Critical Review. What Controls TOR? Estela Jacinto Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ

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1 IUBMB Life, 60(8): , August 2008 Critical Review What Controls TOR? Estela Jacinto Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ Summary 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. Ó 2008 IUBMB IUBMB Life, 60(8): , 2008 Keywords enzyme mechanisms; eukaryotic gene expression; molecular genetics; phosphoinositides; protein function; protein synthesis; signal transduction. RAPAMYCIN AND THE NOT-SO-SIMPLE TOR COMPLEX Rapamycin, a bacterial macrolide, is currently used as an immunosuppressant and its analogues (rapalogs) are undergoing clinical trials as possible anticancer drugs (1 3). In addition to its clinical importance, it has been valuable in the elucidation of a highly conserved signaling pathway among eukaryotes that is essential for cell growth. Studies in both yeast and mammals revealed that rapamycin binds and inhibits two enzymes, one is the peptidyl prolyl isomerase FKBP12 (FK506 binding protein) and the other is the protein kinase target of rapamycin (TOR) (4). Rapamycin, in association with FKBP12, binds TOR at a region termed the FRB (FKBP12-rapamycin binding) (Fig. 1). Received 23 January 2008; accepted 23 January 2008 Address correspondence to: Estela Jacinto, UMDNJ-Robert Wood Johnson Medical School, Physiology and Biophysics, 675 Hoes Lane, Piscataway, NJ 08854, USA. Tel: Fax: jacintes@umdnj.edu The FRB is adjacent to the kinase domain of TOR, but the evidence that the binding of rapamycin/fkbp12 to FRB inhibits the catalytic activity of TOR is rather controversial. Nevertheless, genetic and biochemical studies have revealed that the growth-inhibitory properties of rapamycin are primarily due to TOR inhibition. TOR, first identified in the budding yeast, Saccharomyces cerevisiae, is a central regulator of a signaling pathway that responds to the presence of growth stimuli such as nutrients. Fortunately, the initial characterization of TOR happened to be in this organism that possesses two related but distinct TOR genes, TOR1 and TOR2. Both TOR1 and TOR2 can bind rapamycin/fkbp12, but genetic studies in the budding yeast have also uncovered a rapamycin-insensitive function of TOR2 (5). These early studies recognized that TOR has several functions and that only a subset of these functions can be inhibited by rapamycin. In other organisms such as mammals, there is only one TOR (mtor in mammals). However, both the rapamycinsensitive and -insensitive functions have been conserved in mammals as well. The identification of two distinct TOR/ mtor protein complexes has shed light as to how TOR may perform functions that have different rapamycin sensitivities (4, 6). TOR is part of two distinct protein complexes, TORC1 and TORC2. In the budding yeast, TOR1 associates with the conserved proteins KOG1 (kontroller of growth 1) and LST8 (lethal with sec thirteen) to form TORC1 (Fig. 2) (7, 8). TOR2 is found mostly as a component of TORC2, but it can bind to TORC1 components upon deletion of TOR1. In mammals, mtor binds to raptor (regulatory associated protein of mtor) and mlst8 as part of mtorc1 and associates with rictor (rapamycin-insensitive companion of mtor), SIN1 (SAPKinteracting protein), and mlst8 as part of mtorc2 (7, 9 15). Each TORC is believed to have distinct substrates or targets, but while both TORCs function to ultimately promote cell growth or survival, how extracellular signals control the activity of each complex is poorly understood. In both yeast and mammals, TORC1 is driven by the presence of nutrients. Exactly how this occurs is still a mystery. The events that are ISSN print/issn online DOI: /iub.56

2 484 JACINTO Figure 1. Structural domains of mtor. See text for description. Asterisk indicates phosphorylated residues. Figure 2. Comparison of homologous TORC components and regulators in Saccharomyces cerevisiae (budding yeast), Schizosaccharomyces pombe (fission yeast), and Homo sapiens. Question mark denotes lack of experimental data on linkage to TORCs. coupled to the TORC1-dependent, nutrient-regulated signals culminate in increased protein synthesis that is required for cell growth. In mammals, mtorc1 mediates the phosphorylation of the translational regulators S6K and 4E-BP. These events are largely inhibited by rapamycin. The association of raptor and mtor is only weakened but not disrupted in vivo upon rapamycin treatment (13). Hence, the current model for how rapamycin/fkbp12 may inhibit mtorc1 activity is based on this perturbed interaction of raptor and mtor, which may affect substrate recognition by or presentation to mtor. However, yeast KOG1 (raptor orthologue) and TOR1 interaction is unaffected by rapamycin treatment (7). An alternative model would be that the intrinsic kinase activity of mtor could be inhibited by rapamycin based on the observation that the in vitro phosphorylation of mtor, as part of mtorc1, is decreased in the presence of rapamycin (9). The proximity of the TOR1 FRB to the kinase domain and to the TOS motif of KOG1 based on modeling studies is consistent with both possibilities (16). The extracellular stimuli that TORC2 respond to remain to be elucidated, but so far in mammals, TORC2 functions are regulated by growth factors such as insulin. Stress responses that promote cell survival or viability also require the presence of TORC2. mtorc2 mediates the phosphorylation of Akt and PKC, proteins that belong to the AGC (protein kinases A/G/C) family of kinases and are important for cell proliferation and survival (10, 17 19). Phosphorylation of these kinases by mtor at homologous sites may control their substrate specificity (10). The mtorc2-mediated phosphorylation of Akt is important for glucose transport and cell survival (10, 20). The mtorc2 components, rictor and SIN1 (along with their budding yeast orthologues), do not bind the rapamycin/fkbp12 complex (7, 9). Thus, it is thought that rictor and/or SIN1 that are bound to mtor may hinder the binding of the drug com-

3 TARGET OF RAPAMYCIN 485 plex to the FRB of mtor making this complex insensitive to rapamycin treatment. The structural assembly of TORC2 is not understood, but both rictor and SIN1 are required for the integrity of this complex (10, 21). Some intriguing findings on the inhibition of TORC2 functions by rapamycin under particular conditions were recently reported. While the TORC2-mediated phosphorylation of Akt is not acutely sensitive to rapamycin, prolonged rapamycin treatment led to a dramatic decrease in its phosphorylation but only in certain normal and cancer cell lines (22). The diminished Akt phosphorylation correlated with the decreased association between mtor and rictor. In particular, rapamycin prevented the binding of newly synthesized mtor to rictor in the responsive cells and decreased this association in the less-responsive cells. These findings indicate that although rapamycin does not bind to pre-formed mtorc2, it can inhibit the assembly of mtorc2 and thereby block mtorc2 function. This is somewhat contradictory to previous models proposing that inhibition of mtorc1, which dephosphorylates and inactivates S6K, leads to a feedback mechanism whereby mtorc2 signals become upregulated (23 25). However, Sarbassov et al. have also observed this phenomenon in cell lines that are not responsive to the prolonged rapamycin treatment. In these cells, the remaining intact mtorc2 was sufficient to allow Akt phosphorylation and was not susceptible to rapamycin inhibition. It was proposed that an unidentified protein or posttranslational modification could block the rapamycin/fkbp12 binding site during the assembly of a fraction of these mtorc2 complexes. Indeed, in cell lines responsive to prolonged rapamycin treatment, rictor migrates faster on SDS-PAGE, suggesting defective modification (22, 26). While these findings in mammals reiterate that rapamycin/fkbp12 binds and inhibits mtorc1 and that its effects on mtorc2 are likely only indirect, recent studies in the fission yeast, Schizosaccharomyces pombe, blur the rapamycin specificity between the two TORCs. In this organism, Tor1 is part of TORC2 and forms a complex with the corresponding TORC2 components, Ste20 (rictor orthologue), Sin1, and Wat1 (LST8 orthologue) (Fig. 2) (27). Tor2 associates with Mip1 (raptor orthologue) and Wat1 to constitute TORC1 (27). However, rapamycin/fkbp12 does not inhibit the vegetative growth of prototrophic fission yeast. Furthermore, under normal growing conditions, rapamycin-treated cells do not have a similar phenotype as nitrogen-starved cells (28). This is opposed to the similar, although not identical, effect of rapamycin on nitrogen-starved budding yeast or amino acidstarved mammalian cells (29 31). Instead, rapamycin inhibits fission yeast sexual development by blocking an early event during this differentiation process that occurs upon starvation (28). Interestingly, leucine-auxotrophic strains are rapamycinsensitive even during vegetative growth and this sensitivity is rescued by a tor1 allele that can no longer bind to FKBP-rapamycin (32). These findings have two curious implications. First, they suggest that rapamycin sensitivity is due to effects on TORC2, insinuating that rapamycin/fkbp12 can bind this complex and inhibit its functions. Second, that the effectiveness of inhibition by rapamycin may be due to a somewhat compromised Tor signaling that occurs in starved cells or in leucine auxotrophic strains. Speculatively, under starved or auxotrophic conditions, TORC2 assembly may be perturbed or weakened, making it more sensitive to rapamycin inhibition. This might be similar to the proposed model in mammals in which prolonged rapamycin treatment may affect the complex assembly due to defective modification or binding of a subunit. Hence, the signals that modulate TORC assembly would provide important clues as to how rapamycin may specifically inhibit one complex over the other. In a more recent S. pombe study, rapamycintreated cells can mimic nitrogen starvation if cells are grown in glutamate medium (33). The loss of Tor2 (TORC1) mimics nitrogen starvation, consistent with the function of TORC1 in budding yeast (27, 34, 35). Taken together, these findings suggest that TORC1 may after all also be inhibited by rapamycin in fission yeast. A temperature sensitive strain containing a mutant tor2 allele is hypersensitive to rapamycin at the nonpermissive temperature (36). It was proposed that in this mutant strain, the hypersensitivity could be due to the direct effect of rapamycin on Tor1 (TORC2), which becomes inactivated in the presence of rapamycin. But it could also be that this tor2 mutant is capable to bind rapamycin/fkbp12 and thus inhibit TORC1 directly. The mutation is located in the catalytic domain (L2048 in S. pombe) and this residue could be important in stabilizing the hydrophobic interaction with the adenine base of ATP. Thus, it is possible that this hypersensitive mutant, but not wild-type Tor2, may effectively bind rapamycin. To resolve whether rapamycin indeed inhibits TORC2 and TORC1 directly in this organism, the role of the complex components and how rapamycin may affect TORC assembly and known TORC targets such as Gad8 (37), a TORC2 target, would need to be evaluated. If indeed Tor2 as part of TORC1 is capable to bind rapamycin/fkbp12, one possibility as discussed earlier is that rapamycin can effectively inhibit a TORC (TORC1 or TORC2) under conditions of weakened TORC signals or assembly. Rapamycin may effectively inhibit mtorc1 in mammals due to the presence of only one mtor gene that may be expressed in limiting amounts particularly in certain cell types. In light of these recent findings, the model that rapamycin/fkbp12 inhibits TORC1 turns out to be a not so simple one. REGULATING TOR CATALYTIC ACTIVITY TOR belongs to a family of protein kinases termed the phosphatidyl inositol 3 kinase-related kinases (PIKK). This family of kinases, which has been grouped with atypical protein kinases, is a small subset of protein kinases that do not share sequence homology with conventional protein kinases but have been demonstrated experimentally to possess protein kinase activity (38, 39). Conventional eukaryotic protein kinases are phosphorylated at the activation loop (also called T loop), the flexible polypep-

4 486 JACINTO tide segment connecting the N and C lobes of the kinase domain. This segment, usually around residues and bounded by the conserved DFG residues at the N-terminus and APE at the C-terminus, contains a conserved phosphorylatable residue. Phosphorylation of this residue activates the kinase. This segment in atypical protein kinases like TOR deviates slightly from the canonical sequence (40). They do not contain a phosphorylatable Ser, Thr, or Tyr residue at the activation segment but instead contain Asp or Glu in the middle of this loop (Fig. 1), hence mimicking a phosphorylated or activated state. Therefore, it is not surprising that TOR and other members of the PIKK family are regulated by other means. Most protein kinases are additionally phosphorylated outside of the activation loop to regulate activity and substrate recognition. Very little is known as to how TOR may be regulated by phosphorylation. Currently, three phosphorylation sites have been identified in mtor. Autophosphorylation occurs at Ser2481 of the C-terminus (41). This autophosphorylation was absent in a kinase dead mutant of mtor or when cells were treated with high concentrations of wortmannin, which can bind and inhibit the active site of mtor (42). Thus, phosphorylation at this site is dependent on the presence of an active mtor. This phosphorylation is not blocked by rapamycin or upon withdrawal of growth stimuli (41). These findings imply that rapamycin and withdrawal of growth stimuli may not inhibit the intrinsic kinase activity of mtor, but it is also possible that this autophosphorylation is due to mtorc2, which is not acutely sensitive to rapamycin. Interestingly, the residues surrounding this phosphorylation site bear an uncanny resemblance to the residues around the activation loop phosphorylation sites of PDK1 and other AGC kinases (43). Hence, this region may be recognized by a common regulator such as a phosphatase. Because PDK1 phosphorylates this sequence motif in AGC kinases, an alternative would be that PDK1 may also regulate mtor at this site. mtor is also phosphorylated at Thr2446 and Ser2448. These sites that conform to the AGC kinase recognition motif are phosphorylated by S6K (44, 45). Thr2446 is also phosphorylated by AMPK (46). These phosphorylation sites are contained within a repressor domain. Deletion of this domain increased the phosphorylation of mtorc1 substrates and increased the growth factor-independent, nutrient-dependent cell survival (47, 48). Because S6K is an mtor target, it is conceivable that phosphorylation at these sites may be a feedback mechanism to regulate mtor. The homologous sites are also phosphorylated in S. pombe Tor (36). Additionally, S. pombe Tor2 contains a phosphorylation site within the FAT domain. The functions of these phosphorylation sites in S. pombe are unknown at the moment. The FRB domain of TOR is perceived as a critical regulatory region owing to its association with rapamycin. More recent in vitro analyses have shown that rapamycin can alone bind to the FRB, albeit with modest affinity. However, in complex with FKBP12, the affinity for FRB increases 2000-fold (49). Another study also found that higher levels of rapamycin can inhibit the kinase activity of mtor even in the absence of FKBP12 and that this inhibition is dependent on the binding to the FRB domain (50). Interestingly, in this same study, small molecular compounds that potently bind to FRB (with the same affinity similar to rapamycin in the absence of FKBP12) but do not bind to FKBP12 did not significantly inhibit phosphorylation of the mtorc1 substrate S6K1. These findings suggest that binding of small molecules to the FRB region may not be sufficient to inhibit mtor function. It would be interesting to determine if the mtor-raptor association was unperturbed in the presence of these small molecules. The lipophilic character of rapamycin also hints that the FRB may bind a similar endogenous molecule. Based on structural and biochemical data, the FRB domain has been proposed to interact with phosphatidic acid at overlapping sites with rapamycin (51 54). Phosphatidic acid (PA) is a lipid second messenger and is upregulated upon activation of phospholipase D during mitogenic stimulation. It is not clear how PA may activate mtor, but it is speculated that it could mediate membrane localization of mtor as recently reported for the PA-mediated activation of Ras (51, 55). Several findings infer that TOR/mTOR is active at the membrane periphery or vesicles, but the mechanism as to how TOR and its substrates congregate at the membrane is not known (56). TOR and some of its complex components have also been found localized to membrane structures (57 60). Hence, a lipid-mediated localization of TOR is an attractive model for TOR regulation. Other motifs in the TOR sequence, which could regulate TOR activity, are discussed later. GROWTH STIMULI ACTIVATE TOR: TRUE OR FALSE? The TOR signaling pathway responds to nutrients and growth factors. Early studies in yeast have shown that tor mutants phenotypically resemble starved cells (61). In mammals, amino acid starvation or rapamycin treatment both lead to dephosphorylation of the mtorc1 translation targets S6K and 4E-BP (62, 63). Withdrawal of growth factors also leads to dephosphorylation of these mtor targets along with the dephosphorylation of the mtorc2 target Akt. Corollary to these findings, the addition of growth stimuli to starved cells potently elevates the phosphorylation of these mtor targets. Somewhat disappointingly, kinase assays using mtor immunoprecipitated from stimulated cells (versus starved) to phosphorylate these substrates in vitro only modestly increase their phosphorylation (13, 17, 64) (our unpublished results). It was proposed that mtor forms noncovalent interactions and the loss of interaction with these critical regulators during cell breakage may account for the lack of robust in vitro kinase activity (13, 15). Indeed, by modifying the detergents used in cell lysis, the interaction between mtor and its partners can be preserved. Thus, understanding how mtor kinase activity is controlled would require the elucidation of exact conditions that will allow purification of intact mtor complexes and the identification of other

5 TARGET OF RAPAMYCIN 487 regulators, particularly those that positively regulate mtor activity. Two other possible explanations as to why mtor kinase activity is uninducible in vitro are: 1) that these growth stimuli do not regulate the kinase activity of mtor and 2) the activity of mtor complexes is subject to negative regulation. First, we will consider how growth factors such as insulin and the signaling cascade it triggers may regulate mtor, because the components of this pathway are more well characterized in contrast to the nutrient signaling pathway. Insulin triggers the activation of the class I phosphatidylinositol 3-kinase (PI3K). PI3K generates the phosphoinositides, PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3, both important second messengers that can bind signaling molecules (65). Generation of these phosphoinositides is important for the phosphorylation and activation of Akt, among other targets. Akt activation promotes cell proliferation or enhances cell survival (66). Akt is phosphorylated at the hydrophobic motif (HM) site Ser473, which optimally activates Akt when Thr308 of the activation loop is also phosphorylated by PDK1. mtorc2 can mediate the phosphorylation at the HM site (17, 64). These findings suggest that mtor may be regulated by a PI3K-generated signal. A parallel mechanism may also exist for the regulation of mtorc1. A class III PI3K, hvps34, is modulated by amino acid availability (67, 68). How amino acids can activate hvps34 is currently unknown, but its orthologue in yeast was recently shown to be activated by a Ga protein at the endosome to promote pheromone signaling (69). Speculatively, a similar mechanism may signal the presence of amino acids to TOR. The product of hvps34 activation, phosphatidylinositol 3-phosphate, is known to mediate recruitment of proteins to endosomal membranes (70). The activation of the mtorc1 target S6K1 requires hvps34, but whether the hvps34-generated phospholipids can regulate S6K remain to be elucidated. Although there is no class I PI3K in yeast, the class III Vps34 exist in S. cerevisiae and functions in autophagy (71), a process that is negatively regulated by TORC1. Hence, this primitive PI3K may have retained its function in coupling nutrient signals to TORC1. Thus, a model for TOR activation would be that growth stimuli activate PI3K, which generates phospholipids to regulate TOR/mTOR. However, this model may be incomplete. mtorc2 also mediates the phosphorylation of the AGC kinase PKCa at the homologous HM site (11, 18). Phosphorylation of PKC at the activation loop and presumably at other sites in its carboxyl tail, which includes the HM site, leading to protein maturation does not require phosphoinositides (72). Furthermore, the mtorc2-dependent HM site phosphorylation of PKCa is not abrogated upon withdrawal of growth stimuli (18). The TORC2-mediated HM site phosphorylation of AGC kinases is also conserved in yeast (37, 73, 74). A requirement for PI3K in TORC2 (nor TORC1) function in yeast has not been demonstrated either. There are some clues, however, that sphingolipids may regulate the TORCs in this organism. Phytosphingosine activates PKH1, which in turn phosphorylates the AGC kinases such as YPK1/2 and SCH9 at their activation loop (75). TOR can optimally activate these kinases by phosphorylation of YPK1 (via TORC2) and SCH9 (via TORC1) at their HM site. Phosphorylation of these kinases is increased by the presence of phytosphingosine despite the absence of phosphorylation by PKH1, suggesting that this sphingolipid may exert its effect via the TORCs (75, 76). Therefore, although PI3K may contribute to the regulation of TORC functions, it is probably not the sole regulator of the TORCs and may not even regulate TOR kinase activity per se. Lipids, including phospholipids, sphingolipids, and phosphatidic acid (as described earlier), may regulate the TORCs. It is possible that lipids or lipid-dependent signals promote TOR recognition or accessibility to substrates. For example, upon PI3K activation, Akt, via its pleckstrin homology (PH) domain, localizes to the membrane, where it becomes phosphorylated by mtor at the HM site. A parallel scenario may occur for S6K1 and 4E-BP. Cell stimulation promotes the binding of mtorc1 to the translation initiation complex eif3, where it phosphorylates S6K and 4E-BP (77). Thus, mtor activity may depend on the signaling complex or compartment it associates with. Whether mtor or any of its complex partners undergo compartmentalization in response to the generation of phosphoinositides or other lipids remain to be demonstrated. The presence of intracellular amino acids has also been examined as a possible mechanism to regulate mtor (78). If mtor activity were regulated by intracellular amino acid levels (as opposed to amino acid surface transporter/permease-coupled activation), it would be predicted that increasing this intracellular pool should activate mtor. Indeed, when various protein synthesis inhibitors that block either translation initiation or elongation were used to increase amino acid levels intracellularly, insulin stimulation increased phosphorylation of the mtorc1 targets S6K and 4EBP in amino acid-deprived cells (79). In support for a role of mtorc1 in this response, upon inhibition of protein synthesis, the expression of the mtorc1 repressor REDD1 (repressor regulated in development and DNA damage responses; also known as RTP801/Dig2/DDIT4) was decreased (80). REDD1 is known to inhibit mtorc1 signaling via positive regulation of the tumor suppressor proteins TSC1/2 (see later). However, these studies also found that there could be other proteins other than REDD1 involved in this response (80). Hence, several negative regulators may critically control mtorc1 (and mtorc2) signaling. To date, the receptor(s) or sensor(s) for intracellular amino acids or their metabolite that couples these signals to TOR or its negative regulators remain elusive. Several negative regulators that form interactions with TOR or its complex partners are being uncovered (Fig. 3). In both yeast and mammals, phosphatases have been demonstrated to play a role in downregulating TOR signals. The yeast phosphatase regulator TAP42 associates with TORC1 on membrane structures and is likely a direct inhibitor of TORC signaling (81). The TAP42/phosphatase complex is released into the cytosol upon rapamycin treatment or nutrient deprivation. These findings suggest that the phosphatases are tethered to and are

6 488 JACINTO Figure 3. The two mtor protein complexes (mtorc1 and mtorc2). The model depicts that these protein complexes function in the membrane periphery. While mtorc functions are promoted by growth stimuli, it is not clear how these signals affect mtor activity or assembly. Distinct mtor complexes are predicted to function in different cellular compartments with perhaps specific regulators to mediate local functions. mtorc1 is in dark green while mtorc2 is in light green. Negative regulators are in red. Nonintegral complex interactors are in hexagon. AGC kinases that are known to be phosphorylated via mtorcs are in blue. Arrow indicates positive regulation; bars denote negative regulation. It is currently unknown if PRR5 can positively or negatively regulate rictor. kept inactive by TORC1 in the membrane when nutrients are abundant. The nature of the TAP42/phosphatase association with TORC1 remains to be resolved. Also, there is currently no evidence that the mammalian homologue of TAP42 functions in the mtor pathway. Identification of a phosphatase that can bind either TORC1 or TORC2 would provide an important link as to how TORC activity toward its substrate could be regulated. If indeed phosphatases become excluded from the TORCs when growth signals are limiting, this may explain why in vitro TOR activity is similar in induced and noninduced conditions. Several proteins that inhibit TORC1 activity (such as PRAS40, FKBP38) were recently identified and discussed later. Because these nonconserved negative regulators are not integral components of mtorcs, these findings suggest that specific negative regulators could couple mtorc signals that lead to distinct functions. Hence, the presence (or absence) of a specific negative regulator may be required to assay TOR in vitro kinase activity accurately toward a particular substrate. REVVED UP BY RHEB An upstream activator of mtor (at least as part of mtorc1) is the small GTPase Rheb, a member of the Ras superfamily of GTP-binding proteins. Members of this superfamily become activated by their ability to bind GTP and are inactivated by a GTPase activating protein (GAP) that converts it to its GDP-bound inactive form. Rheb binds directly to the amino terminal small lobe of the mtor catalytic domain (82). This association is strongly inhibited by withdrawal of extracellular amino acids (82). mtor can bind to both the active (Rheb-GTP) and inactive (Rheb-GDP) form of Rheb. However, the active form of Rheb, when bound to mtor, potentiates protein kinase activity of mtor (83). Interestingly, the mtor partner LST8, a small G beta-like protein, binds to the segment of mtor overlapping with the Rheb binding region. The binding of LST8 and Rheb to this segment is not competitive or cooperative (84). Overexpression of LST8 also stimulates mtor kinase activity but does not rescue S6K phosphorylation from amino acid deprivation. In LST8 -/- cells, only the functions of mtorc2 were disrupted. Thus, while Rheb may not activate mtorc2 (85), it is plausible that LST8 may perform a similar function in the activation of mtor as part of this complex. If Rheb can activate mtor, either Rheb or the signal regulating Rheb is presumably modulated by growth stimuli. The GTP loading of Rheb was moderately increased upon addition of amino acids to starved cells (86), suggesting that the pres-

7 TARGET OF RAPAMYCIN 489 ence of amino acids can be sensed at this level or upstream of it. Rheb activity is negatively regulated by TSC2, a tumor suppressor protein that contains a GAP domain in its C-terminus (84). TSC2 is found in complex and functions together with TSC1. The TSC proteins serve as a major hub to convey the presence of stress conditions or diminishing growth signals and thereby downregulate TORC1 signaling via modulation of Rheb activity. TSC2 is phosphorylated by numerous kinases that play a role in the regulation of growth, including Akt, Rsk, and ERK (87 89). The presence of growth signals, which activates these kinases, leads to the phosphorylation and inactivation of TSC2 concomittantly relieving its negative inhibition of Rheb. In the case of Akt, growth factors such as insulin activate Akt via PI3K and mtorc2 signals. Akt, by phosphorylating TSC2, upregulates Rheb activity leading to increased mtorc1 signaling. The phosphorylation of TSC2 by Akt was proposed to promote the translocation of TSC2 to the cytosol and partitions it away from Rheb (90). This is consistent with the findings that Rheb farnesylation, a modification that allows their membrane localization, is required for its ability to promote mtor signaling (91). Under stress or suboptimal growth conditions, TSC2 becomes activated either directly or indirectly, to consequently diminish TORC1 signals and inhibit protein synthesis. AMPK, a protein kinase that is activated under conditions of low energy (high AMP/ATP ratio), can phosphorylate and activate TSC2 leading to decreased TORC1 signaling (92). AMPK (S. pombe Ssp2) and Tsc1/Tsc2 are both present in fission yeast and may function in a similar or parallel pathway as TORC1 to regulate amino acid uptake (35) (A. Lorberg, personal communication). In budding yeast, AMPK (S. cerevisiae SNF1) orthologue also responds to energy conditions, but how it conveys this to TORC1, if at all, is not understood because the TSC proteins are absent in this organism. Other proteins that regulate the Akt/TSC/Rheb branch have been identified, which ultimately modulate TORC1 signals. REDD1, as discussed earlier, regulates TSC1/TSC2 to inhibit mtorc1 signaling in response to hypoxia and other stress conditions (93). Bnip3 (Bcl2-homology 3 domain containing protein) also inhibits mtorc1 activity by directly binding to and inhibiting the GTPase Rheb in response to hypoxia (94). TRB3 binds to and inhibits Akt activation and consequently diminishes mtorc1 signaling in hepatocytes (95). Cumulatively, these findings strongly link Rheb and TSC as critical mtor regulators to communicate the presence of growth signals. TSC1/TSC2 are not conserved in budding yeast, although a Rheb homologue (RHB1) is present and functions in amino acid uptake (96). The role of RHB1 in S. cerevisiae TOR regulation remains to be studied. TORC1 in fission yeast is also regulated by Rheb, which in turn is also controlled by a Tsc1/Tsc2 complex. Several activating mutations clustered in the FAT and kinase domains of fission yeast Tor2 (part of TORC1), which conferred Rheb-independent growth were identified (97). Mutation of two analogous sites in mammals also led to nutrient-independent mtor signaling (97). Whether these mutations relieve TOR from an inhibitory regulator/ constraint or if they promote an active conformation or positive regulation of TOR remain to be demonstrated. Clearly, how Rheb may couple the signals from nutrients would provide important clues on the mechanism of mtor complex activity regulation. To further understand Rheb action, a screen for Rheb-interacting proteins was conducted (98). The GTP-bound form of Rheb was found to interact with FKBP38, a member of the FK506-binding protein family. This prolyl isomerase can also bind a region of mtor that encompasses the FRB and thereby inhibits mtor (98). FKBP38 is highly related to FKBP12 but additionally contains other motifs that include a transmembrane domain in its C-terminal half (99). However, the FKBP12-like region of FKBP38 was sufficient to bind mtor (98). This inhibition of mtor by FKBP38 is antagonized by Rheb, which binds to FKBP38 in response to growth stimuli and in a GTPdependent manner. The finding that Rheb-GTP may squelch the inhibitory effect of FKBP38 could explain previous observations that Rheb-GTP binds to mtor less efficiently than Rheb-GDP. How FKBP38 may inhibit TOR activity remains to be examined, but so far it was also shown to bind mtorc1 components. Because FKBP12 has not been found to bind mtor without rapamycin, it is curious why the FKBP12 homologous region of FKBP38 itself binds to mtor. FKBP38 seems to be an integral mitochondrial membrane protein (99). Future studies should reveal if it plays a specific role in mtor regulation in this compartment. PARTNERING TOR Conserved and nonconserved subunits associate with TOR/ mtor via its different domains and regulate its activity. The N-terminal region of TOR, like other members of the PIKK family, consists of several conserved domains that have been shown to interact with other proteins (6). The bulk of the TOR N-terminus is comprised of HEAT (Huntingtin, elongation factor 3, regulatory A subunit of PP2A, TOR) repeats. In both yeast and mammals, KOG1 and raptor, respectively, binds to this domain of TOR. KOG1 in budding yeast is the product of an essential gene and contains four internal HEAT repeats and seven C-terminal WD40 repeats. Three phosphorylated sites of unknown function located distal to the HEAT repeats were identified in both human raptor and fission yeast Mip1 (36, 100). In mammals, raptor also binds to mtor (7, 13, 15), and this association was shown to be weakened by rapamycin (13, 101). The association has also been reported to be nutrient regulated, such that a loose association of raptor to mtor is found in the presence of nutrients (13). Another group reported that the raptor-mtor interaction is not nutrient sensitive (101), consistent with the KOG1-TOR1 interaction in budding yeast (7). Raptor is thought to serve as a scaffold to present substrates to mtor but does not alter intrinsic catalytic activity of mtor (15). It binds the mtorc1 substrates, S6K and 4E-BP, via their TOS motif (102). By electron microscopy and structural analy-

8 490 JACINTO sis, the TOR HEAT repeats were shown to form a curved tubular-shaped domain that associates with the C-terminal WD40 repeat domain of KOG1 (16). These findings suggest that the N-terminus of KOG1, which also contains HEAT repeats, is in close proximity to the TOR kinase domain and could regulate substrate binding to TOR. The HEAT repeats are followed by the FAT region (FRAP/ TOR, ATM, TRRAP), a domain that is conserved among PIKK members. The FAT domain occurs in tandem with the FATC domain, an 30 amino acid residue at the tail end. Together, the FAT and FATC domains of mtor flank the kinase domain and have been shown to be important for its catalytic activity. Mutations in these domains abolished mtor autophosphorylation activity as well as mtor-dependent phosphorylation of 4E-BP and S6K (41, 103). It was earlier proposed that these domains may play a role in the folding or proper organization of the kinase domain (104). In other PIKK family members, this domain binds to histone acetyltransferase Tip60 to regulate the activation of ATM and DNA-PK in response to DNA damage (105). Proteins that directly bind to the FAT/FATC domains of TOR have yet to be identified. The kinase domain of TOR has been shown to bind to several proteins that regulate TOR activity. Among the conserved TOR partners, LST8, an essential G beta-like protein made up entirely of seven WD40 repeats, has been shown to bind to this region in both yeast and mammals (14). In budding yeast, LST8 is found in both TORC1 and TORC2 and is required for kinase activity (21). In mammals, by overexpression studies, it was shown that mlst8 can bind to mtor and raptor (7). The sensitivity of the raptor and mtor interaction to nutrients and rapamycin occurs in complexes that contain mlst8 (14). Whereas raptor binding to mtor is nutrient sensitive and negatively regulates mtor kinase activity, mlst8 association with mtor is insensitive to nutrients but positively regulates mtor kinase activity. How mlst8 can promote mtor kinase activity is still unclear, but it was speculated that it could contribute to the stability and folding of the mtor kinase domain or could also play a role in the recognition and recruitment of substrates to mtor (14). mlst8 (previously named GbL) protein and mrna levels are upregulated by insulin in adipocytes (106). Interestingly, expression of another G beta-like protein in yeast, CPC2, is induced during utilization of glucose in a manner dependent on the transcriptional regulators FHL1 (forkhead-like transciption factor) and IFH1 (107). These transcriptional regulators are important for transcription of ribosomal protein genes in a TOR-dependent manner (108). Thus, G beta-like proteins, such as LST8, may be required for responses to growth signals and their expression may also be TOR-dependent. In yeast, LST8 plays a role in amino acid biosynthesis and permease transport, both TORregulated processes (109, 110). LST8, in yeast and mammals, is also found in TORC2 and mediates TORC2 functions in actin organization and phosphorylation of AGC kinases (7, 9, 11, 18). mlst8 knockout cells from mice have defective phosphorylation of the mtorc2 targets Akt and PKCa, but surprisingly had normal phosphorylation of the mtorc1 target S6K (18). These results suggest that LST8/mLST8 could play distinct roles in the regulation of TOR/mTOR activity as part of either TORC1 or TORC2. Less is known as to how the TORC2 components rictor (AVO3 in S. cerevisiae) and SIN1 (S. cerevisiae AVO1) can physically bind and regulate mtor. In yeast and mammals, rictor and SIN1 are required for TORC2 structural integrity (10, 12, 21). The kinase activity of S. cerevisiae TOR2 in vitro does not seem to be dependent on AVO3 or AVO1. Rictor contains homology domains among its orthologues, but the functions of these domains are unknown. At least three phosphorylation sites were identified outside of the homology domains in both human rictor and fission yeast Ste20, although their functions remain to be investigated (36, 100). Rictor and SIN1 form a relatively stable complex. Furthermore, their expression levels are dependent on each other (12). In cells with diminished or abolished expression of rictor, SIN1 expression also becomes downregulated. Rictor and SIN1, as part of mtorc2, are required for phosphorylation of Akt at Ser473 of the HM site, but whether they act in a similar manner as raptor in presenting substrates to mtor is not clear. While so far the functions of TORC2 require both rictor and SIN1, SIN1 has other functions that have not been shown to require rictor. In fact, SIN1 was originally identified as a human protein that suppressed the heat shock-sensitive phenotype of an S. cerevisiae strain expressing an activated Ras2 (Val19) (111). The S. pombe Sin1 was later identified to bind the SAPK Sty1/Spc1. Like Sty1, Sin1 is required for stress responses via induction of a number of genes that are under control of Sty1 and its targets, Atf1 and Pap1 transcription factors (112). Although a role for Sin1 was not demonstrated, Tor1 (TORC2) was shown to modulate Sty1/ Spc1 via the phosphatase Pyp2 to inhibit the Sty1-regulated mitotic onset (33). These recent findings further suggest that there is crosstalk between the TOR and MAPK pathways wherein Sin1 may be a critical regulator between the two pathways. In two independent studies, the mammalian SIN1 was identified as a JNK and MEKK2 binding protein (113, 114). SIN1 formed a complex with inactive and nonphosphorylated MEKK2. By binding MEKK2, it prevents MEKK2 activation and subsequent activation of JNKK2, JNK1, and AP-1 reporter gene transcription. Altogether, these studies on SIN1 in different organisms suggest that SIN1 may regulate the MAPK module at different levels. Recent studies in mammals show that knockdown of SIN1 suppressed the responses to osmotic stress-induced phosphorylation of ATF2 and ATF2-mediated transcription, a response known to be mediated by p38/jnk pathway (115). On the other hand, decreasing SIN1 expression did not affect serum-induced phosphorylation of ATF2, a response mediated via the Ras pathway. In these studies, SIN1 was shown to bind directly to both ATF2 and p38. Hence, whether SIN1 has a general function in regulating kinases involved in stress responses remains to be validated.

9 TARGET OF RAPAMYCIN 491 PARTNERING THE TOR PARTNERS Nonconserved proteins were also found to form a complex with either TORC1 or TORC2. Currently, little is known as to how these interactors can regulate the TORCs and if they affect TOR activity. TCO89 (89 kda subunit of TOR complex), the product of a nonessential S. cerevisiae gene originally identified as a protein involved in glycerol uptake under osmotic stress conditions (116), is found in TORC1 (117). Loss of TCO89 leads to rapamycin hypersensitivity and defects in cell wall integrity. Like the other components of TORC1, it localizes to the inner side of the plasma membrane. However, it has distinct localization in vacuolar structures and associates with the vacuolar armadillo repeat protein VAC8 (118), suggesting it performs an additional cellular role that is independent of TORC1 (117). Whether it functions together with TORC1 under particular conditions in the vacuole remains to be examined. TORC2 also physically interacts with the yeast proteins AVO2 (a voracious TOR2 interactor) and BIT61 (61 kda binding partner of TOR2) (7, 117), encoded by nonessential genes. Although the function of these proteins remains to be determined, they were found to associate with SLM1 and SLM2 (synthetic lethal with MSS4), two proteins that mediate TORC2 function in actin cytoskeleton organization (119, 120). SLM1 and SLM2 bind the phospholipids, PtdIns(4,5)P 2, via their PH domains and SLM1 is coordinately targeted to the plasma membrane ( ). The SLMs are phosphoproteins and their phosphorylation requires TORC2 kinase activity in vitro and in vivo (120). SLMs are also phosphorylated during heat stress, which is accompanied by production of sphingolipids (122). Taken together, these findings suggest that the SLMs are putative TORC2 substrates whose phosphorylation may specifically require AVO2 and BIT61. It will be interesting to determine how these TORC2 interactors may regulate TORC2 activity via AVO1, AVO3, and LST8. Several groups have recently identified a protein that binds to and negatively regulates mtorc1. PRAS40 (proline-rich Akt substrate of 40 kda) was previously characterized as a protein phosphorylated by Akt and upon phosphorylation binds to (123). However, its relevance remained unknown until recently. PRAS40 contains a putative TOS motif and this sequence was shown to be required for binding to raptor (124, 125). PRAS40 can interfere with the binding of substrates to mtor and it is phosphorylated by mtorc1 at Ser183 in a rapamycin-sensitive fashion. It inhibits mtor autophosphorylation and kinase activity (126). Phosphorylation of PRAS40 was also inhibited by withdrawal of amino acids (124, 125) and promoted by Rheb (125). PRAS40 binds to raptor in insulindeprived cells. Phosphorylation of PRAS40 at Thr246 by Akt and subsequent association of PRAS40 to is critical for the insulin stimulation of mtor (127). In the presence of insulin and Akt-mediated phosphorylation of PRAS40, the association of PRAS40 and mtorc1 is prevented, which then promotes mtorc1 signaling (128). Although silencing PRAS40 expression can enhance mtorc1 signaling (125, 129), it downregulates IRS-1 and Akt signals (127). These studies provide compelling evidence for the presence of a negative regulatory protein that can directly bind to and inhibit mtorc1. Thus, Akt, via phosphorylation and inhibition of two negative regulators of mtorc1 (TSC2 and PRAS40), communicates the presence of growth signals (insulin) to mtorc1. Unlike TSC2, which antagonizes the positive role of Rheb on mtorc1, PRAS40 can directly inhibit mtorc1. Further work should elucidate the physiological function of PRAS40, although initial studies have shown that its phosphorylation by Akt is required for cell survival (126). These findings on PRAS40 provide a prime example of how mtor activity can be regulated via the regulation of its mtorc partner, raptor. Another proline rich protein, Protor (protein observed with rictor), also called PRR5 (Proline-rich protein 5), was recently identified to bind to rictor, independently of mtor. PRR5/Protor is alternatively spliced with three splice variants. It belongs to the same family of proteins as the S. cerevisiae BIT61, a TORC2 interactor (36). The function of PRR5 is unknown but may play a role in PDGFR signaling (130). It is not required for binding of rictor and SIN1 to mtor (130, 131). The expression or protein stability of PRR5/Protor appears to be controlled by rictor. Knockdown of PRR5/Protor does not significantly reduce phosphorylation of Akt at Ser473. A PRR5- like (PRR5L) protein was also identified to bind to mtorc2 but is not required for mtorc2 integrity (126). PRR5L promotes apoptosis when dissociated from mtorc2. Future studies should reveal if the PRR5 proteins inhibit or activate mtor by regulation of mtorc2 components rictor and SIN1. LOCATION, LOCATION, LOCATION The many functions attributed to TOR may suggest that it is found in different cellular compartments. TOR/mTOR is found in both the cytosol and nucleus and has been localized to specific cellular compartments as well. A significant portion of yeast TOR1 and TOR2 are membrane associated. Both TORC1 and TORC2 fractionate with detergent-resistant membranes, and a number of proteins involved in the regulation of endocytosis and actin cytoskeleton were present in these fractions (57). The HEAT repeats of TOR were shown to be essential for membrane association (58). It is not clear if this interaction is intrinsic or if a HEAT repeat binding protein may mediate membrane interaction. The distal HEAT repeats of mtor were characterized to contain a Golgi and endoplasmic reticulum localization sequence (GLS and ELS) (132). The two HEAT repeats HT18 and HT19 and two intervening interunit spacers (IUS17 and IUS18) may mediate mtor targeting to the Golgi (Fig. 1). Deletion of IUS17 or HT19 led to mislocalization of mtor and the latter mutation could inhibit its function in S6K phosphorylation. These putative targeting sequences do not share similarity to the canonical ER- and Golgi-targeting sequences and could

10 492 JACINTO therefore mediate interaction with ER or Golgi resident proteins instead. The functions of mtor in these compartments remain to be further examined but likely include nutrient permease transport (133). Yeast TOR was also found to localize in the nucleus in a nutrient-dependent and rapamycin-sensitive manner (134). This nuclear localization is essential for 35S rrna synthesis but not for regulation of the TORC1-dependent, PolIItranscribed genes and FHL1/IFH1-dependent ribosomal protein genes (108, ). TOR1 binds the 35S rdna promoter via a helix-turn-helix (HTH) motif found at residues (134). mtor and raptor were also shown to bind the mitochondrial transcription factor yin-yang 1 (YY1) (137). Inhibition of mtorc1 by rapamycin led to defective YY1 activation and interaction with PGC-1a (peroxisome-proliferator-activated receptor coactivator), which then inhibits transcription of genes required for mitochondrial oxidative function (137). These studies suggest that mtor and its complex components may be more physically associated with the genes they regulate. Along these lines, mtor and the fission yeast Tor was found to interact with Tel2, a DNA binding protein implicated in DNA repair and telomere length regulation (36, 138). Mammalian Tel2 was found to regulate the stability of mtor and other PIKK (138). Because fission yeast Tel2 was found in both Tor1 and Tor2 immunoprecipitates, these findings suggest that Tel2 is a common regulator of the two TORCs and that both TORCs may perform a common function. TOR complex components may also play a role in TORC compartmentalization. In budding yeast, LST8 is found in endosomal membranes (139), consistent with its known function in amino acid permease transport. Whether it mediates the interaction of TORC in this compartment remains to be examined. In mammals, at least five alternatively spliced SIN1 isoforms could be generated (113, 114). Three of these isoforms were reported to form three distinct mtorc2 complexes (140). Interestingly, the shorter isoform (55 kda), in complex with mtorc2, can phosphorylate Akt Ser473 even in the absence of insulin stimulation, implying that these complexes may be regulated differently. Among the mtorc components, only SIN1 has been identified to contain a putative PH domain that speculatively may function to colocalize TORC2 with its substrates in the membrane (141). In addition to the core complex members, specific subunits of the complex may couple signals in distinct compartments. Identification of the specific localization of various regulators of TORC signaling should yield important clues as to how distinct TOR complexes mediate a local function. CONCLUSION The identification of two distinct TOR complexes in both yeast and mammals has provided critical insights as to how rapamycin can potently inhibit a subset of TOR/mTOR functions. While we have some clues as to how it may inhibit mtorc1, which are based on findings that it can perturb the raptor-mtor association or mtor kinase activity, the picture is far from being complete. In light of recent findings in fission yeast where rapamycin inhibits TORC2 functions and the finding in mammals that it can also inhibit mtorc2 functions under particular conditions, we need to reassess how this drug can affect the assembly of either TORC1 or TORC2. Furthermore, the dynamics of assembly of these two complexes are also poorly understood at the moment. In yeast, flies, and mammals, it was shown that these complexes form multimers (21, 142, 143). Hypothetically, a fully functional TORC, in the presence of optimal growth signals, may depend on an intact multimer. As growth signals diminish or stress conditions increase, assembly of this multimer may be perturbed (due to changes in modifications and subunit binding) and TORC signals become attenuated. As an analogy, this multimer is probably not like a house of cards wherein it collapses abruptly when one subunit is withdrawn. Instead, it is probably more like a pyramid of blocks where TOR forms the base. When this pyramid is destabilized (while the base is still intact), such as upon removal of upper blocks, TOR may become more susceptible to rapamycin inhibition. Identification of other specific TORC interactors (blocks closer to the top of the pyramid) that do not form stable interactions with TOR but can interact with TOR core complex components and specifically couple the TORC to a cellular function will also provide further insights as to how TOR activity is regulated. These specific interactors, which themselves are regulated, can modulate the expression and modification of the TOR complex components to consequently regulate TOR activity. The role of lipids in this signaling pathway is also a recurring theme and while TOR may not phosphorylate a lipid, it may require these molecules for its activity. Finally, future studies will reveal if compartmentalization of the TORCs and its substrates could critically regulate the activity of TOR. If indeed this were the case, it would suggest that there are several distinct TOR complexes that contain compartment-specific regulators in addition to the core TORC components to mediate the numerous cellular functions of TOR. ACKNOWLEDGEMENTS I thank Anja Lorberg for helpful discussions and communication of unpublished results and Monica Finlan for editing the manuscript. This work was funded in part by grants from the American Heart Association, New Jersey Commission on Cancer Research, and American Cancer Society. REFERENCES 1. Easton, J. B. and Houghton, P. J. (2006) mtor and cancer therapy. Oncogene 25, Chiang, G. G. and Abraham, R. T. 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