The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays a fundamental role in regulating cellular homeostasis and metabolism. In a two-part review, we examine the complex molecular events involved in the regulation and downstream effects of mTOR, as well as the pivotal role played by this kinase in many renal diseases, particularly acute kidney injury, diabetic nephropathy, and polycystic kidney diseases. Here, in the first part of the review, we provide an overview of the complex signaling events and pathways governing mTOR activity and action. mTOR is a key component of two multiprotein complexes, known as mTOR complex 1 (mTORC1) and 2 (mTORC2). Some proteins are found in both mTORC1 and mTORC2, while others are unique to one or the other complex. Activation of mTORC1 promotes cell growth (increased cellular mass or size) and cell proliferation (increased cell number). mTORC1 acts as a metabolic “sensor,” ensuring that conditions are optimal for both cell growth and proliferation. Its activity is tightly regulated by the availability of amino acids, growth factors, energy stores, and oxygen. The effects of mTORC2 activation are distinct from those of mTORC1. Cellular processes modulated by mTORC2 include cell survival, cell polarity, cytoskeletal organization, and activity of the aldosterone-sensitive sodium channel. Upstream events controlling mTORC2 activity are less well understood than those controlling mTORC1, although growth factors appear to stimulate both complexes. Rapamycin and its analogs inhibit the activity of mTORC1 only, and not that of mTORC2, while the newer “catalytic” mTOR inhibitors affect both complexes.
- cell mass, proliferation
the “target of rapamycin” (TOR) is a serine/threonine kinase initially cloned in yeast (53). A few years later, its mammalian ortholog, the “mammalian target of rapamycin” (mTOR), was cloned (7, 17, 101). The identification of TOR and mTOR was the direct result of the discovery of rapamycin, a highly specific inhibitor of both TOR and mTOR produced by the soil bacterium Streptomyces hygroscopicus (53, 73). Rapamycin was essential not only for the identification of mTOR, but also for elucidating mTOR-dependent signaling events and their role in metabolism and disease. The mTOR kinase forms a part of two distinct protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), that have separate downstream targets and functions. Upon entering cells, rapamycin binds to an intracellular receptor, the FK506-binding protein (FKBP12) (18). The resulting complex then binds to and inhibits mTOR kinase activity (18). Sensitivity to rapamycin varies widely among mammalian cells. The relatively high susceptibility of lymphocytes to rapamycin has led to its clinical use as an immunosuppressive therapy for recipients of organ transplantation (5, 40).
mTOR lies at the center of an intricate signaling network with profound implications for a large number of diverse and important biological events, including embryonic development, metabolism, regenerative processes, and ageing (132). Moreover, an inappropriately high level of mTOR activity plays a central role in the progression of several highly prevalent diseases including metabolic syndrome, diabetes, and cancer (35, 51, 132). It is not surprising, therefore, that elucidation of the pathways governing the activity and effects of mTOR is currently a rapidly expanding area of research.
It has been clear for many years that mTOR has a fundamental role in the regulation of two cardinal cellular events: cell proliferation and cell growth. Throughout this review, we will use “proliferation” to refer to an increase in cell number and “growth” to refer to an increase in cell size or mass. Since mTOR promotes an increase in both cell number and cell size, it is also a major determinant of organ and total body mass (132). The role of mTOR is to act as a metabolic “sensor.” mTOR monitors the cell and its immediate environment for adequacy of the conditions and ingredients necessary for proliferation and growth. Factors that stimulate cell growth and proliferation include growth factors, nutrients, cellular energy stores, and oxygen availability (4, 28, 35, 76, 112, 132). On the other hand, conditions unfavorable to proliferation and growth, for example, a deficiency of cellular energy stores or DNA damage, suppress mTOR activity and slow both processes.
In this two-part review, we will focus on four major areas. First, we will provide an overview of the signaling events and pathways governing mTOR activity and action. Second, we will describe the critical regulatory role of mTOR in the pathophysiology of a wide range of renal diseases including acute kidney injury (AKI), diabetic nephropathy (DN), nondiabetic forms of chronic glomerular disease, and polycystic kidney disease (PKD). Third, we will discuss the potential use of rapamycin, or its analogs, in the treatment of mTOR-related renal diseases in humans. Finally, we will consider the properties and utility of a new generation of mTOR inhibitors, which have potential advantages over rapamycin (19, 31, 38, 118, 130).
Structure of the mTOR Kinase and Components of mTORC1 and mTORC2 Complexes
The mTOR kinase.
mTOR is a large (∼289-kDa) multidomain protein that belongs to the phosphatidylinositol 3-kinase (PI3K)-related family of kinases (66) (Fig. 1A). In general, members of this family of kinases are proteins that facilitate the ability of the organism to cope with metabolic, environmental, and genetic forms of stress. mTOR has a C-terminal PI3K-like catalytic domain, flanked on its N- and C-terminal sides by FRAP, ATM, and TRRAP (FAT) and FAT C-terminal (FATC) domains, respectively (Fig. 1A). The FAT domain is believed to mediate interactions with other proteins, while the FATC domain senses cytosolic redox potential and probably influences the rate of degradation of mTOR (24). The N terminus of mTOR contains tandem repeats of Huntington, elongation factor 3, regulatory A subunit of protein phosphatase 2A, and TOR (HEAT) domains. The HEAT domain is an α-helical structure that mediates protein-protein interactions (2) and membrane localization (74). HEAT domains also contribute to the formation of the protein complexes, mTORC1 and mTORC2, and appear necessary for mTOR function (117). A domain found exclusively in the mTOR molecule is the FKBP12-rapamycin binding (FRB) domain, to which the ligand-receptor complex between rapamycin and FKBP12 binds (5, 15, 40) (Fig. 1A). Phosphatidic acid (PA), a phospholipid metabolite produced by phospholipase D (PLD) that activates mTOR, also binds to the FRB domain (116, 119, 127). The opposing effects of rapamycin and PA on mTOR activity may arise through competition for binding to the FRB domain on mTOR.
The mTORC1 and mTORC2 complexes.
Each of the two mTOR complexes, mTORC1 (27, 28, 34, 112) and mTORC2 (23, 63, 81), contains multiple proteins. Some proteins are found in only one of the two complexes, while others are common to both (Fig. 1B).
Two proteins are unique to mTORC1 (Fig. 1B). These are regulatory-associated protein of mTOR (Raptor) (34, 44, 67, 93, 110) and proline-rich Akt substrate of 40 kDa (PRAS40) (56, 104, 120, 121). Activation of mTORC1 is initiated by phosphorylation of Raptor, a scaffold protein necessary for the assembly of mTORC1 as well as for interaction of mTORC1 with its various substrates and regulators (34, 44, 67). Binding of rapamycin-FKBP12 to the FRB domain of mTOR does not directly affect mTOR's kinase activity. Although the molecular mechanisms underlying rapamycin's effect remain poorly understood, inhibition appears to depend, in part, on interference with the association between mTOR and Raptor, thereby preventing mTORC1 from interacting with and phosphorylating its downstream targets (34, 67, 96). PRAS40, when unphosphorylated, binds to and inhibits mTORC1. Phosphorylation of PRAS40 by Akt causes its release from mTORC1, thus relieving mTORC1 from inhibition by PRAS40 (56, 104, 120).
Proteins unique to mTORC2 include rapamycin-insensitive companion of mTOR (Rictor) (55, 105, 126), mammalian stress-activated protein kinase interacting protein 1 (mSIN1), and protein observed with Rictor (Protor) (97) (Fig. 1B). Like Raptor within mTORC1, Rictor acts as a scaffold protein and facilitates both the assembly of mTORC2 and the interaction of mTORC2 with its substrates and regulators. mSIN1, in addition to a role in the assembly of mTORC2, contributes to the membrane targeting of mTORC2, and ultimately the phosphorylation of Akt, a downstream substrate of mTORC2 (36, 128) (Fig. 1B). The function of Protor has not yet been clearly defined.
Two proteins are found in both mTORC1 and mTORC2 (Fig. 1B). The first is mammalian lethal with SEC13 protein 8 (mLST8), which stabilizes the interaction between Raptor and mTOR in mTORC1 (68) and between Rictor and mTOR in mTORC2 (41). The second is DEPTOR, named for its tandem Dishevelled, EGL-10, and pleckstrin (DEP) domains and specific interaction with mTOR. DEPTOR is a negative regulator of the activity of both mTORC1 and mTORC2 (98).
Copp and his colleagues (21) have recently shown that mTOR is phosphorylated differently when part of mTORC1 vs. part of mTORC2. Phosphorylation of mTOR at Ser2448 is seen predominantly, although not exclusively, when mTOR is part of mTORC1, whereas phosphorylation of mTOR at Ser2481 occurs when it is part of mTORC2. Thus phosphorylation at residues Ser2448 and Ser2481 can be used as a biomarker to distinguish activation of mTORC1 and mTORC2, respectively (21).
Besides having distinct downstream targets, mTORC1 and mTORC2 respond differently to rapamycin. While rapamycin inhibits the catalytic activity of mTORC1 by interfering with the association between mTOR and Raptor, the drug has no direct effect on the catalytic activity of mTORC2 (81). Despite rapamycin's lack of direct effect on mTORC2, prolonged exposure to rapamycin in some cell types can diminish mTORC2 activity. Inhibition is indirect and occurs through interference with the assembly of new mTORC2 complexes (106).
The mTORC1 Signaling Pathway
Downstream effects of mTORC1.
CELL GROWTH, CELL PROLIFERATION, AND PROTEIN SYNTHESIS.
mTORC1 stimulates both cell growth (increase in cell size or mass) and cell proliferation (49). In terms of the cell cycle, the major effect of mTORC1 is to promote progression through the G0/G1 phase of the cell cycle (51). The best-characterized targets of mTORC1 are the ribosomal S6 kinases S6K1 and S6K2 [also known as p70S6K1 (phosphoprotein 70 S6K1/2)] and the 4E-binding proteins (4E-BPs), including 4E-BP1, 4E-BP2, and 4E-BP3, which bind to and regulate the activity of the eukaryotic translation initiation factor (eIF) eIF-4E (9, 39, 51, 84) (Fig. 2A). Raptor and another eIF, eIF-3, play critical roles in mTOR-mediated phosphorylation of S6K1/2 and the 4E-BPs (84, 93, 110). eIF-3 acts as a scaffold, recruiting mTORC1 to the ribosomal translation initiation complex (54). Raptor then binds to conserved TOR signaling (TOS) motifs in both S6K1/2 and 4E-BPs, thereby bringing these substrates within range of mTOR (84, 93, 110). Once phosphorylated, S6K1/2 and 4E-BPs augment protein synthesis by increasing the initiation and progression of mRNA translation (84). In general, S6K1/2 is responsible for stimulating protein synthesis and cell growth, while the 4E-BPs play a predominant role in cell proliferation (26).
In its unphosphorylated state, 4E-BP1 binds to and inhibits eIF-4E (43, 45). Upon phosphorylation by mTORC1, 4E-BP1 dissociates from eIF-4E. This allows eIF-4E to interact with the translation initiation factor eIF-4G and recruit eIF-4G to the 5′-ends of a subset of mRNA transcripts known as “eIF-4E-sensitive mRNAs” (43, 45). Two other proteins, eIF-4A and eIF-4B, also play key roles in translation initiation. eIF-4B modulates the activity of eIF-4A, an RNA helicase that facilitates translation by unwinding the 5′ untranslated regions of many RNAs (100). S6K1/2, when phosphorylated by mTORC1, also promote mRNA translation by phosphorylating multiple key proteins including eIF-4B, eukaryotic elongation factor-2 kinase (eEF-2K) (122), S6K1 Aly/REF-like target (SKAR) (85), and 80-kDa nuclear cap-binding protein (CBP80), all of which are necessary for translation initiation and/or elongation (54).
AUTOPHAGY AND OTHER EFFECTS.
Although regulation of protein synthesis is the best understood role of mTORC1, the complex also contributes to the regulation of other critical cellular functions. These include autophagy, ribosomal biogenesis, mitochondrial metabolism, lipid synthesis, and activity of the transcription factor hypoxia-inducible factor-1α (HIF-1α).
Autophagy is a ubiquitous process that mediates the bulk removal of abnormal or aged macromolecules and cell organelles from the cytosol (71, 80). Autophagy also provides an essential source of energy during periods of nutrient deficiency. Autophagy is mediated by the formation of autophagosomes, double-membraned structures that envelop intracellular components destined for degradation and then fuse with lysosomes to form autolysosomes, which degrade their contents (89, 123, 129).
mTORC1 is an important upstream inhibitor of autophagy (80, 132) (Fig. 2A). Many factors, including nutrient deprivation, ATP depletion, hypoxia, and rapamycin, stimulate autophagy by inhibiting mTORC1. mTORC1 phosphorylates and inhibits the kinases Unc-51-like kinase-1 and -2 (ULK1/2), catalytic subunits of the multisubunit protein kinase complex that initiates the autophagic cascade. ULK1/2 is also known as human autophagy-1 (hATG1) (48, 52).
While cell growth and autophagic degradation are generally regarded as oppositely regulated, exceptions may occur. Narita et. al. (90) have recently described a mechanism by which the cell's catabolic and anabolic machineries can be coupled. In senescent cells, autolysosomes and mTOR were shown to accumulate within a distinct cellular compartment located at the trans-side of the Golgi apparatus. The authors named this structure the TOR-autophagy spatial coupling compartment (TASCC). Colocalization of autolysosomes and mTOR within the TASCC allows amino acids generated by autolysosomes to activate mTORC1 in a RAG GTPase-dependent manner (see below) and thereby enhance the synthesis of proteins targeted for secretion. Interestingly, the TASCC has been shown to increase protein secretion in a number of cell types, including glomerular podocytes (90).
In addition to protein synthesis, mTORC1 also stimulates lipid synthesis (99). These effects are mediated in part through activation of lipogenic transcription factors such as sterol responsive element binding protein (SREBP1) (99) and peroxisome proliferator-activated receptor-γ (PPAR-γ) (70).
As a further means to increase protein synthesis, mTORC1 also stimulates ribosomal biogenesis, increasing the number of ribosomes within each cell (88). mTORC1 has also been shown to modulate mitochondrial function. Inhibition of mTORC1 by rapamycin leads to decreases in several mitochondrial parameters, including transmembrane potential, oxygen consumption, and ATP synthetic capacity (111). Finally, mTORC1 plays an important role in determining the balance between mitochondrial and nonmitochondrial sources of ATP generation (111).
Upstream regulation of mTORC1.
mTORC1 acts as a sensor of the adequacy of factors and conditions necessary for cell growth and proliferation. Information is derived by continuous monitoring of parameters within four major areas: the availability of growth factors such as insulin, insulin-like growth factor-1 (IGF-1), and epidermal growth factor (EGF); the adequacy of nutrients such as glucose, lipids, and amino acids; the sufficiency of cellular energy stores; and the presence and extent of cell damage (Fig. 2, A and B). Input from these multiple sources can either raise or lower the overall activity of mTORC1, thereby enabling each cell to adjust its mTORC1 activity to a level appropriate for the prevailing environment (4, 27, 29, 35, 76, 84, 132). This “fine-tuning” is achieved through a complex network of signaling events and pathways that for the most part converge on two upstream regulators of mTORC1. These are Ras homolog enriched in brain (Rheb) and the tuberous sclerosis complex (TSC) (4, 28, 35, 76, 112, 132) (Fig. 2, A and B).
The immediate upstream regulator of mTORC1 is Rheb, a cytoplasmic G protein with intrinsic GTPase activity (Fig. 2, A and B). Rheb acts as a molecular switch, depending upon whether it is bound to GTP (“on” position) or GDP (“off” position). In its on position, Rheb stimulates the kinase activity of mTORC1 (109, 115). While the precise mechanism by which Rheb-GTP controls mTORC1 remains poorly defined, part of Rheb's effect is to facilitate binding of mTORC1 to its substrates, such as S6K1/2 and the 4E-BPs (108).
TSC consists of two proteins, TSC1 (hamartin) and TSC2 (tuberin) (58, 59, 95) (Fig. 2A). TSC is a tumor suppressor that negatively regulates Rheb. TSC2 contains a GTPase-activating protein (GAP) domain that catalyzes the conversion of Rheb from an active GTP-bound state to an inactive GDP-bound state (35, 108). TSC1 is necessary for the function of TSC2's GAP domain.
TSC-mediated regulation of Rheb comprises the final common pathway for all known upstream regulators of mTORC1 (Fig. 2, A and B), with several exceptions. The two most important are PA, which binds to and activates mTOR directly, and amino acids, which bypass TSC and activate Rheb (Fig. 2C). Since TSC is a negative regulator of Rheb, activators of mTORC1, such as growth factors, do so by inhibiting TSC. In contrast, inhibitors of mTORC1, such as energy depletion, hypoxia, and DNA damage, do so by activating TSC (4, 35, 76).
ACTIVATION OF MTORC1 BY GROWTH FACTORS.
Growth factors, like insulin and IGF-1, activate mTORC1 predominantly via the kinase Akt (Fig. 2A). Activation of mTORC1 begins with activation of the lipid kinase PI3K. PI3K phosphorylates the membrane-associated lipid, phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] to yield PtdIns(3,4,5)P3 (87). PtdIns(3,4,5)P3 then activates 3-phosphoinositide-dependent protein kinase 1 (PDK1), which in turn activates Akt by phosphorylating it at Thr308.
Akt activates mTORC1 through at least two mechanisms. Both involve release of inhibition through phosphorylation of a negative regulator of mTORC1. The first target of Akt is TSC2 (25, 60). Interestingly, phosphorylation of TSC2 by Akt does not alter its intrinsic GAP activity. Rather, phosphorylation results in translocation of TSC2 from the cell membrane to the cytosol, where it is sequestered by 14-3-3 proteins, thereby separating TSC2 from both its partner (TSC1) and its target (Rheb) (11). The second target of Akt is PRAS40, a negative regulator within the mTORC1 complex. Phosphorylation of PRAS40 by Akt causes its dissociation from mTORC1 (104, 120). Relief of inhibition from these two sources, the first upstream of mTORC1 and the second within the mTORC1 complex itself, leads to activation of mTOR (Fig. 2A).
The activity of PI3K is opposed by the tumor suppressor phosphatase and tensin homolog on chromosome 10 (PTEN), which dephosphorylates PtdIns(3,4,5)P3 back to PtdIns(4,5)P2 (50, 65) (Fig. 2A). Since PtdIns(3,4,5)P3 is the major activator of Akt, the predominant antiapoptotic kinase in cells (8, 9), loss of PTEN function leads to excessive Akt activity and conditions conducive to tumorigenesis (72).
Growth factors also modulate mTORC1 activity via pathways independent of Akt. For example, EGF activates mTORC1 via extracellular signal-regulated kinase-1 and -2 (ERK1/2), which phosphorylates and inhibits TSC2 (83) (Fig. 2B). The Wnt pathway also activates mTORC1 through inhibition of TSC2, but in this case inhibition involves decreased phosphorylation of TSC2. Activation of the Wnt pathway leads to inhibition of glycogen synthase kinase-3β (GSK3β), which in its uninhibited state phosphorylates and activates TSC2 at a site distinct from that of Akt and ERK1/2 (13, 61). Lack of phosphorylation by GSK3β leads to decreased TSC2 activity and therefore activation of mTORC1 (Fig. 2B).
ACTIVATION OF MTORC1 BY AMINO ACIDS.
Amino acids are absolutely required for mTORC1 activation, and a deficiency of amino acids cannot be overcome by other activating stimuli (132). The availability of amino acids, especially the essential amino acids leucine and arginine, was the earliest identified activator of mTORC1 (3, 46). Notably, as opposed to other known upstream regulators of mTORC1, amino acids act directly on Rheb and independently of TSC (113) (Fig. 2C). Recently, the RAG family of small cytoplasmic G proteins has been identified as important mediators of amino acid-induced activation of mTORC1 (69, 103). Sabatini and coworkers (69, 102, 103) have proposed a model in which amino acids convert inactive GDP-loaded RAG heterodimers to their active GTP-loaded form. A protein complex which has been termed the “Ragulator,” interacts with the RAG GTPases, recruits them to lysosomes, and is essential for mTORC1 activation (102).
Amino acids signal their availability from within lysosomes through their interaction with the luminal aspect of vacuolar H+-adenosine triphosphatase (v-ATPase). This interaction leads to dissociation of v-ATPase from the “Ragulator” (102), which then activates the RAG heterodimers (131). Active RAG heterodimers bind Raptor and recruit mTORC1 to the membrane of lysosomes where Rheb is situated (Fig. 2C).
The role of other putative mediators of amino acid-induced activation of mTORC1, such as the class III PI3K hVPS34 (10, 92), mitogen-activated protein kinase kinase kinase kinase-3 (MAP4K3) (33), and the cytoplasmic G protein RalA (86), remains to be elucidated. Intracellular availability of leucine and arginine, the most important amino acid regulators of mTORC1, is determined by an interconnected system of amino acid transporters, in which glutamine also plays a key role (20, 91).
INHIBITION OF MTORC1 BY DEPLETION OF CELL ENERGY STORES.
The adequacy of cellular energy stores represents a second major determinant of mTORC1 activity (Fig. 2B). Decreases in cellular energy stores inhibit mTORC1. The ability of cells to “sense” energy status is dependent largely on the activity of AMP-activated protein kinase (AMPK) (47, 49). ADP and AMP both activate AMPK, whereas ATP inhibits AMPK. ADP and AMP activate AMPK through an allosteric effect on AMPK that facilitates its phosphorylation at Thr172 by liver kinase B1 (LKB1), a constitutively active kinase. (124). ADP and AMP also maintain AMPK in an active state through blocking its dephosphorylation by phosphatases (49, 94). When ATP stores are replete, dephosphorylation of AMPK is uninhibited and AMPK remains inactive (49, 94). The net effect of these regulatory events is that AMPK activity is related inversely to a cell's energy charge, as reflected by the ratio of ATP to either AMP or ADP (49, 94).
When active, AMPK inhibits mTORC1 in at least two ways. First, like GSK3β, AMPK phosphorylates and activates TSC2 (22, 61) (Fig. 2, A and B). Second, AMPK phosphorylates Raptor. Phosphorylation of Raptor by AMPK leads to Raptor's sequestration by 14-3-3 proteins, rendering Raptor incapable of fulfilling its role as a scaffolding molecule for mTORC1 and its downstream substrates (42). Thus, through coupling of AMPK activity to a cell's energy status, AMPK ensures that cell growth and proliferation cannot proceed unless the energy required to fuel these processes are in adequate supply.
In addition to inhibiting cell growth and proliferation, AMPK also promotes autophagy. AMPK does this in both an mTORC1-dependent and an mTORC1-independent manner. Via phosphorylation and inhibition of mTORC1, AMPK promotes autophagy by releasing ULK1/2 from mTORC1-mediated inhibition. AMPK also more directly promotes autophagy by phosphorylating and activating ULK1/2 (30) Thus AMPK delivers two independent signals, both leading to activation of the autophagic program (30, 48).
OTHER UPSTREAM MODULATORS OF MTORC1.
Hypoxia, if severe enough to reduce cellular ATP levels, will inhibit mTORC1 via activation of AMPK. Hypoxia can also inhibit mTORC1 independently of AMPK. Regulated in development and DNA damage responses (REDD1), an HIF-1α-inducible protein, suppresses mTORC1 by activating TSC2 (8) (Fig. 2B). PA, a metabolite of PLD, has also been implicated in the regulation of mTORC1. PA activates mTORC1 by direct interaction with mTOR and may compete with rapamycin for binding to mTOR's FRB domain. DNA damage inhibits mTORC1 through activation of p53, which, in turn, activates AMPK (32) (Fig. 2B) and also stimulates increased transcription of PTEN (114) (Fig. 2A). Finally, tumor necrosis factor-α, a mediator of inflammation, can activate mTORC1 through IκB kinase-β (IKKβ), which phosphorylates and inhibits TSC1 (78) (Fig. 2B).
The mTORC2 Signaling Pathway
While substantial progress has been made in our understanding of the regulation and function of mTORC1, far less is known about mTORC2. Progress in this area has been slowed by two main factors: lack of a specific inhibitor of mTORC2, and the early embryologic lethality that follows deletion of components of this complex (4, 34, 35, 76).
Downstream effects of mTORC2.
Among the downstream targets of mTORC2 are Akt, protein kinase Cα (PKCα), and serum and glucocorticoid-induced protein kinase 1 (SGK1) (75) (Fig. 3). These kinases are all members of the so-called AGC family (protein kinases A, G, and C), which contribute to the regulation of diverse cell functions including cell survival, cell polarity, cytoskeletal rearrangement, and aldosterone-mediated sodium reabsorption by the distal nephron (1, 64). S6K1/2, a major downstream target of mTORC1, is also a member of the AGC family of kinases (64).
mTORC2 activates Akt by phosphorylating it at Ser473 (41, 62, 107) (Fig. 2A). While phosphorylation of Akt at Thr308 by PDK1 is required for phosphorylation and inactivation of many proapoptotic mediators, including BAD, Bcl-xL, and GSK3β (6), phosphorylation of Akt at Ser473 by mTORC2 is not required for these events (6, 41, 62). Rather, phosphorylation of Akt at Ser473 by mTORC2 is essential for the phosphorylation and activation of a distinct subset of Akt substrates, which includes the FoxO family of Forkhead transcription factors (12) (Fig. 3). These transcription factors induce the expression of a number of genes important in ameliorating age-dependent diseases, including diabetes, cancer, and neurodegenerative processes (6, 12). SGK1, another target of mTORC2 (37), which is activated by growth factors and shares homology with Akt (75), has also been shown to phosphorylate and activate the FoxO family of transcription factors (1, 37) (Fig. 3).
Activation of mTORC2 leads to changes in the actin cytoskeleton and cell polarity (Fig. 3). Although the specific mechanism for these changes has not been clearly identified (23, 63, 105), cytoskeletal rearrangement is mediated in part by activation of PKCα, which, in turn, activates Rho and Rac1, two G proteins and major regulators of the cytoskeleton. PKCα also phosphorylates paxillin and induces its localization to focal adhesions (105).
Besides its prosurvival effects, SGK1 has been known for many years to stimulate sodium reabsorption in the distal nephron by increasing activity of the aldosterone-regulated epithelial sodium channel (ENaC) (16, 125). Activation of SGK1 requires phosphorylation within a C-terminal domain known as the hydrophobic motif. Recently, Lu and colleagues (82) have shown that mTORC2 is the kinase responsible for activation of SGK1. This suggests that mTOR, in addition to its other effects on renal function, is involved in sodium homeostasis and perhaps also regulation of blood pressure (Fig. 3).
Upstream regulation of mTORC2.
It is generally believed that mTORC2, like mTORC1, is activated by growth factors. In support of this concept, stimulation with IGF-1 induces phosphorylation of Akt at Ser473 (107). Given that activation of Akt, SGK1, and PKCα occurs in response to a wide range of growth factors, the number of growth factors that activate mTORC2 may be quite large. In addition to activating mTORC1, PA may also activate mTORC2 (119).
Since phosphorylation of Akt by mTORC2 occurs at the cell membrane (77), membrane localization of mTORC2 may be critical to its activation. It has been suggested that translocation of mTORC2 to the cell membrane is mediated by mSIN1 via its C-terminal pleckstrin homology (PH) domain (76) (Fig. 1B). Additional studies, however, are needed to confirm this model. Alternatively, mSIN1 may function as an adaptor between mTORC2 and certain growth factor receptors (14, 79). Finally, an intriguing report suggests that TSC may lie upstream not only of mTORC1, but also mTORC2 (57). As opposed to its effects on mTORC1, activated TSC would promote rather than inhibit mTORC2 activity, and do so in a Rheb-independent manner.
NOTE ADDED IN PROOF
Until recently, as discussed in this review, it has been believed that rapamycin predominantly inhibits mTORC1. However, a recent study by Lamming and associates (Lamming DW, Ye L, Katajisto P, Goncalves MD, Saitoh M, Stevens DM, Davis JG, Salmon AB, Richardson A, Ahima RS, Guertin DA, Sabatini DM, Bauer JA. Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity. Science 335: 1638–1643, 2012) shows conclusively that rapamycin inhibits both complexes. Rapamycin inhibits mTORC2 by disrupting the mTORC2 complex, rather than through interference with its assembly (as suggested by earlier work). Lamming et al. also demonstrate that the downstream effects of rapamycin on mTORC1 and mTORC2 can be dissociated. Whereas rapamycin-mediated inhibition of mTORC1 activity promotes longevity, its disruption of mTORC2 induces insulin resistance and impaired glucose homeostasis.
No conflicts of interest, financial or otherwise. are declared by the authors.
Author contributions: W.L. prepared figures; W.L. drafted manuscript; J.S.L. edited and revised manuscript; W.L. and J.S.L. approved final version of manuscript.