabidopsis thaliana TOR phosphorylated recombinant Tap46 protein in vitro, supporting a functional link between Tap46 and TOR. Tap46 depletion reproduced the signature phenotypes of TOR inactivation, such as dramatic repression of global translation and activation of autophagy and nitrogen mobilization, indicating that Tap46 may act as a positive effector of TOR signaling in controlling those processes. Additionally, Tap46 silencing in tobacco (Nicotiana tabacum) BY-2 cells caused chromatin bridge formation at anaphase, indicating its role in sister chromatid segregation. These findings suggest that Tap46, in conjunction with associated phosphatases, plays an essential role in plant growth and development as a component of the TOR signaling pathway.     

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 binds and regulates the mTOR kinase

BACKGROUND: The target of rapamycin (TOR), in complex with the proteins raptor and LST8 (TOR complex 1), phosphorylates the p70S6K and 4E-BP1 to promote mRNA translation. Genetic evidence establishes that TOR complex activity in vivo requires the small GTPase Rheb, and overexpression of Rheb can rescue TOR from inactivation in vivo by amino-acid withdrawal. The Tuberous Sclerosis heterodimer (TSC1/TSC2) functions as a Rheb GTPase activator and inhibits TOR signaling in vivo. RESULTS: Here, we show that Rheb binds to the TOR complex specifically, independently of its ability to bind TSC2, through separate interactions with the mTOR catalytic domain and with LST8. Rheb binding to the TOR complex in vivo and in vitro does not require Rheb guanyl nucleotide charging but is modulated by GTP and impaired by certain mutations (Ile39Lys) in the switch 1 loop. Nucleotide-deficient Rheb mutants, although capable of binding mTOR in vivo and in vitro, are inhibitory in vivo, and the mTOR polypeptides that associate with nucleotide-deficient Rheb in vivo lack kinase activity in vitro. Reciprocally, mTOR polypeptides bound to Rheb(Gln64Leu), a mutant that is nearly 90% GTP charged, exhibit substantially higher protein kinase specific activity than mTOR bound to wild-type Rheb. CONCLUSIONS: The TOR complex 1 is a direct target of Rheb-GTP, whose binding enables activation of the TOR kinase.     

Review series: TOR kinase complexes and cell migration

Cell migration is a fundamental process in a wide array of biological and pathological responses. It is regulated by complex signal transduction pathways in response to external cues that couple to growth factor and chemokine receptors. In recent years, the target of rapamycin (TOR) kinase, as part of either TOR complex 1 (TORC1) or TOR complex 2 (TORC2), has been shown to be an important signaling component linking external signals to the cytoskeletal machinery in a variety of cell types and organisms. Thus, these complexes have emerged as key regulators of cell migration and chemotaxis.     

A Highlights from MBoC Selection: Identification of defined structural elements within TOR2 kinase required for TOR complex 2 assembly and function in Saccharomyces cerevisiae

The mammalian target of rapamycin (mTOR) is a large protein kinase that assembles into two multisubunit protein complexes, mTORC1 and mTORC2, to regulate cell growth in eukaryotic cells. While significant progress has been made in our understanding of the composition and structure of these complexes, important questions remain regarding the role of specific sequences within mTOR important for complex formation and activity. To address these issues, we have used a molecular genetic approach to explore TOR complex assembly in budding yeast, where two closely related TOR paralogues, TOR1 and TOR2, partition preferentially into TORC1 versus TORC2, respectively. We previously identified an ∼500-amino-acid segment within the N-terminal half of each protein, termed the major assembly specificity (MAS) domain, which can govern specificity in formation of each complex. In this study, we have extended the use of chimeric TOR1-TOR2 genes as a “sensitized” genetic system to identify specific subdomains rendered essential for TORC2 function, using synthetic lethal interaction analyses. Our findings reveal important design principles underlying the dimeric assembly of TORC2 as well as identifying specific segments within the MAS domain critical for TORC2 function, to a level approaching single-amino-acid resolution. Together these findings highlight the complex and cooperative nature of TOR complex assembly and function.     

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.