mTOR signaling: RAG GTPases transmit the amino acid signal

mTOR (mammalian target of rapamycin) is a highly conserved nutrient-responsive regulator of cell growth that is found in all eukaryotes. The mechanism by which amino acids signal to mTOR has remained one of the largest outstanding questions in the field. Two recent complimentary studies provide compelling evidence that the Rag family of small GTPases is both necessary and sufficient to transmit a positive signal from amino acids to mTOR.     

Regulation of TOR by small GTPases

TOR is a conserved serine/threonine kinase that responds to nutrients, growth factors, the bioenergetic status of the cell and cellular stress to control growth, metabolism and ageing. A diverse group of small GTPases including Rheb, Rag, Rac1, RalA and Ryh1 play a variety of roles in the regulation of TOR. For example, while Rheb binds to and activates TOR directly, Rag and Rac1 regulate its localization and RalA activates it indirectly through the production of phosphatidic acid. Here, we review recent findings on the regulation of TOR by small GTPases.     

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.     

Plant TOR signaling components

Cell growth is a process that needs to be tightly regulated. Cells must be able to sense environmental factors like nutrient abundance, the energy level or stress signals and coordinate growth accordingly. The Target Of Rapamycin (TOR) pathway is a major controller of growth-related processes in all eukaryotes. If environmental conditions are favorable, the TOR pathway promotes cell and organ growth and restrains catabolic processes like autophagy. Rapamycin is a specific inhibitor of the TOR kinase and acts as a potent inhibitor of TOR signaling. As a consequence, interfering with TOR signaling has a strong impact on plant development. This review summarizes the progress in the understanding of the biological significance and the functional analysis of the TOR pathway in plants.     

Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway

In many species, reducing nutrient intake without causing malnutrition extends lifespan. Like DR (dietary restriction), modulation of genes in the insulin-signaling pathway, known to alter nutrient sensing, has been shown to extend lifespan in various species. In Drosophila, the target of rapamycin (TOR) and the insulin pathways have emerged as major regulators of growth and size. Hence we examined the role of TOR pathway genes in regulating lifespan by using Drosophila. We show that inhibition of TOR signaling pathway by alteration of the expression of genes in this nutrient-sensing pathway, which is conserved from yeast to human, extends lifespan in a manner that may overlap with known effects of dietary restriction on longevity. In Drosophila, TSC1 and TSC2 (tuberous sclerosis complex genes 1 and 2) act together to inhibit TOR (target of rapamycin), which mediates a signaling pathway that couples amino acid availability to S6 kinase, translation initiation, and growth. We find that overexpression of dTsc1, dTsc2, or dominant-negative forms of dTOR or dS6K all cause lifespan extension. Modulation of expression in the fat is sufficient for the lifespan-extension effects. The lifespan extensions are dependent on nutritional condition, suggesting a possible link between the TOR pathway and dietary restriction.     

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.     

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.     

Carbon catabolite repression regulates amino acid permeases in Saccharomyces cerevisiae via the TOR signaling pathway

We have identified carbon catabolite repression (CCR) as a regulator of amino acid permeases in Saccharomyces cerevisiae, elucidated the permeases regulated by CCR, and identified the mechanisms involved in amino acid permease regulation by CCR. Transport of l-arginine and l-leucine was increased by approximately 10-25-fold in yeast grown in carbon sources alternate to glucose, indicating regulation by CCR. In wild type yeast the uptake (pmol/10(6) cells/h), in glucose versus galactose medium, of l-[(14)C]arginine was (0.24 +/- 0.04 versus 6.11 +/- 0.42) and l-[(14)C]leucine was (0.30 +/- 0.02 versus 3.60 +/- 0.50). The increase in amino acid uptake was maintained when galactose was replaced with glycerol. Deletion of gap1Delta and agp1Delta from the wild type strain did not alter CCR induced increase in l-leucine uptake; however, deletion of further amino acid permeases reduced the increase in l-leucine uptake in the following manner: 36% (gnp1Delta), 62% (bap2Delta), 83% (Delta(bap2-tat1)). Direct immunofluorescence showed large increases in the expression of Gnp1 and Bap2 proteins when grown in galactose compared with glucose medium. By extending the functional genomic approach to include major nutritional transducers of CCR in yeast, we concluded that SNF/MIG, GCN, or PSK pathways were not involved in the regulation of amino acid permeases by CCR. Strikingly, the deletion of TOR1, which regulates cellular response to changes in nitrogen availability, from the wild type strain abolished the CCR-induced amino acid uptake. Our results provide novel insights into the regulation of yeast amino acid permeases and signaling mechanisms involved in this regulation.     

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.     

The TOR pathway comes of age

Studies in a variety of model organisms indicate that nutrient signaling is tightly coupled to longevity. In nutrient replete conditions, organisms develop, grow, and age quickly. When nutrients become sparse as with dietary restriction, growth and development decline, stress response pathways become induced and organisms live longer. Considerable effort has been devoted to understanding the molecular events mediating lifespan extension by dietary restriction. One central focus has been on nutrient-responsive signal transduction pathways including insulin/IGF-1, AMP kinase, protein kinase A and the TOR pathway. Here we describe the increasingly prominent links between TOR signaling and aging in invertebrates. Longevity studies in mammals are not published to date. Instead, we highlight studies in mouse models, which indicate that dampening the TOR pathway leads to widespread protection from an array of age-related diseases.     

Mammalian TOR signaling to the AGC kinases

The mechanistic (or mammalian) target of rapamycin (mTOR), an evolutionarily conserved protein kinase, orchestrates cellular responses to growth, metabolic and stress signals. mTOR processes various extracellular and intracellular inputs as part of two mTOR protein complexes, mTORC1 or mTORC2. The mTORCs have numerous cellular targets but members of a family of protein kinases, the protein kinase (PK)A/PKG/PKC (AGC) family are the best characterized direct mTOR substrates. The AGC kinases control multiple cellular functions and deregulation of many members of this family underlies numerous pathological conditions. mTOR phosphorylates conserved motifs in these kinases to allosterically augment their activity, influence substrate specificity, and promote protein maturation and stability. Activation of AGC kinases in turn triggers the phosphorylation of diverse, often overlapping, targets that ultimately control cellular response to a wide spectrum of stimuli. This review will highlight recent findings on how mTOR regulates AGC kinases and how mTOR activity is feedback regulated by these kinases. We will discuss how this regulation can modulate downstream targets in the mTOR pathway that could account for the varied cellular functions of mTOR.     

Mammalian TOR signaling to the AGC kinases

The mechanistic (or mammalian) target of rapamycin (mTOR), an evolutionarily conserved protein kinase, orchestrates cellular responses to growth, metabolic and stress signals. mTOR processes various extracellular and intracellular inputs as part of two mTOR protein complexes, mTORC1 or mTORC2. The mTORCs have numerous cellular targets but members of a family of protein kinases, the protein kinase (PK)A/PKG/PKC (AGC) family are the best characterized direct mTOR substrates. The AGC kinases control multiple cellular functions and deregulation of many members of this family underlies numerous pathological conditions. mTOR phosphorylates conserved motifs in these kinases to allosterically augment their activity, influence substrate specificity, and promote protein maturation and stability. Activation of AGC kinases in turn triggers the phosphorylation of diverse, often overlapping, targets that ultimately control cellular response to a wide spectrum of stimuli. This review will highlight recent findings on how mTOR regulates AGC kinases and how mTOR activity is feedback regulated by these kinases. We will discuss how this regulation can modulate downstream targets in the mTOR pathway that could account for the varied cellular functions of mTOR.     

TOR signaling regulates microtubule structure and function

The functional diversity and structural heterogeneity of microtubules are largely determined by microtubule-associated proteins (MAPs) [1] [2]. Bik1p (bilateral karyogamy defect protein) is one of the MAPs required for microtubule assembly, stability and function in cell processes such as karyogamy and nuclear migration and positioning in the yeast Saccharomyces cerevisiae [3]. The macrocyclic immunosuppressive antibiotic rapamycin, complexed with its binding protein FKBP12, binds to and inhibits the target of rapamycin protein (TOR) in yeast [4] [5]. We report here that TOR physically interacts with Bik1p, the yeast homolog of human CLIP-170/Restin [6] [7]. Inhibition of TOR by rapamycin significantly affects microtubule assembly, elongation and stability. This function of TOR is independent of new protein synthesis. Rapamycin also causes defects in spindle orientation, nuclear movement and positioning, karyogamy and chromosomal stability, defects also found in the bikDelta mutant. Our data suggest a role for TOR signaling in regulating microtubule stability and function, possibly through Bik1p.