mTORC1-Activated S6K1 Phosphorylates Rictor on Threonine 1135 and Regulates mTORC2 Signaling

The mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that forms two functionally distinct complexes important for nutrient and growth factor signaling. While mTOR complex 1 (mTORC1) regulates mRNA translation and ribosome biogenesis, mTORC2 plays an important role in the phosphorylation and subsequent activation of Akt. Interestingly, mTORC1 negatively regulates Akt activation, but whether mTORC1 signaling directly targets mTORC2 remains unknown. Here we show that growth factors promote the phosphorylation of Rictor (rapamycin-insensitive companion of mTOR), an essential subunit of mTORC2. We found that Rictor phosphorylation requires mTORC1 activity and, more specifically, the p70 ribosomal S6 kinase 1 (S6K1). We identified several phosphorylation sites in Rictor and found that Thr1135 is directly phosphorylated by S6K1 in vitro and in vivo, in a rapamycin-sensitive manner. Phosphorylation of Rictor on Thr1135 did not affect mTORC2 assembly, kinase activity, or cellular localization. However, cells expressing a Rictor T1135A mutant were found to have increased mTORC2-dependent phosphorylation of Akt. In addition, phosphorylation of the Akt substrates FoxO1/3a and glycogen synthase kinase 3α/β (GSK3α/β) was found to be increased in these cells, indicating that S6K1-mediated phosphorylation of Rictor inhibits mTORC2 and Akt signaling. Together, our results uncover a new regulatory link between the two mTOR complexes, whereby Rictor integrates mTORC1-dependent signaling.The mammalian target of rapamycin (mTOR) is an evolutionarily conserved phosphatidylinositol 3-kinase (PI3K)-related Ser/Thr kinase that integrates signals from nutrients, energy sufficiency, and growth factors to regulate cell growth as well as organ and body size in a variety of organisms (reviewed in references 4, 38, 49, and 77). mTOR was discovered as the molecular target of rapamycin, an antifungal agent used clinically as an immunosuppressant and more recently as an anticancer drug (5, 20). Recent evidence indicates that deregulation of the mTOR pathway occurs in a majority of human cancers (12, 18, 25, 46), suggesting that rapamycin analogs may be potent antineoplastic therapeutic agents.mTOR forms two distinct multiprotein complexes, the rapamycin-sensitive and -insensitive mTOR complexes 1 and 2 (mTORC1 and mTORC2), respectively (6, 47). In cells, rapamycin interacts with FKBP12 and targets the FKBP12-rapamycin binding (FRB) domain of mTORC1, thereby inhibiting some of its function (13, 40, 66). mTORC1 is comprised of the mTOR catalytic subunit and four associated proteins, Raptor (regulatory associated protein of mTOR), mLST8 (mammalian lethal with sec13 protein 8), PRAS40 (proline-rich Akt substrate of 40 kDa), and Deptor (28, 43, 44, 47, 59, 73, 74). The small GTPase Rheb (Ras homolog enriched in brain) is a key upstream activator of mTORC1 that is negatively regulated by the tuberous sclerosis complex 1 (TSC1)/TSC2 GTPase-activating protein complex (reviewed in reference 35). mTORC1 is activated by PI3K and Ras signaling through direct phosphorylation and inactivation of TSC2 by Akt, extracellular signal-regulated kinase (ERK), and p90 ribosomal protein S6 kinase (RSK) (11, 37, 48, 53, 63). mTORC1 activity is also regulated at the level of Raptor. Whereas low cellular energy levels negatively regulate mTORC1 activity through phosphorylation of Raptor by AMP-activated protein kinase (AMPK) (27), growth signaling pathways activating the Ras/ERK pathway positively regulate mTORC1 activity through direct phosphorylation of Raptor by RSK (10). More recent evidence has also shown that mTOR itself positively regulates mTORC1 activity by directly phosphorylating Raptor at proline-directed sites (20a, 75). Countertransport of amino acids (55) and amino acid signaling through the Rag GTPases were also shown to regulate mTORC1 activity (45, 65). When activated, mTORC1 phosphorylates two main regulators of mRNA translation and ribosome biogenesis, the AGC (protein kinase A, G, and C) family kinase p70 ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and thus stimulates protein synthesis and cellular growth (50, 60).The second mTOR complex, mTORC2, is comprised of mTOR, Rictor (rapamycin-insensitive companion of mTOR), mSin1 (mammalian stress-activated mitogen-activated protein kinase-interacting protein 1), mLST8, PRR5 (proline-rich region 5), and Deptor (21, 39, 58, 59, 66, 76, 79). Rapamycin does not directly target and inhibit mTORC2, but long-term treatment with this drug was shown to correlate with mTORC2 disassembly and cytoplasmic accumulation of Rictor (21, 39, 62, 79). Whereas mTORC1 regulates hydrophobic motif phosphorylation of S6K1, mTORC2 has been shown to phosphorylate other members of the AGC family of kinases. Biochemical and genetic evidence has demonstrated that mTORC2 phosphorylates Akt at Ser473 (26, 39, 68, 70), thereby contributing to growth factor-mediated Akt activation (6, 7, 52). Deletion or knockdown of the mTORC2 components mTOR, Rictor, mSin1, and mLST8 has a dramatic effect on mTORC2 assembly and Akt phosphorylation at Ser473 (26, 39, 79). mTORC2 was also shown to regulate protein kinase Cα (PKCα) (26, 66) and, more recently, serum- and glucocorticoid-induced protein kinase 1 (SGK1) (4, 22). Recent evidence implicates mTORC2 in the regulation of Akt and PKCα phosphorylation at their turn motifs (19, 36), but whether mTOR directly phosphorylates these sites remains a subject of debate (4).Activation of mTORC1 has been shown to negatively regulate Akt phosphorylation in response to insulin or insulin-like growth factor 1 (IGF1) (reviewed in references 30 and 51). This negative regulation is particularly evident in cell culture models with defects in the TSC1/TSC2 complex, where mTORC1 and S6K1 are constitutively activated. Phosphorylation of insulin receptor substrate-1 (IRS-1) by mTORC1 (72) and its downstream target S6K1 has been shown to decrease its stability and lead to an inability of insulin or IGF1 to activate PI3K and Akt (29, 69). Although the mechanism is unknown, platelet-derived growth factor receptor β (PDGF-Rβ) has been found to be downregulated in TSC1- and TSC2-deficient murine embryonic fibroblasts (MEFs), contributing to a reduction of PI3K signaling (80). Interestingly, inhibition of Akt phosphorylation by mTORC1 has also been observed in the presence of growth factors other than IGF-1, insulin, or PDGF, suggesting that there are other mechanisms by which mTORC1 activation restricts Akt activity in cells (reviewed in references 6 and 31). Recent evidence demonstrates that rapamycin treatment causes a significant increase in Rictor electrophoretic mobility (2, 62), suggesting that phosphorylation of the mTORC2 subunit Rictor may be regulated by mTORC1 or downstream protein kinases.Herein, we demonstrate that Rictor is phosphorylated by S6K1 in response to mTORC1 activation. We demonstrate that Thr1135 is directly phosphorylated by S6K1 and found that a Rictor mutant lacking this phosphorylation site increases Akt phosphorylation induced by growth factor stimulation. Cells expressing the Rictor T1135A mutant were found to have increased Akt signaling to its substrates compared to Rictor wild-type- and T1135D mutant-expressing cells. Together, our results suggest that Rictor integrates mTORC1 signaling via its phosphorylation by S6K1, resulting in the inhibition of mTORC2 and Akt signaling.     

Akt regulates skeletal development through GSK3, mTOR, and FoxOs

Although Akt plays key roles in various cellular processes, the functions of Akt and Akt downstream signaling pathways in the cellular processes of skeletal development remain to be clarified. By analyzing transgenic embryos that expressed constitutively active Akt (myrAkt) or dominant-negative Akt in chondrocytes, we found that Akt positively regulated the four processes of chondrocyte maturation, chondrocyte proliferation, cartilage matrix production, and cell growth in skeletal development. As phosphorylation of GSK3β, S6K, and FoxO3a was enhanced in the growth plates of myrAkt transgenic mice, we examined the Akt downstream signaling pathways by organ culture. The Akt-mTOR pathway was responsible for positive regulation of the four cellular processes. The Akt-FoxO pathway enhanced chondrocyte proliferation but inhibited chondrocyte maturation and cartilage matrix production, while the Akt-GSK3 pathway negatively regulated three of the cellular processes in limb skeletons but not in vertebrae due to less GSK3 expression in vertebrae. These findings indicate that Akt positively regulates the cellular processes of skeletal growth and endochondral ossification, that the Akt-mTOR, Akt-FoxO, and Akt-GSK3 pathways positively or negatively regulate the cellular processes, and that Akt exerts its function in skeletal development by tuning the three pathways in a manner dependent on the skeletal part.     

Mdm20 Modulates Actin Remodeling through the mTORC2 Pathway via Its Effect on Rictor Expression

NatB is an N-terminal acetyltransferase consisting of a catalytic Nat5 subunit and an auxiliary Mdm20 subunit. In yeast, NatB acetylates N-terminal methionines of proteins during de novo protein synthesis and also regulates actin remodeling through N-terminal acetylation of tropomyosin (Trpm), which stabilizes the actin cytoskeleton by interacting with actin. However, in mammalian cells, the biological functions of the Mdm20 and Nat5 subunits are not well understood. In the present study, we show for the first time that Mdm20-knockdown (KD), but not Nat5-KD, in HEK293 and HeLa cells suppresses not only cell growth, but also cellular motility. Although stress fibers were formed in Mdm20-KD cells, and not in control or Nat5-KD cells, the localization of Trpm did not coincide with the formation of stress fibers in Mdm20-KD cells. Notably, knockdown of Mdm20 reduced the expression of Rictor, an mTORC2 complex component, through post-translational regulation. Additionally, PKCα^S657 phosphorylation, which regulates the organization of the actin cytoskeleton, was also reduced in Mdm20-KD cells. Our data also suggest that FoxO1 phosphorylation is regulated by the Mdm20-mTORC2-Akt pathway in response to serum starvation and insulin stimulation. Taken together, the present findings suggest that Mdm20 acts as a novel regulator of Rictor, thereby controlling mTORC2 activity, and leading to the activation of PKCα^S657 and FoxO1.     

Multiple site acetylation of Rictor stimulates mammalian target of rapamycin complex 2 (mTORC2)-dependent phosphorylation of Akt protein

The serine/threonine protein kinase Akt is a critical regulator of cell growth and survival in response to growth factors. A key step in Akt activation is phosphorylation at Ser-473 by the mammalian target of rapamycin (mTOR) complex 2 (mTORC2). Although Rictor is required for the stability and activity of mTORC2, little is known about functional regions or post-translational modifications within Rictor that are responsible for regulating mTORC2. Here, we demonstrate that Rictor contains two distinct central regions critical for mTORC2 function. One we refer to as the stability region because it is critical for interaction with Sin1.1 and LST8, and a second adjacent region is required for multisite acetylation. p300-mediated acetylation of Rictor increases mTORC2 activity toward Akt, whereas site-directed mutants within the acetylation region of Rictor exhibit reduced insulin-like growth factor 1 (IGF-1)-stimulated mTORC2 kinase activity. Inhibition of deacetylases, including the NAD+-dependent sirtuins, promotes Rictor acetylation and IGF-1-mediated Akt phosphorylation. These results suggest that multiple-site acetylation of Rictor signals for increased activation of mTORC2, providing a critical link between nutrient-sensitive deacetylases and mTORC2 signaling to Akt.     

PI3K/Akt Cooperates with Oncogenic Notch by Inducing Nitric Oxide-Dependent Inflammation

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The hVps34‐SGK3 pathway alleviates sustained PI3K/Akt inhibition by stimulating mTORC1 and tumour growth

We explore mechanisms that enable cancer cells to tolerate PI3K or Akt inhibitors. Prolonged treatment of breast cancer cells with PI3K or Akt inhibitors leads to increased expression and activation of a kinase termed SGK3 that is related to Akt. Under these conditions, SGK3 is controlled by hVps34 that generates PtdIns(3)P, which binds to the PX domain of SGK3 promoting phosphorylation and activation by its upstream PDK1 activator. Furthermore, under conditions of prolonged PI3K/Akt pathway inhibition, SGK3 substitutes for Akt by phosphorylating TSC2 to activate mTORC1. We characterise 14h, a compound that inhibits both SGK3 activity and activation in vivo, and show that a combination of Akt and SGK inhibitors induced marked regression of BT‐474 breast cancer cell‐derived tumours in a xenograft model. Finally, we present the kinome‐wide analysis of mRNA expression dynamics induced by PI3K/Akt inhibition. Our findings highlight the importance of the hVps34‐SGK3 pathway and suggest it represents a mechanism to counteract inhibition of PI3K/Akt signalling. The data support the potential of targeting both Akt and SGK as a cancer therapeutic.     

HIV-1 Nef Binds the DOCK2–ELMO1 Complex to Activate Rac and Inhibit Lymphocyte Chemotaxis

The infectious cycle of primate lentiviruses is intimately linked to interactions between cells of the immune system. Nef, a potent virulence factor, alters cellular environments to increase lentiviral replication in the host, yet the mechanisms underlying these effects have remained elusive. Since Nef likely functions as an adaptor protein, we exploited a proteomic approach to directly identify molecules that Nef targets to subvert the signaling machinery in T cells. We purified to near homogeneity a major Nef-associated protein complex from T cells and identified by mass spectroscopy its subunits as DOCK2–ELMO1, a key activator of Rac in antigen- and chemokine-initiated signaling pathways, and Rac. We show that Nef activates Rac in T cell lines and in primary T cells following infection with HIV-1 in the absence of antigenic stimuli. Nef activates Rac by binding the DOCK2–ELMO1 complex, and this interaction is linked to the abilities of Nef to inhibit chemotaxis and promote T cell activation. Our data indicate that Nef targets a critical switch that regulates Rac GTPases downstream of chemokine- and antigen-initiated signaling pathways. This interaction enables Nef to influence multiple aspects of T cell function and thus provides an important mechanism by which Nef impacts pathogenesis by primate lentiviruses.     

ERK and Akt signaling pathways function through parallel mechanisms to promote mTORC1 signaling

The mammalian target of rapamycin (mTOR) is a protein kinase that, when present in a complex referred to as mTOR complex 1 (mTORC1), acts as an important regulator of growth and metabolism. The activity of the complex is regulated through multiple upstream signaling pathways, including those involving Akt and the extracellular-regulated kinase (ERK). Previous studies have shown that, in part, Akt and ERK promote mTORC1 signaling through phosphorylation of a GTPase activator protein (GAP), referred to as tuberous sclerosis complex 2 (TSC2), that acts as an upstream inhibitor of mTORC1. In the present study we extend the earlier studies to show that activation of the Akt and ERK pathways acts in a synergistic manner to promote mTORC1 signaling. Moreover, we provide evidence that the Akt and ERK signaling pathways converge on TSC2, and that Akt phosphorylates residues on TSC2 distinct from those phosphorylated by ERK. The results also suggest that leucine-induced stimulation of mTORC1 signaling occurs through a mechanism distinct from TSC2 and the Akt and ERK signaling pathways. Overall, the results are consistent with a model in which Akt and ERK phosphorylate distinct sites on TSC2, leading to greater repression of its GAP activity, and consequently a magnified stimulation of mTORC1 signaling, when compared with either input alone. The results further suggest that leucine acts through a mechanism distinct from TSC2 to stimulate mTORC1 signaling.     

Phosphorylation of Rictor at Thr1135 impairs the Rictor/Cullin-1 complex to ubiquitinate SGK1

The Rictor/mTOR complex plays a pivotal role in a variety of cellular functions including cellular metabolism, cell proliferation and survival by phosphorylating Akt at Ser473 to fully activate the Akt kinase. However, its upstream regulatory pathways as well as whether it has additional function(s) remain largely unknown. We recently reported that Rictor contains a novel ubiquitin E3 ligase activity by forming a novel complex with Cullin-1, but not with other Cullin family members. Furthermore, we identified SGK1 as its downstream target. Interestingly, Rictor, but not Raptor or mTOR, promotes SGK1 ubiquitination. As a result, SGK1 expression is elevated in Rictor^--/-- MEFs. We further defined that as a feedback mechanism, Rictor can be phosphorylated by multiple AGC family kinases including Akt, S6K and SGK1. Phosphorylation of Rictor at the Thr1135 site did not affect its kinase activity towards phosphorylating its conventional substrates including Akt and SGK1. On the other hand, it disrupted the interaction between Rictor and Cullin-1. Consequently, T1135E Rictor was defective in promoting SGK1 ubiquitination and destruction. This finding further expands our knowledge of Rictor’s function. Furthermore, our work also illustrates that Rictor E3 ligase activity could be governed by specific signaling kinase cascades, and that misregulation of this process might contribute to SGK overexpression which is frequently observed in various types of cancers.     

Livers with Constitutive mTORC1 Activity Resist Steatosis Independent of Feedback Suppression of Akt

Insulin resistance is an important contributing factor in non-alcoholic fatty liver disease. AKT and mTORC1 are key components of the insulin pathway, and play a role in promoting de novo lipogenesis. However, mTORC1 hyperactivity per se does not induce steatosis in mouse livers, but instead, protects against high-fat diet induced steatosis. Here, we investigate the in vivo mechanism of steatosis-resistance secondary to mTORC1 activation, with emphasis on the role of S6K1-mediated feedback inhibition of AKT. Mice with single or double deletion of Tsc1 and/or S6k1 in a liver-specific or whole-body manner were generated to study glucose and hepatic lipid metabolism between the ages of 6–14 weeks. Following 8 weeks of high-fat diet, the Tsc1-/-;S6k1-/- mice had lower body weights but higher liver TG levels compared to that of the Tsc1-/- mice. However, the loss of S6k1 did not relieve feedback inhibition of Akt activity in the Tsc1-/- livers. To overcome Akt suppression, Pten was deleted in Tsc1-/- livers, and the resultant mice showed improved glucose tolerance compared with the Tsc1-/- mice. However, liver TG levels were significantly reduced in the Tsc1-/-;Pten-/- mice compared to the Pten-/- mice, which was restored with rapamycin. We found no correlation between liver TG and serum NEFA levels. Expression of lipogenic genes (Srebp1c, Fasn) were elevated in the Tsc1-/-;Pten-/- livers, but this was counter-balanced by an up-regulation of Cpt1a involved in fatty acid oxidation and the anti-oxidant protein, Nrf2. In summary, our in vivo models showed that mTORC1-induced resistance to steatosis was dependent on S6K1 activity, but not secondary to AKT suppression. These findings confirm that AKT and mTORC1 have opposing effects on hepatic lipid metabolism in vivo.     

Hepatic mTORC2 activates glycolysis and lipogenesis through Akt, glucokinase, and SREBP1c

Mammalian target of rapamycin complex 2 (mTORC2) phosphorylates and activates AGC kinase family members, including Akt, SGK1, and PKC, in response to insulin/IGF1. The liver is a key organ in insulin-mediated regulation of metabolism. To assess the role of hepatic mTORC2, we generated liver-specific rictor knockout (LiRiKO) mice. Fed LiRiKO mice displayed loss of Akt Ser473 phosphorylation and reduced glucokinase and SREBP1c activity in the liver, leading to constitutive gluconeogenesis, and impaired glycolysis and lipogenesis, suggesting that the mTORC2-deficient liver is unable to sense satiety. These liver-specific defects resulted in systemic hyperglycemia, hyperinsulinemia, and hypolipidemia. Expression of constitutively active Akt2 in mTORC2-deficient hepatocytes restored both glucose flux and lipogenesis, whereas glucokinase overexpression rescued glucose flux but not lipogenesis. Thus, mTORC2 regulates hepatic glucose and lipid metabolism via insulin-induced Akt signaling to control whole-body metabolic homeostasis. These findings have implications for emerging drug therapies that target mTORC2.     

Transient and rapid activation of Akt/GSK‐3β and mTORC1 signaling by D3 dopamine receptor stimulation in dorsal striatum and nucleus accumbens

D2/D3 dopamine receptors (D2R/D3R) agonists regulate Akt, but their effects display a complex time‐course. In addition, the respective roles of D2R and D3R are not defined and downstream targets remain poorly characterized, especially in vivo. These issues were addressed here for D3R. Systemic administration of quinelorane, a D2R/D3R agonist, transiently increased phosphorylation of Akt and GSK‐3β in rat nucleus accumbens and dorsal striatum with maximal effects 10 min after injection. Akt activation was associated with phosphorylation of several effectors of the mammalian target of rapamycin complex 1 (mTORC1): p70S6 kinase, ribosomal protein‐S6 (Ser240/244), and eukaryotic initiation factor‐4E binding protein‐1. The action of quinelorane was antagonized by a D2/D3R antagonist, raclopride, and the selective D3R antagonist S33084, inactive by themselves. Furthermore, no effect of quinerolane was seen in knock‐out mice lacking D3R. In drd1a‐EGFP transgenic mice, quinelorane activated Akt/GSK‐3β in both neurons expressing and lacking D1 receptor. Thus, the stimulation of D3R transiently activates the Akt/GSK‐3β pathway in the two populations of medium‐size spiny neurons of the nucleus accumbens and dorsal striatum. This effect may contribute to the influence of D3R ligands on reward, cognition, and processes disrupted in schizophrenia, drug abuse, and Parkinson's disease.