- Email: [email protected]
Regulation of TOR Signaling in Mammals
2 Regulation of TOR Signaling in Mammals DUDLEY W. LAMMING
DAVID M. SABATINI
Whitehead Institute Cambridge, Massachusetts, USA
I.
Abstract
Since the initial discovery of rapamycin in the soils of Easter Island, there has been significant interest in determining the biological mechanism underlying the effects of rapamycin on mammalian tissues. These effects include changes in cell size and proliferation, as well as decreased and altered mRNA translation, and changes in autophagy. It was thus immediately clear that the mammalian target of rapamycin (mTOR) was a key regulator of cell growth and proliferation, and further work has shown that mTOR serves as a central regulator of cell processes in response to nutrients and environmental stimuli. In this chapter, we will discuss the mechanisms behind the many different functions of mTOR, with emphasis on the proteins that make up two mTOR-containing complexes and regulate mTOR signaling in response to insulin and amino acids. We will also discuss the medical relevance of these findings and the potential clinical significance of inhibiting mTOR.
II.
One Enzyme,Two Complexes
Rapamycin functions by binding to the immunophilin FKBP12, and this complex then binds to and inhibits many functions of mTOR. The mTOR protein kinase was identified in 1994–1995 by three separate groups that THE ENZYMES, Vol. XXVII # 2010 Elsevier Inc. All rights reserved.
21
ISSN NO: 1874-6047 DOI: 10.1016/S1874-6047(10)27002-6
22
DUDLEY W. LAMMING AND DAVID M. SABATINI
isolated a 289-kD protein with a rapamycin-dependent interaction with FKBP12 [1–3]. mTOR was found to have approximately 40% homology to the two Saccharomyces cerevisiae TOR proteins, as well as high homology to the TOR proteins that would later be found in other eukaryotes. Indeed, many of the proteins that complex with mTOR to regulate and target mTOR are conserved in diverse eukaryotes, including fungi such as S. cerevisiae, metazoans such as Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and mammals including mice and humans (see Table 2.1). Most early work on mTOR used rapamycin extensively as a tool. However, work with kinase-dead mTOR and RNAi knockdown of mTOR made it apparent that mTOR participates in both rapamycin-dependent and independent processes. Organisms spanning complexity from humans to yeast have now been shown to have both rapamycin-sensitive and insensitive TOR-dependent processes (see Figure 2.1). Higher eukaryotes, including mammals, incorporate the same mTOR protein into both a rapamycinsensitive mTOR complex I (mTORC1) and a complex that is relatively resistant to the effects of rapamycin, mTOR complex II (mTORC2). The core mTORC1 proteins are mTOR, Raptor, and mLST8/GbL, while the core mTORC2 proteins are mTOR, Rictor, and mLST8/GbL; the components of mTORC1 show a higher degree of evolutionary conservation than do the components of mTORC1. Yeast actually has two distinct TOR TABLE 2.1 MTORC1 AND MTORC2 PROTEINS IN HUMANS,
DROSOPHILA MELANOGASTER, CAENORHABDITIS ELEGANS, AND SACCHAROMYCES CEREVISIAE H. sapiens
D. melanogaster
C. elegans
S. cerevisiae
mTORC1 (rapamycin sensitive)
mTOR mLST8 Raptor
dTOR CG3004 Raptor
ceTOR
mTORC2 (rapamycin insensitive)
PRAS40 DEPTOR RagA, RagB RagC, RagD mTOR mLST8 mSin1
Lobe dRagA dRagC dTOR CG3004
raga-1 ragc-1 ceTOR
Rictor
Rictor
CeRictor
Protor DEPTOR
Tor1, Tor2 Lst8 Kog1 Tco89 Gtr1 Gtr2 Tor2 Lst8 Avo1 Avo2 Avo3 Bit61
ceRaptor
23
2. REGULATION OF TOR SIGNALING IN MAMMALS
Growth factors (insulin and IGF-1)
Amino acids
Hypoxia Energy deficit Rapamycin
mTORC1
Autophagy
mTORC2
Proliferation Cell volume Cytoskeletal structure
FIG. 2.1. The regulation of growth and proliferation by mTOR. mTORC1 is positively regulated by amino acids and growth factors, including insulin and IGF-1, and is inhibited by hypoxia, low levels of ATP, and rapamycin, while mTORC2 is positively regulated by growth factors. Both mTORC1 and mTORC2 signaling promote growth and proliferation, but only mTORC1 inhibits autophagy.
proteins; TOR1, which is TORC1 specific, and TOR2, which participates in both TORC1 and TORC2 (discussed in Chapter 1) [4]. Depending on cell type and nutrient conditions, numerous other proteins also associate with mTORC1, mTORC2, or both (see Table 2.1). In this chapter, we will focus on the regulation of mTOR signaling by PRAS40, DEPTOR, and the Rag family of small GTP-binding proteins.
III.
Raptor Defines mTORC1
The identification of Raptor as an mTOR associated protein came about as researchers in many labs were attempting to discover the mechanism by which nutrients and environmental stimuli regulate the phosphorylation of the mTOR targets S6K-1 and 4E-BP1. In vivo, these targets are phosphorylated by mTOR in response to such stimuli as amino acids, but researchers observed that the activity of the immunoprecipitated mTOR kinase against S6K-1 or 4E-BP in vitro was unchanged. One possible explanation for these results is that mTOR exists in a complex with additional regulatory proteins, but that these proteins were disassociating during mTOR purification. Two separate groups simultaneously investigated the properties of mTOR that was immunoprecipitated using different lysis conditions. One group approached the problem using the reversible cross-linker dithiobis
24
DUDLEY W. LAMMING AND DAVID M. SABATINI
(succinimidyl propionate) (DSP) [5], while another lab used an approach utilizing chromatography [6]. Both groups identified a 150-kD protein that immunoprecipitated with mTOR and showed high conservation within all eukaryotes (see Table 2.1 for a list of mTOR associated proteins and their homologues in various organisms). Both labs subsequently found that Raptor (for regulatory associated protein of mTOR), can also be isolated in the absence of cross-linker, indicating that the interaction is real and not an artifact of the cross-linker, so long as appropriate and gentle lysis conditions are used. Specifically, Raptor remains bound to mTOR when low concentrations of the detergent CHAPS was used instead of the more commonly used Triton X-100 or NP-40 [5]. To identify the role of Raptor in the regulation of mTOR, RNAi was used to decrease endogenous levels of Raptor in mammalian cells. Cells in which Raptor expression was thus lowered showed decreased phosphorylation of the mTOR target S6K-1, decreased proliferation, and smaller volume, all phenotypes that also occur when cells are treated with rapamycin or mTOR itself is knocked down with RNAi. RNAi against C. elegans homologues of Raptor and mTOR demonstrated that RNAi against either resulted in a similar set of developmental phenotypes, including delayed gonadal development, the development of large gut lysosomes, and smaller intestinal cells [6]. These experiments demonstrated that many of the phenotypes regulated by mTOR signaling are Raptor dependent. The initial question of how nutrients and environmental stimuli regulate the phosphorylation of mTOR targets was at least partially answered by observing the properties of the mTOR–Raptor interaction. Researchers found that the stability of the mTOR–Raptor interaction was regulated by specific nutrients; specifically, the in vivo complex was destabilized by the addition of amino acids, leucine, and glucose, treatments that result in increased mTOR activity. Other treatments that decrease mTOR signaling to S6K-1, such as treatment with valinomycin, 2-deoxyglucose, and H2O2, also stabilized the mTOR–Raptor interaction. This led to the hypothesis that in the absence of nutrients, Raptor regulates mTOR by inhibiting its kinase activity, and that nutrients increase mTOR signaling by destabilizing the mTOR–Raptor interaction. This theory was supported by the finding that overexpression of Raptor suppresses the catalytic activity mTOR and inhibits phosphorylation of S6K1 and 4E-BP1 in vivo. However, this hypothesis does not explain everything; treatment of serum-starved cells with insulin, which results in increased mTOR signaling and increased phosphorylation of S6K1 by mTOR, does not destabilize the mTOR–Raptor interaction, suggesting that the regulation of mTOR activity by insulin and other growth factors may proceed via a different mechanism [5].
2. REGULATION OF TOR SIGNALING IN MAMMALS
IV.
25
Rictor Defines a Rapamycin-Insensitive mTOR Complex
Two additional proteins that complex with mTOR, Rictor and mLST8, were also discovered independently by different groups and at roughly the same time. These groups used different approaches to the same problem, some working with immunoprecipitation of mTOR followed by mass spectrometry or alternatively working up from yeast TOR and using homology to find mTOR-interacting proteins. As mentioned above, it was discovered that yeast TOR could be found in two complexes, only one of which was rapamycin sensitive (see Table 2.1 and [4]). An additional protein of unknown function, Lst8, could be found in both complexes, and shared homology with a relatively small human protein known originally as GbL and now dubbed mLST8 due to its homology to the yeast protein [7]. They found that overexpressed mLST8 and mTOR coprecipitated. A study conducted by a different group using mass spectrometry upon immunoprecipitated mTOR found that mTOR coprecipitates with a 36-kD protein known as GbL that acts to strongly increase mTOR kinase activity toward S6K1 and 4E-BP1 [8]. Yeast TOR functions in two complexes, one of which is rapamycin insensitive, and it was the subject of substantial interest to determine if mTOR similarly functioned in two complexes. Rictor was identified in 2004 as a defining component of mTORC2, a rapamycin-insensitive mTOR complex [9, 10]. Rictor was difficult to identify by homology due to its relatively low sequence conservation between mammals and lower eukaryotes such as yeast, and a lack of any domains of known function [9]. Indeed, the C. elegans Rictor homologue was only recently identified due to the low sequence conservation [11, 12]. The percentage of mTOR that complexes with Rictor also varies widely by cell type and is inversely correlated with the expression of Raptor. For instance, HEK293T cells contain mostly mTOR–Raptor complexes, while HeLa cells contain more Rictor than Raptor [9]. Because mTORC2 is relatively insensitive to rapamycin, the function of mTORC2 has been less clear until recently. One initial observation was that mTORC2 might regulate the cytoskeleton, although the precise mechanism behind this effect was unclear [4, 9, 10]. The deletion of mTORC2-specific subunits leads to the dephosphorylation of the hydrophobic motif site in Protein Kinase C (PKC)a. Subsequent experiments demonstrated that siRNA knockdown of either mTOR or Rictor, but not Raptor or treatment with rapamycin, also inhibited the kinase activity of PKCa, demonstrating the importance of this phosphorylation. Unfortunately, no environmental stimuli that regulate PKCa phosphorylation by mTOR have been
26
DUDLEY W. LAMMING AND DAVID M. SABATINI
identified, but siRNA knockdown of either Rictor or PKCa has similar effects on the actin cytoskeleton, implicating the regulation of PKCa phosphorylation as one mechanism by which mTOR signaling regulates the cytoskeleton [9]. While the effects of mTOR on the cytoskeleton remain an interesting area of study, an important step in understanding the effects of mTORC2 signaling occurred the following year with the identification of Akt as an mTORC2 substrate [13]. It had long been known that active Akt was phosphorylated on two key residues, Threonine 308 and Serine 473. The Thr308 kinase had previously been identified as PDK1, but the kinase for Serine 473 had remained elusive. While numerous kinases had been proposed as the Serine 473 kinase, including PDK1, Akt, and DNA-PK, none of these studies were fully convincing. However, in the course of experiments in Drosophila S2 cells and mammalian cell lines, it was observed that siRNA against mTOR or Raptor slightly increased the phosphorylation of Akt Ser473, while siRNA against Rictor resulted in the almost complete inhibition of Akt Ser473 phosphorylation. This was seen even in cell lines in which PTEN, an antagonist of PI3K signaling, had been knocked down or deleted. Subsequent experiments confirmed that mTORC2 directly phosphorylates Akt Ser473 both in vitro and in vivo, and that mTORC2 was the long-sought ‘‘PDK2’’ for Akt Ser473 [13]. Interestingly, mTORC2 appears to also regulate the activity of a related kinase, SGK1 [14]. mTORC2 regulates activation of SGK1 via direct phosphorylation of SGK1 Ser422, and may thus act as a regulator of numerous downstream targets of SGK1, including NDRG1 and FoxO proteins [14].
V.
Additional mTORC1and mTORC2 Proteins
We can therefore define the two major mTOR complexes as the rapamycin-sensitive mTORC1, containing mTOR, Raptor, and mLST8, while the remainder of mTOR is in the relatively rapamycin-insensitive mTORC2, containing mTOR, Rictor, and mLST8 (see Figure 2.2 and Table 2.1). Subsequent work by a variety of labs has identified additional proteins that complex with either mTORC1, mTORC2, or both and isoforms of many of these proteins also exist. All or most of these isoforms can be combined together, which together likely define several different ‘‘flavors’’ of mTORC1 and mTORC2 that may vary between cell types and may have different sensitivity to stimuli [15]. There are at least two isoforms of mTOR; an alternative isoform of mTOR dubbed mTORb demonstrates increased signaling to S6K, 4EBP1, and Akt, and when overexpressed is tumorigenic in nude mice [16].
27
2. REGULATION OF TOR SIGNALING IN MAMMALS
Growth factor receptor (insulin, IGF-1) IRS1
mTORC2
PTEN
mSin1 Protor (Vertebrates only)
mTOR
DEPTOR
Rictor mLST8
Low ATP
PKC-α AMPK
Akt
(Cytoskeleton)
SGK
TSC 2 FoxO (activation of FoxO dependent genes)
PRAS40 Rheb-GDP
Rheb-GTP
Raptor
mTORC1
mTOR mLST8
Autophagy
DEPTOR
4E-BP1 Cap-dependent translation
(Vertebrates only)
S6K Ribosomal biogenesis
FIG. 2.2 Regulation of mTOR signaling by insulin. mTORC1 and mTORC2 signaling are activated by insulin. mTORC1 signaling via insulin is in part regulated by the tuberous sclerosis complex (TSC 1/2), which acts to inhibit mTOR by acting as the GTPase-activating protein (GAP) for Rheb-GTP, which is essential for mTORC1 activity. Akt activity is positively regulated by the activity of insulin and mTORC2, and Akt activates mTOR by (1) inhibiting the action of TSC 1/2 and (2) phosphorylating the mTORC1 inhibitor PRAS40 and thus relieving the inhibitory effects of PRAS40 on mTORC1. Finally, mTORC1 activity regulates the strength of insulin signaling at the cell surface via a feedback pathway (dashed line) by which S6K activity promotes the phosphorylation and degradation of IRS1.
28
DUDLEY W. LAMMING AND DAVID M. SABATINI
There is an alternate cell-type specific isoform of Raptor; one group recently identified an isoform of Raptor which may not be able to interact with normal mTOR substrates [17]. There are several mTORC2-specific three isoforms of mSin1 [15] as well as the Protor-1 and Protor-2 proteins [18]. Finally, there are two isoforms of the mTORC1 and mTORC2 interacting protein DEPTOR, but the smaller isoform may not be significantly expressed (M. Laplante, unpublished data). However, environmental signals which may regulate mTORC2 are largely unknown, although mTORC2 immunoprecipitated from insulinstimulated cells has increased activity against Akt Ser473 in vitro, demonstrating that mTORC2 activity is regulated by insulin [15]. The proteins involved in the regulation of mTORC2 activity likewise largely remain mysterious. One exception is the recent discovery of Deptor, which interacts with and inhibits the function of both mTORC1 and mTORC2 [19]. In contrast, substantial progress has been made in understanding and defining the mechanisms by which mTORC1 is regulated. These mechanisms include the activation of mTORC1 by Rheb, the inhibition of mTOR by TSC1/2, and the inhibition of mTORC1 function by the Akt substrate PRAS40, which binds to and inhibits mTORC1 function in the absence of insulin [20–23]. Finally, while it has long been known that mTORC1 is regulated by amino acids, it was only recently discovered that the Rag proteins mediate this effect [24, 25].
VI.
The Regulation of mTOR Signaling by Insulin and PRAS40
Insulin signaling via mTORC1 is positively stimulated by the GTPbinding protein Rheb, which is itself negatively regulated by the action of the tuberous sclerosis tumor suppressor proteins TSC1/TSC2 [26]. TSC2 is a GTPase-activating protein, and the loss or mutation of TSC2 results in the constitutive loading of Rheb with GTP and the constitutive activation of mTORC1 signaling. The TSC1/2 complex serves as a signal-integration hub for a variety of nutrient-related signaling to mTORC1. Energy deprivation by AMPK, MAPK signaling, Wnt signaling, and hypoxia all regulate mTORC1 signaling via the regulation of TSC1/2 and Rheb [27–29]. AMPK also regulates mTOR signaling by directly phosphorylating Raptor and inhibiting its binding to mTOR [30]. TSC1/2 is also regulated by Akt, and as mentioned above the activity of Akt is itself regulated by mTORC2 [13, 23]. Inhibition of mTORC1 signaling by rapamycin leads to the stabilization of the interaction between IRS1 and the insulin receptor due to a feedback-loop mediated via S6K1 [31, 32]
2. REGULATION OF TOR SIGNALING IN MAMMALS
29
which in many cell types leads to increased signaling through mTORC2, and as mentioned above the phosphorylation of Ser473 on Akt and its activation [13, 32]. Akt then acts at three levels to regulate mTORC1 activity. First, it directly phosphorylates TSC2, disrupting the formation of a TSC1/2 complex and thus positively regulating mTORC1 activity [33]. Secondly, it again potentiates mTORC1 activity by phosphorylating and inhibiting the mTORC1-inhibitor PRAS40 [20]. Figure 2.2 provides a simplified diagram of the signaling between mTORC1, mTORC2, Akt, and TSC2. Finally, activated Akt stabilizes the surface expression of nutrient transporters, including Glut1 and amino acid transporters, which in turn promotes the uptake of nutrients and activates mTOR signaling [34]. The role of PRAS40 in the regulation of mTORC1 signaling was, much like many of the other core components of mTORC1, discovered at approximately the same time by different teams of researchers. A massspectrometry-based approach was used to examine mTOR immunoprecipitates [20, 21]. The team of Vander Haar et al. then used a direct approach to discover additional proteins bound to mTOR, while Sancak et al. discovered PRAS40 as a consequence of the development of an in vitro kinase assay for mTORC1. They found that mTORC1 immunoprecipitated from either insulin-stimulated or serum-starved cells was equally active, leading to the hypothesis that perhaps an additional factor that conferred insulin sensitivity was being lost during the purification process. They discovered that washing with low-salt buffers during the immunoprecipitation enabled them to recover complexes that had an insulin-induced activity difference, and subsequently identified the Aktsubstrate PRAS40 as a salt-sensitive factor that inhibits mTORC1 during insulin deprivation [20]. While PRAS40 is an mTORC1 inhibitor, its action can be overcome in vitro by Rheb loaded with GTP, demonstrating that this is likely how insulin signaling to mTORC1 overcomes the effect of PRAS40 in vivo. While mTOR signaling is highly conserved, PRAS40 appears to be a more recent evolutionary development, as it is not found in yeast. However, a Drosophila homologue of PRAS40, Lobe, also functions as an mTORC1 inhibitor, demonstrating that this protein has been an important mTORC1 regulator for a substantial period of evolutionary time [20]. Subsequent work shed additional light on how PRAS40 functions and its potential clinical relevance. PRAS40 is now believed to function as a director inhibitor of substrate binding to Raptor and may itself also be an mTOR substrate [35–37]. PRAS40 has been identified as a target of Akt3 activity during malignant melanomas, and phosphorylated PRAS40 is believed to protect cancer cells from apoptosis [38]. However, this same property of PRAS40 may be beneficial in some contexts, and
30
DUDLEY W. LAMMING AND DAVID M. SABATINI
transfection of PRAS40 protects motor neurons from death in a mouse model of spinal cord injury [39].
VII.
DEPTOR: A Regulator of mTOR Signaling Found Only inVertebrates
While PRAS40 homologues are found in flies, the same is not true of the recently identified DEPTOR protein, an inhibitor of mTORC1 and mTORC2 activity that is found only in vertebrates [19]. DEPTOR was discovered via mass spectrometry of mTOR immunoprecipitates in the same, low-salt conditions used to discover PRAS40. However, unlike PRAS40, DEPTOR interacts with both mTORC1 and mTORC2, via a Cterminal portion of mTOR [19]. The interplay between DEPTOR and mTOR is complex; DEPTOR inhibits the activity of both mTORC1 and mTORC2, and loss of DEPTOR results in the activation of mTORC1 and mTORC2 both in vivo and in vitro. However, mTORC1 and mTORC2 activity both negatively regulate DEPTOR at a transcriptional and posttranslational level. DEPTOR thus serves as a mechanism for stabilizing a given level of mTOR activity; when mTOR activity is low, DEPTOR makes it harder to turn mTOR signaling on, but once mTOR signaling is on, DEPTOR makes it harder to turn off. As DEPTOR functions as an inhibitor of both mTORC1 and mTORC2, it might be predicted that overexpression of DEPTOR would shut down all mTOR signaling. Interestingly, this is not the case, and overexpression of DEPTOR actually leads to increased mTORC2 signaling and the activation of Akt. DEPTOR has 13 serine and threonine phosphorylation sites, and DEPTOR is phosphorylated in an mTOR-dependent manner. Work with a nonphosphorylatable DEPTOR mutant indicates that phosphorylated DEPTOR binds preferentially to mTORC1, and that phosphorylation of DEPTOR reverses the inhibitory effects of DEPTOR on mTORC2 activity. Thus, overexpression of phosphorylated DEPTOR will inactivate mTORC1, while promoting insulin signaling via a negative feedback loop [40, 41]. Phosphorylation of IRS1, which is promoted by mTORC1 via S6K1, results in the disassociation of IRS1 from the insulin receptor and inhibition of insulin signaling. Conversely, inhibition of mTORC1 signaling by rapamycin results in increased interaction between IRS1 and the insulin receptor, and thus activates mTORC2 signaling and promotes the phosphorylation and activation of Akt. One might predict that because overexpression of DEPTOR leads to constitutive activation of Akt, DEPTOR might be involved in certain types of cancer. Using transcriptional profiles of different cancer types, it was
31
2. REGULATION OF TOR SIGNALING IN MAMMALS
Growth factor receptor (insulin, IGF-1) IRS1
c-MAF
MAFB
mTORC1
mTORC2
inactive
active
PRAS40
Rheb-GTP DEPTOR
Raptor
mTOR DEPTOR
mLST8
mSin1 Protor
Rictor
mTOR DEPTOR
mLST8 PKC-α SGK
Akt
(Cytoskeleton)
FoxO (activation of FoxO dependent genes)
Growth and proliferation
FIG. 2.3. The Roll of DEPTOR in multiple myeloma. Multiple myelomas with translocations involving the transcription factors c-MAF and MAFB have increased transcription of DEPTOR and high levels of DEPTOR protein. This leads to the inactivation of mTORC1 and the inactivation of the feedback loop between mTORC1 activity and signaling through IRS1. As a result, IRS1 is stabilized, and the increased signaling through the insulin receptor drives increased mTORC2 activity and the activation of Akt, leading to growth and proliferation.
observed that elevated DEPTOR mRNA was seen in a subset of human multiple myeloma (MM) samples. Specifically, elevated DEPTOR is expressed in nonhyperdiploid MM, including those which had translocations involving the c-MAF or MAFB transcription factors. Additional study demonstrated that increased DEPTOR expression promotes survival in MM cell lines and that the high levels of DEPTOR were driven by overexpression of c-MAF or MAFB, and resulted in hyperactivation of PI3K signaling in general and Akt in particular. As shown in Figure 2.3, although c-MAF or MAFB results in the upregulation of DEPTOR, only mTORC1 is inactivated, and increased signaling through mTORC2 leads to unchecked growth and proliferation.
VIII.
The Rag Proteins: Regulation of mTOR Signaling byAminoAcids
In the preceding section, we have discussed the organization of mTOR signaling in higher eukaryotes, with particular emphasis on PRAS40 and DEPTOR, proteins which are found only in higher eukaryotes. Raptor,
32
DUDLEY W. LAMMING AND DAVID M. SABATINI
Rictor, and mLST8, which are conserved from yeast to humans, serve to target the activity of mTOR against specific endogenous targets, including S6K1, 4E-BP1, Akt1, and SGK1. However, these proteins do not link insulin signaling to mTOR activity, and this is achieved by the eukaryotic specific proteins PRAS40 and DEPTOR. However, while it has long been known that mTOR activity in yeast, flies, and humans is responsive to amino acid signaling, the mechanism behind this response has remained a mystery until recently. As we have discussed above, mTORC1 signaling is responsive to a variety of stresses and environmental stimuli. Many of these inputs are integrated by the mTORC1 inhibitor TSC1/2; however, mTORC1 signaling remains sensitive to amino acid withdrawal even in cells lacking TSC1/2. Amino acids were therefore thought likely to regulate the mTORC1 complex directly, but previous attempts to immunoprecipitated mTORC1 in the presence or absence of amino acids did not succeed in isolating a difference. However, examination of immunoprecipitated protein lysates is complicated by the presence of a heavy-chain band that serves to obscure proteins that are similar in size. Sancak et al. chose to isolate mTORC1 using an approach that did not rely on antibodies; instead, Flag-tagged Raptor was isolated using a Flag affinity gel and the proteins were then eluted using Flag peptide. One of the proteins that bound only in the presence of amino acids was RagC [24]. Kim et al. also identified the Rag GTPase family as essential for mTORC1 activity by conducting an RNAi screen for GTPases that regulate S6 phosphorylation [25]. RagC is a member of a family of four small Ras-related GTP-binding proteins in humans (RagA through RagD). RagA and RagB are homologous to each other as well as to yeast Gtr1, while RagC and RagD are homologous to each other as well as yeast Gtr2. The mode of action of these proteins is conserved from yeast to humans, and in both Rag proteins function as heterodimers consisting of one Gtr1-like protein (Rag A or RagB) and one Gtr2-like protein (RagC or Rag D). In yeast, the Gtr1 and Gtr2 proteins have been linked to control of amino acid permeases and microautophagy, processes that are regulated by TOR signaling. Sancak et al. showed that Raptor copurified with a heterodimer of Rag proteins, and both Sancak et al. and Kim et al. found that the Rag proteins were essential for mTOR signaling by amino acids [24, 25]. Sancak et al. went on to show that in the presence of amino acids, Rag proteins function by localizing mTORC1 to a subcellular vesicular compartment marked by Rab7, indicating that mTOR is localized to the late endosomal and lysosomal compartments [24]. Therefore, in the presence of amino acids, both mTOR and its activator Rheb are present in Rab7-positive structures (see Figure 2.4) [24, 42]. Recently, it was demonstrated that TORC1 in yeast may be regulated in a similar manner by GTP-bound Gtr1, and that the
33
2. REGULATION OF TOR SIGNALING IN MAMMALS
PRAS40
Raptor mTOR
Rheb-GTP
/D
A/ B
C
ag
ag
R
Rag A/B
Rag C/D
R
r pto Ra
GTP
40 AS PR
OR PT DE
GTP
R
Rheb-GTP
TO
8 ST mL
+ Amino acids GDP
m
mLST8 DEPTOR
GDP
Rab7
Late endosome/lysosome
Rab7
Late endosome/lysosome
FIG. 2.4. Activation of mTORC1 in response to amino acids. In the absence of amino acids, mTORC1 is distributed throughout the cell, while its activator Rheb-GTP is localized to a late endosomal or lysosomal compartment marked by Rab7. Heterodimeric pairs of Rag proteins in Rab7-positive compartments are inactive, with Rag A/B bound to GDP and Rag C/D bound to GTP. In the presence of amino acids, the Rag proteins become active, with Rag A/B bound to GTP and Rag C/D bound to GDP. mTORC1 then relocalizes to the Rab7-positive endosomal compartments and is united with Rheb-GTP.
nucleotide binding of status of Gtr1 was regulated by the guanine nucleotide exchange factor Vam6 [43].
IX.
The Future: Remaining Mysteries of mTOR Signaling and Clinical Significance of mTOR
In this chapter, we have explored the function and structure of the two mammalian mTOR complexes, mTORC1 and mTORC2, and their control in higher eukaryotes such as human via PRAS40, DEPTOR, and the Rag proteins. We might, looking at a complex diagram of mTOR signaling, think that we now fully know everything about mTOR signaling, but in fact substantial work remains in understanding the function of the mTOR pathway. We will discuss some of these mysteries below and then discuss briefly the clinical significance of the mTOR pathway in light of recent discoveries about mTOR, cancer, and aging. Above, we discussed how the Rag proteins activate mTORC1 in response to amino acids by localizing it to a compartment containing Rheb. However, it is still not clear how signals from various amino acids activate this relocalization, and GEFs for the mammalian Rag proteins have not yet been identified. It is also unclear exactly how localization of mTORC1 may contribute to the localization (and phosphorylation) of its substrates. In yeast, the S6K1 homologue Sch9 localizes to the vacuole and
34
DUDLEY W. LAMMING AND DAVID M. SABATINI
its localization is nutrient sensitive [44], so perhaps S6K1 (and other mTORC1 substrates) localize to the same compartment as mTORC1 during periods of abundant nutrients. We also do not have a good understanding of what other substrates there may be for mTORC1, or of how mTORC1 signaling regulates autophagy. Turning our attention to mTORC2, there is still much we do not understand. At the most basic level, while we know that increased PI3K activity results in increased mTORC2 activity, we do not understand how this signal is transmitted to mTORC2. While rapamycin has allowed researchers around the world to study mTORC1 signaling, mTORC2 substrates have only been identified in the last few years. This includes such key regulatory proteins as Akt, which regulates the activity of forkhead transcription factors as well as the activity of mTORC1, and SGK [13, 14]. It is likely that as work progresses, additional substrates for mTORC2 activity will also be found. It has also recently been appreciated that rapamycin can inhibit mTORC2 function in some cell types, likely by inhibiting mTORC2 assembly [45]. As mTORC2 signaling may be important in some types of cancer, including prostate cancer and AML, the regulation of mTORC2 function by rapamycin may not only lead to the discovery of interesting biology but may also be clinically relevant [46, 47]. One factor that will help drive the discovery of more mTORC2 biology is the development of drugs that specifically inhibit mTORC2. While no mTORC2-specific drugs have yet been identified, several groups have identified compounds that will directly inhibit the activity of the mTOR kinase and thus inhibit both mTORC1 and mTORC2 [48–50]. From a clinical perspective, rapamycin has always been viewed as a promising drug candidate for the treatment of cancer—it specifically inhibits mTORC1 at nanomolar concentrations, and mTORC1 is hyperactive in many types of cancer. However, despite numerous clinical trials, rapamycin has had less of a clinical impact than hoped, although recent work suggests that rapamycin may be of use in treating some types of AML, renal and prostate cancers [46, 47, 51]. However, when applied to cancer cell lines in a tissue culture model, rapamycin often only has modest effects on proliferation. This may in part be due to the feedback loop between mTORC1 and IRS1 discussed above, resulting in the activation of PI3K signaling and subsequent phosphorylation and activation of Akt. Drugs that inhibit the mTOR kinase itself, possibly in combination with inhibition of PI3K activity, may fare better and at least one new inhibitor of mTORC1 function has been reported to induce cell cycle arrest [50]. Finally, a great deal of attention from both the scientific community and the popular press has recently been focused on the mTOR pathway as possible means of regulating the aging process. The influence of mTOR signaling on lifespan was first observed in 2003 in C. elegans, and was soon duplicated in
2. REGULATION OF TOR SIGNALING IN MAMMALS
35
Drosophila and yeast [52–54]. Treatment of an organism with rapamycin similarly extends lifespan in yeast, and was recently shown to extend the lifespan of mice started on a diet containing rapamycin at 20 months of age [55, 56]. While the mechanisms behind this extension of lifespan in mammals have not yet been determined, it seems likely that the effect relies in part on decreased or altered translation. Several studies in C. elegans have demonstrated that RNAi against certain ribosomal proteins, S6K, the translation initiation factor eIF2, or other proteins required for translation such as eIF4E and eiF4G all extend lifespan and decrease protein translation [57]. However, the lifespan of worms with RNAi against ceTOR can be further extended by RNAi against ribosomal proteins, S6K, and transcription initiation factors, indicating that perhaps inhibition of TOR engages a different life extension pathway than the other interventions. Indeed, the Kenyon lab found that RNAi against many of these factors induced daf-16/ FOXO signaling, while RNAi against ceTOR did not; also, RNAi against TOR in C. elegans subjected to dietary restriction (DR) reduced translation but did not further extend lifespan [57]. A recent study of S6K1 / mice by Selman et al. found that female S6K1 / mice had extended lifespan despite not demonstrating decreased protein synthesis or rates of translation, and the researchers noted that these mice had a gene expression pattern similar to that of mice subjected to CR or with an activated AMPK pathway [58]. Clearly, inhibition of mTOR signaling and DR is likely to involve the engagement of separate, but somewhat overlapping sets of pathways.
ACKNOWLEDGMENTS We thank D.E. Cohen, M. Laplante, T. Peterson, Y. Sancak, and A. Strohecker for helpful discussions and critical reading of the manuscript. DWL was supported by a Ruth L. Kirschstein National Research Service Award (NRSA) Postdoctoral Fellowship (F32AG032833) from the National Institute of Aging, but the preceding content is solely the responsibility of the authors and does not necessarily represent the official views of the NIA or the NIH. DMS was supported by grants from the NIH (R01-AI47389 and R01-CA103866) and awards from the American Federation of Aging Research, the Keck Foundation and the LAM (Lymphangioleiomyomatosis) Foundation and is an investigator of the Howard Hughes Medical Institute.
REFERENCES 1. Brown, E.J., et al. (1994). A mammalian protein targeted by G1-arresting rapamycinreceptor complex. Nature 369:756–758. 2. Sabatini, D.M., Erdjument-Bromage, H., Lui, M., Tempst, P., and Snyder, S.H. (1994). RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell 78:35–43.
36
DUDLEY W. LAMMING AND DAVID M. SABATINI
3. Sabers, C.J., et al. (1995). Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells. J Biol Chem 270:815–822. 4. 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. 5. Kim, D.H., et al. (2002). mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110:163–175. 6. Hara, K., et al. (2002). Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 110:177–189. 7. Chen, E.J., and Kaiser, C.A. (2003). LST8 negatively regulates amino acid biosynthesis as a component of the TOR pathway. J Cell Biol 161:333–347. 8. Kim, D.H., et al. (2003). GbetaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR. Mol Cell 11:895–904. 9. 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. 10. Jacinto, E., et al. (2004). Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol 6:1122–1128. 11. Jones, K.T., Greer, E.R., Pearce, D., and Ashrafi, K. (2009). Rictor/TORC2 regulates Caenorhabditis elegans fat storage, body size, and development through sgk-1. PLoS Biol 7:e60. 12. Soukas, A.A., Kane, E.A., Carr, C.E., Melo, J.A., and Ruvkun, G. (2009). Rictor/TORC2 regulates fat metabolism, feeding, growth, and life span in Caenorhabditis elegans. Genes Dev 23:496–511. 13. Sarbassov, D.D., Guertin, D.A., Ali, S.M., and Sabatini, D.M. (2005). Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307:1098–1101. 14. Garcia-Martinez, J.M., and Alessi, D.R. (2008). mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1). Biochem J 416:375–385. 15. Frias, M.A., et al. (2006). mSin1 is necessary for Akt/PKB phosphorylation, and its isoforms define three distinct mTORC2s. Curr Biol 16:1865–1870. 16. Panasyuk, G., et al. (2009). The mTORbeta splicing isoform promotes cell proliferation and tumorigenesis. J Biol Chem 284(45):30807–30814. 17. Sun, C., Southard, C., and Di Rienzo, A. (2009). Characterization of a novel splicing variant in the RAPTOR gene. Mutat Res 662:88–92. 18. Pearce, L.R., et al. (2007). Identification of Protor as a novel Rictor-binding component of mTOR complex-2. Biochem J 405:513–522. 19. Peterson, T.R., et al. (2009). DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival. Cell 137:873–886. 20. Sancak, Y., et al. (2007). PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase. Mol Cell 25:903–915. 21. Vander Haar, E., Lee, S.I., Bandhakavi, S., Griffin, T.J., and Kim, D.H. (2007). Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat Cell Biol 9:316–323. 22. Long, X., Ortiz-Vega, S., Lin, Y., and Avruch, J. (2005). Rheb binding to mammalian target of rapamycin (mTOR) is regulated by amino acid sufficiency. J Biol Chem 280:23433–23436. 23. Inoki, K., Li, Y., Zhu, T., Wu, J., and Guan, K.L. (2002). TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol 4:648–657.
2. REGULATION OF TOR SIGNALING IN MAMMALS
37
24. Sancak, Y., et al. (2008). The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320:1496–1501. 25. Kim, E., Goraksha-Hicks, P., Li, L., Neufeld, T.P., and Guan, K.L. (2008). Regulation of TORC1 by Rag GTPases in nutrient response. Nat Cell Biol 10:935–945. 26. Zhang, Y., Gao, X., Saucedo, L.J., Ru, B., Edgar, B.A., and Pan, D. (2003). Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins. Nat Cell Biol 5:578–581. 27. Inoki, K., et al. (2006). TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth. Cell 126:955–968. 28. Inoki, K., Zhu, T., and Guan, K.L. (2003). TSC2 mediates cellular energy response to control cell growth and survival. Cell 115:577–590. 29. 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. 30. Gwinn, D.M., et al. (2008). AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell 30:214–226. 31. 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. 32. Tremblay, F., Gagnon, A., Veilleux, A., Sorisky, A., and Marette, A. (2005). Activation of the mammalian target of rapamycin pathway acutely inhibits insulin signaling to Akt and glucose transport in 3T3-L1 and human adipocytes. Endocrinology 146:1328–1337. 33. Potter, C.J., Pedraza, L.G., and Xu, T. (2002). Akt regulates growth by directly phosphorylating Tsc2. Nat Cell Biol 4:658–665. 34. Edinger, A.L., and Thompson, C.B. (2002). Akt maintains cell size and survival by increasing mTOR-dependent nutrient uptake. Mol Biol Cell 13:2276–2288. 35. Oshiro, N., et al. (2007). The proline-rich Akt substrate of 40 kDa (PRAS40) is a physiological substrate of mammalian target of rapamycin complex 1. J Biol Chem 282:20329–20339. 36. Wang, L., Harris, T.E., Roth, R.A., and Lawrence, J.C., Jr (2007). PRAS40 regulates mTORC1 kinase activity by functioning as a direct inhibitor of substrate binding. J Biol Chem 282:20036–20044. 37. Fonseca, B.D., Smith, E.M., Lee, V.H., MacKintosh, C., and Proud, C.G. (2007). PRAS40 is a target for mammalian target of rapamycin complex 1 and is required for signaling downstream of this complex. J Biol Chem 282:24514–24524. 38. Madhunapantula, S.V., Sharma, A., and Robertson, G.P. (2007). PRAS40 deregulates apoptosis in malignant melanoma. Cancer Res 67:3626–3636. 39. Yu, F., Narasimhan, P., Saito, A., Liu, J., and Chan, P.H. (2008). Increased expression of a proline-rich Akt substrate (PRAS40) in human copper/zinc-superoxide dismutase transgenic rats protects motor neurons from death after spinal cord injury. J Cereb Blood Flow Metab 28:44–52. 40. Ozes, O.N., et al. (2001). A phosphatidylinositol 3-kinase/Akt/mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate-1. Proc Natl Acad Sci USA 98:4640–4645. 41. Shah, O.J., Wang, Z., and Hunter, T. (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. 42. Avruch, J., Long, X., Ortiz-Vega, S., Rapley, J., Papageorgiou, A., and Dai, N. (2009). Amino acid regulation of TOR complex 1. Am J Physiol Endocrinol Metab 296: E592–E602. 43. Binda, M., et al. (2009). The Vam6 GEF controls TORC1 by activating the EGO complex. Mol Cell 35:563–573.
38
DUDLEY W. LAMMING AND DAVID M. SABATINI
44. Jorgensen, P., Rupes, I., Sharom, J.R., Schneper, L., Broach, J.R., and Tyers, M. (2004). A dynamic transcriptional network communicates growth potential to ribosome synthesis and critical cell size. Genes Dev 18:2491–2505. 45. Sarbassov, D.D., et al. (2006). Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell 22:159–168. 46. Zeng, Z., et al. (2007). Rapamycin derivatives reduce mTORC2 signaling and inhibit AKT activation in AML. Blood 109:3509–3512. 47. Guertin, D.A., et al. (2009). mTOR complex 2 is required for the development of prostate cancer induced by Pten loss in mice. Cancer Cell 15:148–159. 48. Feldman, M.E., et al. (2009). Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2. PLoS Biol 7:e38. 49. Thoreen, C.C., et al. (2009). An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1. J Biol Chem 284:8023–8032. 50. Garcia-Martinez, J.M., et al. (2009). Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR). Biochem J 421:29–42. 51. Oudard, S., et al. (2009). Everolimus (RAD001): an mTOR inhibitor for the treatment of metastatic renal cell carcinoma. Expert Rev Anticancer Ther 9:705–717. 52. Vellai, T., Takacs-Vellai, K., Zhang, Y., Kovacs, A.L., Orosz, L., and Muller, F. (2003). Genetics: influence of TOR kinase on lifespan in C. elegans. Nature 426:620. 53. Kaeberlein, M., et al. (2005). Regulation of yeast replicative life span by TOR and Sch9 in response to nutrients. Science 310:1193–1196. 54. Kapahi, P., Zid, B.M., Harper, T., Koslover, D., Sapin, V., and Benzer, S. (2004). Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway. Curr Biol 14:885–890. 55. Medvedik, O., Lamming, D.W., Kim, K.D., and Sinclair, D.A. (2007). MSN2 and MSN4 Link Calorie Restriction and TOR to Sirtuin-Mediated Lifespan Extension in Saccharomyces cerevisiae. PLoS Biol 5:e261. 56. Harrison, D.E., et al. (2009). Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature 460:392–395. 57. Hansen, M., Taubert, S., Crawford, D., Libina, N., Lee, S.J., and Kenyon, C. (2007). Lifespan extension by conditions that inhibit translation in Caenorhabditis elegans. Aging Cell 6:95–110. 58. Selman, C., et al. (2009). Ribosomal protein S6 kinase 1 signaling regulates mammalian life span. Science 326:140–144.
DAVID M. SABATINI
Whitehead Institute Cambridge, Massachusetts, USA
I.
Abstract
Since the initial discovery of rapamycin in the soils of Easter Island, there has been significant interest in determining the biological mechanism underlying the effects of rapamycin on mammalian tissues. These effects include changes in cell size and proliferation, as well as decreased and altered mRNA translation, and changes in autophagy. It was thus immediately clear that the mammalian target of rapamycin (mTOR) was a key regulator of cell growth and proliferation, and further work has shown that mTOR serves as a central regulator of cell processes in response to nutrients and environmental stimuli. In this chapter, we will discuss the mechanisms behind the many different functions of mTOR, with emphasis on the proteins that make up two mTOR-containing complexes and regulate mTOR signaling in response to insulin and amino acids. We will also discuss the medical relevance of these findings and the potential clinical significance of inhibiting mTOR.
II.
One Enzyme,Two Complexes
Rapamycin functions by binding to the immunophilin FKBP12, and this complex then binds to and inhibits many functions of mTOR. The mTOR protein kinase was identified in 1994–1995 by three separate groups that THE ENZYMES, Vol. XXVII # 2010 Elsevier Inc. All rights reserved.
21
ISSN NO: 1874-6047 DOI: 10.1016/S1874-6047(10)27002-6
22
DUDLEY W. LAMMING AND DAVID M. SABATINI
isolated a 289-kD protein with a rapamycin-dependent interaction with FKBP12 [1–3]. mTOR was found to have approximately 40% homology to the two Saccharomyces cerevisiae TOR proteins, as well as high homology to the TOR proteins that would later be found in other eukaryotes. Indeed, many of the proteins that complex with mTOR to regulate and target mTOR are conserved in diverse eukaryotes, including fungi such as S. cerevisiae, metazoans such as Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and mammals including mice and humans (see Table 2.1). Most early work on mTOR used rapamycin extensively as a tool. However, work with kinase-dead mTOR and RNAi knockdown of mTOR made it apparent that mTOR participates in both rapamycin-dependent and independent processes. Organisms spanning complexity from humans to yeast have now been shown to have both rapamycin-sensitive and insensitive TOR-dependent processes (see Figure 2.1). Higher eukaryotes, including mammals, incorporate the same mTOR protein into both a rapamycinsensitive mTOR complex I (mTORC1) and a complex that is relatively resistant to the effects of rapamycin, mTOR complex II (mTORC2). The core mTORC1 proteins are mTOR, Raptor, and mLST8/GbL, while the core mTORC2 proteins are mTOR, Rictor, and mLST8/GbL; the components of mTORC1 show a higher degree of evolutionary conservation than do the components of mTORC1. Yeast actually has two distinct TOR TABLE 2.1 MTORC1 AND MTORC2 PROTEINS IN HUMANS,
DROSOPHILA MELANOGASTER, CAENORHABDITIS ELEGANS, AND SACCHAROMYCES CEREVISIAE H. sapiens
D. melanogaster
C. elegans
S. cerevisiae
mTORC1 (rapamycin sensitive)
mTOR mLST8 Raptor
dTOR CG3004 Raptor
ceTOR
mTORC2 (rapamycin insensitive)
PRAS40 DEPTOR RagA, RagB RagC, RagD mTOR mLST8 mSin1
Lobe dRagA dRagC dTOR CG3004
raga-1 ragc-1 ceTOR
Rictor
Rictor
CeRictor
Protor DEPTOR
Tor1, Tor2 Lst8 Kog1 Tco89 Gtr1 Gtr2 Tor2 Lst8 Avo1 Avo2 Avo3 Bit61
ceRaptor
23
2. REGULATION OF TOR SIGNALING IN MAMMALS
Growth factors (insulin and IGF-1)
Amino acids
Hypoxia Energy deficit Rapamycin
mTORC1
Autophagy
mTORC2
Proliferation Cell volume Cytoskeletal structure
FIG. 2.1. The regulation of growth and proliferation by mTOR. mTORC1 is positively regulated by amino acids and growth factors, including insulin and IGF-1, and is inhibited by hypoxia, low levels of ATP, and rapamycin, while mTORC2 is positively regulated by growth factors. Both mTORC1 and mTORC2 signaling promote growth and proliferation, but only mTORC1 inhibits autophagy.
proteins; TOR1, which is TORC1 specific, and TOR2, which participates in both TORC1 and TORC2 (discussed in Chapter 1) [4]. Depending on cell type and nutrient conditions, numerous other proteins also associate with mTORC1, mTORC2, or both (see Table 2.1). In this chapter, we will focus on the regulation of mTOR signaling by PRAS40, DEPTOR, and the Rag family of small GTP-binding proteins.
III.
Raptor Defines mTORC1
The identification of Raptor as an mTOR associated protein came about as researchers in many labs were attempting to discover the mechanism by which nutrients and environmental stimuli regulate the phosphorylation of the mTOR targets S6K-1 and 4E-BP1. In vivo, these targets are phosphorylated by mTOR in response to such stimuli as amino acids, but researchers observed that the activity of the immunoprecipitated mTOR kinase against S6K-1 or 4E-BP in vitro was unchanged. One possible explanation for these results is that mTOR exists in a complex with additional regulatory proteins, but that these proteins were disassociating during mTOR purification. Two separate groups simultaneously investigated the properties of mTOR that was immunoprecipitated using different lysis conditions. One group approached the problem using the reversible cross-linker dithiobis
24
DUDLEY W. LAMMING AND DAVID M. SABATINI
(succinimidyl propionate) (DSP) [5], while another lab used an approach utilizing chromatography [6]. Both groups identified a 150-kD protein that immunoprecipitated with mTOR and showed high conservation within all eukaryotes (see Table 2.1 for a list of mTOR associated proteins and their homologues in various organisms). Both labs subsequently found that Raptor (for regulatory associated protein of mTOR), can also be isolated in the absence of cross-linker, indicating that the interaction is real and not an artifact of the cross-linker, so long as appropriate and gentle lysis conditions are used. Specifically, Raptor remains bound to mTOR when low concentrations of the detergent CHAPS was used instead of the more commonly used Triton X-100 or NP-40 [5]. To identify the role of Raptor in the regulation of mTOR, RNAi was used to decrease endogenous levels of Raptor in mammalian cells. Cells in which Raptor expression was thus lowered showed decreased phosphorylation of the mTOR target S6K-1, decreased proliferation, and smaller volume, all phenotypes that also occur when cells are treated with rapamycin or mTOR itself is knocked down with RNAi. RNAi against C. elegans homologues of Raptor and mTOR demonstrated that RNAi against either resulted in a similar set of developmental phenotypes, including delayed gonadal development, the development of large gut lysosomes, and smaller intestinal cells [6]. These experiments demonstrated that many of the phenotypes regulated by mTOR signaling are Raptor dependent. The initial question of how nutrients and environmental stimuli regulate the phosphorylation of mTOR targets was at least partially answered by observing the properties of the mTOR–Raptor interaction. Researchers found that the stability of the mTOR–Raptor interaction was regulated by specific nutrients; specifically, the in vivo complex was destabilized by the addition of amino acids, leucine, and glucose, treatments that result in increased mTOR activity. Other treatments that decrease mTOR signaling to S6K-1, such as treatment with valinomycin, 2-deoxyglucose, and H2O2, also stabilized the mTOR–Raptor interaction. This led to the hypothesis that in the absence of nutrients, Raptor regulates mTOR by inhibiting its kinase activity, and that nutrients increase mTOR signaling by destabilizing the mTOR–Raptor interaction. This theory was supported by the finding that overexpression of Raptor suppresses the catalytic activity mTOR and inhibits phosphorylation of S6K1 and 4E-BP1 in vivo. However, this hypothesis does not explain everything; treatment of serum-starved cells with insulin, which results in increased mTOR signaling and increased phosphorylation of S6K1 by mTOR, does not destabilize the mTOR–Raptor interaction, suggesting that the regulation of mTOR activity by insulin and other growth factors may proceed via a different mechanism [5].
2. REGULATION OF TOR SIGNALING IN MAMMALS
IV.
25
Rictor Defines a Rapamycin-Insensitive mTOR Complex
Two additional proteins that complex with mTOR, Rictor and mLST8, were also discovered independently by different groups and at roughly the same time. These groups used different approaches to the same problem, some working with immunoprecipitation of mTOR followed by mass spectrometry or alternatively working up from yeast TOR and using homology to find mTOR-interacting proteins. As mentioned above, it was discovered that yeast TOR could be found in two complexes, only one of which was rapamycin sensitive (see Table 2.1 and [4]). An additional protein of unknown function, Lst8, could be found in both complexes, and shared homology with a relatively small human protein known originally as GbL and now dubbed mLST8 due to its homology to the yeast protein [7]. They found that overexpressed mLST8 and mTOR coprecipitated. A study conducted by a different group using mass spectrometry upon immunoprecipitated mTOR found that mTOR coprecipitates with a 36-kD protein known as GbL that acts to strongly increase mTOR kinase activity toward S6K1 and 4E-BP1 [8]. Yeast TOR functions in two complexes, one of which is rapamycin insensitive, and it was the subject of substantial interest to determine if mTOR similarly functioned in two complexes. Rictor was identified in 2004 as a defining component of mTORC2, a rapamycin-insensitive mTOR complex [9, 10]. Rictor was difficult to identify by homology due to its relatively low sequence conservation between mammals and lower eukaryotes such as yeast, and a lack of any domains of known function [9]. Indeed, the C. elegans Rictor homologue was only recently identified due to the low sequence conservation [11, 12]. The percentage of mTOR that complexes with Rictor also varies widely by cell type and is inversely correlated with the expression of Raptor. For instance, HEK293T cells contain mostly mTOR–Raptor complexes, while HeLa cells contain more Rictor than Raptor [9]. Because mTORC2 is relatively insensitive to rapamycin, the function of mTORC2 has been less clear until recently. One initial observation was that mTORC2 might regulate the cytoskeleton, although the precise mechanism behind this effect was unclear [4, 9, 10]. The deletion of mTORC2-specific subunits leads to the dephosphorylation of the hydrophobic motif site in Protein Kinase C (PKC)a. Subsequent experiments demonstrated that siRNA knockdown of either mTOR or Rictor, but not Raptor or treatment with rapamycin, also inhibited the kinase activity of PKCa, demonstrating the importance of this phosphorylation. Unfortunately, no environmental stimuli that regulate PKCa phosphorylation by mTOR have been
26
DUDLEY W. LAMMING AND DAVID M. SABATINI
identified, but siRNA knockdown of either Rictor or PKCa has similar effects on the actin cytoskeleton, implicating the regulation of PKCa phosphorylation as one mechanism by which mTOR signaling regulates the cytoskeleton [9]. While the effects of mTOR on the cytoskeleton remain an interesting area of study, an important step in understanding the effects of mTORC2 signaling occurred the following year with the identification of Akt as an mTORC2 substrate [13]. It had long been known that active Akt was phosphorylated on two key residues, Threonine 308 and Serine 473. The Thr308 kinase had previously been identified as PDK1, but the kinase for Serine 473 had remained elusive. While numerous kinases had been proposed as the Serine 473 kinase, including PDK1, Akt, and DNA-PK, none of these studies were fully convincing. However, in the course of experiments in Drosophila S2 cells and mammalian cell lines, it was observed that siRNA against mTOR or Raptor slightly increased the phosphorylation of Akt Ser473, while siRNA against Rictor resulted in the almost complete inhibition of Akt Ser473 phosphorylation. This was seen even in cell lines in which PTEN, an antagonist of PI3K signaling, had been knocked down or deleted. Subsequent experiments confirmed that mTORC2 directly phosphorylates Akt Ser473 both in vitro and in vivo, and that mTORC2 was the long-sought ‘‘PDK2’’ for Akt Ser473 [13]. Interestingly, mTORC2 appears to also regulate the activity of a related kinase, SGK1 [14]. mTORC2 regulates activation of SGK1 via direct phosphorylation of SGK1 Ser422, and may thus act as a regulator of numerous downstream targets of SGK1, including NDRG1 and FoxO proteins [14].
V.
Additional mTORC1and mTORC2 Proteins
We can therefore define the two major mTOR complexes as the rapamycin-sensitive mTORC1, containing mTOR, Raptor, and mLST8, while the remainder of mTOR is in the relatively rapamycin-insensitive mTORC2, containing mTOR, Rictor, and mLST8 (see Figure 2.2 and Table 2.1). Subsequent work by a variety of labs has identified additional proteins that complex with either mTORC1, mTORC2, or both and isoforms of many of these proteins also exist. All or most of these isoforms can be combined together, which together likely define several different ‘‘flavors’’ of mTORC1 and mTORC2 that may vary between cell types and may have different sensitivity to stimuli [15]. There are at least two isoforms of mTOR; an alternative isoform of mTOR dubbed mTORb demonstrates increased signaling to S6K, 4EBP1, and Akt, and when overexpressed is tumorigenic in nude mice [16].
27
2. REGULATION OF TOR SIGNALING IN MAMMALS
Growth factor receptor (insulin, IGF-1) IRS1
mTORC2
PTEN
mSin1 Protor (Vertebrates only)
mTOR
DEPTOR
Rictor mLST8
Low ATP
PKC-α AMPK
Akt
(Cytoskeleton)
SGK
TSC 2 FoxO (activation of FoxO dependent genes)
PRAS40 Rheb-GDP
Rheb-GTP
Raptor
mTORC1
mTOR mLST8
Autophagy
DEPTOR
4E-BP1 Cap-dependent translation
(Vertebrates only)
S6K Ribosomal biogenesis
FIG. 2.2 Regulation of mTOR signaling by insulin. mTORC1 and mTORC2 signaling are activated by insulin. mTORC1 signaling via insulin is in part regulated by the tuberous sclerosis complex (TSC 1/2), which acts to inhibit mTOR by acting as the GTPase-activating protein (GAP) for Rheb-GTP, which is essential for mTORC1 activity. Akt activity is positively regulated by the activity of insulin and mTORC2, and Akt activates mTOR by (1) inhibiting the action of TSC 1/2 and (2) phosphorylating the mTORC1 inhibitor PRAS40 and thus relieving the inhibitory effects of PRAS40 on mTORC1. Finally, mTORC1 activity regulates the strength of insulin signaling at the cell surface via a feedback pathway (dashed line) by which S6K activity promotes the phosphorylation and degradation of IRS1.
28
DUDLEY W. LAMMING AND DAVID M. SABATINI
There is an alternate cell-type specific isoform of Raptor; one group recently identified an isoform of Raptor which may not be able to interact with normal mTOR substrates [17]. There are several mTORC2-specific three isoforms of mSin1 [15] as well as the Protor-1 and Protor-2 proteins [18]. Finally, there are two isoforms of the mTORC1 and mTORC2 interacting protein DEPTOR, but the smaller isoform may not be significantly expressed (M. Laplante, unpublished data). However, environmental signals which may regulate mTORC2 are largely unknown, although mTORC2 immunoprecipitated from insulinstimulated cells has increased activity against Akt Ser473 in vitro, demonstrating that mTORC2 activity is regulated by insulin [15]. The proteins involved in the regulation of mTORC2 activity likewise largely remain mysterious. One exception is the recent discovery of Deptor, which interacts with and inhibits the function of both mTORC1 and mTORC2 [19]. In contrast, substantial progress has been made in understanding and defining the mechanisms by which mTORC1 is regulated. These mechanisms include the activation of mTORC1 by Rheb, the inhibition of mTOR by TSC1/2, and the inhibition of mTORC1 function by the Akt substrate PRAS40, which binds to and inhibits mTORC1 function in the absence of insulin [20–23]. Finally, while it has long been known that mTORC1 is regulated by amino acids, it was only recently discovered that the Rag proteins mediate this effect [24, 25].
VI.
The Regulation of mTOR Signaling by Insulin and PRAS40
Insulin signaling via mTORC1 is positively stimulated by the GTPbinding protein Rheb, which is itself negatively regulated by the action of the tuberous sclerosis tumor suppressor proteins TSC1/TSC2 [26]. TSC2 is a GTPase-activating protein, and the loss or mutation of TSC2 results in the constitutive loading of Rheb with GTP and the constitutive activation of mTORC1 signaling. The TSC1/2 complex serves as a signal-integration hub for a variety of nutrient-related signaling to mTORC1. Energy deprivation by AMPK, MAPK signaling, Wnt signaling, and hypoxia all regulate mTORC1 signaling via the regulation of TSC1/2 and Rheb [27–29]. AMPK also regulates mTOR signaling by directly phosphorylating Raptor and inhibiting its binding to mTOR [30]. TSC1/2 is also regulated by Akt, and as mentioned above the activity of Akt is itself regulated by mTORC2 [13, 23]. Inhibition of mTORC1 signaling by rapamycin leads to the stabilization of the interaction between IRS1 and the insulin receptor due to a feedback-loop mediated via S6K1 [31, 32]
2. REGULATION OF TOR SIGNALING IN MAMMALS
29
which in many cell types leads to increased signaling through mTORC2, and as mentioned above the phosphorylation of Ser473 on Akt and its activation [13, 32]. Akt then acts at three levels to regulate mTORC1 activity. First, it directly phosphorylates TSC2, disrupting the formation of a TSC1/2 complex and thus positively regulating mTORC1 activity [33]. Secondly, it again potentiates mTORC1 activity by phosphorylating and inhibiting the mTORC1-inhibitor PRAS40 [20]. Figure 2.2 provides a simplified diagram of the signaling between mTORC1, mTORC2, Akt, and TSC2. Finally, activated Akt stabilizes the surface expression of nutrient transporters, including Glut1 and amino acid transporters, which in turn promotes the uptake of nutrients and activates mTOR signaling [34]. The role of PRAS40 in the regulation of mTORC1 signaling was, much like many of the other core components of mTORC1, discovered at approximately the same time by different teams of researchers. A massspectrometry-based approach was used to examine mTOR immunoprecipitates [20, 21]. The team of Vander Haar et al. then used a direct approach to discover additional proteins bound to mTOR, while Sancak et al. discovered PRAS40 as a consequence of the development of an in vitro kinase assay for mTORC1. They found that mTORC1 immunoprecipitated from either insulin-stimulated or serum-starved cells was equally active, leading to the hypothesis that perhaps an additional factor that conferred insulin sensitivity was being lost during the purification process. They discovered that washing with low-salt buffers during the immunoprecipitation enabled them to recover complexes that had an insulin-induced activity difference, and subsequently identified the Aktsubstrate PRAS40 as a salt-sensitive factor that inhibits mTORC1 during insulin deprivation [20]. While PRAS40 is an mTORC1 inhibitor, its action can be overcome in vitro by Rheb loaded with GTP, demonstrating that this is likely how insulin signaling to mTORC1 overcomes the effect of PRAS40 in vivo. While mTOR signaling is highly conserved, PRAS40 appears to be a more recent evolutionary development, as it is not found in yeast. However, a Drosophila homologue of PRAS40, Lobe, also functions as an mTORC1 inhibitor, demonstrating that this protein has been an important mTORC1 regulator for a substantial period of evolutionary time [20]. Subsequent work shed additional light on how PRAS40 functions and its potential clinical relevance. PRAS40 is now believed to function as a director inhibitor of substrate binding to Raptor and may itself also be an mTOR substrate [35–37]. PRAS40 has been identified as a target of Akt3 activity during malignant melanomas, and phosphorylated PRAS40 is believed to protect cancer cells from apoptosis [38]. However, this same property of PRAS40 may be beneficial in some contexts, and
30
DUDLEY W. LAMMING AND DAVID M. SABATINI
transfection of PRAS40 protects motor neurons from death in a mouse model of spinal cord injury [39].
VII.
DEPTOR: A Regulator of mTOR Signaling Found Only inVertebrates
While PRAS40 homologues are found in flies, the same is not true of the recently identified DEPTOR protein, an inhibitor of mTORC1 and mTORC2 activity that is found only in vertebrates [19]. DEPTOR was discovered via mass spectrometry of mTOR immunoprecipitates in the same, low-salt conditions used to discover PRAS40. However, unlike PRAS40, DEPTOR interacts with both mTORC1 and mTORC2, via a Cterminal portion of mTOR [19]. The interplay between DEPTOR and mTOR is complex; DEPTOR inhibits the activity of both mTORC1 and mTORC2, and loss of DEPTOR results in the activation of mTORC1 and mTORC2 both in vivo and in vitro. However, mTORC1 and mTORC2 activity both negatively regulate DEPTOR at a transcriptional and posttranslational level. DEPTOR thus serves as a mechanism for stabilizing a given level of mTOR activity; when mTOR activity is low, DEPTOR makes it harder to turn mTOR signaling on, but once mTOR signaling is on, DEPTOR makes it harder to turn off. As DEPTOR functions as an inhibitor of both mTORC1 and mTORC2, it might be predicted that overexpression of DEPTOR would shut down all mTOR signaling. Interestingly, this is not the case, and overexpression of DEPTOR actually leads to increased mTORC2 signaling and the activation of Akt. DEPTOR has 13 serine and threonine phosphorylation sites, and DEPTOR is phosphorylated in an mTOR-dependent manner. Work with a nonphosphorylatable DEPTOR mutant indicates that phosphorylated DEPTOR binds preferentially to mTORC1, and that phosphorylation of DEPTOR reverses the inhibitory effects of DEPTOR on mTORC2 activity. Thus, overexpression of phosphorylated DEPTOR will inactivate mTORC1, while promoting insulin signaling via a negative feedback loop [40, 41]. Phosphorylation of IRS1, which is promoted by mTORC1 via S6K1, results in the disassociation of IRS1 from the insulin receptor and inhibition of insulin signaling. Conversely, inhibition of mTORC1 signaling by rapamycin results in increased interaction between IRS1 and the insulin receptor, and thus activates mTORC2 signaling and promotes the phosphorylation and activation of Akt. One might predict that because overexpression of DEPTOR leads to constitutive activation of Akt, DEPTOR might be involved in certain types of cancer. Using transcriptional profiles of different cancer types, it was
31
2. REGULATION OF TOR SIGNALING IN MAMMALS
Growth factor receptor (insulin, IGF-1) IRS1
c-MAF
MAFB
mTORC1
mTORC2
inactive
active
PRAS40
Rheb-GTP DEPTOR
Raptor
mTOR DEPTOR
mLST8
mSin1 Protor
Rictor
mTOR DEPTOR
mLST8 PKC-α SGK
Akt
(Cytoskeleton)
FoxO (activation of FoxO dependent genes)
Growth and proliferation
FIG. 2.3. The Roll of DEPTOR in multiple myeloma. Multiple myelomas with translocations involving the transcription factors c-MAF and MAFB have increased transcription of DEPTOR and high levels of DEPTOR protein. This leads to the inactivation of mTORC1 and the inactivation of the feedback loop between mTORC1 activity and signaling through IRS1. As a result, IRS1 is stabilized, and the increased signaling through the insulin receptor drives increased mTORC2 activity and the activation of Akt, leading to growth and proliferation.
observed that elevated DEPTOR mRNA was seen in a subset of human multiple myeloma (MM) samples. Specifically, elevated DEPTOR is expressed in nonhyperdiploid MM, including those which had translocations involving the c-MAF or MAFB transcription factors. Additional study demonstrated that increased DEPTOR expression promotes survival in MM cell lines and that the high levels of DEPTOR were driven by overexpression of c-MAF or MAFB, and resulted in hyperactivation of PI3K signaling in general and Akt in particular. As shown in Figure 2.3, although c-MAF or MAFB results in the upregulation of DEPTOR, only mTORC1 is inactivated, and increased signaling through mTORC2 leads to unchecked growth and proliferation.
VIII.
The Rag Proteins: Regulation of mTOR Signaling byAminoAcids
In the preceding section, we have discussed the organization of mTOR signaling in higher eukaryotes, with particular emphasis on PRAS40 and DEPTOR, proteins which are found only in higher eukaryotes. Raptor,
32
DUDLEY W. LAMMING AND DAVID M. SABATINI
Rictor, and mLST8, which are conserved from yeast to humans, serve to target the activity of mTOR against specific endogenous targets, including S6K1, 4E-BP1, Akt1, and SGK1. However, these proteins do not link insulin signaling to mTOR activity, and this is achieved by the eukaryotic specific proteins PRAS40 and DEPTOR. However, while it has long been known that mTOR activity in yeast, flies, and humans is responsive to amino acid signaling, the mechanism behind this response has remained a mystery until recently. As we have discussed above, mTORC1 signaling is responsive to a variety of stresses and environmental stimuli. Many of these inputs are integrated by the mTORC1 inhibitor TSC1/2; however, mTORC1 signaling remains sensitive to amino acid withdrawal even in cells lacking TSC1/2. Amino acids were therefore thought likely to regulate the mTORC1 complex directly, but previous attempts to immunoprecipitated mTORC1 in the presence or absence of amino acids did not succeed in isolating a difference. However, examination of immunoprecipitated protein lysates is complicated by the presence of a heavy-chain band that serves to obscure proteins that are similar in size. Sancak et al. chose to isolate mTORC1 using an approach that did not rely on antibodies; instead, Flag-tagged Raptor was isolated using a Flag affinity gel and the proteins were then eluted using Flag peptide. One of the proteins that bound only in the presence of amino acids was RagC [24]. Kim et al. also identified the Rag GTPase family as essential for mTORC1 activity by conducting an RNAi screen for GTPases that regulate S6 phosphorylation [25]. RagC is a member of a family of four small Ras-related GTP-binding proteins in humans (RagA through RagD). RagA and RagB are homologous to each other as well as to yeast Gtr1, while RagC and RagD are homologous to each other as well as yeast Gtr2. The mode of action of these proteins is conserved from yeast to humans, and in both Rag proteins function as heterodimers consisting of one Gtr1-like protein (Rag A or RagB) and one Gtr2-like protein (RagC or Rag D). In yeast, the Gtr1 and Gtr2 proteins have been linked to control of amino acid permeases and microautophagy, processes that are regulated by TOR signaling. Sancak et al. showed that Raptor copurified with a heterodimer of Rag proteins, and both Sancak et al. and Kim et al. found that the Rag proteins were essential for mTOR signaling by amino acids [24, 25]. Sancak et al. went on to show that in the presence of amino acids, Rag proteins function by localizing mTORC1 to a subcellular vesicular compartment marked by Rab7, indicating that mTOR is localized to the late endosomal and lysosomal compartments [24]. Therefore, in the presence of amino acids, both mTOR and its activator Rheb are present in Rab7-positive structures (see Figure 2.4) [24, 42]. Recently, it was demonstrated that TORC1 in yeast may be regulated in a similar manner by GTP-bound Gtr1, and that the
33
2. REGULATION OF TOR SIGNALING IN MAMMALS
PRAS40
Raptor mTOR
Rheb-GTP
/D
A/ B
C
ag
ag
R
Rag A/B
Rag C/D
R
r pto Ra
GTP
40 AS PR
OR PT DE
GTP
R
Rheb-GTP
TO
8 ST mL
+ Amino acids GDP
m
mLST8 DEPTOR
GDP
Rab7
Late endosome/lysosome
Rab7
Late endosome/lysosome
FIG. 2.4. Activation of mTORC1 in response to amino acids. In the absence of amino acids, mTORC1 is distributed throughout the cell, while its activator Rheb-GTP is localized to a late endosomal or lysosomal compartment marked by Rab7. Heterodimeric pairs of Rag proteins in Rab7-positive compartments are inactive, with Rag A/B bound to GDP and Rag C/D bound to GTP. In the presence of amino acids, the Rag proteins become active, with Rag A/B bound to GTP and Rag C/D bound to GDP. mTORC1 then relocalizes to the Rab7-positive endosomal compartments and is united with Rheb-GTP.
nucleotide binding of status of Gtr1 was regulated by the guanine nucleotide exchange factor Vam6 [43].
IX.
The Future: Remaining Mysteries of mTOR Signaling and Clinical Significance of mTOR
In this chapter, we have explored the function and structure of the two mammalian mTOR complexes, mTORC1 and mTORC2, and their control in higher eukaryotes such as human via PRAS40, DEPTOR, and the Rag proteins. We might, looking at a complex diagram of mTOR signaling, think that we now fully know everything about mTOR signaling, but in fact substantial work remains in understanding the function of the mTOR pathway. We will discuss some of these mysteries below and then discuss briefly the clinical significance of the mTOR pathway in light of recent discoveries about mTOR, cancer, and aging. Above, we discussed how the Rag proteins activate mTORC1 in response to amino acids by localizing it to a compartment containing Rheb. However, it is still not clear how signals from various amino acids activate this relocalization, and GEFs for the mammalian Rag proteins have not yet been identified. It is also unclear exactly how localization of mTORC1 may contribute to the localization (and phosphorylation) of its substrates. In yeast, the S6K1 homologue Sch9 localizes to the vacuole and
34
DUDLEY W. LAMMING AND DAVID M. SABATINI
its localization is nutrient sensitive [44], so perhaps S6K1 (and other mTORC1 substrates) localize to the same compartment as mTORC1 during periods of abundant nutrients. We also do not have a good understanding of what other substrates there may be for mTORC1, or of how mTORC1 signaling regulates autophagy. Turning our attention to mTORC2, there is still much we do not understand. At the most basic level, while we know that increased PI3K activity results in increased mTORC2 activity, we do not understand how this signal is transmitted to mTORC2. While rapamycin has allowed researchers around the world to study mTORC1 signaling, mTORC2 substrates have only been identified in the last few years. This includes such key regulatory proteins as Akt, which regulates the activity of forkhead transcription factors as well as the activity of mTORC1, and SGK [13, 14]. It is likely that as work progresses, additional substrates for mTORC2 activity will also be found. It has also recently been appreciated that rapamycin can inhibit mTORC2 function in some cell types, likely by inhibiting mTORC2 assembly [45]. As mTORC2 signaling may be important in some types of cancer, including prostate cancer and AML, the regulation of mTORC2 function by rapamycin may not only lead to the discovery of interesting biology but may also be clinically relevant [46, 47]. One factor that will help drive the discovery of more mTORC2 biology is the development of drugs that specifically inhibit mTORC2. While no mTORC2-specific drugs have yet been identified, several groups have identified compounds that will directly inhibit the activity of the mTOR kinase and thus inhibit both mTORC1 and mTORC2 [48–50]. From a clinical perspective, rapamycin has always been viewed as a promising drug candidate for the treatment of cancer—it specifically inhibits mTORC1 at nanomolar concentrations, and mTORC1 is hyperactive in many types of cancer. However, despite numerous clinical trials, rapamycin has had less of a clinical impact than hoped, although recent work suggests that rapamycin may be of use in treating some types of AML, renal and prostate cancers [46, 47, 51]. However, when applied to cancer cell lines in a tissue culture model, rapamycin often only has modest effects on proliferation. This may in part be due to the feedback loop between mTORC1 and IRS1 discussed above, resulting in the activation of PI3K signaling and subsequent phosphorylation and activation of Akt. Drugs that inhibit the mTOR kinase itself, possibly in combination with inhibition of PI3K activity, may fare better and at least one new inhibitor of mTORC1 function has been reported to induce cell cycle arrest [50]. Finally, a great deal of attention from both the scientific community and the popular press has recently been focused on the mTOR pathway as possible means of regulating the aging process. The influence of mTOR signaling on lifespan was first observed in 2003 in C. elegans, and was soon duplicated in
2. REGULATION OF TOR SIGNALING IN MAMMALS
35
Drosophila and yeast [52–54]. Treatment of an organism with rapamycin similarly extends lifespan in yeast, and was recently shown to extend the lifespan of mice started on a diet containing rapamycin at 20 months of age [55, 56]. While the mechanisms behind this extension of lifespan in mammals have not yet been determined, it seems likely that the effect relies in part on decreased or altered translation. Several studies in C. elegans have demonstrated that RNAi against certain ribosomal proteins, S6K, the translation initiation factor eIF2, or other proteins required for translation such as eIF4E and eiF4G all extend lifespan and decrease protein translation [57]. However, the lifespan of worms with RNAi against ceTOR can be further extended by RNAi against ribosomal proteins, S6K, and transcription initiation factors, indicating that perhaps inhibition of TOR engages a different life extension pathway than the other interventions. Indeed, the Kenyon lab found that RNAi against many of these factors induced daf-16/ FOXO signaling, while RNAi against ceTOR did not; also, RNAi against TOR in C. elegans subjected to dietary restriction (DR) reduced translation but did not further extend lifespan [57]. A recent study of S6K1 / mice by Selman et al. found that female S6K1 / mice had extended lifespan despite not demonstrating decreased protein synthesis or rates of translation, and the researchers noted that these mice had a gene expression pattern similar to that of mice subjected to CR or with an activated AMPK pathway [58]. Clearly, inhibition of mTOR signaling and DR is likely to involve the engagement of separate, but somewhat overlapping sets of pathways.
ACKNOWLEDGMENTS We thank D.E. Cohen, M. Laplante, T. Peterson, Y. Sancak, and A. Strohecker for helpful discussions and critical reading of the manuscript. DWL was supported by a Ruth L. Kirschstein National Research Service Award (NRSA) Postdoctoral Fellowship (F32AG032833) from the National Institute of Aging, but the preceding content is solely the responsibility of the authors and does not necessarily represent the official views of the NIA or the NIH. DMS was supported by grants from the NIH (R01-AI47389 and R01-CA103866) and awards from the American Federation of Aging Research, the Keck Foundation and the LAM (Lymphangioleiomyomatosis) Foundation and is an investigator of the Howard Hughes Medical Institute.
REFERENCES 1. Brown, E.J., et al. (1994). A mammalian protein targeted by G1-arresting rapamycinreceptor complex. Nature 369:756–758. 2. Sabatini, D.M., Erdjument-Bromage, H., Lui, M., Tempst, P., and Snyder, S.H. (1994). RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell 78:35–43.
36
DUDLEY W. LAMMING AND DAVID M. SABATINI
3. Sabers, C.J., et al. (1995). Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells. J Biol Chem 270:815–822. 4. 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. 5. Kim, D.H., et al. (2002). mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110:163–175. 6. Hara, K., et al. (2002). Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 110:177–189. 7. Chen, E.J., and Kaiser, C.A. (2003). LST8 negatively regulates amino acid biosynthesis as a component of the TOR pathway. J Cell Biol 161:333–347. 8. Kim, D.H., et al. (2003). GbetaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR. Mol Cell 11:895–904. 9. 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. 10. Jacinto, E., et al. (2004). Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol 6:1122–1128. 11. Jones, K.T., Greer, E.R., Pearce, D., and Ashrafi, K. (2009). Rictor/TORC2 regulates Caenorhabditis elegans fat storage, body size, and development through sgk-1. PLoS Biol 7:e60. 12. Soukas, A.A., Kane, E.A., Carr, C.E., Melo, J.A., and Ruvkun, G. (2009). Rictor/TORC2 regulates fat metabolism, feeding, growth, and life span in Caenorhabditis elegans. Genes Dev 23:496–511. 13. Sarbassov, D.D., Guertin, D.A., Ali, S.M., and Sabatini, D.M. (2005). Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307:1098–1101. 14. Garcia-Martinez, J.M., and Alessi, D.R. (2008). mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1). Biochem J 416:375–385. 15. Frias, M.A., et al. (2006). mSin1 is necessary for Akt/PKB phosphorylation, and its isoforms define three distinct mTORC2s. Curr Biol 16:1865–1870. 16. Panasyuk, G., et al. (2009). The mTORbeta splicing isoform promotes cell proliferation and tumorigenesis. J Biol Chem 284(45):30807–30814. 17. Sun, C., Southard, C., and Di Rienzo, A. (2009). Characterization of a novel splicing variant in the RAPTOR gene. Mutat Res 662:88–92. 18. Pearce, L.R., et al. (2007). Identification of Protor as a novel Rictor-binding component of mTOR complex-2. Biochem J 405:513–522. 19. Peterson, T.R., et al. (2009). DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival. Cell 137:873–886. 20. Sancak, Y., et al. (2007). PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase. Mol Cell 25:903–915. 21. Vander Haar, E., Lee, S.I., Bandhakavi, S., Griffin, T.J., and Kim, D.H. (2007). Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat Cell Biol 9:316–323. 22. Long, X., Ortiz-Vega, S., Lin, Y., and Avruch, J. (2005). Rheb binding to mammalian target of rapamycin (mTOR) is regulated by amino acid sufficiency. J Biol Chem 280:23433–23436. 23. Inoki, K., Li, Y., Zhu, T., Wu, J., and Guan, K.L. (2002). TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol 4:648–657.
2. REGULATION OF TOR SIGNALING IN MAMMALS
37
24. Sancak, Y., et al. (2008). The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320:1496–1501. 25. Kim, E., Goraksha-Hicks, P., Li, L., Neufeld, T.P., and Guan, K.L. (2008). Regulation of TORC1 by Rag GTPases in nutrient response. Nat Cell Biol 10:935–945. 26. Zhang, Y., Gao, X., Saucedo, L.J., Ru, B., Edgar, B.A., and Pan, D. (2003). Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins. Nat Cell Biol 5:578–581. 27. Inoki, K., et al. (2006). TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth. Cell 126:955–968. 28. Inoki, K., Zhu, T., and Guan, K.L. (2003). TSC2 mediates cellular energy response to control cell growth and survival. Cell 115:577–590. 29. 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. 30. Gwinn, D.M., et al. (2008). AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell 30:214–226. 31. 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. 32. Tremblay, F., Gagnon, A., Veilleux, A., Sorisky, A., and Marette, A. (2005). Activation of the mammalian target of rapamycin pathway acutely inhibits insulin signaling to Akt and glucose transport in 3T3-L1 and human adipocytes. Endocrinology 146:1328–1337. 33. Potter, C.J., Pedraza, L.G., and Xu, T. (2002). Akt regulates growth by directly phosphorylating Tsc2. Nat Cell Biol 4:658–665. 34. Edinger, A.L., and Thompson, C.B. (2002). Akt maintains cell size and survival by increasing mTOR-dependent nutrient uptake. Mol Biol Cell 13:2276–2288. 35. Oshiro, N., et al. (2007). The proline-rich Akt substrate of 40 kDa (PRAS40) is a physiological substrate of mammalian target of rapamycin complex 1. J Biol Chem 282:20329–20339. 36. Wang, L., Harris, T.E., Roth, R.A., and Lawrence, J.C., Jr (2007). PRAS40 regulates mTORC1 kinase activity by functioning as a direct inhibitor of substrate binding. J Biol Chem 282:20036–20044. 37. Fonseca, B.D., Smith, E.M., Lee, V.H., MacKintosh, C., and Proud, C.G. (2007). PRAS40 is a target for mammalian target of rapamycin complex 1 and is required for signaling downstream of this complex. J Biol Chem 282:24514–24524. 38. Madhunapantula, S.V., Sharma, A., and Robertson, G.P. (2007). PRAS40 deregulates apoptosis in malignant melanoma. Cancer Res 67:3626–3636. 39. Yu, F., Narasimhan, P., Saito, A., Liu, J., and Chan, P.H. (2008). Increased expression of a proline-rich Akt substrate (PRAS40) in human copper/zinc-superoxide dismutase transgenic rats protects motor neurons from death after spinal cord injury. J Cereb Blood Flow Metab 28:44–52. 40. Ozes, O.N., et al. (2001). A phosphatidylinositol 3-kinase/Akt/mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate-1. Proc Natl Acad Sci USA 98:4640–4645. 41. Shah, O.J., Wang, Z., and Hunter, T. (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. 42. Avruch, J., Long, X., Ortiz-Vega, S., Rapley, J., Papageorgiou, A., and Dai, N. (2009). Amino acid regulation of TOR complex 1. Am J Physiol Endocrinol Metab 296: E592–E602. 43. Binda, M., et al. (2009). The Vam6 GEF controls TORC1 by activating the EGO complex. Mol Cell 35:563–573.
38
DUDLEY W. LAMMING AND DAVID M. SABATINI
44. Jorgensen, P., Rupes, I., Sharom, J.R., Schneper, L., Broach, J.R., and Tyers, M. (2004). A dynamic transcriptional network communicates growth potential to ribosome synthesis and critical cell size. Genes Dev 18:2491–2505. 45. Sarbassov, D.D., et al. (2006). Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell 22:159–168. 46. Zeng, Z., et al. (2007). Rapamycin derivatives reduce mTORC2 signaling and inhibit AKT activation in AML. Blood 109:3509–3512. 47. Guertin, D.A., et al. (2009). mTOR complex 2 is required for the development of prostate cancer induced by Pten loss in mice. Cancer Cell 15:148–159. 48. Feldman, M.E., et al. (2009). Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2. PLoS Biol 7:e38. 49. Thoreen, C.C., et al. (2009). An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1. J Biol Chem 284:8023–8032. 50. Garcia-Martinez, J.M., et al. (2009). Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR). Biochem J 421:29–42. 51. Oudard, S., et al. (2009). Everolimus (RAD001): an mTOR inhibitor for the treatment of metastatic renal cell carcinoma. Expert Rev Anticancer Ther 9:705–717. 52. Vellai, T., Takacs-Vellai, K., Zhang, Y., Kovacs, A.L., Orosz, L., and Muller, F. (2003). Genetics: influence of TOR kinase on lifespan in C. elegans. Nature 426:620. 53. Kaeberlein, M., et al. (2005). Regulation of yeast replicative life span by TOR and Sch9 in response to nutrients. Science 310:1193–1196. 54. Kapahi, P., Zid, B.M., Harper, T., Koslover, D., Sapin, V., and Benzer, S. (2004). Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway. Curr Biol 14:885–890. 55. Medvedik, O., Lamming, D.W., Kim, K.D., and Sinclair, D.A. (2007). MSN2 and MSN4 Link Calorie Restriction and TOR to Sirtuin-Mediated Lifespan Extension in Saccharomyces cerevisiae. PLoS Biol 5:e261. 56. Harrison, D.E., et al. (2009). Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature 460:392–395. 57. Hansen, M., Taubert, S., Crawford, D., Libina, N., Lee, S.J., and Kenyon, C. (2007). Lifespan extension by conditions that inhibit translation in Caenorhabditis elegans. Aging Cell 6:95–110. 58. Selman, C., et al. (2009). Ribosomal protein S6 kinase 1 signaling regulates mammalian life span. Science 326:140–144.