The TOR Kinases Link Nutrient Sensing to Cell Growth*

Rapamycin is an immunosuppressive natural product that inhibits the proliferation of T-cells in response to and growth Rapamycin binds to the peptidyl-prolyl isomerase FKBP12 and forms protein-drug complexes that inhibit signal transduction by the TOR kinases. The FKBP12 and TOR proteins are conserved from fungi to and in both organisms the TOR signaling pathway plays a role in nutrient sensing. an drug inva-sive

is correlated with cell growth and proliferation and sensitive to rapamycin, indicating a role as downstream targets of mTOR (41,42).
Genetic studies reveal that the TOR protein kinase activity is essential for in vivo TOR functions in yeast (16,17,33,43). Furthermore, Tor1 displays an intrinsic, rapamycin-sensitive protein kinase activity with the mammalian translation repressor PHAS-I as a substrate (33). Finally, chimeric proteins with the kinase domain of mTOR fused to the N-terminal regions of yeast Tor1 or Tor2 provide TOR activity in yeast, underscoring the conservation of this domain throughout evolution (44).
Recently, a repressor domain has been identified in mTOR, which is located on the C-terminal end of the kinase domain (45). This repressor domain contains an amino acid residue, Ser-2448, which appears to be phosphorylated by protein kinase B (AKT/ PKB). Although an S2448A mutation did not affect signaling by mTOR, a deletion of 30 amino acids, including Ser-2448, enhances mTOR kinase activity and signaling. This repressor domain is absent in the yeast TOR proteins and may be unique to mTOR.

TOR Controls Cellular Responses to Nutrients:
Regulation of Translation TOR is part of a signal transduction pathway that senses nutrients and regulates transcription, translation, and protein degradation. TOR mutations or inhibition by rapamycin elicits cellular responses characteristic of nutrient starvation, including inhibition of protein synthesis, transcriptional changes, cell cycle arrest, and autophagy (1,3,8,10,11,17,46).
The TOR proteins play a role in controlling translation initiation in both mammalian and yeast cells (Fig. 2). The mammalian TOR protein regulates translation by two independent mechanisms involving direct or indirect activation of p70 S6 kinase and inactivation of the translational repressor PHAS-I (Fig. 2). In mammalian cells different growth stimuli, such as growth factors or amino acids, result in phosphorylation and activation of p70 S6 kinase, which in turn phosphorylates the ribosomal protein S6 (47)(48)(49)(50)(51)(52). Phosphorylation of the S6 protein facilitates translation of mRNAs containing a 5Ј-polypyrimidine tract, including those encoding ribosomal proteins, translation elongation factors, and growth control proteins (53). PHAS-I binds to and inhibits eIF-4E, which is part of the multiprotein complex eIF-4F that functions in CAP recognition and recruitment of ribosomes to the mRNA. Phosphorylation of PHAS-I by mTOR prevents the association of PHAS-I with eIF-4E, thereby promoting translation initiation (for reviews see Refs. 54 and 55). Rapamycin treatment prevents activation of p70 S6 kinase and results in PHAS-I dephosphorylation, suggesting rapamycin blocks translation initiation by two mechanisms (47,48,54,55) (Fig. 2). Yeast contains no direct structural homolog of the mammalian eIF-4E binding protein PHAS-I. Recently, however, the yeast Eap1 protein was identified based on its ability to interact with translation eIF-4E. The Eap1 protein inhibits CAPdependent translation, and deletion of the EAP1 gene confers partial rapamycin resistance, suggesting Eap1 functions similar to mammalian PHAS-I (56).
The Drosophila TOR homolog, dTOR, plays a prominent role in cell growth and development (24,25). Null mutations in the dTOR gene impair larval growth and reduce endoreplicating tissue. The phenotypic arrest point of dTOR mutant larvae is similar to wildtype larvae deprived of amino acids. dTOR mutations also mimic amino acid withdrawal in adult tissues. As in yeast and mammalian cells, p70 S6K kinase is a key effector of dTOR, and constitutive overexpression of p70 S6K rescues viability of dTOR mutant flies. In addition, dTOR was found to be required for PI-3K signal-ing, possibly linking mitogen-induced PI-3K signaling to nutrients (24,25).
TOR control of translation in yeast cells involves regulation of PP2A catalytic subunits, including Pph21, Pph22, and Sit4, which are known to associate with Tap42 (57) (Fig. 2). The target of TOR in this process appears to be Tap42. In accord with this view, certain tap42 mutations confer resistance to rapamycin, and the association of Tap42 with Pph21/Pph22 and Sit4 is prevented by entry into stationary growth phase or by rapamycin (57). Furthermore, direct phosphorylation of Tap42 by TOR has recently been reported (58). Mammalian cells also contain a homolog of Tap42, the ␣4 protein, which associates with PP2A phosphatases and modifies the substrate specificity of PP2A (59,60). However, the rapamycin sensitivity of the ␣4-PP2A association is at present controversial (59,60). Furthermore, the regulation of this complex may differ from that in yeast as a recent study has suggested that PP2A is the target of mTOR (61). Although evidence for direct phosphorylation of p70 S6 kinase by mTOR has been presented, other studies failed to find significant kinase activity of recombinant mTOR toward p70 S6 kinase (40,61). Instead, p70 S6 kinase was found in a complex with a fraction of PP2A, and a model by which TOR phosphorylation of PP2A results in phosphatase inactivation and thereby prevents S6 kinase dephosphorylation has been proposed (61).

Transcriptional Regulation by TOR
Recent studies have uncovered a central role of TOR signaling in the regulation of transcription (Fig. 3). Earlier work established a role for TOR in rRNA and tRNA synthesis (7,62). Although the targets of this regulation are not known, the TOR pathway may act via PP2A in a manner analogous to translational control. Mutations that affect PP2A function also impair rRNA and tRNA gene expression (63,64).
Genome array studies reveal that ribosome biosynthetic genes expressed by PolII are also repressed by the addition of rapamycin in a manner that mimics nutritional limitation (8, 10, 65) (Fig. 3). Ribosomal protein (RP) genes are coordinately regulated in response to many environmental changes; however, the molecular details involving the transcription of RP genes are as yet unclear. Most RP gene promoters that contain binding sites for the activator/repressor protein, Rap1, and the transactivator, Abf1 (reviewed in Ref. 66). Studies have linked Rap1-mediated activation of RP genes to the cAMP pathway although the signaling events resulting in cAMP-induced transcription are at this time unknown (67,68). The identification of Tor kinases as upstream regulators of RP genes provides a starting point to dissect regulatory events governing these genes and should provide insight to the interplay of the TOR and cAMP nutrient-stimulated signaling pathways.
Recently, the TOR pathway was shown to control the expression of the nitrogen catabolite repressed (NCR) genes, underscoring the central role of TOR in nitrogen sensing (8 -10, 65). The NCR genes are repressed by preferred nitrogen sources, such as glutamine or ammonia, and derepressed by limiting or poor nitrogen sources, such as proline or urea. Regulatory factors involved in the repression or activation of these genes were tested as targets of the TOR kinases (8 -10, 69). Many NCR genes are regulated by a set of GATA transcription factors including the transactivators Gln3 and Nil1 and by their inhibitor, Ure2. Both Gln3 and Ure2 were shown to be phosphoproteins; furthermore, these factors are rapidly dephosphorylated upon nitrogen limitation or rapamycin treatment (8 -10).
Recent studies have shown that Tor, Gln3, and Ure2 form a complex and that Gln3 is directly phosphorylated by Tor in vitro (69). Mechanistically, this complex controls the activity of Gln3 by retaining it in the cytoplasm (9). Nitrogen limitation or rapamycin causes Gln3 to translocate into the nucleus, whereas Ure2 remains cytoplasmic. tap42-11 or sit4 mutations prevent rapamycin-induced nuclear import of Gln3, indicating that PP2A is involved in this function of TOR (9). In the resulting model, the TOR kinases and PP2A share the same substrates, providing stringent and dynamic control whereby the TOR kinase regulates the dephosphorylation of its direct substrates by also inhibiting the relevant phosphatase (Fig. 3).
A particular set of genes subject to nitrogen catabolite repression

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includes genes required for the accumulation of precursors of ␣-ketoglutarate when yeast are grown on poor nitrogen sources such as urea. Expression of these genes is controlled by the transactivators Rtg1 and Rtg3 and their positive regulator Rtg2. The TOR signaling pathway controls the activity of the Rtg proteins (65). Rapamycin or poor nitrogen sources induce rapid nuclear import of Rtg1 and Rtg3 in an Rtg2-dependent process. In this case, the importin ␤ family member, Msn5, is required for the export of Rtg1 and Rtg3 as msn5 mutations result in constitutive nuclear accumulation of these factors. Interestingly, an msn5 mutation does not cause constitutive activation of the target genes for Rtg1 and Rtg3; instead, addition of rapamycin is still required for Rtg-directed gene expression. Thus, the TOR pathway controls both nuclear localization of the transactivators and downstream signaling events required for gene expression.
The control of nuclear import/export appears to be a general mechanism by which TOR regulates transcription (Fig. 3). Two additional transcription factors, Msn2 and Msn4, are constrained to the cytoplasm through interaction with a negative regulator (the 14-3-3 proteins Bmh1 and Bmh2) (9). The addition of rapamycin induces nuclear import of Msn2 and Msn4 and induction of stressinducible genes regulated by these factors.
The emerging theme of Tor-regulated nuclear localization of transcription factors may also extend to mammalian cells. The signal transducer and activator of transcription, STAT3, is activated in response to cytokines and translocates into the nucleus where it directs transcription of its target genes. Recent studies indicate both the nuclear localization and the ability to activate transcription may be regulated by both mTor kinase and PP2A (70,71).

Regulation of TOR Kinase Activity by Nutrients
Cells control the rate of translation in response to energy and amino acids. Amino acid levels control amino acid biosynthesis, transport, and expression of the translation machinery in yeast and mammalian cells. Yeast cells use multiple mechanisms to determine the quality and abundance of amino acids and other nitrogen sources. Ammonium availability is sensed by an ammonium-specific permease, Mep2 (72). External amino acids are sensed by the amino acid receptor Ssy1 in a manner analogous to glucose sensing via the Snf3 and Rgt2 glucose sensors (73,74). Internal amino acid availability is sensed by the general control response that detects uncharged tRNAs through the protein kinase Gcn2, and this pathway is conserved in mammals (70).
In mammalian cells, amino acids such as L-leucine stimulate TOR kinase activity. Both the activity and phosphorylation states FIG. 2. The rapamycin-sensitive TOR signaling pathway senses nutrients and growth factors and regulates cellular responses. In yeast cells, the TOR pathway responds to nutrient availability and promotes Tap42 association with the protein phosphatase catalytic subunit. The Tap42-PP2A interaction results in a modified protein phosphatase activity that regulates translation and transcription. Similarly, the TOR pathway also acts via the mammalian Tap42 homolog ␣4 to alter PP2A activity and thereby regulate translation and transcription. In mammalian and Drosophila cells, the TOR pathway responds to growth factors and amino acids and stimulates translation by activating p70 S6 kinase and inactivating the translational repressor PHAS-I. In yeast, Eap1 may have functions similar to mammalian PHAS-I. Growth factors may act on TOR via the PI-3K-protein kinase B (PKB) pathway.

FIG. 3. TOR controls nuclear import of transcription factors.
In the presence of nutrients, the TOR signaling pathway is required for the expression of genes necessary for ribosomal biogenesis and restricts nuclear import of transcription factors responsible for expression of genes induced by nitrogen limitation. Upon nutrient limitation or rapamycin addition, genes required for ribosomal biogenesis are repressed, and transcription factors required to express stress-induced genes, nitrogen catabolite-repressed genes, and certain genes of the trichloroacetic acid (TCA) cycle are all imported into the nucleus (represented by dashed lines).

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of the mTOR downstream effector p70 S6K are decreased in response to amino acid limitation and stimulated upon their readdition (50,52). Moreover, a rapamycin-resistant allele of p70 S6K causes cells to be unresponsive to amino acid depletion (52). Amino acid alcohols that inhibit amino acid charging of tRNA were found to suppress p70 S6K activity, and a temperature-sensitive mutation of histidyl-tRNA synthetase also impaired p70 S6K activity at the non-permissive temperature (71). These results suggest aminoacylation of tRNAs may regulate TOR in response to amino acids.
Clinical Perspective Rapamycin was originally identified as a potent antifungal agent with an undesired side effect involving bone marrow suppression. The structural resemblance of rapamycin with the immunosuppressant FK506 prompted clinical studies to develop rapamycin as an immunosuppressive drug. During the last decade, the basic mechanisms of rapamycin drug action were elucidated and the targets FKBP12 and TOR identified, and rapamycin was approved by the Food and Drug Administration as an immunosuppressant in renal transplant recipients in August 1999.
Ongoing clinical studies address further uses of rapamycin, alone and in combination with other immunosuppressants and in other transplant settings. Rapamycin is synergistic with cyclosporin A and FK506 and lacks the nephrotoxic effects of cyclosporin A or FK506, providing renal sparing drug combinations. The rapamycin analog everlimus is in phase III clinical trials as a immunosuppressant. Phase II clinical trials of the rapamycin analog CCI-779 as a novel chemotherapy agent for a variety of different solid tumors are ongoing. Finally, rapamycin may also find a novel use in cardiology. Clinical studies in human patients reveal that impregnating cardiac stents with rapamycin inhibits proliferation and restenosis that commonly occur after treatment of coronary artery disease. These clinical advances illustrate the dramatic impact of rapamycin on modern medicine.