Regulation of mTORC1 and mTORC2 Complex Assembly by Phosphatidic Acid: Competition with Rapamycin

mTOR, the mammalian target of rapamycin, is a critical node for control of cell growth and survival and has widely been implicated in cancer survival signals. mTOR exists in two complexes: mTORC1 and mTORC2. Phospholipase D (PLD) and its metabolite phosphatidic acid (PA) have been implicated in the regulation of mTOR; however, their role has been controversial. We report here that suppression of PLD prevents phosphorylation of the mTORC1 substrate S6 kinase (S6K) at Thr389 and the mTORC2 substrate Akt at Ser473. Suppression of PLD also blocked insulin-stimulated Akt phosphorylation at Ser473 and the mTORC2-dependent phosphorylation of PRAS40. Importantly, PA was required for the association of mTOR with Raptor to form mTORC1 and that of mTOR with Rictor to form mTORC2. The effect of PA was competitive with rapamycin—with much higher concentrations of rapamycin needed to compete with the PA-mTORC2 interaction than with PA-mTORC1. Suppressing PA production substantially increased the sensitivity of mTORC2 to rapamycin. Data provided here demonstrate a PA requirement for the stabilization of both mTORC1 and mTORC2 complexes and reveal a mechanism for the inhibitory effect of rapamycin on mTOR. This study also suggests that by suppressing PLD activity, mTORC2 could be targeted therapeutically with rapamycin.It has become apparent during the past decade that a critical aspect of tumor progression is the suppression of default apoptotic programs that constitute what is likely the most important protection against cancer. Cellular signals that suppress apoptosis have come to be known as “survival signals.” A common node for survival signals is mTOR, the mammalian target of rapamycin (5, 13, 14, 25). mTOR exists in two distinct complexes, mTORC1 and mTORC2 (21), that differ in their subunit composition and sensitivity to rapamycin. mTORC1 consists of a complex that includes mTOR and a protein known as Raptor (regulatory associated protein of mTOR), whereas mTORC2 consists of a complex that includes mTOR and a protein known as Rictor (rapamycin-insensitive companion of mTOR) (13, 14). There are also mTORC2 complexes that can be distinguished by association with different isoforms of mSin1 (9). While much is known about the regulation of mTORC1 (21), very little is known about the regulation of mTORC2.mTORC1 is highly sensitive to rapamycin, whereas mTORC2 is relatively insensitive to rapamycin (21). However, it was recently reported that long-term exposure to rapamycin prevented the formation of mTORC2 complexes and blocked the phosphorylation of the mTORC2 substrate Akt at Ser473 (24, 38). Rapamycin, in association with FK506 binding protein 12 (FKBP12), has been reported to interact with mTOR in a manner that is competitive with phosphatidic acid (PA), the metabolic product of phospholipase D (PLD) (2, 4). PLD, like mTOR, has been implicated in survival signals in several human cancer cell lines (1, 10, 11, 27, 32, 39). Since rapamycin-FKBP12 competes with PA for binding to mTOR, the sensitivity of mTORC2 complex formation to rapamycin suggests that PA facilitates the assembly of mTORC2—and ultimately the activation of mTORC2. We report here that the assembly of both mTORC1 and mTORC2 complexes is dependent upon PLD and its metabolite PA. The study also provides mechanistic insight into how rapamycin impacts on mTOR-mediated signals and how PLD regulates mTOR by facilitating the formation of mTOR complexes.     

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.     

Mammalian Target of Rapamycin Complex 1 (mTORC1) Activity Is Associated with Phosphorylation of Raptor by mTOR

mTORC1 contains multiple proteins and plays a central role in cell growth and metabolism. Raptor (regulatory-associated protein of mammalian target of rapamycin (mTOR)), a constitutively binding protein of mTORC1, is essential for mTORC1 activity and critical for the regulation of mTORC1 activity in response to insulin signaling and nutrient and energy sufficiency. Herein we demonstrate that mTOR phosphorylates raptor in vitro and in vivo. The phosphorylated residues were identified by using phosphopeptide mapping and mutagenesis. The phosphorylation of raptor is stimulated by insulin and inhibited by rapamycin. Importantly, the site-directed mutation of raptor at one phosphorylation site, Ser⁸⁶³, reduced mTORC1 activity both in vitro and in vivo. Moreover, the Ser⁸⁶³ mutant prevented small GTP-binding protein Rheb from enhancing the phosphorylation of S6 kinase (S6K) in cells. Therefore, our findings indicate that mTOR-mediated raptor phosphorylation plays an important role on activation of mTORC1.Mammalian target of rapamycin (mTOR)² has been shown to function as a critical controller in cellular growth, survival, metabolism, and development (1). mTOR, a highly conserved Ser-Thr phosphatidylinositol 3-kinase-related protein kinase, structurally forms two distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), each of which catalyzes the phosphorylation of different substrates (1). The best characterized substrates for mTORC1 are eIF4E-binding protein (4E-BP, also known as PHAS) and p70 S6 kinase (S6K) (1), whereas mTORC2 phosphorylates the hydrophobic and turn motifs of protein kinase B (Akt/protein kinase B) (2) and protein kinase C (3, 4). mTORC1 constitutively consists of mTOR, raptor, and mLst8/GβL (1), whereas the proline-rich Akt substrate of 40 kDa (PRAS40) is a regulatory component of mTORC1 that disassociates after growth factor stimulation (5, 6). Raptor is essential for mTORC1 activity by providing a substrate binding function (7) but also plays a regulatory role on mTORC1 with stimuli of growth factors and nutrients (8). In response to insulin, raptor binding to substrates is elevated through the release of the competitive inhibitor PRAS40 from mTORC1 (9, 10) because PRAS40 and the substrates of mTORC1 (4E-BP and S6K) appear to bind raptor through a consensus sequence, the TOR signaling (TOS) motif (10–14). In response to amino acid sufficiency, raptor directly interacts with a heterodimer of Rag GTPases and promotes mTORC1 localization to the Rheb-containing vesicular compartment (15).mTORC1 integrates signaling pathways from growth factors, nutrients, energy, and stress, all of which generally converge on the tuberous sclerosis complex (TSC1-TSC2) through the phosphorylation of TSC2 (1). Growth factors inhibit the GTPase-activating protein activity of TSC2 toward the small GTPase Rheb via the PI3K/Akt pathway (16, 17), whereas energy depletion activates TSC2 GTPase-activating protein activity by stimulating AMP-activated protein kinase (AMPK) (18). Rheb binds directly to mTOR, albeit with very low affinity (19), and upon charging with GTP, Rheb functions as an mTORC1 activator (6). mTORC1 complexes isolated from growth factor-stimulated cells show increased kinase activity yet do not contain detectable levels of associated Rheb. Therefore, how Rheb-GTP binding to mTOR leads to an increase in mTORC1 activity toward substrates, and what the role of raptor is in this activation is currently unknown. More recently, the AMPK and p90 ribosomal S6 kinase (RSK) have been reported to directly phosphorylate raptor and regulate mTORC1 activity. The phosphorylation of raptor directly by AMPK reduced mTORC1 activity, suggesting an alternative regulation mechanism independent of TSC2 in response to energy supply (20). RSK-mediated raptor phosphorylation enhances mTORC1 activity and provides a mechanism whereby stress may activate mTORC1 independent of the PI3K/Akt pathway (21). Therefore, the phosphorylation status of raptor can be critical for the regulation of mTORC1 activity.In this study, we investigated phosphorylation sites in raptor catalyzed by mTOR. Using two-dimensional phosphopeptide mapping, we found that Ser⁸⁶³ and Ser⁸⁵⁹ in raptor were phosphorylated by mTOR both in vivo and in vitro. mTORC1 activity in vitro and in vivo is associated with the phosphorylation of Ser⁸⁶³ in raptor.     

Site-Specific mTOR Phosphorylation Promotes mTORC1-Mediated Signaling and Cell Growth

The mammalian target of rapamycin (mTOR) complex 1 (mTORC1) functions as a rapamycin-sensitive environmental sensor that promotes cellular biosynthetic processes in response to growth factors and nutrients. While diverse physiological stimuli modulate mTORC1 signaling, the direct biochemical mechanisms underlying mTORC1 regulation remain poorly defined. Indeed, while three mTOR phosphorylation sites have been reported, a functional role for site-specific mTOR phosphorylation has not been demonstrated. Here we identify a new site of mTOR phosphorylation (S1261) by tandem mass spectrometry and demonstrate that insulin-phosphatidylinositol 3-kinase signaling promotes mTOR S1261 phosphorylation in both mTORC1 and mTORC2. Here we focus on mTORC1 and show that TSC/Rheb signaling promotes mTOR S1261 phosphorylation in an amino acid-dependent, rapamycin-insensitive, and autophosphorylation-independent manner. Our data reveal a functional role for mTOR S1261 phosphorylation in mTORC1 action, as S1261 phosphorylation promotes mTORC1-mediated substrate phosphorylation (e.g., p70 ribosomal protein S6 kinase 1 [S6K1] and eukaryotic initiation factor 4E binding protein 1) and cell growth to increased cell size. Moreover, Rheb-driven mTOR S2481 autophosphorylation and S6K1 phosphorylation require S1261 phosphorylation. These data provide the first evidence that site-specific mTOR phosphorylation regulates mTORC1 function and suggest a model whereby insulin-stimulated mTOR S1261 phosphorylation promotes mTORC1 autokinase activity, substrate phosphorylation, and cell growth.The mammalian target of rapamycin (mTOR), an evolutionarily conserved serine/threonine protein kinase, senses and integrates signals from diverse environmental cues (14, 31, 50, 74). mTOR associates with different partner proteins to form functionally distinct signaling complexes (4). The immunosuppressive drug rapamycin acutely inhibits signaling by mTOR complex 1 (mTORC1) (22), which contains mTOR, mLST8/GβL, raptor, and PRAS40 (24, 33, 34, 54, 67). Rapamycin fails to acutely inhibit signaling by mTORC2, which contains mTOR, mLST8/GβL, rictor, mSin1, and PRR5/Protor (18, 32, 47, 55, 73, 76). mTORC1 promotes various biosynthetic processes, including protein synthesis, cell growth (an increase in cell mass and size), and cell proliferation (an increase in cell number) (14, 40, 74). During growth factor (e.g., insulin) and nutrient (e.g., amino acids and glucose) sufficiency, mTORC1 phosphorylates the translational regulators p70 ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4EBP1) to coordinately upregulate protein biosynthesis (40). Both S6K1 and 4EBP1 contain a TOR signaling motif, which mediates their interaction with raptor and thus facilitates their recruitment to the mTOR kinase (10, 44, 57, 58). In addition to regulating protein synthesis, mTORC1-mediated phosphorylation of S6K1 and 4EBP also promotes cell growth and cell cycle progression (15, 16). While more recently identified and thus less well characterized than mTORC1, mTORC2 mediates the phosphorylation of AGC kinase family members (e.g., Akt [also known as protein kinase B, PKB], PKCα, and SGK1) on their hydrophobic motifs and modulates the organization of the actin cytoskeleton (20, 26, 32, 55, 56).The insulin pathway represents the best-characterized activator of mTORC1 signaling to date, and thus many signaling intermediates that link insulin receptor activation to mTORC1 have been identified (12, 31). Complementary work using Drosophila melanogaster genetics and mammalian cell culture identified TSC1 (hamartin) and TSC2 (tuberin) as upstream negative regulators of mTORC1 (27). Inactivation of either the TSC1 or TSC2 genes, whose protein products heterodimerize to form a tumor suppressor complex, causes the development of benign tumors in diverse organs in both humans and rodents, a disease known as tuberous sclerosis complex (TSC) (36). TSC2 contains a GTPase-activating protein domain that acts on Rheb, a Ras-like GTP binding protein that activates mTORC1 (27). Thus, in TSC-deficient cells, constitutive Rheb-GTP leads to chronically high mTORC1 signaling. While the mechanism by which Rheb-GTP activates mTORC1 remains incompletely understood, Rheb coimmunoprecipitates with mTOR and directly activates mTORC1 kinase activity in vivo and in vitro when GTP bound (2, 38, 54). Rheb has been reported to augment the activity of PLD1, an enzyme that catalyzes the production of the lipid second messenger phosphatidic acid, which contributes to the mitogenic activation of mTORC1 signaling (13, 62). Additionally, Rheb-GTP was reported to induce the dissociation of the endogenous mTOR inhibitor FKBP38 (3), although aspects of this model have been questioned (72). Insulin/phosphatidylinositol 3-kinase (PI3K) signaling reduces the inhibitory effect of TSC on mTORC1 via Akt-mediated phosphorylation of TSC2 (29, 42, 64). Additionally, Ras-regulated signaling via mitogen-activated protein kinase (MAPK) and RSK also inhibits TSC via PI3K/Akt-independent phosphorylation of TSC2 (39, 51, 63). In contrast, glucose deprivation enhances TSC''s inhibitory effect on mTORC1 signaling via AMP-activated protein kinase (AMPK)-mediated phosphorylation of TSC2 (on different sites) (30). Thus, TSC functions as a central nexus of diverse physiological signals to fine-tune mTORC1 signaling depending on environmental conditions (27). While the mechanism by which amino acids promote mTORC1 signaling has remained elusive, compelling new data reveal that the Rag GTPases link amino acid sensing to mTORC1 activation (35, 52, 53). During amino acid sufficiency, GTP-bound Rag heterodimers bind raptor and recruit mTORC1 to an endomembrane compartment that contains the mTORC1 activator Rheb; thus, amino acid sufficiency may function to prime mTORC1 for subsequent growth factor-mediated activation via a dynamic subcellular redistribution mechanism (52).Despite the well-characterized regulation of mTORC1 signaling by growth factors (e.g., insulin), nutrients (e.g., amino acids and glucose), and cellular stress (e.g., hypoxia) and the identification of numerous signaling mediators of these pathways, the direct molecular mechanisms by which cellular signals modulate mTORC1 action remain obscure (31). While three phosphorylation sites (P-sites) on mTOR have been reported to date (T2446, S2448, and S2481), no function has yet been ascribed to any site (7, 43, 49, 59). Here we identify S1261 as a novel mTOR phosphorylation site in vivo in cultured mammalian cells and provide the first evidence that site-specific mTOR phosphorylation regulates mTORC1 function. We show that insulin signals via the PI3K/TSC/Rheb pathway in an amino acid-dependent and rapamycin-insensitive manner to promote mTOR S1261 phosphorylation, which regulates mTORC1 autokinase activity, biochemical signaling to downstream substrates, and cell growth to increased cell size, a major cellular function of mTORC1. Elucidation of the molecular mechanisms underlying mTORC1 regulation will enable us to better understand how mTORC1 senses environmental stimuli to control cellular physiology. As aberrantly upregulated mTORC1 signaling likely contributes to cancer, insulin-resistant diabetes, and cardiovascular diseases, understanding mTORC1 regulation may aid in the development of novel therapeutics for these prevalent human diseases (11, 21, 28).     

Specific Activation of mTORC1 by Rheb G-protein in Vitro Involves Enhanced Recruitment of Its Substrate Protein

Rheb G-protein plays critical roles in the TSC/Rheb/mTOR signaling pathway by activating mTORC1. The activation of mTORC1 by Rheb can be faithfully reproduced in vitro by using mTORC1 immunoprecipitated by the use of anti-raptor antibody from mammalian cells starved for nutrients. The low in vitro kinase activity against 4E-BP1 of this mTORC1 preparation is dramatically increased by the addition of recombinant Rheb. On the other hand, the addition of Rheb does not activate mTORC2 immunoprecipitated from mammalian cells by the use of anti-rictor antibody. The activation of mTORC1 is specific to Rheb, because other G-proteins such as KRas, RalA/B, and Cdc42 did not activate mTORC1. Both Rheb1 and Rheb2 activate mTORC1. In addition, the activation is dependent on the presence of bound GTP. We also find that the effector domain of Rheb is required for the mTORC1 activation. FKBP38, a recently proposed mediator of Rheb action, appears not to be involved in the Rheb-dependent activation of mTORC1 in vitro, because the preparation of mTORC1 that is devoid of FKBP38 is still activated by Rheb. The addition of Rheb results in a significant increase of binding of the substrate protein 4E-BP1 to mTORC1. PRAS40, a TOR signaling (TOS) motif-containing protein that competes with the binding of 4EBP1 to mTORC1, inhibits Rheb-induced activation of mTORC1. A preparation of mTORC1 that is devoid of raptor is not activated by Rheb. Rheb does not induce autophosphorylation of mTOR. These results suggest that Rheb induces alteration in the binding of 4E-BP1 with mTORC1 to regulate mTORC1 activation.Rheb defines a unique member of the Ras superfamily G-proteins (1). We have shown that Rheb proteins are conserved and are found from yeast to human (2). Although yeast and fruit fly have one Rheb, mouse and human have two Rheb proteins termed Rheb1 (or simply Rheb) and Rheb2 (RhebL1) (2). Structurally, these proteins contain G1-G5 boxes, short stretches of amino acids that define the function of the Ras superfamily G-proteins including guanine nucleotide binding (1, 3, 4). Rheb proteins have a conserved arginine at residue 15 that corresponds to residue 12 of Ras (1). The effector domain required for the binding with downstream effectors encompasses the G2 box and its adjacent sequences (1, 5). Structural analysis by x-ray crystallography further shows that the effector domain is exposed to solvent, is located close to the phosphates of GTP especially at residues 35–38, and undergoes conformational change during GTP/GDP exchange (6). In addition, all Rheb proteins end with the CAAX (C is cysteine, A is an aliphatic amino acid, and X is the C-terminal amino acid) motif that signals farnesylation. In fact, we as well as others have shown that these proteins are farnesylated (7–9).Rheb plays critical roles in the TSC/Rheb/mTOR signaling, a signaling pathway that plays central roles in regulating protein synthesis and growth in response to nutrient, energy, and growth conditions (10–14). Rheb is down-regulated by a TSC1·TSC2 complex that acts as a GTPase-activating protein for Rheb (15–19). Recent studies established that the GAP domain of TSC2 defines the functional domain for the down-regulation of Rheb (20). Mutations in the Tsc1 or Tsc2 gene lead to tuberous sclerosis whose symptoms include the appearance of benign tumors called hamartomas at different parts of the body as well as neurological symptoms (21, 22). Overexpression of Rheb results in constitutive activation of mTOR even in the absence of nutrients (15, 16). Two mTOR complexes, mTORC1 and mTORC2, have been identified (23, 24). Whereas mTORC1 is involved in protein synthesis activation mediated by S6K and 4EBP1, mTORC2 is involved in the phosphorylation of Akt in response to insulin. It has been suggested that Rheb is involved in the activation of mTORC1 but not mTORC2 (25).Although Rheb is clearly involved in the activation of mTOR, the mechanism of activation has not been established. We as well as others have suggested a model that involves the interaction of Rheb with the TOR complex (26–28). Rheb activation of mTOR kinase activity using immunoprecipitated mTORC1 was reported (29). Rheb has been shown to interact with mTOR (27, 30), and this may involve direct interaction of Rheb with the kinase domain of mTOR (27). However, this Rheb/mTOR interaction is a weak interaction and is not dependent on the presence of GTP bound to Rheb (27, 28). Recently, a different model proposing that FKBP38 (FK506-binding protein 38) mediates the activation of mTORC1 by Rheb was proposed (31, 32). In this model, FKBP38 binds mTOR and negatively regulates mTOR activity, and this negative regulation is blocked by the binding of Rheb to FKBP38. However, recent reports dispute this idea (33).To further characterize Rheb activation of mTOR, we have utilized an in vitro system that reproduces activation of mTORC1 by the addition of recombinant Rheb. We used mTORC1 immunoprecipitated from nutrient-starved cells using anti-raptor antibody and have shown that its kinase activity against 4E-BP1 is dramatically increased by the addition of recombinant Rheb. Importantly, the activation of mTORC1 is specific to Rheb and is dependent on the presence of bound GTP as well as an intact effector domain. FKBP38 is not detected in our preparation and further investigation suggests that FKBP38 is not an essential component for the activation of mTORC1 by Rheb. Our study revealed that Rheb enhances the binding of a substrate 4E-BP1 with mTORC1 rather than increasing the kinase activity of mTOR.     

Rapamycin-enhanced mitomycin C-induced apoptotic death is mediated through the S6K1–Bad–Bak pathway in peritoneal carcinomatosis

Peritoneal carcinomatosis (PC) is the most common secondary cancerous disease, and more effective novel regimens are needed. In this study, we identified a novel combination treatment for PC, chemotherapeutic agent mitomycin C in combination with mTOR (mammalian target of rapamycin) inhibitor rapamycin. We observed that the combination of mitomycin C and rapamycin induced synergistic cytotoxicity and apoptosis, which was mediated through an increase in caspase activation. The combination of mitomycin C and rapamycin inactivated p70 S6 ribosomal kinase (S6K1) and dephosphorylated Bad, leading to dissociation of Bcl-xL from Bak, which resulted in Bak oligomerization, mitochondria dysfunction and cytochrome c release. PF-4708671, a S6K1-specific inhibitor, enhanced the combination treatment-induced apoptosis, whereas S6K1 E389 DeltaCT-HA (S6K1 active form) dramatically decreased the induction of apoptosis. In addition, the combination treatment significantly inhibited LS174T intraperitoneal tumor growth in vivo. This study provides a preclinical rationale for apoptosis induction linked with the mTOR pathway through a combination of chemotherapeutic agents and mTOR inhibitor, and will support this combinatorial strategy to PC patients.Peritoneal carcinomatosis (PC) is the most common secondary cancerous disease to affect the peritoneal cavity following colorectal cancer, appendiceal cancer, ovarian cancer, gastric cancer, or diffuse malignant peritoneal mesothelioma, and is regarded as a lethal condition having poor prognosis with a median survival of ∼6 months.^{1, 2} Systemic chemotherapy prolongs survival in PC patients, but is less effective than for patients with hepatic metastasis; thus, an increasing number of patients are treated with cytoreductive surgery followed by hyperthermic intraperitoneal chemoperfusion (HIPEC).³ Nevertheless, more effective novel regimens are still needed to improve the survival of PC patients.Mitomycin C, a potent DNA crosslinker, was used as an anticancer drug and led to pronounced induction of apoptosis, especially through the mitochondrial-dependent pathway.^{4, 5} It has received a great number of preclinical and clinical studies and is mostly widely used for PC.⁶ Combination of cytotoxic drugs, such as mitomycin C with novel biological agents, has achieved improvement in animal models and provides additional survival benefits for patients.^{7, 8, 9}mTOR (mammalian target of rapamycin), a serine/threonine kinase, is ubiquitously involved in cell growth and proliferation. The fact that hyperactivation of mTOR is a common feature of human cancers makes it an attractive target in cancer therapy.¹⁰ It has been reported that some agents inactivate mTOR signaling as an early response prior to the execution of apoptosis.^{11, 12} Recently, it has been found that mTOR can directly control mitochondrial function,¹³ which raises the possibility that targeting mTOR may regulate the apoptotic pathway. Sirolimus, known as rapamycin, an mTOR inhibitor, potently inhibits S6K1 (S6 ribosomal kinase) activity¹⁴ and has been generally well tolerated in clinical trials, often inducing prolonged stable disease or tumor regression, and can be considered as an option for combination with other anticancer agents.^{15, 16}In this study we observed that the combination of chemotherapy agent mitomycin C and mTOR inhibitor rapamycin induced synergistic cytotoxicity and apoptosis in vitro and in vivo. The combination of mitomycin C and rapamycin inactivated S6K1, not Akt, and resulted in dephosphorylation of Bad, which inactivated its pro-apoptotic function, inducing Bak oligomerization and mitochondrial-dependent apoptotic cell death.     

Glycolytic Flux Signals to mTOR through Glyceraldehyde-3-Phosphate Dehydrogenase-Mediated Regulation of Rheb

The mammalian target of rapamycin (mTOR) interacts with raptor to form the protein complex mTORC1 (mTOR complex 1), which plays a central role in the regulation of cell growth in response to environmental cues. Given that glucose is a primary fuel source and a biosynthetic precursor, how mTORC1 signaling is coordinated with glucose metabolism has been an important question. Here, we found that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) binds Rheb and inhibits mTORC1 signaling. Under low-glucose conditions, GAPDH prevents Rheb from binding to mTOR and thereby inhibits mTORC1 signaling. High glycolytic flux suppresses the interaction between GAPDH and Rheb and thus allows Rheb to activate mTORC1. Silencing of GAPDH or blocking of the Rheb-GAPDH interaction desensitizes mTORC1 signaling to changes in the level of glucose. The GAPDH-dependent regulation of mTORC1 in response to glucose availability occurred even in TSC1-deficient cells and AMPK-silenced cells, supporting the idea that the GAPDH-Rheb pathway functions independently of the AMPK axis. Furthermore, we show that glyceraldehyde-3-phosphate, a glycolytic intermediate that binds GAPDH, destabilizes the Rheb-GAPDH interaction even under low-glucose conditions, explaining how high-glucose flux suppresses the interaction and activates mTORC1 signaling. Taken together, our results suggest that the glycolytic flux regulates mTOR''s access to Rheb by regulating the Rheb-GAPDH interaction, thereby allowing mTORC1 to coordinate cell growth with glucose availability.The mTOR complex 1 (mTORC1) signal transduction pathway acts as a central controller of cell growth in mammals (20, 23, 29). mTORC1 integrates a wide range of intracellular and extracellular signals, including insulin, availability of nutrients (glucose and amino acids), cellular energy status, and hypoxia, to regulate protein synthesis and cell growth (11, 12, 17, 36, 46). Many of these environmental cues are integrated into tuberous sclerosis complex (TSC1-TSC2), the major upstream regulator of mTORC1. In response to the absence of insulin and to the low-energy status of cells, the TSC1-TSC2 complex stimulates the GTPase function of Rheb, a small GTPase that acts as a proximal key activator of mTORC1, which leads to the inhibition of Rheb-mediated mTORC1 activation. In contrast, inactivation of the TSC1-TSC2 complex results in the accumulation of GTP-bound Rheb and thus activation of mTORC1 (3, 13, 21, 27, 32, 39). For this reason, both the loss of TSC proteins and the overexpression of Rheb cause hyperactivation of mTORC1 signaling, which is frequently observed in many common human cancers (2, 5, 19, 25, 33). Therefore, a tight regulation of Rheb activity is critical for the proper operation of the mTORC1 pathway in response to environmental cues.Rheb is an atypical member of the Ras superfamily of GTPases (1, 10, 47). As with other small GTPases, the activity of Rheb is regulated by its guanine nucleotide binding status. However, the negative control of GTP-bound Rheb by the TSC1-TSC2 complex has only recently been investigated, and the regulation of the nucleotide binding status of Rheb is not fully understood. A recent study proposed that translationally controlled tumor protein may function as a guanine nucleotide exchange factor for Rheb that causes the accumulation of GTP-bound Rheb (18). GTP-bound Rheb is essential for activating mTOR kinase (21, 28, 38). However, the interaction between Rheb and mTOR does not depend on the GTP binding status of Rheb (30), raising questions regarding the mechanism by which Rheb activates mTORC1. Recently, FKBP38 (immunophilin FK506-binding protein, 38 kDa) was found to be a direct binding partner of Rheb and an inhibitor of mTORC1 (4). GTP-bound Rheb binds FKBP38 and releases FKBP38 from mTORC1, resulting in activation of the mTORC1 pathway. However, there have been conflicting results regarding the effects of nutrient availability on Rheb activity (31, 37, 42, 50) and the effect of these newly identified regulators of Rheb function (44, 45). Thus, the precise molecular mechanisms underlying Rheb regulation and Rheb-mediated mTORC1 activation have remained unclear.In this study, we identified glyceraldehyde-3-phosphate (Gly-3-P) dehydrogenase (GAPDH) as a novel Rheb binding protein and a negative regulator of Rheb. We found that the interaction between GAPDH and Rheb is induced when the glycolytic flux is suppressed under low-glucose conditions to inhibit mTORC1. Here, we provide a molecular mechanism underlying the cross talk between the glycolytic flux and the mTORC1 signaling.     

Mitotic Raptor Promotes mTORC1 Activity,G2/M Cell Cycle Progression,and Internal Ribosome Entry Site-Mediated mRNA Translation

The mTOR signaling complex integrates signals from growth factors and nutrient availability to control cell growth and proliferation, in part through effects on the protein-synthetic machinery. Protein synthesis rates fluctuate throughout the cell cycle but diminish significantly during the G₂/M transition. The fate of the mTOR complex and its role in coordinating cell growth and proliferation signals with protein synthesis during mitosis remain unknown. Here we demonstrate that the mTOR complex 1 (mTORC1) pathway, which stimulates protein synthesis, is actually hyperactive during mitosis despite decreased protein synthesis and reduced activity of mTORC1 upstream activators. We describe previously unknown G₂/M-specific phosphorylation of a component of mTORC1, the protein raptor, and demonstrate that mitotic raptor phosphorylation alters mTORC1 function during mitosis. Phosphopeptide mapping and mutational analysis demonstrate that mitotic phosphorylation of raptor facilitates cell cycle transit through G₂/M. Phosphorylation-deficient mutants of raptor cause cells to delay in G₂/M, whereas depletion of raptor causes cells to accumulate in G₁. We identify cyclin-dependent kinase 1 (cdk1 [cdc2]) and glycogen synthase kinase 3 (GSK3) pathways as two probable mitosis-regulated protein kinase pathways involved in mitosis-specific raptor phosphorylation and altered mTORC1 activity. In addition, mitotic raptor promotes translation by internal ribosome entry sites (IRES) on mRNA during mitosis and is demonstrated to be associated with rapamycin resistance. These data suggest that this pathway may play a role in increased IRES-dependent mRNA translation during mitosis and in rapamycin insensitivity.Cell growth and cell division are tightly coordinated processes required for cells to remain equal in size after division. In unicellular organisms, cell growth and proliferation are coordinated by nutrient availability, whereas their multicellular counterparts must also respond to growth factor input. Both processes lead to organismal growth as well as to increased cell number and cell mass. Cell growth and cell proliferation are also linked via the mTOR signaling pathway (16, 17). The mTOR kinase forms a distinct signaling complex (mTORC1) that participates in the coordination of nutrient and growth factor signaling. mTORC1 is composed of the kinase mTOR, the adaptor protein raptor, and the regulatory protein LST8 (25, 33, 34, 72).Accumulation of cellular proteins leads to cell growth and cell division. However, cell growth occurs only during certain phases of the cell cycle, necessitating that protein synthesis rates oscillate during cell cycling (40). In addition, in quiescent cells in G₀, protein synthesis rates are significantly reduced, whereas a select group of mRNAs maintain active translation (20, 68). During the G₁ phase, overall protein synthesis rates increase through S phase to allow cells to grow and enter another round of cell division while maintaining cell size (2, 3, 42, 45). As with G₀, entrance into mitosis (G₂/M phase) results in a global downregulation by as much as 60 to 80% of cap-dependent mRNA translation in primary, immortalized, and some transformed cells (5, 14, 29).Studies report several possible mechanisms for inhibition of protein synthesis during mitosis. Translation initiation requires the formation of an initiation factor complex known as eukaryotic translation initiation factor 4F (eIF4F), which consists of cap binding protein eIF4E, molecular scaffold protein eIF4G, and RNA helicase eIF4A. Together, they recruit ribosomes to mRNAs via bridging interactions between the 7-methyl-GTP (m⁷GTP) 5′ cap and the small 40S ribosomal subunit. Downregulation of protein synthesis during G₂/M was first ascribed to hypophosphorylation of eIF4E and the eIF4E binding proteins (4E-BPs) (5, 46). 4E-BPs are activated by hypophosphorylation, which allows them to bind and sequester eIF4E, preventing it from binding eIF4G and thereby blocking cap-dependent mRNA translation. More recently, several studies suggest that 4E-BP1, the major 4E-BP and a key target of mTORC1, is actually hyperphosphorylated (inactivated) during mitosis (26, 49). It is puzzling, then, that the phosphatidylinositol 3-kinase (PI3K)/AKT network and AKT itself (which modulate mTORC1 activity) are reportedly inactivated during late mitosis (1, 9, 22). In addition, phosphorylation of another mTORC1 target, ribosomal S6 kinase 1 (S6K1), and its activity are actually highest during G₂/M phase, consistent with elevated mTORC1 activity during mitosis (6).In this study we show that, despite repression of AKT and other activators of mTORC1 activity in mitosis, mTORC1 remains active and phosphorylates 4E-BP1 and S6K1 during G₂/M. We describe the multisite phosphorylation of raptor during mitosis, and we identify seven mitosis-specific raptor phosphorylation sites. By developing phosphomimetic and phosphorylation-deficient mutants of raptor, we show that hyperphosphorylated raptor promotes cell cycle transit through G₂/M, whereas hypophosphorylated raptor promotes transit through G₁. Raptor phosphorylation is shown to involve kinase pathways that are known to be active during mitosis, including cyclin-dependent kinase 1 (cdk1 [cdc2]) and glycogen synthase kinase 3 (GSK3) pathways that are also upregulated in certain human cancers, including breast cancers. These and other findings disclose a novel regulatory network for mTORC1 that is active during mitosis, important for G₂/M progression and increased internal ribosome entry site (IRES)-dependent translation during mitosis, and indirectly associated with rapamycin resistance.     

CSF-1 Receptor Signaling in Myeloid Cells

The CSF-1 receptor (CSF-1R) is activated by the homodimeric growth factors colony-stimulating factor-1 (CSF-1) and interleukin-34 (IL-34). It plays important roles in development and in innate immunity by regulating the development of most tissue macrophages and osteoclasts, of Langerhans cells of the skin, of Paneth cells of the small intestine, and of brain microglia. It also regulates the differentiation of neural progenitor cells and controls functions of oocytes and trophoblastic cells in the female reproductive tract. Owing to this broad tissue expression pattern, it plays a central role in neoplastic, inflammatory, and neurological diseases. In this review we summarize the evolution, structure, and regulation of expression of the CSF-1R gene. We review, the structures of CSF-1, IL-34, and the CSF-1R and the mechanism of ligand binding to and activation of the receptor. We further describe the pathways regulating macrophage survival, proliferation, differentiation, and chemotaxis downstream from the CSF-1R.The glycoprotein, colony-stimulating factor-1 (CSF-1), also known as macrophage-CSF (M-CSF), was the first of the CSFs to be purified (Stanley and Heard 1977) and was shown to stimulate the formation of colonies of macrophages (Stanley et al. 1978). This led to the identification (Guilbert and Stanley 1980) and purification (Yeung et al. 1987) of the CSF-1 receptor (CSF-1R) and the demonstration that it possessed intrinsic tyrosine kinase activity (Yeung et al. 1987). It was subsequently shown to be identical to the c-fms proto-oncoprotein (Sherr et al. 1985) previously studied by Sherr and colleagues (Rettenmier et al. 1985). The c-fms cDNA was cloned and shown to encode a typical class III receptor tyrosine kinase (RTK) (Coussens et al. 1986).The CSF-1R plays a central role in many diseases. Dominant inactivating mutations in the CSF-1R lead to adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (Rademakers et al. 2011; Nicholson et al. 2013). Inappropriate expression of the CSF-1R contributes to the development of leukemias and lymphomas, and autocrine and paracrine regulation of the CSF-1R enhances the progression and metastasis of solid tumors (reviewed in Pollard 2009; Chitu and Stanley 2014). In addition, regulation through the CSF-1R contributes to chronic inflammatory diseases (reviewed in Chitu and Stanley 2006; Chitu et al. 2012). This review focuses on the CSF-1R regulation and signaling in cells of the myeloid lineage.     

Molecular Mechanisms of Fibroblast Growth Factor Signaling in Physiology and Pathology

Fibroblast growth factors (FGFs) signal in a paracrine or endocrine fashion to mediate a myriad of biological activities, ranging from issuing developmental cues, maintaining tissue homeostasis, and regulating metabolic processes. FGFs carry out their diverse functions by binding and dimerizing FGF receptors (FGFRs) in a heparan sulfate (HS) cofactor- or Klotho coreceptor-assisted manner. The accumulated wealth of structural and biophysical data in the past decade has transformed our understanding of the mechanism of FGF signaling in human health and development, and has provided novel concepts in receptor tyrosine kinase (RTK) signaling. Among these contributions are the elucidation of HS-assisted receptor dimerization, delineation of the molecular determinants of ligand–receptor specificity, tyrosine kinase regulation, receptor cis-autoinhibition, and tyrosine trans-autophosphorylation. These structural studies have also revealed how disease-associated mutations highjack the physiological mechanisms of FGFR regulation to contribute to human diseases. In this paper, we will discuss the structurally and biophysically derived mechanisms of FGF signaling, and how the insights gained may guide the development of therapies for treatment of a diverse array of human diseases.Fibroblast growth factor (FGF) signaling fulfills essential roles in metazoan development and metabolism. A wealth of literature has documented the requirement for FGF signaling in multiple processes during embryogenesis, including implantation (Feldman et al. 1995), gastrulation (Sun et al. 1999), somitogenesis (Dubrulle and Pourquie 2004; Wahl et al. 2007; Lee et al. 2009; Naiche et al. 2011; Niwa et al. 2011), body plan formation (Martin 1998; Rodriguez Esteban et al. 1999; Tanaka et al. 2005; Mariani et al. 2008), morphogenesis (Metzger et al. 2008; Makarenkova et al. 2009), and organogenesis (Goldfarb 1996; Kato and Sekine 1999; Sekine et al. 1999; Sun et al. 1999; Colvin et al. 2001; Serls et al. 2005; Vega-Hernandez et al. 2011). Recent clinical and biochemical data have uncovered unexpected roles for FGF signaling in metabolic processes, including phosphate/vitamin D homeostasis (Consortium 2000; Razzaque and Lanske 2007; Nakatani et al. 2009; Gattineni et al. 2011; Kir et al. 2011), cholesterol/bile acid homeostasis (Yu et al. 2000a; Holt et al. 2003), and glucose/lipid metabolism (Fu et al. 2004; Moyers et al. 2007). Highlighting its diverse biology, deranged FGF signaling contributes to many human diseases, such as congenital craniosynostosis and dwarfism syndromes (Naski et al. 1996; Wilkie et al. 2002, 2005), Kallmann syndrome (Dode et al. 2003; Pitteloud et al. 2006a), hearing loss (Tekin et al. 2007, 2008), and renal phosphate wasting disorders (Shimada et al. 2001; White et al. 2001), as well as many acquired forms of cancers (Rand et al. 2005; Pollock et al. 2007; Gartside et al. 2009; di Martino et al. 2012). Endocrine FGFs have also been implicated in the progression of acquired metabolic disorders, including chronic kidney disease (Fliser et al. 2007), obesity (Inagaki et al. 2007; Moyers et al. 2007; Reinehr et al. 2012), and insulin resistance (Fu et al. 2004; Chen et al. 2008b; Chateau et al. 2010; Huang et al. 2011), giving rise to many opportunities for drug discovery in the field of FGF biology (Beenken and Mohammadi 2012).Based on sequence homology and phylogeny, the 18 mammalian FGFs are grouped into six subfamilies (Ornitz and Itoh 2001; Popovici et al. 2005; Itoh and Ornitz 2011). Five of these subfamilies act in a paracrine fashion, namely, the FGF1 subfamily (FGF1 and FGF2), the FGF4 subfamily (FGF4, FGF5, and FGF6), the FGF7 subfamily (FGF3, FGF7, FGF10, and FGF22), the FGF8 subfamily (FGF8, FGF17, and FGF18), and the FGF9 subfamily (FGF9, FGF16, and FGF20). In contrast, the FGF19 subfamily (FGF19, FGF21, and FGF23) signals in an endocrine manner (Beenken and Mohammadi 2012). FGFs exert their pleiotropic effects by binding and activating the FGF receptor (FGFR) subfamily of receptor tyrosine kinases that are coded by four genes (FGFR1, FGFR2, FGFR3, and FGFR4) in mammals (Johnson and Williams 1993; Mohammadi et al. 2005b). The extracellular domain of FGFRs consists of three immunoglobulin (Ig)-like domains (D1, D2, and D3), and the intracellular domain harbors the conserved tyrosine kinase domain flanked by the flexible amino-terminal juxtamembrane linker and carboxy-terminal tail (Lee et al. 1989; Dionne et al. 1991; Givol and Yayon 1992). A unique feature of FGFRs is the presence of a contiguous segment of glutamic and aspartic acids in the D1–D2 linker, termed the acid box (AB). The two-membrane proximal D2 and D3 and the intervening D2–D3 linker are necessary and sufficient for ligand binding/specificity (Dionne et al. 1990; Johnson et al. 1990), whereas D1 and the D1–D2 linker are implicated in receptor autoinhibition (Wang et al. 1995; Roghani and Moscatelli 2007; Kalinina et al. 2012). Alternative splicing and translational initiation further diversify both ligands and receptors. The amino-terminal regions of FGF8 and FGF17 can be differentially spliced to yield FGF8a, FGF8b, FGF8e, FGF8f (Gemel et al. 1996; Blunt et al. 1997), and FGF17a and FGF17b isoforms (Xu et al. 1999), whereas cytosine-thymine-guanine (CTG)-mediated translational initiation gives rise to multiple high molecular weight isoforms of FGF2 and FGF3 (Florkiewicz and Sommer 1989; Prats et al. 1989; Acland et al. 1990). The tissue-specific alternative splicing in D3 of FGFR1, FGFR2, and FGFR3 yields “b” and “c” receptor isoforms which, along with their temporal and spatial expression patterns, is the major regulator of FGF–FGFR specificity/promiscuity (Orr-Urtreger et al. 1993; Ornitz et al. 1996; Zhang et al. 2006). A large body of structural data on FGF–FGFR complexes has begun to reveal the intricate mechanisms by which different FGFs and FGFRs combine selectively to generate quantitatively and qualitatively different intracellular signals, culminating in distinct biological responses. In addition, these structural data have unveiled how pathogenic mutations hijack the normal physiological mechanisms of FGFR regulation to lead to pathogenesis. We will discuss the current state of the structural biology of the FGF–FGFR system, lessons learned from studying the mechanism of action of pathogenic mutations, and how the structural data are beginning to shape and advance the translational research.     

Receptor Tyrosine Kinases: Legacy of the First Two Decades

Receptor tyrosine kinases (RTKs) and their cellular signaling pathways play important roles in normal development and homeostasis. Aberrations in their activation or signaling leads to many pathologies, especially cancers, motivating the development of a variety of drugs that block RTK signaling that have been successfully applied for the treatment of many cancers. As the current field of RTKs and their signaling pathways are covered by a very large amount of literature, spread over half a century, I am focusing the scope of this review on seminal discoveries made before tyrosine phosphorylation was discovered, and on the early days of research into RTKs and their cellular signaling pathways. I review the history of the early days of research in the field of RTKs. I emphasize key early findings, which provided conceptual frameworks for addressing the questions of how RTKs are activated and how they regulate intracellular signaling pathways.The family of cell-surface receptors designated receptor tyrosine kinases (RTK) received their name more that a decade after the same molecules were already known as the cell-surface receptors for insulin (insulin receptor), epidermal growth factor (EGFR), and many other growth factor receptors. Following the pioneering discoveries of nerve growth factor and epidermal growth factor (EGF; Levi-Montalcini and Booker 1960; Cohen 1962) and the establishment of the important roles of these two growth factors in the control of neuronal differentiation and cell proliferation in vivo and in vitro, it became clear that these cytokines bind specifically to cell-surface receptors. Insulin had already been discovered by this time, and had been applied successfully to treat diabetes patients since the early twentieth century. The resulting homogenous preparations of pure insulin enabled the quantitative characterization of insulin binding to its receptor on intact cells or to solubilized insulin receptor preparations using radiolabeled insulin (De Meyts et al. 1973). These studies greatly advanced understanding of the ligand binding characteristics of insulin receptor and, later on EGFR (Carpenter et al. 1975), including the establishment of negative cooperativity in insulin binding to its receptor expressed on the surface of living cells (De Meyts et al. 1973). Moreover, these studies shed important light on the dynamic nature of the cellular behavior of these receptors. The capacities of insulin receptor and EGFR to undergo ligand-dependent down-regulation and desensitization through receptor-mediated internalization and degradation (Carpenter and Cohen 1976; Gordon et al. 1978; Schlessinger et al. 1978a,b; Carpentier et al. 1979; Haigler et al. 1979) were also established well before the realization that growth factors receptors are endowed with intrinsic protein tyrosine kinase activities (Fig. 1).Open in a separate windowFigure 1.A time line of key findings during the history of RTKs, with emphasis on findings and discoveries that produced the conceptual framework in the development of the RTK field and its application for cancer therapy. References for the key findings are also presented in the text (Lee et al. 1985; Libermann et al. 1985; Margolis et al. 1990; Bottaro et al. 1991; Bae et al. 2009).Progress was also made in elucidating the role of growth factors in normal embryonic development, wound healing, and pathological conditions such as cancer. Early studies in the 1960s and 1970s showed that growth factors play an important role in oncogenesis induced by retroviruses and in the proliferation of tumor-derived cancer cells. Pioneering studies performed by Howard Temin (1966, 1967) showed that cancer cells need less insulin and serum growth factors for cell proliferation compared with normal cells, suggesting that cancer cells produce and use their own growth factors and/or use cellular processes that in normal cells are regulated by exogenously supplied growth factors; both predictions were subsequently confirmed. A variety of new polypeptide growth factors that stimulate cell proliferation by binding to receptors at the cell surface were subsequently discovered. Those include a growth factor isolated from human platelets designated platelet-derived growth factor (PDGF; Antoniades et al. 1979; Heldin et al. 1979), a growth factor isolated from bovine brain designated fibroblast growth factor (FGF; Gospodarowicz et al. 1978), a growth factor isolated from rat platelets that stimulates the proliferation of mature hepatocytes, designated hepatocyte growth factor (HGF; Nakamura et al. 1986). In addition to EGF, another growth factor that binds selectively to cells expressing EGFR was isolated from virally and chemically transformed cells, suggesting that this growth factor—designated transforming growth factor α—may play a role in oncogenesis by an autocrine mechanism (Roberts et al. 1980, 1982). This discovery provided further support to the earlier finding that transformation by murine and feline sarcoma viruses selectively interferes with EGF binding to EGFR in transformed cells (Todaro et al. 1976). Together with many other studies published since the 1980s, this work showed that growth factors and their receptors play numerous important roles during development and in many normal cellular processes as well as in pathologies such as cancer, diabetes, atherosclerosis, severe bone disorders, and tumor angiogenesis.Visualization of dynamic cellular redistribution of ligand/receptor complexes, and rapid receptor-mediated internalization of growth factors such as insulin or EGF, led to the proposal that cell-surface receptors for these ligands may play a passive role in delivering them to intracellular compartments in which internalized EGF or insulin molecules exert their actions (Vigneri et al. 1978; Podlecki et al. 1986; Jiang and Schindler 1990). In other words, according to this hypothesis, the biological signals induced by insulin or EGF were thought to be mediated by binding of the ligands themselves to intracellular target(s) in the cytoplasm or nucleus, with the role of the cell-surface receptor being to act as a “carrier” that delivers them directly to these targets. An alternative hypothesis was that insulin or EGF activates their cognate receptors at the cell surface, which in turn stimulate the production of an intracellular second messenger molecule analogous to cAMP in signaling by the G-protein-activating β-adrenergic receptor. Indeed, several potential second messengers that are generated in cells on stimulation with insulin or other growth factors were proposed before (and even after) it became clear that insulin receptor, EGFR, and other RTKs are endowed with intrinsic tyrosine kinase activity (Larner et al. 1979; Das 1980; Saltiel and Cuatrecasas 1986).A demonstration that anti-insulin receptor antibodies from the serum of certain diabetic patients could mimic cellular responses of insulin (Flier et al. 1977; Van Obberghen et al. 1979) provided the first conclusive answer to the question of whether the biological activity of growth factors is mediated directly or indirectly through their membrane receptors. This experiment ruled out the possibility that insulin receptor functions as a passive carrier that delivers insulin to an intracellular target to induce cellular responses. Studies showing that intact, bivalent antibodies against the insulin receptor can activate its signaling, whereas monovalent Fab fragments of the same antibodies cannot further argued that ligand-induced receptor dimerization or stimulation of a particular arrangement between two receptor molecules in a dimer can activate the insulin receptor (Kahn et al. 1978).A similar conclusion was reached using certain monoclonal antibodies that bind to the extracellular region of EGFR and block ligand binding (Schreiber et al. 1981). Whereas intact antibodies were able to mimic EGF in stimulating a variety of EGF-like responses including cell proliferation, monovalent Fab fragments of the same monoclonal EGFR antibodies failed to do so—and acted instead as EGFR antagonists (Schreiber et al. 1981, 1983). These experiments provided strong evidence both that EGFR plays a crucial role in mediating EGF-induced cellular responses and that EGFR is activated by ligand-induced receptor dimerization (Schreiber 1981, 1983).     

Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC)

Many biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer''s disease, and autism spectrum disorder—neurological disorders that exhibit elevated mTORC1 activity. Through a protein–protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson''s disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and colocalizes with the postsynaptic marker postsynaptic density-95. Our work offers a comprehensive view of mTORC1 and its role in regulating regional protein expression in normal and diseased states.The mechanistic/mammalian target of rapamycin complex 1 (mTORC1)¹ is a serine/threonine protein kinase that is highly expressed in many cell types (1). In the brain, mTORC1 tightly coordinates different synaptic plasticities — long-term potentiation (LTP) and long-term depression (LTD) — the molecular correlates of learning and memory (2–5). Because mTORC1 is at the core of many synaptic signaling pathways downstream of glutamate and neurotrophin receptors, many hypothesize that dysregulated mTORC1 signaling underlies cognitive deficits observed in several neurodegenerative diseases (3, 6–17). For example, mTORC1 and its downstream targets are hyperactive in human brains diagnosed with Alzheimer''s disease (AD) (18–20). Additionally in animal models of autism spectrum disorder (ASD), altered mTORC1 signaling contributes to the observed synaptic dysfunction and aberrant network connectivity (13, 15, 21–27). Furthermore, epilepsy, which is common in AD and ASD, has enhanced mTORC1 activity (28–32).Phosphorylation of mTORC1, considered the active form, is generally regarded to promote protein synthesis (33). Thus, many theorize that diseases with overactive mTORC1 arise from excessive protein synthesis (14). Emerging data, however, show that suppressing mTORC1 activation can trigger local translation in neurons (34, 35). Pharmacological antagonism of N-methyl-d-aspartate (NMDA) receptors, a subtype of glutamate receptors that lies upstream of mTOR activation, promotes the synthesis of the voltage-gated potassium channel, Kv1.1, in dendrites (34, 35). Consistent with these results, in models of temporal lobe epilepsy there is a reduction in the expression of voltage-gated ion channels including Kv1.1 (30, 31, 36). Interestingly in a model of focal neocortical epilepsy, overexpression of Kv1.1 blocked seizure activity (37). Because both active and inactive mTORC1 permit protein synthesis, we sought to determine the proteins whose expression is altered when mTORC1 phosphorylation is reduced in vivo.Rapamycin is an FDA-approved, immunosuppressive drug that inhibits mTORC1 activity (38). We capitalized on the ability of rapamycin to reduce mTORC1 activity in vivo and the unbiased approach of mass spectrometry to identify changes in protein expression. Herein, we provide evidence that mTORC1 activation bidirectionally regulates protein expression, especially in the PSD where roughly an equal distribution of proteins dynamically appear and disappear. Remarkably, using protein–protein interaction networks facilitated the novel discovery that PARK7, a protein thus far only implicated in Parkinson''s disease, (1) is up-regulated by increased mTORC1 activity, (2) resides in the PSD only when mTORC1 is active, and (3) is aberrantly expressed in a rodent model of TSC, an mTORC1-related disease that has symptoms of epilepsy and autism. Collectively, these data provide the first comprehensive list of proteins whose abundance or subcellular distributions are altered with acute changes in mTORC1 activity in vivo.     

NF2/Merlin Is a Novel Negative Regulator of mTOR Complex 1, and Activation of mTORC1 Is Associated with Meningioma and Schwannoma Growth

Inactivating mutations of the neurofibromatosis 2 (NF2) gene, NF2, result predominantly in benign neurological tumors, schwannomas and meningiomas, in humans; however, mutations in murine Nf2 lead to a broad spectrum of cancerous tumors. The tumor-suppressive function of the NF2 protein, merlin, a membrane-cytoskeleton linker, remains unclear. Here, we identify the mammalian target of rapamycin complex 1 (mTORC1) as a novel mediator of merlin''s tumor suppressor activity. Merlin-deficient human meningioma cells and merlin knockdown arachnoidal cells, the nonneoplastic cell counterparts of meningiomas, exhibit rapamycin-sensitive constitutive mTORC1 activation and increased growth. NF2 patient tumors and Nf2-deficient mouse embryonic fibroblasts demonstrate elevated mTORC1 signaling. Conversely, the exogenous expression of wild-type merlin isoforms, but not a patient-derived L64P mutant, suppresses mTORC1 signaling. Merlin does not regulate mTORC1 via the established mechanism of phosphoinositide 3-kinase-Akt or mitogen-activated protein kinase/extracellular signal-regulated kinase-mediated TSC2 inactivation and may instead regulate TSC/mTOR signaling in a novel fashion. In conclusion, the deregulation of mTORC1 activation underlies the aberrant growth and proliferation of NF2-associated tumors and may restrain the growth of these lesions through negative feedback mechanisms, suggesting that rapamycin in combination with phosphoinositide 3-kinase inhibitors may be therapeutic for NF2.Meningiomas are mesenchymal tumors that arise from the arachnoid layer covering the brain and spinal cord and account for approximately 30% of all primary intracranial neoplasms (30). Most sporadic meningiomas (60%) display somatic inactivation of the NF2 gene. Germ line mutations of NF2 are associated with neurofibromatosis 2 (NF2), a dominantly inherited disorder characterized by multiple nervous system tumors, including schwannomas and meningiomas (33). Although most meningiomas are benign (WHO grade I), they often cause significant morbidity due to compression of the adjacent brain or spinal cord. Benign meningiomas also have recurrence rates of up to 20% over 10 years. Ten percent of meningiomas are classified as atypical (WHO grade II) or anaplastic (WHO grade III) and display more aggressive clinical behavior, with rapid growth and increased recurrence rates (6, 21). The current standard of care is maximal surgical resection, with adjuvant radiation reserved for progressive tumors or those with aggressive features (e.g., WHO grade II or III). The treatment strategy for meningiomas that progress despite surgery and radiation remains limited, and currently there is no effective chemotherapy.The development of effective therapies has been hampered, in part, by our incomplete understanding of the signals influencing meningioma cell growth. Enhanced expression of certain peptide and steroid growth factors and receptors in meningioma tissue suggests that specific autocrine growth-stimulatory loops may be functionally important in meningioma cell proliferation (20, 38). The scarcity of established meningioma models that would allow for the assessment of growth-regulatory mechanisms has also hampered progress. Recently, we have developed reliable meningioma models that overcome the challenges of the low growth rates and senescence of primary benign meningioma cells (19).Biallelic inactivation of the NF2 gene is detected in the majority of sporadic meningiomas and nearly all schwannomas (11). The tumor suppressor gene NF2 encodes merlin (also called schwannomin), a member of the ezrin-radixin-moesin (ERM) protein family that functions to link membrane proteins to the cortical actin cytoskeleton (31, 41). Like the ERM proteins, merlin has been implicated in the regulation of membrane organization and cytoskeleton-based cellular processes such as adhesion, migration, cell-cell contact, spreading, proliferation, and signal transduction (27). The loss of contact-dependent inhibition of proliferation is seen in several types of NF2-deficient cells (23, 29). Merlin controls cell proliferation in response to cell contact via CD44 (28) and functions together with the related tumor suppressor Expanded via the Hippo/Mst pathway in both Drosophila and some types of mammalian cells (14, 49). Although merlin is implicated in a wide range of cellular activities, the precise mechanism by which merlin mediates growth-inhibitory functions in human arachnoidal and Schwann cells and the way in which its loss results in tumor formation in NF2 remain poorly understood.We recently reported that primary human merlin-deficient meningioma cells exhibit a striking, enlarged-cell phenotype compared to nonneoplastic arachnoidal cell counterparts derived from the same patient (19). Interestingly, the tuberous sclerosis complex (TSC) tumor suppressor syndrome is characterized by widespread benign tumors that possess abnormally large cells (22). Mutations in the tumor suppressor genes TSC1 and TSC2 result in TSC syndrome, and the corresponding protein products, hamartin and tuberin (referred to as TSC1 and TSC2), function together as a complex that potently inhibits mammalian target of rapamycin complex 1 (mTORC1) (17). mTOR is an evolutionarily conserved Ser/Thr kinase that exists in one of two distinct functional complexes, TORC1 and TORC2. TORC1, which regulates autophagy, protein translation, and ribosome biogenesis, is potently and specifically inhibited by rapamycin (10, 46). TORC2, which is less sensitive to rapamycin, is important for cytoskeletal regulation and Akt/protein kinase B activation (16, 18, 36).The TSC1-TSC2 complex inhibits mTORC1 by acting as a GTPase-activating protein for the small GTPase Rheb (Ras homolog enriched in brain). Inactivation of the TSC1-TSC2 complex results in the accumulation of GTP-bound Rheb, which activates mTORC1 (10). In addition to naturally occurring mutations in the TSC1 and TSC2 genes, growth factor stimulation of the phosphoinositide 3-kinase (PI3K)-Akt pathway, as well as Ras/mitogen-activated protein kinase (MAPK) pathways, leads to the phosphorylation and inactivation of the TSC1-TSC2 complex and consequent activation of mTORC1 (17). The activation of mTORC1 results in the phosphorylation of two well-characterized effectors, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and S6 kinase 1 (S6K1), leading to an increase in ribosomal biogenesis and the selective translation of specific mRNA populations. As a critical regulator of cell growth and proliferation, the mTORC1 pathway is dysregulated in several hamartoma syndromes, as well as in many cancers (10).In this report, we identify the NF2 tumor suppressor protein, merlin, as a novel negative regulator of the mTORC1 pathway to control cell growth (cell size). We show that mTORC1 is constitutively activated in merlin-deficient human meningioma cells, leading to increased cell size. Furthermore, we suggest that the slow growth of merlin-deficient meningioma cells is due to a rapamycin-sensitive, mTORC1-S6K-dependent negative feedback loop that diminishes PI3K-Akt signaling in response to growth factor stimulation. The findings of these studies provide insight into the mechanism of merlin tumor suppressor activity and, moreover, indicate that rapamycin or rapamycin analogs in combination with PI3K inhibitors may provide promise as new therapeutics in the treatment of meningiomas and schwannomas.     

Cadherin-mediated Intercellular Adhesion and Signaling Cascades Involving Small GTPases

Epithelia form physical barriers that separate the internal milieu of the body from its external environment. The biogenesis of functional epithelia requires the precise coordination of many cellular processes. One of the key events in epithelial biogenesis is the establishment of cadherin-dependent cell–cell contacts, which initiate morphological changes and the formation of other adhesive structures. Cadherin-mediated adhesions generate intracellular signals that control cytoskeletal reorganization, polarity, and vesicle trafficking. Among such signaling pathways, those involving small GTPases play critical roles in epithelial biogenesis. Assembly of E-cadherin activates several small GTPases and, in turn, the activated small GTPases control the effects of E-cadherin-mediated adhesions on epithelial biogenesis. Here, we focus on small GTPase signaling at E-cadherin-mediated epithelial junctions.Cell–cell adhesions are involved in a diverse range of physiological processes, including morphological changes during tissue development, cell scattering, wound healing, and synaptogenesis (Adams and Nelson 1998; Gumbiner 2000; Halbleib and Nelson 2006; Takeichi 1995; Tepass et al. 2000). In epithelial cells, cell–cell adhesions are classified into three kinds of adhesions: adherens junction, tight junction, and desmosome (for more details, see Meng and Takeichi 2009, Furuse 2009, and Delva et al. 2009, respectively). A key event in epithelial polarization and biogenesis is the establishment of cadherin-dependent cell–cell contacts. Cadherins belong to a large family of adhesion molecules that require Ca²⁺ for their homophilic interactions (Adams and Nelson 1998; Blanpain and Fuchs 2009; Gumbiner 2000; Hartsock and Nelson 2008; Takeichi 1995; Tepass et al. 2000). Cadherins form transinteraction on the surface of neighboring cells (for details, see Shapiro and Weis 2009). For the development of strong and rigid adhesions, cadherins are clustered concomitantly with changes in the organization of the actin cytoskeleton (Tsukita et al. 1992). Classical cadherins are required, but not sufficient, to initiate cell–cell contacts, and other adhesion protein complexes subsequently assemble (for details, see Green et al. 2009). These complexes include the tight junction, which controls paracellular permeability, and desmosomes, which support the structural continuum of epithelial cells. A fundamental problem is to understand how these diverse cellular processes are regulated and coordinated. Intracellular signals, generated when cells attach with one another, mediate these complicated processes.Several signaling pathways upstream or downstream of cadherin-mediated cell–cell adhesions have been identified (Perez-Moreno et al. 2003) (see also McCrea et al. 2009). Among these pathways, small GTPases including the Rho and Ras family GTPases play critical roles in epithelial biogenesis and have been studied extensively. Many key morphological and functional changes are induced when these small GTPases act at epithelial junctions, where they mediate an interplay between cell–cell adhesion molecules and fundamental cellular processes including cytoskeletal activity, polarity, and vesicle trafficking. In addition to these small GTPases, Ca²⁺ signaling and phosphorylation of cadherin complexes also play pivotal roles in the formation and maintenance of cadherin-mediated adhesions. Here, we focus on signaling pathways involving the small GTPases in E-cadherin-mediated cell–cell adhesions. Other signaling pathways are described in recent reviews (Braga 2002; Fukata and Kaibuchi 2001; Goldstein and Macara 2007; McLachlan et al. 2007; Tsukita et al. 2008; Yap and Kovacs 2003; see also McCrea et al. 2009).