PI3-kinase and TOR: PIKTORing cell growth

Regulation of growth and proliferation in higher eukaryotic cells results from an integration of nutritional, energy, and mitogenic signals. Biochemical processes underlying cell growth and proliferation are governed by the phosphatidylinositol 3-kinase (PI3K) and target of rapamycin (TOR) signaling pathways. The importance of the interplay between these two pathways is underscored by the discovery that the TOR inhibitor rapamycin is effective against tumors caused by misregulation of the PI3K pathway. We review here recent data concerning the convergence of the PI3K and TOR pathways, the role of these pathways in cell growth and proliferation, and the regulation of growth by downstream TOR targets.     

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.     

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.     

<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.     

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.     

TOR coordinates bulk and targeted endocytosis in the Drosophila melanogaster fat body to regulate cell growth

Target of rapamycin (TOR) is a central regulator of cellular and organismal growth in response to nutrient conditions. In a genetic screen for novel TOR interactors in Drosophila melanogaster, we have identified the clathrin-uncoating ATPase Hsc70-4, which is a key regulator of endocytosis. We present genetic evidence that TOR signaling stimulates bulk endocytic uptake and inhibits the targeted endocytic degradation of the amino acid importer Slimfast. Thus, TOR simultaneously down-regulates aspects of endocytosis that inhibit growth and up-regulates potential growth-promoting functions of endocytosis. In addition, we find that disruption of endocytosis leads to changes in TOR and phosphatidylinositol-3 kinase activity, affecting cell growth, autophagy, and rapamycin sensitivity. Our data indicate that endocytosis acts both as an effector function downstream of TOR and as a physiologically relevant regulator of TOR signaling.     

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.     

Plant target of rapamycin signaling network: Complexes,conservations, and specificities

Target of rapamycin (TOR) is an evolutionarily conserved protein kinase that functions as a central signaling hub to integrate diverse internal and external cues to precisely orchestrate cellular and organismal physiology. During evolution, TOR both maintains the highly conserved TOR complex compositions, and cellular and molecular functions, but also evolves distinctive roles and strategies to modulate cell growth, proliferation, metabolism, survival, and stress responses in eukaryotes. Here, we review recent discoveries on the plant TOR signaling network. We present an overview of plant TOR complexes, analyze the signaling landscape of the plant TOR signaling network from the upstream signals that regulate plant TOR activation to the downstream effectors involved in various biological processes, and compare their conservation and specificities within different biological contexts. Finally, we summarize the impact of dysregulation of TOR signaling on every stage of plant growth and development, from embryogenesis and seedling growth, to flowering and senescence.     

The amino acid sensitive TOR pathway from yeast to mammals

The target of rapamycin (TOR) is an ancient effector of cell growth that integrates signals from growth factors and nutrients. Two downstream effectors of mammalian TOR, the translational components S6K1 and 4EBP1, are commonly used as reporters of mTOR activity. The conical signaling cascade initiated by growth factors is mediated by PI3K, PKB, TSC1/2 and Rheb. However, the process through which nutrients, i.e., amino acids, activate mTOR remains largely unknown. Evidence exists for both an intracellular and/or a membrane bound sensor for amino acid mediated mTOR activation. Research in eukaryotic models, has implicated amino acid transporters as nutrient sensors. This review describes recent advances in nutrient signaling that impinge on mTOR and its targets including hVps34, class III PI3K, a transducer of nutrient availability to mTOR.     

Regulation of the cell integrity pathway by rapamycin-sensitive TOR function in budding yeast

The TOR (target of rapamycin) pathway controls cell growth in response to nutrient availability in eukaryotic cells. Inactivation of TOR function by rapamycin or nutrient exhaustion is accompanied by triggering various cellular mechanisms aimed at overcoming the nutrient stress. Here we report that in Saccharomyces cerevisiae the protein kinase C (PKC)-mediated mitogen-activated protein kinase pathway is regulated by TOR function because upon specific Tor1 and Tor2 inhibition by rapamycin, Mpk1 is activated rapidly in a process mediated by Sit4 and Tap42. Osmotic stabilization of the plasma membrane prevents both Mpk1 activation by rapamycin and the growth defect that occurs upon the simultaneous absence of Tor1 and Mpk1 function, suggesting that, at least partially, TOR inhibition is sensed by the PKC pathway at the cell envelope. This process involves activation of cell surface sensors, Rom2, and downstream elements of the mitogen-activated protein kinase cascade. Rapamycin also induces depolarization of the actin cytoskeleton through the TOR proteins, Sit4 and Tap42, in an osmotically suppressible manner. Finally, we show that entry into stationary phase, a physiological situation of nutrient depletion, also leads to the activation of the PKC pathway, and we provide further evidence demonstrating that Mpk1 is essential for viability once cells enter G(0).     

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.     

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.     

mTOR regulation of autophagy

Nutrient starvation induces autophagy in eukaryotic cells through inhibition of TOR (target of rapamycin), an evolutionarily-conserved protein kinase. TOR, as a central regulator of cell growth, plays a key role at the interface of the pathways that coordinately regulate the balance between cell growth and autophagy in response to nutritional status, growth factor and stress signals. Although TOR has been known as a key regulator of autophagy for more than a decade, the underlying regulatory mechanisms have not been clearly understood. This review discusses the recent advances in understanding of the mechanism by which TOR regulates autophagy with focus on mammalian TOR (mTOR) and its regulation of the autophagy machinery.     

植物TOR激酶响应上游信号的研究进展

雷帕霉素靶蛋白(TOR)是真核生物中高度保守的丝氨酸/苏氨酸蛋白激酶, 能整合营养、能量、生长因子及环境信号, 协调细胞增殖、生长和代谢等过程, 是真核生物生长发育的核心调控因子。近年来, 随着相关研究系统的建立, 植物TOR的功能和机制研究取得了众多突破, 发现其进化上保守的生物学功能及植物中特有的信号通路。该文概述了TOR蛋白复合体的构成, 以及植物TOR响应糖、营养元素(氮、磷和硫)、激素及逆境胁迫信号来调控下游基因转录、蛋白翻译、代谢、细胞自噬和胁迫应答等生物学过程的分子机制, 并提出了植物TOR领域一些亟待解决的科学问题, 以期为全面揭示植物TOR的生物学功能提供参考。     

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.     

Rapamycin treatment causes developmental delay, pigmentation defects, and gastrointestinal malformation on Xenopus embryogenesis

Rapamycin is a drug working as an inhibitor of the TOR (target of rapamycin) signaling pathway and influences various life phenomena such as cell growth, proliferation, and life span extension in eukaryote. However, the extent to which rapamycin controls early developmental events of amphibians remains to be understood. Here we report an examination of rapamycin effects during Xenopus early development, followed by a confirmation of suppression of TOR downstream kinase S6K by rapamycin treatment. First, we found that developmental speed was declined in dose-dependent manner of rapamycin. Second, black pigment spots located at dorsal and lateral skin in tadpoles were reduced by rapamycin treatment. Moreover, in tadpole stages severe gastrointestinal malformations were observed in rapamycin-treated embryos. Taken together with these results, we conclude that treatment of the drug rapamycin causes enormous influences on early developmental period.     

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.