- Email: [email protected]
Complexity of the TOR signaling network
Review
TRENDS in Cell Biology
Vol.16 No.4 April 2006
Complexity of the TOR signaling network Ken Inoki and Kun-Liang Guan Life Sciences Institute, Department of Biological Chemistry, Institute of Gerontology, University of Michigan, Ann Arbor, MI 48109, USA
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. Rapamycin is an antifungal agent isolated from Streptomyces hygroscopicus [1]. Rapamycin analogs are clinically used as immunosuppressants in organ transplantation and as inhibitors of restenosis of arteries after angioplasty [2,3]. Current clinical trials indicate the potential of rapamycin as an anti-cancer drug because it inhibits cell growth [4]. The target of rapamycin (TOR) was initially isolated by genetic screens in yeast [5] and is a member of the phosphatidylinositol kinase-related kinase (PIKK) family [6], which consists of large molecular weight protein kinases, including TOR, ATM (ataxia-telangiectasia mutated), ATR (ATM and Rad3-related), DNA-PK (DNA-dependent protein kinase) and hSMG1 (suppressor with morphological effect on genitalia) [7]. Although the sequence of the catalytic domain of PIKK family members is closer to that of phosphatidylinositol kinases, PIKK enzymes have only protein kinase activity [7]. Despite the name ‘target of rapamycin’, rapamycin binds to the immunophilin FKBP12 [8]. Together, the rapamycin– FKBP12 complex inhibits TOR function by binding TOR specifically [5,9]. Because of its high potency and specificity to TOR, rapamycin has been used to probe the biological functions of TOR. TOR is structurally and functionally conserved from yeast to mammals. Yeast have two TOR genes, TOR1 and TOR2 [10], whereas higher eukaryotes have a single TOR gene [11]. TOR exists in multiprotein complexes and has an essential role in regulation of cell growth and size by modulating transcription, translation, ribosomal biogenesis and cell morphology [11]. Rapamycin treatment causes a significant size reduction of mammalian cells in Corresponding author: Guan, K.-L. ([email protected]). Available online 3 March 2006
culture [12]. Genetic inactivation of the Drosophila TOR gene (dTOR) also causes a dramatic reduction in cell size [13,14]. The TOR pathway is regulated by many intracellular and extracellular signals. For example, mammalian TOR (mTOR) is activated by growth factors and nutrients, such as amino acids [15]. Conversely, it is inhibited by numerous stress conditions, such as cellular energy depletion, hypoxia and osmotic stress [16–18]. Therefore, the TOR pathway must integrate both positive and negative signals to regulate cell growth in a coordinated manner. Several recent reviews have covered the regulation and function of TOR in cell growth [11,19,20]. Here, we focus mainly on new developments in the TOR field, specifically those regarding the TOR complexes (TORC) and their functions. TOR complexes in yeast Early studies in Saccharomyces cerevisiae indicated that TOR has at least two separable cellular activities because not all of its functions are sensitive to inhibition by rapamycin [21]. Mutations in either TOR1 or TOR2 confer resistance to growth inhibition by rapamycin [5], indicating that these genes overlap in a function required for cell growth at G1. However, despite this convergence in function, TOR1 and TOR2 are not completely redundant with respect to each other. Moreover, rapamycin treatment causes a G1-specific arrest in yeast, whereas deletion of TOR2 is lethal but does not cause the same arrest [21,22], suggesting that some functions of TOR2 are not inhibited by rapamycin. Significantly, TOR2, but not TOR1, is involved in cellular polarization and cytoskeletal reorganization [23]. These observations indicate that the yeast TOR genes have two distinct functions, one of which is sensitive to rapamycin. The molecular mechanism for the two distinct TOR functions in yeast was not appreciated until the purification of two separate TOR complexes (TORC1 and TORC2) by Hall and colleagues [24]. TORC1 contains Kog1 (Kontroller of growth 1), Lst8 (Lethal with SEC thirteen) and either Tor1 or Tor2 (Table 1). The purified TORC2 contains Avo1 (Adheres voraciously to Tor2), Avo2, Avo3, Lst8 and Tor2 (Table 1) [24]. In vitro biochemical characterization shows that the rapamycin–FKBP12 complex directly binds to TORC1 but not to TORC2; therefore, only TORC1 function is inhibited by rapamycin. More recently, Tco89 and Bit61 were found to be novel
www.sciencedirect.com 0962-8924/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2006.02.002
Review
TRENDS in Cell Biology
Vol.16 No.4 April 2006
207
Table 1. Components of TOR complexes
TORC1
TORC2
Saccharomyces cerevisiae Tor1, Tor2 Lst8 Kog1 Tco89 Tor2 Lst8 Avo1 Avo2 Avo3 Bit61
Caenorhabditis elegans ceTOR AAB42347.1 ceRaptor
Drosophila melanogaster dTOR dGbL dRaptor
Homo sapiens
ceTOR AAB42347.1
dTOR dGbL
mTOR mLST8/GbL
CAB042201.1 CAB54288.1
dRictor
Rictor/mAVO3
components of TORC1 and TORC2, respectively [25]. Consistent with the protein interaction data, deletion of TCO89 increases sensitivity to rapamycin, a phenotype similar to TOR1 deletion. Two additional proteins, Slm1 and Slm2, interact with components of TORC2 [26,27]. However, further studies are needed to conclude whether these proteins are integral components of TORC2. Taken together, these studies demonstrate that TOR functions in two distinct multi-molecular complexes and this explains early observations that rapamycin only inhibits part of yeast TOR2 function. In yeast, TORC1 controls cell growth by regulating multiple processes including transcription, translation, ribosomal biogenesis and autophagy [24,28,29]. Recent studies have shown a molecular mechanism by which TORC1 stimulates expression of genes encoding ribosomal proteins [30–32]. The yeast forkhead transcription factor Fhl1 is important for ribosomal gene expression, as it binds directly to ribosomal gene promoters [33]. To stimulate ribosomal gene expression effectively, Fhl1 must associate with Ifh1, a transcription co-factor; however, the interaction between Fhl1 and Ifh1 can be competed for by Crf1, a transcriptional repressor. Crf1 is regulated by Yak1, which is indirectly inhibited by TORC1 [32]. Under nutrient-deficient conditions, the Yak1 protein kinase is active and phosphorylates Crf1, which then relocates into the nucleus and displaces Ifh1 from Fhl1 at the ribosomal gene promoters; therefore, transcription of ribosomal genes is inhibited. Under nutrient-sufficient conditions, active TORC1 inhibits Yak1 through Pka. Inhibition of Yak1 leads to dephosphorylation and the relief of inhibition of Fhl1 by Crf1; therefore, ribosomal gene transcription is increased. Taken together, these studies reveal a biochemical mechanism connecting ribosomal gene regulation and TOR pathway activity [32]. TORC1 in cell-growth regulation Rapamycin treatment inhibits mammalian cell growth [12]. Expression of a rapamycin-resistant mTOR mutant confers resistance to growth inhibition by rapamycin [34,35]. Therefore, the growth-inhibitory effect of rapamycin on mammalian cells is mediated solely through mTOR inhibition. The best-known targets of mTOR are ribosomal S6 kinase 1 (S6K) and the eukaryotic initiation factor 4E binding protein 1 (4EBP1) [11]; rapamycin potently and rapidly induces dephosphorylation of these proteins [11]. mTOR directly phosphorylates Thr389 of S6K, which is essential for S6K kinase activity and S6K, in www.sciencedirect.com
mTOR mLST8/GbL Raptor
turn, phosphorylates ribosomal S6 protein [36]. A knockin of S6 protein with the S6K phosphorylation site eliminated (rpS6pK/K) does not affect 5 0 -TOP (track of polypyrimidine) mRNA translation but instead enhances total protein synthesis [37]. Interestingly, rpS6pK/K mouse embryo fibroblasts (MEFs) are significantly smaller than wild-type MEFs. Furthermore, the size of rpS6pK/K cells, unlike wild-type MEFs, is not decreased by rapamycin. These observations indicate that S6 phosphorylation has a crucial role in mediating the effect of mTOR on mammalian cell-size regulation. Phosphorylation of 4EBP1 by mTOR relieves the inhibitory activity of 4EBP1 towards eukaryotic initiation factor 4E (eIF4E) [38]. eIF4E recognizes the cap structure at the 5 0 end of most eukaryotic mRNA; this provides a potential mechanism by which mTOR can stimulate translation initiation [39,40]. Together, regulation of cell growth and protein translation are the best-characterized functions of mTOR in mammalian cells. Using chemical crosslinking and immunoaffinity purification, Raptor was isolated as an mTOR-associated protein [41,42]. Interestingly, Raptor is homologous to the yeast Kog1 protein in TORC1 that was identified by Loewith et al. [24]. These authors have also isolated Lst8 as a component of TORC1 and TORC2 in yeast. They identified the mammalian counterpart of yeast Lst8 (mLST8) and demonstrated its interaction with mTOR. mLST8/GbL has an essential role in the phosphorylation of S6K and 4EBP1 by TORC1 and in the regulation of cell size [43]. These studies demonstrate the structural and functional conservation of TORC1 in eukaryotes (Table 1). Both S6K and 4EBP1 contain a short sequence termed a TOR signaling (TOS) motif that is important for phosphorylation by mTOR [44] and, interestingly, is important for TORC1 substrates to bind to Raptor [45–47]. This demonstrates that mTOR is the catalytic subunit in TORC1 and that Raptor is involved in substrate recognition. However, the function of Raptor is more complex than being a simple substrate-recruiting component in TORC1. Knockdown of Raptor also significantly reduces mTOR protein levels, suggesting that Raptor stabilizes mTOR [41]. Furthermore, the binding between Raptor and mTOR is sensitive to stimulations that affect mTOR kinase activity, such as by amino acids [41]. One group has proposed that Raptor and mTOR form a nutrient-sensitive complex [41]. Based on this model, nutrient deprivation, such as leucine starvation, results in
208
Review
TRENDS in Cell Biology
a tight mTOR–Raptor complex that has low activity towards S6K although the interaction between mLST8 and mTOR is unaffected by nutrients. By contrast, stimulation with leucine results in a loose mTOR–Raptor complex that has high activity. Consistently, removal of Raptor from immunoprecipitated mTOR by washing with Triton X-100 increased mTOR kinase activity towards S6K [41]. However, contradictory results were reported when Raptor was removed from immunoprecipitated mTOR by washing with NP-40 [42]. This yielded decreased mTOR kinase activity towards 4EBP1. In the study [42], nutrient starvation did not affect the interaction between Raptor and mTOR. One explanation for this discrepancy is that the first study analyzed the interaction of endogenous proteins, whereas the second tested overexpressed proteins, possibly indicating that nutrients regulate the interaction between mTOR and Raptor under physiological conditions but not under overexpression conditions. Moreover, the two groups used different substrates in the mTOR kinase assays. Further work is needed to clarify whether Raptor complexes with mTOR in two states in vivo, one tight with low activity and the other loose with high activity. The function of mLST8/GbL in TORC1 is less well defined. Co-expression of mLST8/GbL with mTOR enhances mTOR kinase activity [43], and mLST8/GbL is required for mTOR to be activated by amino acids. This indicates an important role of mLST8/GbL in mediating upstream signaling to mTOR activation. Furthermore, knockdown of mLST8/GbL by RNA interference decreases phosphorylation of S6K and 4EBP1, supporting the functional importance of mLST8/GbL in TORC1 function [41,43]. In addition to regulating cell growth, TOR functions in cell-size regulation, and mutations in dTOR cause a dramatic reduction in organ and cell size [13,48]. Rapamycin treatment decreases the size of many cell types [12]. The functions of Raptor and mLST8/GbL in cell-size regulation have also been confirmed [41,43]. Knockdown of either Raptor or mLST8/GbL by RNA interference causes a cell-size reduction in HEK293 cells that is similar to that caused by mTOR knockdown. These studies further establish that Raptor and mLST8/GbL are essential components of TORC1 in cell-growth regulation. TORC2 and new functions of TOR mTOR exists in two different complexes in mammalian cells. A TORC2 complex that is similar to that in yeast has been recently identified in mammalian cells and contains mTOR, Rictor/mAVO3 and mLST8/GbL (Table 1) [49,50]. The mammalian TORC2 is also resistant to rapamycin inhibition. Co-immunoprecipitation and co-purification experiments show that Raptor and Rictor/mAVO3 exist in two mutually exclusive complexes, TORC1 and TORC2, respectively [49,50]. Knockdown of Rictor does not affect phosphorylation of S6K, whereas knockdown of any TORC1 component, including mTOR, Raptor and mLST8/GbL, decreases S6K phosphorylation. Similar to the results observed in yeast, the TORC2 complex has an important role in regulation of cell morphology, possibly by modulating actin polymerization [49,50]. Knockdown of www.sciencedirect.com
Vol.16 No.4 April 2006
any component of the TORC2 complex, including mTOR, mLST8/GbL and Rictor/mAVO3, causes a similar alteration of cell morphology and cell adhesion. By contrast, downregulation of Raptor has little effect on cell morphology. However, Sarbassov et al. showed that knockdown of Rictor/mAVO3 and mTOR results in an increase in stress-fiber formation in HeLa cells [49]. By contrast, Jacinto et al. reported that knockdown of mTOR, mLST8/ GbL and Rictor/mAVO3 decreased stress fibers in NIH3T3 cells [50]. There is no apparent explanation for these contradictory results except that different cell lines were used in the two studies. Collectively, it is clear that TORC2 has a rapamycin-insensitive function that regulates cell morphology and cytoskeletal reorganization; however, further investigation is required to elucidate the precise role of TORC2 in stress-fiber formation. TORC2 modulates cytoskeletal structure through the Rho family small GTPases, including Rho, Rac and Cdc42 [23,50] (Figure 1). Expression of active Rac or Rho suppresses the actin defects caused by the downregulation of TORC2 [50]. Consistent with this model, knockdown of mTOR, mLST8/GbL or Rictor/mAVO3 decreases Rac GTP levels, suggesting that TORC2 functions upstream of Rac. Recent studies have also suggested that TORC2 regulates protein kinase C a (PKCa) activity to control the actin cytoskeleton [23,49]. Knockdown of either mTOR or Rictor/mAVO3, but not Raptor, significantly decreases PKCa phosphorylation [49]. PKCa has an important role in cytoskeleton organization [51]. Interestingly, studies in yeast have already demonstrated a pathway in which TOR regulates cytoskeleton organization through Rho and the yeast PKCa equivalent, PKC1 [23]. Therefore, TORC2 uses an evolutionary conserved pathway involving Rho and PKCa to regulate cell morphology. AKT [also known as protein kinase B (PKB)] has been shown to act upstream of TORC1 and indirectly stimulates TORC1 activity. Unexpectedly, TORC2 phosphorylates and activates AKT [52]. AKT has essential roles in many aspects of cellular regulation, including stimulation of cell growth, proliferation, survival and metabolism [53]. Its activation requires the phosphorylation of Thr308 in its activation loop by PDK1 (3-phosphoinositide-dependent protein kinase-1) and the phosphorylation of Ser473 in its C-terminal hydrophobic region by a second kinase activity termed PDK2 [54,55] (Figure 1). The identity of PDK2 remained unknown although several kinases had been implicated [56,57]. Interestingly, Sarbassov et al. showed that TORC2 has a crucial role in the phosphorylation of the motif on AKT that is targeted by PDK2 [52]. Using an RNA interference-based study in Drosophila S2 cells, Sarbassov discovered that knockdown of dTOR significantly reduces phosphorylation of the PDK2 site on dAKT [52]. Importantly, knockdown of Rictor/AVO3, but not Raptor, decreased AKT phosphorylation in Drosophila and mammalian cells. In vitro kinase assays with immunoprecipitated mTOR complexes provide direct biochemical evidence that TORC2, but not TORC1, can phosphorylate Ser473 of AKT in vitro and that phosphorylation of AKT by TORC2 promotes the in vitro phosphorylation of AKT by purified PDK1 [52]. These observations demonstrate that TORC2 phosphorylates the
Review
TRENDS in Cell Biology
Insulin IGF1
IRS
Vol.16 No.4 April 2006
209
Insulin IGF1
Amino acids
PI3K
PIP3
PIP3
PDK1
AKT
PIP3 PDK1
S6K
IRS Negative feedback
mTORC2 Rho
TSC2 TSC1
Cytoskeleton
Rheb 4EBP1
Cell survival Cell growth Glycogen metabolism
PTEN
mTORC1
Rac PKCα
PI3K
S6
Protein translation Cell growth regulation
TRENDS in Cell Biology
Figure 1. Signaling network of TORC1 and TORC2. TORC1 (consisting of mTOR, Raptor and mLST8) phosphorylates S6K and 4EBP1, and therefore regulates translation and cell growth. Rheb stimulates TORC1 activity and itself is inhibited by the TSC1/TSC2 complex. TSC2 is phosphorylated and inhibited by AKT, which is activated by growth factors through the PI3K and PDK1 cascade. Therefore, the mTOR pathway has an important role in mediating growth factor signals to cell growth. PTEN is a tumor suppressor gene that antagonizes the action of PI3K by hydrolyzing PIP3, therefore, PTEN inhibits the mTOR pathway. S6K is involved in the feedback inhibition of AKT by phosphorylating and inactivating IRS, which has a key positive role in PI3K activation in response to insulin. In addition, TORC2 (consisting of mTOR, Rictor and mLST8) can directly phosphorylate and activate AKT, although it is unclear how TORC2 is regulated. TORC2 has also been implicated in modulating cytoskeleton organization through PKC and the Rho family GTPases. Red diamonds indicate phosphorylation. Broken arrows indicate undefined regulation. The circled minus signs indicate negative regulatory effects.
PDK2 site of AKT in vitro and in vivo. Therefore, TORC2 acts at the center of cell-growth regulation because AKT is a key protein in regulation of cell growth and survival (Figure 1). The PDK2-equivalent site in AKT is also present in many of the AGC kinase family members. SGK and AKT are closely related to each other and are regulated by similar stimuli. It has been shown that yeast YPK2, a homolog of mammalian SGK, was phosphorylated and activated by TORC2 [58]. Therefore, TORC2 might display a much broader cellular function by phosphorylating the presumed PDK2 sites in other AGC family kinases [6]. Because higher eukaryotes have only one TOR gene, the TORC1 and TORC2 complexes must share the same pool of TOR proteins. It has been postulated that Raptor and Rictor compete for binding to mTOR and direct competition between Raptor and Rictor/mAVO3 in the binding of mTOR has been observed in overexpression studies [49]. Moreover, the relationship between TORC1 and TORC2 is complicated by the observation that TORC1 activation results in a negative feedback loop that inhibits the phosophotidylinositol 3 kinase (PI3K) pathway. PI3K activity is essential for phosphorylation of AKT by TORC2, suggesting that TORC2 is activated by PI3K. However, the effect of PI3K on the TORC2 site phosphorylation in AKT could be due solely to the membrane localization of AKT to phosphotidylinositol 3,4,5 trisphosphate (PIP3) generated by PI3K, rather than the activation of TORC2. S6K, a downstream effector of TORC1, is a key component of the feedback loop because S6K phosphorylates and www.sciencedirect.com
inhibits the insulin receptor substrate-1 (IRS1), which is upstream of the PI3K pathway [59,60] (Figure 1). The effect of this negative feedback loop is apparent in TSC (tuberous sclerosis complex) mutant cells. Constitutive activation of TORC1 in TSC mutant cells decreases insulin- or insulin-like growth factor 1- (IGF1-)induced AKT phosphorylation [59,61]. Consistent with this, inhibition of TORC1 with rapamycin restores insulininduced AKT phosphorylation in TSC mutant cells. Importantly, several pathological features, including hamartomas in tuberous sclerosis, can be explained by this negative-feedback mechanism. For instance, inhibition of AKT by the constitutively activated TORC1 is a possible reason why TSC-associated hamartoma syndromes are benign rather than malignant, because AKT has crucial roles in cell growth, survival and migration [62,63]. Activation of TOR by Rheb Several key components that function upstream of mTOR have recently been identified, including TSC1, TSC2 and Rheb (Ras homolog enriched in brain) [64,65]. TSC1 and TSC2 are tumor suppressor genes mutated in tuberous sclerosis [64] and their products form a physical and functional complex. Mutations in either TSC1 or TSC2 induce hamartoma formation in multiple organs and a significant increase in cell size, a phenotype similar to mTOR activation. TSC1 or TSC2 mutant cells have high TORC1 activity [64]. These results establish that an important physiological function of TSC1 and TSC2 is to
210
Review
TRENDS in Cell Biology
inhibit mTOR. Biochemical data demonstrate that TSC2 is a GTPase activating protein (GAP) towards Rheb, which is a small GTPase related to Ras [65]. Interestingly, Rheb stimulates phosphorylation of S6K and 4EBP1, and Rhebinduced S6K phosphorylation is blocked by rapamycin and dominant-negative mTOR. Furthermore, genetic data support that dTOR functions downstream of dRheb. Taken together, these studies establish a signaling pathway from TSC2 to Rheb to mTOR (Figure 1). TSC2 is a physiological substrate of AKT [11] and therefore AKT might activate the mTOR pathway by phosphorylating and inhibiting TSC2 (Figure 1). However, the direct inhibition of TSC2 GAP activity by AKT phosphorylation has not been reported. The regulation of TOR by AKT might not be evolutionarily conserved because two studies have demonstrated that dAKT phosphorylates dTSC2. Potter et al. showed that the phosphorylation of dTSC2 by dAKT has an important role in mediating the growth-stimulating signal from insulin [66]. By contrast, Dong and Pan showed that mutation of the dAKT phosphorylation sites in dTSC2 does not compromise the essential function of dTSC2 in Drosophila development, indicating that TSC2 is not a crucial target of AKT during normal development [67]. A noticeable difference is that one group used overexpression [66] whereas the other did not [67]. Whether dAKT promotes dS6K phosphorylation in Drosophila cells is controversial. Two groups have reported that knockdown of dAKT has little effect on dS6K phosphorylation [52,68], whereas another study shows conflicting results – a strong inhibition of dS6K phosphorylation after dAKT knockdown [69]. We have observed that knockdown of dAKT had little effect on the phosphorylation of dS6K (K. Inoki and K-L. Guan, unpublished). The underlying reasons for this apparent conflict are not clear. The mechanism by which Rheb activates TORC1 is not fully understood. However, a potential mechanism that operates through a direct interaction between Rheb and TORC1 has been implicated [70]. Interestingly, the binding between Rheb and mTOR is inhibited by amino acid depletion [71] and Rheb also associates with Raptor and mLST8/GbL, suggesting that Rheb activates TORC1 through a direct interaction [70]. Rheb–GTP precipitates more mTOR activity than does Rheb–GDP. However, the interaction between Rheb and components of TORC1 is not nucleotide dependent: the GDP-bound or nucleotidefree Rheb interacts with mTOR more strongly than does the GTP-bound Rheb [70,72]. This is perplexing because only Rheb bound to GTP activates TORC1. Recently, an interaction between Rhb1 (the homolog of Rheb) and Tor2 in Schizosaccharomyces pombe has been reported, and the interaction appears to be nucleotide dependent [73]. The interaction between Rhb1 and Tor2 is only detected in a Tsc2–/– strain. Furthermore, constitutively active Rhb1 mutants display a strong interaction with Tor2, whereas the wild-type Rhb1 shows little interaction with Tor2 in wild-type S. pombe. These results strongly imply that Rheb–GTP specifically interacts with TOR, but further studies are needed to understand the molecular mechanism. www.sciencedirect.com
Vol.16 No.4 April 2006
Concluding remarks Recent progress has established and expanded the complexity of the TOR signaling network. A key advance is the identification of the two distinct TOR complexes TORC1 and TORC2, which perform different physiological functions in vivo. Another key development is that TSC2 and Rheb are important upstream regulators of TORC1. These findings have connected the dysregulation of TORC1 to several human diseases and have provided a scientific basis for rapamycin as a potential drug for the treatment of human diseases, especially hamartoma syndromes and cancers [74]. For example, PTEN (protein phosphatase and tensin homolog deleted on chromosome 10) is a tumor suppressor that is frequently mutated in human cancers [4]. PTEN mutations activate AKT and cause elevated TORC1 activity (Figure 1). It has been reported that rapamycin is effective against tumors with PTEN mutations [4]. The identification of TORC2 as the long-sought PDK2 activity further emphasizes the importance of mTOR in cellular growth and survival regulation, and this significantly expands the complexity of the TOR signaling pathway, blurring the up- and downstream relationship between AKT and mTOR. AKT activates TORC1; however, AKT is activated by TORC2. Perhaps, it is necessary to think of TORC1 and TORC2 as two distinct functional entities. Despite exciting and rapid developments in the TOR signaling field, many key questions remain. Little is known about the function of TORC2 except for its role in AKT activation and cytoskeletal regulation. The identification of this new complex raises important questions regarding how or whether TORC2 is activated by the growth factors and how TORC2 regulates cytoskeletal organization. It will be important to determine whether TORC2 is a ‘master kinase’ involved in phosphorylation of many AGC family kinases. It is well established that Rheb stimulates TORC1 function, but little data are available as to whether Rheb also regulates TORC2 and, if so, by what mechanism. Another important question is the mechanism of Rheb regulation. Although Rheb has high basal GTP binding, it is likely to be regulated by a yet-to-be identified GEF. Future studies to identify and characterize the Rheb GEF might provide key information for understanding how mTOR receives and integrates various upstream signals to control cell growth. Furthermore, amino acids are known to be essential regulators of TORC1 activity. However, the precise nature of amino acid sensing is unknown and requires further investigation. Finally, autophagy is an important cellular process and is regulated by TORC1 [28]. Autophagy is the major pathway for degradation of long-lived proteins, especially under nutrient limitation conditions, and is of current interest in cell biology. Future studies are needed to understand the molecular mechanism how mTOR regulates autophagy in mammalian cells. Acknowledgements We apologize to colleagues whose work could not be cited owing to the scope and space limitations. We thank Chung-Han Lee and Michael N. Corradetti for the critical reading of the article. This work is supported by
Review
TRENDS in Cell Biology
grants to K-L.G. from the National Institutes of Health and the Department of Defense.
References 1 Vezina, C. et al. (1975) Rapamycin (AY-22,989), a new antifungal antibiotic. I. Taxonomy of the producing streptomycete and isolation of the active principle. J. Antibiot. 28, 721–726 2 Easton, J.B. and Houghton, P.J. (2004) Therapeutic potential of target of rapamycin inhibitors. Expert Opin. Ther. Targets 8, 551–564 3 Moses, J.W. et al. (2003) Sirolimus-eluting stents versus standard stents in patients with stenosis in a native coronary artery. N. Engl. J. Med. 349, 1315–1323 4 Bjornsti, M.A. and Houghton, P.J. (2004) The TOR pathway: a target for cancer therapy. Nat. Rev. Cancer 4, 335–348 5 Heitman, J. et al. (1991) Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 253, 905–909 6 Manning, G. et al. (2002) The protein kinase complement of the human genome. Science 298, 1912–1934 7 Bakkenist, C.J. and Kastan, M.B. (2004) Initiating cellular stress responses. Cell 118, 9–17 8 Koltin, Y. et al. (1991) Rapamycin sensitivity in Saccharomyces cerevisiae is mediated by a peptidyl-prolyl cis-trans isomerase related to human FK506-binding protein. Mol. Cell. Biol. 11, 1718–1723 9 Chung, J. et al. (1992) Rapamycin-FKBP specifically blocks growthdependent activation of and signaling by the 70 kd S6 protein kinases. Cell 69, 1227–1236 10 Helliwell, S.B. et al. (1994) TOR1 and TOR2 are structurally and functionally similar but not identical phosphatidylinositol kinase homologues in yeast. Mol. Biol. Cell 5, 105–118 11 Hay, N. and Sonenberg, N. (2004) Upstream and downstream of mTOR. Genes Dev. 18, 1926–1945 12 Fingar, D.C. et al. (2002) Mammalian cell size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E. Genes Dev. 16, 1472–1487 13 Montagne, J. et al. (1999) Drosophila S6 kinase: a regulator of cell size. Science 285, 2126–2129 14 Gao, X. et al. (2002) Tsc tumour suppressor proteins antagonize amino-acid-TOR signalling. Nat. Cell Biol. 4, 699–704 15 Hara, K. et al. (1998) Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism. J. Biol. Chem. 273, 14484–14494 16 Dennis, P.B. et al. (2001) Mammalian TOR: a homeostatic ATP sensor. Science 294, 1102–1105 17 Desai, B.N. et al. (2002) FKBP12-rapamycin-associated protein associates with mitochondria and senses osmotic stress via mitochondrial dysfunction. Proc. Natl. Acad. Sci. U. S. A. 99, 4319–4324 18 Brugarolas, J. et al. (2004) Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex. Genes Dev. 18, 2893–2904 19 Harris, T.E. and Lawrence, J.C., Jr. (2003) TOR signaling. Sci. STKE 2003, re15 20 Fingar, D.C. and Blenis, J. (2004) Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression. Oncogene 23, 3151–3171 21 Zheng, X.F. et al. (1995) TOR kinase domains are required for two distinct functions, only one of which is inhibited by rapamycin. Cell 82, 121–130 22 Kunz, J. et al. (1993) Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression. Cell 73, 585–596 23 Schmidt, A. et al. (1997) The yeast phosphatidylinositol kinase homolog TOR2 activates RHO1 and RHO2 via the exchange factor ROM2. Cell 88, 531–542 24 Loewith, R. et al. (2002) Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol. Cell 10, 457–468 25 Reinke, A. et al. (2004) TOR complex 1 (TORC1) includes a novel component, Tco89p (YPL180w), and cooperates with Ssd1p to maintain cellular integrity in S. cerevisiae. J. Biol. Chem. 279, 14752–14756 www.sciencedirect.com
Vol.16 No.4 April 2006
211
26 Fadri, M. et al. (2005) The pleckstrin homology domain proteins Slm1 and Slm2 are required for actin cytoskeleton organization in yeast and bind phosphatidylinositol-4,5-bisphosphate and TORC2. Mol. Biol. Cell 16, 1883–1900 27 Audhya, A. et al. (2004) Genome-wide lethality screen identifies new PI4,5P2 effectors that regulate the actin cytoskeleton. EMBO J. 23, 3747–3757 28 Crespo, J.L. and Hall, M.N. (2002) Elucidating TOR signaling and rapamycin action: lessons from Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 66, 579–591 29 Rudra, D. et al. (2005) Central role of Ifh1p-Fhl1p interaction in the synthesis of yeast ribosomal proteins. EMBO J. 24, 533–542 30 Schawalder, S.B. et al. (2004) Growth-regulated recruitment of the essential yeast ribosomal protein gene activator Ifh1. Nature 432, 1058–1061 31 Wade, J.T. et al. (2004) The transcription factor Ifh1 is a key regulator of yeast ribosomal protein genes. Nature 432, 1054–1058 32 Martin, D.E. et al. (2004) TOR regulates ribosomal protein gene expression via PKA and the Forkhead transcription factor FHL1. Cell 119, 969–979 33 Lee, T.I. et al. (2002) Transcriptional regulatory networks in Saccharomyces cerevisiae. Science 298, 799–804 34 Brown, E.J. et al. (1995) Control of p70 s6 kinase by kinase activity of FRAP in vivo. Nature 377, 441–446 35 Lorenz, M.C. and Heitman, J. (1995) TOR mutations confer rapamycin resistance by preventing interaction with FKBP12rapamycin. J. Biol. Chem. 270, 27531–27537 36 Thomas, G. (2002) The S6 kinase signaling pathway in the control of development and growth. Biol. Res. 35, 305–313 37 Ruvinsky, I. et al. (2005) Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis. Genes Dev. 19, 2199–2211 38 Gingras, A.C. et al. (1998) 4E-BP1, a repressor of mRNA translation, is phosphorylated and inactivated by the Akt(PKB) signaling pathway. Genes Dev. 12, 502–513 39 Haghighat, A. et al. (1995) Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E. EMBO J. 14, 5701–5709 40 Beretta, L. et al. (1996) Rapamycin blocks the phosphorylation of 4EBP1 and inhibits cap-dependent initiation of translation. EMBO J. 15, 658–664 41 Kim, D.H. et al. (2002) mTOR interacts with raptor to form a nutrientsensitive complex that signals to the cell growth machinery. Cell 110, 163–175 42 Hara, K. et al. (2002) Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 110, 177–189 43 Kim, D.H. et al. (2003) GbetaL, a positive regulator of the rapamycinsensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR. Mol. Cell 11, 895–904 44 Schalm, S.S. and Blenis, J. (2002) Identification of a conserved motif required for mTOR signaling. Curr. Biol. 12, 632–639 45 Choi, K.M. et al. (2003) Two motifs in the translational repressor PHAS-I required for efficient phosphorylation by mammalian target of rapamycin and for recognition by raptor. J. Biol. Chem. 278, 19667–19673 46 Nojima, H. et al. (2003) The mammalian target of rapamycin (mTOR) partner, raptor, binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif. J. Biol. Chem. 278, 15461–15464 47 Schalm, S.S. et al. (2003) TOS motif-mediated raptor binding regulates 4E-BP1 multisite phosphorylation and function. Curr. Biol. 13, 797–806 48 Oldham, S. et al. (2000) Genetic and biochemical characterization of dTOR, the Drosophila homolog of the target of rapamycin. Genes Dev. 14, 2689–2694 49 Sarbassov, D.D. et al. (2004) Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr. Biol. 14, 1296–1302 50 Jacinto, E. et al. (2004) Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat. Cell Biol. 6, 1122–1128 51 Nakashima, S. (2002) Protein kinase C a (PKCa): regulation and biological function. J. Biochem. (Tokyo) 132, 669–675
Review
212
TRENDS in Cell Biology
52 Sarbassov, D.D. et al. (2005) Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307, 1098–1101 53 Brazil, D.P. and Hemmings, B.A. (2001) Ten years of protein kinase B signalling: a hard Akt to follow. Trends Biochem. Sci. 26, 657–664 54 Alessi, D.R. et al. (1997) Characterization of a 3-phosphoinositidedependent protein kinase which phosphorylates and activates protein kinase Ba. Curr. Biol. 7, 261–269 55 Chan, T.O. and Tsichlis, P.N. (2001) PDK2: a complex tail in one Akt. Sci. STKE 2001, PE1 56 Rane, M.J. et al. (2001) p38 Kinase-dependent MAPKAPK-2 activation functions as 3-phosphoinositide-dependent kinase-2 for Akt in human neutrophils. J. Biol. Chem. 276, 3517–3523 57 Feng, J. et al. (2004) Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase. J. Biol. Chem. 279, 41189–41196 58 Harrington, L.S. et al. (2004) The TSC1-2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins. J. Cell Biol. 166, 213–223 59 Um, S.H. et al. (2004) Absence of S6K1 protects against age- and dietinduced obesity while enhancing insulin sensitivity. Nature 431, 200–205 60 Shah, O.J. et al. (2004) Inappropriate activation of the TSC/Rheb/mTOR/S6K cassette induces IRS1/2 depletion, insulin resistance, and cell survival deficiencies. Curr. Biol. 14, 1650–1656 61 Manning, B.D. et al. (2005) Feedback inhibition of Akt signaling limits the growth of tumors lacking Tsc2. Genes Dev. 19, 1773–1778 62 Ma, L. et al. (2005) Genetic analysis of Pten and Tsc2 functional interactions in the mouse reveals asymmetrical haploinsufficiency in tumor suppression. Genes Dev. 19, 1779–1786
Vol.16 No.4 April 2006
63 Kwiatkowski, D.J. (2003) Rhebbing up mTOR: new insights on TSC1 and TSC2, and the pathogenesis of tuberous sclerosis. Cancer Biol. Ther. 2, 471–476 64 Manning, B.D. and Cantley, L.C. (2003) Rheb fills a GAP between TSC and TOR. Trends Biochem. Sci. 28, 573–576 65 Potter, C.J. et al. (2002) Akt regulates growth by directly phosphorylating Tsc2. Nat. Cell Biol. 4, 658–665 66 Dong, J. and Pan, D. (2004) Tsc2 is not a critical target of Akt during normal Drosophila development. Genes Dev. 18, 2479–2484 67 Radimerski, T. et al. (2002) dS6K-regulated cell growth is dPKB/dPI(3)K-independent, but requires dPDK1. Nat. Cell Biol. 4, 251–255 68 Lizcano, J.M. et al. (2003) Insulin-induced Drosophila S6 kinase activation requires phosphoinositide 3-kinase and protein kinase B. Biochem. J. 374, 297–306 69 Long, X. et al. (2005) Rheb binds and regulates the mTOR kinase. Curr. Biol. 15, 702–713 70 Long, X. et al. (2005) Rheb binding to mTOR is regulated by amino acid sufficiency. J. Biol. Chem. 280, 23433–23436 71 Smith, E.M. et al. (2005) The tuberous sclerosis protein TSC2 is not required for the regulation of the mammalian target of rapamycin by amino acids and certain cellular stresses. J. Biol. Chem. 280, 18717–18727 72 Urano, J. et al. (2005) Identification of novel single amino acid changes that result in hyperactivation of the unique GTPase, Rheb, in fission yeast. Mol. Microbiol. 58, 1074–1086 73 Inoki, K. et al. (2005) Dysregulation of the TSC-mTOR pathway in human disease. Nat. Genet. 37, 19–24 74 Kamada, Y. et al. (2005) Tor2 directly phosphorylates the AGC kinase Ypk2 to regulate actin polarization. Mol. Cell. Biol. 25, 7239–7248
Reuse of Current Opinion and Trends journal figures in multimedia presentations It’s easy to incorporate figures published in Trends or Current Opinion journals into your PowerPoint presentations or other imagedisplay programs. Simply follow the steps below to augment your presentations or teaching materials with our fine figures! 1. 2. 3. 4. 5.
Locate the article with the required figure in the Science Direct journal collection Click on the ‘Full text + links’ hyperlink Scroll down to the thumbnail of the required figure Place the cursor over the image and click to engage the ‘Enlarge Image’ option On a PC, right-click over the expanded image and select ‘Copy’ from pull-down menu (Mac users: hold left button down and then select the ‘Copy image’ option) 6. Open a blank slide in PowerPoint or other image-display program 7. Right-click over the slide and select ‘paste’ (Mac users hit ‘Apple-V’ or select the ‘Edit-Paste’ pull-down option). Permission of the publisher, Elsevier, is required to re-use any materials in Trends or Current Opinion journals or any other works published by Elsevier. Elsevier authors can obtain permission by completing the online form available through the Copyright Information section of Elsevier’s Author Gateway at http://authors.elsevier.com/. Alternatively, readers can access the request form through Elsevier’s main web site at http://www.elsevier.com/locate/permissions.
www.sciencedirect.com
TRENDS in Cell Biology
Vol.16 No.4 April 2006
Complexity of the TOR signaling network Ken Inoki and Kun-Liang Guan Life Sciences Institute, Department of Biological Chemistry, Institute of Gerontology, University of Michigan, Ann Arbor, MI 48109, USA
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. Rapamycin is an antifungal agent isolated from Streptomyces hygroscopicus [1]. Rapamycin analogs are clinically used as immunosuppressants in organ transplantation and as inhibitors of restenosis of arteries after angioplasty [2,3]. Current clinical trials indicate the potential of rapamycin as an anti-cancer drug because it inhibits cell growth [4]. The target of rapamycin (TOR) was initially isolated by genetic screens in yeast [5] and is a member of the phosphatidylinositol kinase-related kinase (PIKK) family [6], which consists of large molecular weight protein kinases, including TOR, ATM (ataxia-telangiectasia mutated), ATR (ATM and Rad3-related), DNA-PK (DNA-dependent protein kinase) and hSMG1 (suppressor with morphological effect on genitalia) [7]. Although the sequence of the catalytic domain of PIKK family members is closer to that of phosphatidylinositol kinases, PIKK enzymes have only protein kinase activity [7]. Despite the name ‘target of rapamycin’, rapamycin binds to the immunophilin FKBP12 [8]. Together, the rapamycin– FKBP12 complex inhibits TOR function by binding TOR specifically [5,9]. Because of its high potency and specificity to TOR, rapamycin has been used to probe the biological functions of TOR. TOR is structurally and functionally conserved from yeast to mammals. Yeast have two TOR genes, TOR1 and TOR2 [10], whereas higher eukaryotes have a single TOR gene [11]. TOR exists in multiprotein complexes and has an essential role in regulation of cell growth and size by modulating transcription, translation, ribosomal biogenesis and cell morphology [11]. Rapamycin treatment causes a significant size reduction of mammalian cells in Corresponding author: Guan, K.-L. ([email protected]). Available online 3 March 2006
culture [12]. Genetic inactivation of the Drosophila TOR gene (dTOR) also causes a dramatic reduction in cell size [13,14]. The TOR pathway is regulated by many intracellular and extracellular signals. For example, mammalian TOR (mTOR) is activated by growth factors and nutrients, such as amino acids [15]. Conversely, it is inhibited by numerous stress conditions, such as cellular energy depletion, hypoxia and osmotic stress [16–18]. Therefore, the TOR pathway must integrate both positive and negative signals to regulate cell growth in a coordinated manner. Several recent reviews have covered the regulation and function of TOR in cell growth [11,19,20]. Here, we focus mainly on new developments in the TOR field, specifically those regarding the TOR complexes (TORC) and their functions. TOR complexes in yeast Early studies in Saccharomyces cerevisiae indicated that TOR has at least two separable cellular activities because not all of its functions are sensitive to inhibition by rapamycin [21]. Mutations in either TOR1 or TOR2 confer resistance to growth inhibition by rapamycin [5], indicating that these genes overlap in a function required for cell growth at G1. However, despite this convergence in function, TOR1 and TOR2 are not completely redundant with respect to each other. Moreover, rapamycin treatment causes a G1-specific arrest in yeast, whereas deletion of TOR2 is lethal but does not cause the same arrest [21,22], suggesting that some functions of TOR2 are not inhibited by rapamycin. Significantly, TOR2, but not TOR1, is involved in cellular polarization and cytoskeletal reorganization [23]. These observations indicate that the yeast TOR genes have two distinct functions, one of which is sensitive to rapamycin. The molecular mechanism for the two distinct TOR functions in yeast was not appreciated until the purification of two separate TOR complexes (TORC1 and TORC2) by Hall and colleagues [24]. TORC1 contains Kog1 (Kontroller of growth 1), Lst8 (Lethal with SEC thirteen) and either Tor1 or Tor2 (Table 1). The purified TORC2 contains Avo1 (Adheres voraciously to Tor2), Avo2, Avo3, Lst8 and Tor2 (Table 1) [24]. In vitro biochemical characterization shows that the rapamycin–FKBP12 complex directly binds to TORC1 but not to TORC2; therefore, only TORC1 function is inhibited by rapamycin. More recently, Tco89 and Bit61 were found to be novel
www.sciencedirect.com 0962-8924/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2006.02.002
Review
TRENDS in Cell Biology
Vol.16 No.4 April 2006
207
Table 1. Components of TOR complexes
TORC1
TORC2
Saccharomyces cerevisiae Tor1, Tor2 Lst8 Kog1 Tco89 Tor2 Lst8 Avo1 Avo2 Avo3 Bit61
Caenorhabditis elegans ceTOR AAB42347.1 ceRaptor
Drosophila melanogaster dTOR dGbL dRaptor
Homo sapiens
ceTOR AAB42347.1
dTOR dGbL
mTOR mLST8/GbL
CAB042201.1 CAB54288.1
dRictor
Rictor/mAVO3
components of TORC1 and TORC2, respectively [25]. Consistent with the protein interaction data, deletion of TCO89 increases sensitivity to rapamycin, a phenotype similar to TOR1 deletion. Two additional proteins, Slm1 and Slm2, interact with components of TORC2 [26,27]. However, further studies are needed to conclude whether these proteins are integral components of TORC2. Taken together, these studies demonstrate that TOR functions in two distinct multi-molecular complexes and this explains early observations that rapamycin only inhibits part of yeast TOR2 function. In yeast, TORC1 controls cell growth by regulating multiple processes including transcription, translation, ribosomal biogenesis and autophagy [24,28,29]. Recent studies have shown a molecular mechanism by which TORC1 stimulates expression of genes encoding ribosomal proteins [30–32]. The yeast forkhead transcription factor Fhl1 is important for ribosomal gene expression, as it binds directly to ribosomal gene promoters [33]. To stimulate ribosomal gene expression effectively, Fhl1 must associate with Ifh1, a transcription co-factor; however, the interaction between Fhl1 and Ifh1 can be competed for by Crf1, a transcriptional repressor. Crf1 is regulated by Yak1, which is indirectly inhibited by TORC1 [32]. Under nutrient-deficient conditions, the Yak1 protein kinase is active and phosphorylates Crf1, which then relocates into the nucleus and displaces Ifh1 from Fhl1 at the ribosomal gene promoters; therefore, transcription of ribosomal genes is inhibited. Under nutrient-sufficient conditions, active TORC1 inhibits Yak1 through Pka. Inhibition of Yak1 leads to dephosphorylation and the relief of inhibition of Fhl1 by Crf1; therefore, ribosomal gene transcription is increased. Taken together, these studies reveal a biochemical mechanism connecting ribosomal gene regulation and TOR pathway activity [32]. TORC1 in cell-growth regulation Rapamycin treatment inhibits mammalian cell growth [12]. Expression of a rapamycin-resistant mTOR mutant confers resistance to growth inhibition by rapamycin [34,35]. Therefore, the growth-inhibitory effect of rapamycin on mammalian cells is mediated solely through mTOR inhibition. The best-known targets of mTOR are ribosomal S6 kinase 1 (S6K) and the eukaryotic initiation factor 4E binding protein 1 (4EBP1) [11]; rapamycin potently and rapidly induces dephosphorylation of these proteins [11]. mTOR directly phosphorylates Thr389 of S6K, which is essential for S6K kinase activity and S6K, in www.sciencedirect.com
mTOR mLST8/GbL Raptor
turn, phosphorylates ribosomal S6 protein [36]. A knockin of S6 protein with the S6K phosphorylation site eliminated (rpS6pK/K) does not affect 5 0 -TOP (track of polypyrimidine) mRNA translation but instead enhances total protein synthesis [37]. Interestingly, rpS6pK/K mouse embryo fibroblasts (MEFs) are significantly smaller than wild-type MEFs. Furthermore, the size of rpS6pK/K cells, unlike wild-type MEFs, is not decreased by rapamycin. These observations indicate that S6 phosphorylation has a crucial role in mediating the effect of mTOR on mammalian cell-size regulation. Phosphorylation of 4EBP1 by mTOR relieves the inhibitory activity of 4EBP1 towards eukaryotic initiation factor 4E (eIF4E) [38]. eIF4E recognizes the cap structure at the 5 0 end of most eukaryotic mRNA; this provides a potential mechanism by which mTOR can stimulate translation initiation [39,40]. Together, regulation of cell growth and protein translation are the best-characterized functions of mTOR in mammalian cells. Using chemical crosslinking and immunoaffinity purification, Raptor was isolated as an mTOR-associated protein [41,42]. Interestingly, Raptor is homologous to the yeast Kog1 protein in TORC1 that was identified by Loewith et al. [24]. These authors have also isolated Lst8 as a component of TORC1 and TORC2 in yeast. They identified the mammalian counterpart of yeast Lst8 (mLST8) and demonstrated its interaction with mTOR. mLST8/GbL has an essential role in the phosphorylation of S6K and 4EBP1 by TORC1 and in the regulation of cell size [43]. These studies demonstrate the structural and functional conservation of TORC1 in eukaryotes (Table 1). Both S6K and 4EBP1 contain a short sequence termed a TOR signaling (TOS) motif that is important for phosphorylation by mTOR [44] and, interestingly, is important for TORC1 substrates to bind to Raptor [45–47]. This demonstrates that mTOR is the catalytic subunit in TORC1 and that Raptor is involved in substrate recognition. However, the function of Raptor is more complex than being a simple substrate-recruiting component in TORC1. Knockdown of Raptor also significantly reduces mTOR protein levels, suggesting that Raptor stabilizes mTOR [41]. Furthermore, the binding between Raptor and mTOR is sensitive to stimulations that affect mTOR kinase activity, such as by amino acids [41]. One group has proposed that Raptor and mTOR form a nutrient-sensitive complex [41]. Based on this model, nutrient deprivation, such as leucine starvation, results in
208
Review
TRENDS in Cell Biology
a tight mTOR–Raptor complex that has low activity towards S6K although the interaction between mLST8 and mTOR is unaffected by nutrients. By contrast, stimulation with leucine results in a loose mTOR–Raptor complex that has high activity. Consistently, removal of Raptor from immunoprecipitated mTOR by washing with Triton X-100 increased mTOR kinase activity towards S6K [41]. However, contradictory results were reported when Raptor was removed from immunoprecipitated mTOR by washing with NP-40 [42]. This yielded decreased mTOR kinase activity towards 4EBP1. In the study [42], nutrient starvation did not affect the interaction between Raptor and mTOR. One explanation for this discrepancy is that the first study analyzed the interaction of endogenous proteins, whereas the second tested overexpressed proteins, possibly indicating that nutrients regulate the interaction between mTOR and Raptor under physiological conditions but not under overexpression conditions. Moreover, the two groups used different substrates in the mTOR kinase assays. Further work is needed to clarify whether Raptor complexes with mTOR in two states in vivo, one tight with low activity and the other loose with high activity. The function of mLST8/GbL in TORC1 is less well defined. Co-expression of mLST8/GbL with mTOR enhances mTOR kinase activity [43], and mLST8/GbL is required for mTOR to be activated by amino acids. This indicates an important role of mLST8/GbL in mediating upstream signaling to mTOR activation. Furthermore, knockdown of mLST8/GbL by RNA interference decreases phosphorylation of S6K and 4EBP1, supporting the functional importance of mLST8/GbL in TORC1 function [41,43]. In addition to regulating cell growth, TOR functions in cell-size regulation, and mutations in dTOR cause a dramatic reduction in organ and cell size [13,48]. Rapamycin treatment decreases the size of many cell types [12]. The functions of Raptor and mLST8/GbL in cell-size regulation have also been confirmed [41,43]. Knockdown of either Raptor or mLST8/GbL by RNA interference causes a cell-size reduction in HEK293 cells that is similar to that caused by mTOR knockdown. These studies further establish that Raptor and mLST8/GbL are essential components of TORC1 in cell-growth regulation. TORC2 and new functions of TOR mTOR exists in two different complexes in mammalian cells. A TORC2 complex that is similar to that in yeast has been recently identified in mammalian cells and contains mTOR, Rictor/mAVO3 and mLST8/GbL (Table 1) [49,50]. The mammalian TORC2 is also resistant to rapamycin inhibition. Co-immunoprecipitation and co-purification experiments show that Raptor and Rictor/mAVO3 exist in two mutually exclusive complexes, TORC1 and TORC2, respectively [49,50]. Knockdown of Rictor does not affect phosphorylation of S6K, whereas knockdown of any TORC1 component, including mTOR, Raptor and mLST8/GbL, decreases S6K phosphorylation. Similar to the results observed in yeast, the TORC2 complex has an important role in regulation of cell morphology, possibly by modulating actin polymerization [49,50]. Knockdown of www.sciencedirect.com
Vol.16 No.4 April 2006
any component of the TORC2 complex, including mTOR, mLST8/GbL and Rictor/mAVO3, causes a similar alteration of cell morphology and cell adhesion. By contrast, downregulation of Raptor has little effect on cell morphology. However, Sarbassov et al. showed that knockdown of Rictor/mAVO3 and mTOR results in an increase in stress-fiber formation in HeLa cells [49]. By contrast, Jacinto et al. reported that knockdown of mTOR, mLST8/ GbL and Rictor/mAVO3 decreased stress fibers in NIH3T3 cells [50]. There is no apparent explanation for these contradictory results except that different cell lines were used in the two studies. Collectively, it is clear that TORC2 has a rapamycin-insensitive function that regulates cell morphology and cytoskeletal reorganization; however, further investigation is required to elucidate the precise role of TORC2 in stress-fiber formation. TORC2 modulates cytoskeletal structure through the Rho family small GTPases, including Rho, Rac and Cdc42 [23,50] (Figure 1). Expression of active Rac or Rho suppresses the actin defects caused by the downregulation of TORC2 [50]. Consistent with this model, knockdown of mTOR, mLST8/GbL or Rictor/mAVO3 decreases Rac GTP levels, suggesting that TORC2 functions upstream of Rac. Recent studies have also suggested that TORC2 regulates protein kinase C a (PKCa) activity to control the actin cytoskeleton [23,49]. Knockdown of either mTOR or Rictor/mAVO3, but not Raptor, significantly decreases PKCa phosphorylation [49]. PKCa has an important role in cytoskeleton organization [51]. Interestingly, studies in yeast have already demonstrated a pathway in which TOR regulates cytoskeleton organization through Rho and the yeast PKCa equivalent, PKC1 [23]. Therefore, TORC2 uses an evolutionary conserved pathway involving Rho and PKCa to regulate cell morphology. AKT [also known as protein kinase B (PKB)] has been shown to act upstream of TORC1 and indirectly stimulates TORC1 activity. Unexpectedly, TORC2 phosphorylates and activates AKT [52]. AKT has essential roles in many aspects of cellular regulation, including stimulation of cell growth, proliferation, survival and metabolism [53]. Its activation requires the phosphorylation of Thr308 in its activation loop by PDK1 (3-phosphoinositide-dependent protein kinase-1) and the phosphorylation of Ser473 in its C-terminal hydrophobic region by a second kinase activity termed PDK2 [54,55] (Figure 1). The identity of PDK2 remained unknown although several kinases had been implicated [56,57]. Interestingly, Sarbassov et al. showed that TORC2 has a crucial role in the phosphorylation of the motif on AKT that is targeted by PDK2 [52]. Using an RNA interference-based study in Drosophila S2 cells, Sarbassov discovered that knockdown of dTOR significantly reduces phosphorylation of the PDK2 site on dAKT [52]. Importantly, knockdown of Rictor/AVO3, but not Raptor, decreased AKT phosphorylation in Drosophila and mammalian cells. In vitro kinase assays with immunoprecipitated mTOR complexes provide direct biochemical evidence that TORC2, but not TORC1, can phosphorylate Ser473 of AKT in vitro and that phosphorylation of AKT by TORC2 promotes the in vitro phosphorylation of AKT by purified PDK1 [52]. These observations demonstrate that TORC2 phosphorylates the
Review
TRENDS in Cell Biology
Insulin IGF1
IRS
Vol.16 No.4 April 2006
209
Insulin IGF1
Amino acids
PI3K
PIP3
PIP3
PDK1
AKT
PIP3 PDK1
S6K
IRS Negative feedback
mTORC2 Rho
TSC2 TSC1
Cytoskeleton
Rheb 4EBP1
Cell survival Cell growth Glycogen metabolism
PTEN
mTORC1
Rac PKCα
PI3K
S6
Protein translation Cell growth regulation
TRENDS in Cell Biology
Figure 1. Signaling network of TORC1 and TORC2. TORC1 (consisting of mTOR, Raptor and mLST8) phosphorylates S6K and 4EBP1, and therefore regulates translation and cell growth. Rheb stimulates TORC1 activity and itself is inhibited by the TSC1/TSC2 complex. TSC2 is phosphorylated and inhibited by AKT, which is activated by growth factors through the PI3K and PDK1 cascade. Therefore, the mTOR pathway has an important role in mediating growth factor signals to cell growth. PTEN is a tumor suppressor gene that antagonizes the action of PI3K by hydrolyzing PIP3, therefore, PTEN inhibits the mTOR pathway. S6K is involved in the feedback inhibition of AKT by phosphorylating and inactivating IRS, which has a key positive role in PI3K activation in response to insulin. In addition, TORC2 (consisting of mTOR, Rictor and mLST8) can directly phosphorylate and activate AKT, although it is unclear how TORC2 is regulated. TORC2 has also been implicated in modulating cytoskeleton organization through PKC and the Rho family GTPases. Red diamonds indicate phosphorylation. Broken arrows indicate undefined regulation. The circled minus signs indicate negative regulatory effects.
PDK2 site of AKT in vitro and in vivo. Therefore, TORC2 acts at the center of cell-growth regulation because AKT is a key protein in regulation of cell growth and survival (Figure 1). The PDK2-equivalent site in AKT is also present in many of the AGC kinase family members. SGK and AKT are closely related to each other and are regulated by similar stimuli. It has been shown that yeast YPK2, a homolog of mammalian SGK, was phosphorylated and activated by TORC2 [58]. Therefore, TORC2 might display a much broader cellular function by phosphorylating the presumed PDK2 sites in other AGC family kinases [6]. Because higher eukaryotes have only one TOR gene, the TORC1 and TORC2 complexes must share the same pool of TOR proteins. It has been postulated that Raptor and Rictor compete for binding to mTOR and direct competition between Raptor and Rictor/mAVO3 in the binding of mTOR has been observed in overexpression studies [49]. Moreover, the relationship between TORC1 and TORC2 is complicated by the observation that TORC1 activation results in a negative feedback loop that inhibits the phosophotidylinositol 3 kinase (PI3K) pathway. PI3K activity is essential for phosphorylation of AKT by TORC2, suggesting that TORC2 is activated by PI3K. However, the effect of PI3K on the TORC2 site phosphorylation in AKT could be due solely to the membrane localization of AKT to phosphotidylinositol 3,4,5 trisphosphate (PIP3) generated by PI3K, rather than the activation of TORC2. S6K, a downstream effector of TORC1, is a key component of the feedback loop because S6K phosphorylates and www.sciencedirect.com
inhibits the insulin receptor substrate-1 (IRS1), which is upstream of the PI3K pathway [59,60] (Figure 1). The effect of this negative feedback loop is apparent in TSC (tuberous sclerosis complex) mutant cells. Constitutive activation of TORC1 in TSC mutant cells decreases insulin- or insulin-like growth factor 1- (IGF1-)induced AKT phosphorylation [59,61]. Consistent with this, inhibition of TORC1 with rapamycin restores insulininduced AKT phosphorylation in TSC mutant cells. Importantly, several pathological features, including hamartomas in tuberous sclerosis, can be explained by this negative-feedback mechanism. For instance, inhibition of AKT by the constitutively activated TORC1 is a possible reason why TSC-associated hamartoma syndromes are benign rather than malignant, because AKT has crucial roles in cell growth, survival and migration [62,63]. Activation of TOR by Rheb Several key components that function upstream of mTOR have recently been identified, including TSC1, TSC2 and Rheb (Ras homolog enriched in brain) [64,65]. TSC1 and TSC2 are tumor suppressor genes mutated in tuberous sclerosis [64] and their products form a physical and functional complex. Mutations in either TSC1 or TSC2 induce hamartoma formation in multiple organs and a significant increase in cell size, a phenotype similar to mTOR activation. TSC1 or TSC2 mutant cells have high TORC1 activity [64]. These results establish that an important physiological function of TSC1 and TSC2 is to
210
Review
TRENDS in Cell Biology
inhibit mTOR. Biochemical data demonstrate that TSC2 is a GTPase activating protein (GAP) towards Rheb, which is a small GTPase related to Ras [65]. Interestingly, Rheb stimulates phosphorylation of S6K and 4EBP1, and Rhebinduced S6K phosphorylation is blocked by rapamycin and dominant-negative mTOR. Furthermore, genetic data support that dTOR functions downstream of dRheb. Taken together, these studies establish a signaling pathway from TSC2 to Rheb to mTOR (Figure 1). TSC2 is a physiological substrate of AKT [11] and therefore AKT might activate the mTOR pathway by phosphorylating and inhibiting TSC2 (Figure 1). However, the direct inhibition of TSC2 GAP activity by AKT phosphorylation has not been reported. The regulation of TOR by AKT might not be evolutionarily conserved because two studies have demonstrated that dAKT phosphorylates dTSC2. Potter et al. showed that the phosphorylation of dTSC2 by dAKT has an important role in mediating the growth-stimulating signal from insulin [66]. By contrast, Dong and Pan showed that mutation of the dAKT phosphorylation sites in dTSC2 does not compromise the essential function of dTSC2 in Drosophila development, indicating that TSC2 is not a crucial target of AKT during normal development [67]. A noticeable difference is that one group used overexpression [66] whereas the other did not [67]. Whether dAKT promotes dS6K phosphorylation in Drosophila cells is controversial. Two groups have reported that knockdown of dAKT has little effect on dS6K phosphorylation [52,68], whereas another study shows conflicting results – a strong inhibition of dS6K phosphorylation after dAKT knockdown [69]. We have observed that knockdown of dAKT had little effect on the phosphorylation of dS6K (K. Inoki and K-L. Guan, unpublished). The underlying reasons for this apparent conflict are not clear. The mechanism by which Rheb activates TORC1 is not fully understood. However, a potential mechanism that operates through a direct interaction between Rheb and TORC1 has been implicated [70]. Interestingly, the binding between Rheb and mTOR is inhibited by amino acid depletion [71] and Rheb also associates with Raptor and mLST8/GbL, suggesting that Rheb activates TORC1 through a direct interaction [70]. Rheb–GTP precipitates more mTOR activity than does Rheb–GDP. However, the interaction between Rheb and components of TORC1 is not nucleotide dependent: the GDP-bound or nucleotidefree Rheb interacts with mTOR more strongly than does the GTP-bound Rheb [70,72]. This is perplexing because only Rheb bound to GTP activates TORC1. Recently, an interaction between Rhb1 (the homolog of Rheb) and Tor2 in Schizosaccharomyces pombe has been reported, and the interaction appears to be nucleotide dependent [73]. The interaction between Rhb1 and Tor2 is only detected in a Tsc2–/– strain. Furthermore, constitutively active Rhb1 mutants display a strong interaction with Tor2, whereas the wild-type Rhb1 shows little interaction with Tor2 in wild-type S. pombe. These results strongly imply that Rheb–GTP specifically interacts with TOR, but further studies are needed to understand the molecular mechanism. www.sciencedirect.com
Vol.16 No.4 April 2006
Concluding remarks Recent progress has established and expanded the complexity of the TOR signaling network. A key advance is the identification of the two distinct TOR complexes TORC1 and TORC2, which perform different physiological functions in vivo. Another key development is that TSC2 and Rheb are important upstream regulators of TORC1. These findings have connected the dysregulation of TORC1 to several human diseases and have provided a scientific basis for rapamycin as a potential drug for the treatment of human diseases, especially hamartoma syndromes and cancers [74]. For example, PTEN (protein phosphatase and tensin homolog deleted on chromosome 10) is a tumor suppressor that is frequently mutated in human cancers [4]. PTEN mutations activate AKT and cause elevated TORC1 activity (Figure 1). It has been reported that rapamycin is effective against tumors with PTEN mutations [4]. The identification of TORC2 as the long-sought PDK2 activity further emphasizes the importance of mTOR in cellular growth and survival regulation, and this significantly expands the complexity of the TOR signaling pathway, blurring the up- and downstream relationship between AKT and mTOR. AKT activates TORC1; however, AKT is activated by TORC2. Perhaps, it is necessary to think of TORC1 and TORC2 as two distinct functional entities. Despite exciting and rapid developments in the TOR signaling field, many key questions remain. Little is known about the function of TORC2 except for its role in AKT activation and cytoskeletal regulation. The identification of this new complex raises important questions regarding how or whether TORC2 is activated by the growth factors and how TORC2 regulates cytoskeletal organization. It will be important to determine whether TORC2 is a ‘master kinase’ involved in phosphorylation of many AGC family kinases. It is well established that Rheb stimulates TORC1 function, but little data are available as to whether Rheb also regulates TORC2 and, if so, by what mechanism. Another important question is the mechanism of Rheb regulation. Although Rheb has high basal GTP binding, it is likely to be regulated by a yet-to-be identified GEF. Future studies to identify and characterize the Rheb GEF might provide key information for understanding how mTOR receives and integrates various upstream signals to control cell growth. Furthermore, amino acids are known to be essential regulators of TORC1 activity. However, the precise nature of amino acid sensing is unknown and requires further investigation. Finally, autophagy is an important cellular process and is regulated by TORC1 [28]. Autophagy is the major pathway for degradation of long-lived proteins, especially under nutrient limitation conditions, and is of current interest in cell biology. Future studies are needed to understand the molecular mechanism how mTOR regulates autophagy in mammalian cells. Acknowledgements We apologize to colleagues whose work could not be cited owing to the scope and space limitations. We thank Chung-Han Lee and Michael N. Corradetti for the critical reading of the article. This work is supported by
Review
TRENDS in Cell Biology
grants to K-L.G. from the National Institutes of Health and the Department of Defense.
References 1 Vezina, C. et al. (1975) Rapamycin (AY-22,989), a new antifungal antibiotic. I. Taxonomy of the producing streptomycete and isolation of the active principle. J. Antibiot. 28, 721–726 2 Easton, J.B. and Houghton, P.J. (2004) Therapeutic potential of target of rapamycin inhibitors. Expert Opin. Ther. Targets 8, 551–564 3 Moses, J.W. et al. (2003) Sirolimus-eluting stents versus standard stents in patients with stenosis in a native coronary artery. N. Engl. J. Med. 349, 1315–1323 4 Bjornsti, M.A. and Houghton, P.J. (2004) The TOR pathway: a target for cancer therapy. Nat. Rev. Cancer 4, 335–348 5 Heitman, J. et al. (1991) Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 253, 905–909 6 Manning, G. et al. (2002) The protein kinase complement of the human genome. Science 298, 1912–1934 7 Bakkenist, C.J. and Kastan, M.B. (2004) Initiating cellular stress responses. Cell 118, 9–17 8 Koltin, Y. et al. (1991) Rapamycin sensitivity in Saccharomyces cerevisiae is mediated by a peptidyl-prolyl cis-trans isomerase related to human FK506-binding protein. Mol. Cell. Biol. 11, 1718–1723 9 Chung, J. et al. (1992) Rapamycin-FKBP specifically blocks growthdependent activation of and signaling by the 70 kd S6 protein kinases. Cell 69, 1227–1236 10 Helliwell, S.B. et al. (1994) TOR1 and TOR2 are structurally and functionally similar but not identical phosphatidylinositol kinase homologues in yeast. Mol. Biol. Cell 5, 105–118 11 Hay, N. and Sonenberg, N. (2004) Upstream and downstream of mTOR. Genes Dev. 18, 1926–1945 12 Fingar, D.C. et al. (2002) Mammalian cell size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E. Genes Dev. 16, 1472–1487 13 Montagne, J. et al. (1999) Drosophila S6 kinase: a regulator of cell size. Science 285, 2126–2129 14 Gao, X. et al. (2002) Tsc tumour suppressor proteins antagonize amino-acid-TOR signalling. Nat. Cell Biol. 4, 699–704 15 Hara, K. et al. (1998) Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism. J. Biol. Chem. 273, 14484–14494 16 Dennis, P.B. et al. (2001) Mammalian TOR: a homeostatic ATP sensor. Science 294, 1102–1105 17 Desai, B.N. et al. (2002) FKBP12-rapamycin-associated protein associates with mitochondria and senses osmotic stress via mitochondrial dysfunction. Proc. Natl. Acad. Sci. U. S. A. 99, 4319–4324 18 Brugarolas, J. et al. (2004) Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex. Genes Dev. 18, 2893–2904 19 Harris, T.E. and Lawrence, J.C., Jr. (2003) TOR signaling. Sci. STKE 2003, re15 20 Fingar, D.C. and Blenis, J. (2004) Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression. Oncogene 23, 3151–3171 21 Zheng, X.F. et al. (1995) TOR kinase domains are required for two distinct functions, only one of which is inhibited by rapamycin. Cell 82, 121–130 22 Kunz, J. et al. (1993) Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression. Cell 73, 585–596 23 Schmidt, A. et al. (1997) The yeast phosphatidylinositol kinase homolog TOR2 activates RHO1 and RHO2 via the exchange factor ROM2. Cell 88, 531–542 24 Loewith, R. et al. (2002) Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol. Cell 10, 457–468 25 Reinke, A. et al. (2004) TOR complex 1 (TORC1) includes a novel component, Tco89p (YPL180w), and cooperates with Ssd1p to maintain cellular integrity in S. cerevisiae. J. Biol. Chem. 279, 14752–14756 www.sciencedirect.com
Vol.16 No.4 April 2006
211
26 Fadri, M. et al. (2005) The pleckstrin homology domain proteins Slm1 and Slm2 are required for actin cytoskeleton organization in yeast and bind phosphatidylinositol-4,5-bisphosphate and TORC2. Mol. Biol. Cell 16, 1883–1900 27 Audhya, A. et al. (2004) Genome-wide lethality screen identifies new PI4,5P2 effectors that regulate the actin cytoskeleton. EMBO J. 23, 3747–3757 28 Crespo, J.L. and Hall, M.N. (2002) Elucidating TOR signaling and rapamycin action: lessons from Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 66, 579–591 29 Rudra, D. et al. (2005) Central role of Ifh1p-Fhl1p interaction in the synthesis of yeast ribosomal proteins. EMBO J. 24, 533–542 30 Schawalder, S.B. et al. (2004) Growth-regulated recruitment of the essential yeast ribosomal protein gene activator Ifh1. Nature 432, 1058–1061 31 Wade, J.T. et al. (2004) The transcription factor Ifh1 is a key regulator of yeast ribosomal protein genes. Nature 432, 1054–1058 32 Martin, D.E. et al. (2004) TOR regulates ribosomal protein gene expression via PKA and the Forkhead transcription factor FHL1. Cell 119, 969–979 33 Lee, T.I. et al. (2002) Transcriptional regulatory networks in Saccharomyces cerevisiae. Science 298, 799–804 34 Brown, E.J. et al. (1995) Control of p70 s6 kinase by kinase activity of FRAP in vivo. Nature 377, 441–446 35 Lorenz, M.C. and Heitman, J. (1995) TOR mutations confer rapamycin resistance by preventing interaction with FKBP12rapamycin. J. Biol. Chem. 270, 27531–27537 36 Thomas, G. (2002) The S6 kinase signaling pathway in the control of development and growth. Biol. Res. 35, 305–313 37 Ruvinsky, I. et al. (2005) Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis. Genes Dev. 19, 2199–2211 38 Gingras, A.C. et al. (1998) 4E-BP1, a repressor of mRNA translation, is phosphorylated and inactivated by the Akt(PKB) signaling pathway. Genes Dev. 12, 502–513 39 Haghighat, A. et al. (1995) Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E. EMBO J. 14, 5701–5709 40 Beretta, L. et al. (1996) Rapamycin blocks the phosphorylation of 4EBP1 and inhibits cap-dependent initiation of translation. EMBO J. 15, 658–664 41 Kim, D.H. et al. (2002) mTOR interacts with raptor to form a nutrientsensitive complex that signals to the cell growth machinery. Cell 110, 163–175 42 Hara, K. et al. (2002) Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 110, 177–189 43 Kim, D.H. et al. (2003) GbetaL, a positive regulator of the rapamycinsensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR. Mol. Cell 11, 895–904 44 Schalm, S.S. and Blenis, J. (2002) Identification of a conserved motif required for mTOR signaling. Curr. Biol. 12, 632–639 45 Choi, K.M. et al. (2003) Two motifs in the translational repressor PHAS-I required for efficient phosphorylation by mammalian target of rapamycin and for recognition by raptor. J. Biol. Chem. 278, 19667–19673 46 Nojima, H. et al. (2003) The mammalian target of rapamycin (mTOR) partner, raptor, binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif. J. Biol. Chem. 278, 15461–15464 47 Schalm, S.S. et al. (2003) TOS motif-mediated raptor binding regulates 4E-BP1 multisite phosphorylation and function. Curr. Biol. 13, 797–806 48 Oldham, S. et al. (2000) Genetic and biochemical characterization of dTOR, the Drosophila homolog of the target of rapamycin. Genes Dev. 14, 2689–2694 49 Sarbassov, D.D. et al. (2004) Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr. Biol. 14, 1296–1302 50 Jacinto, E. et al. (2004) Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat. Cell Biol. 6, 1122–1128 51 Nakashima, S. (2002) Protein kinase C a (PKCa): regulation and biological function. J. Biochem. (Tokyo) 132, 669–675
Review
212
TRENDS in Cell Biology
52 Sarbassov, D.D. et al. (2005) Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307, 1098–1101 53 Brazil, D.P. and Hemmings, B.A. (2001) Ten years of protein kinase B signalling: a hard Akt to follow. Trends Biochem. Sci. 26, 657–664 54 Alessi, D.R. et al. (1997) Characterization of a 3-phosphoinositidedependent protein kinase which phosphorylates and activates protein kinase Ba. Curr. Biol. 7, 261–269 55 Chan, T.O. and Tsichlis, P.N. (2001) PDK2: a complex tail in one Akt. Sci. STKE 2001, PE1 56 Rane, M.J. et al. (2001) p38 Kinase-dependent MAPKAPK-2 activation functions as 3-phosphoinositide-dependent kinase-2 for Akt in human neutrophils. J. Biol. Chem. 276, 3517–3523 57 Feng, J. et al. (2004) Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase. J. Biol. Chem. 279, 41189–41196 58 Harrington, L.S. et al. (2004) The TSC1-2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins. J. Cell Biol. 166, 213–223 59 Um, S.H. et al. (2004) Absence of S6K1 protects against age- and dietinduced obesity while enhancing insulin sensitivity. Nature 431, 200–205 60 Shah, O.J. et al. (2004) Inappropriate activation of the TSC/Rheb/mTOR/S6K cassette induces IRS1/2 depletion, insulin resistance, and cell survival deficiencies. Curr. Biol. 14, 1650–1656 61 Manning, B.D. et al. (2005) Feedback inhibition of Akt signaling limits the growth of tumors lacking Tsc2. Genes Dev. 19, 1773–1778 62 Ma, L. et al. (2005) Genetic analysis of Pten and Tsc2 functional interactions in the mouse reveals asymmetrical haploinsufficiency in tumor suppression. Genes Dev. 19, 1779–1786
Vol.16 No.4 April 2006
63 Kwiatkowski, D.J. (2003) Rhebbing up mTOR: new insights on TSC1 and TSC2, and the pathogenesis of tuberous sclerosis. Cancer Biol. Ther. 2, 471–476 64 Manning, B.D. and Cantley, L.C. (2003) Rheb fills a GAP between TSC and TOR. Trends Biochem. Sci. 28, 573–576 65 Potter, C.J. et al. (2002) Akt regulates growth by directly phosphorylating Tsc2. Nat. Cell Biol. 4, 658–665 66 Dong, J. and Pan, D. (2004) Tsc2 is not a critical target of Akt during normal Drosophila development. Genes Dev. 18, 2479–2484 67 Radimerski, T. et al. (2002) dS6K-regulated cell growth is dPKB/dPI(3)K-independent, but requires dPDK1. Nat. Cell Biol. 4, 251–255 68 Lizcano, J.M. et al. (2003) Insulin-induced Drosophila S6 kinase activation requires phosphoinositide 3-kinase and protein kinase B. Biochem. J. 374, 297–306 69 Long, X. et al. (2005) Rheb binds and regulates the mTOR kinase. Curr. Biol. 15, 702–713 70 Long, X. et al. (2005) Rheb binding to mTOR is regulated by amino acid sufficiency. J. Biol. Chem. 280, 23433–23436 71 Smith, E.M. et al. (2005) The tuberous sclerosis protein TSC2 is not required for the regulation of the mammalian target of rapamycin by amino acids and certain cellular stresses. J. Biol. Chem. 280, 18717–18727 72 Urano, J. et al. (2005) Identification of novel single amino acid changes that result in hyperactivation of the unique GTPase, Rheb, in fission yeast. Mol. Microbiol. 58, 1074–1086 73 Inoki, K. et al. (2005) Dysregulation of the TSC-mTOR pathway in human disease. Nat. Genet. 37, 19–24 74 Kamada, Y. et al. (2005) Tor2 directly phosphorylates the AGC kinase Ypk2 to regulate actin polarization. Mol. Cell. Biol. 25, 7239–7248
Reuse of Current Opinion and Trends journal figures in multimedia presentations It’s easy to incorporate figures published in Trends or Current Opinion journals into your PowerPoint presentations or other imagedisplay programs. Simply follow the steps below to augment your presentations or teaching materials with our fine figures! 1. 2. 3. 4. 5.
Locate the article with the required figure in the Science Direct journal collection Click on the ‘Full text + links’ hyperlink Scroll down to the thumbnail of the required figure Place the cursor over the image and click to engage the ‘Enlarge Image’ option On a PC, right-click over the expanded image and select ‘Copy’ from pull-down menu (Mac users: hold left button down and then select the ‘Copy image’ option) 6. Open a blank slide in PowerPoint or other image-display program 7. Right-click over the slide and select ‘paste’ (Mac users hit ‘Apple-V’ or select the ‘Edit-Paste’ pull-down option). Permission of the publisher, Elsevier, is required to re-use any materials in Trends or Current Opinion journals or any other works published by Elsevier. Elsevier authors can obtain permission by completing the online form available through the Copyright Information section of Elsevier’s Author Gateway at http://authors.elsevier.com/. Alternatively, readers can access the request form through Elsevier’s main web site at http://www.elsevier.com/locate/permissions.
www.sciencedirect.com