The yoga of Rag GTPases: Dynamic structural poses confer amino acid sensing by mTORC1

Heterodimeric Rag GTPases play a critical role in relaying fluctuating levels of cellular amino acids to the sensor mechanistic target of rapamycin complex 1. Important mechanistic questions remain unresolved, however, regarding how guanine nucleotide binding enables Rag GTPases to transition dynamically between distinct yoga-like structural poses that control activation state. Egri and Shen identified a critical interdomain hydrogen bond within RagA and RagC that stabilizes their GDP-bound states. They demonstrate that this long-distance interaction controls Rag structure and function to confer appropriate amino acid sensing by mechanistic target of rapamycin complex 1.

Mechanistic target of rapamycin complex 1 (mTORC1) integrates diverse cellular cues to promote cell growth and proliferation (1, 2). Sufficient levels of nutrients such as amino acids are required for growth factors and hormones (e.g., IGF-1 and insulin) to activate mTORC1 via PI3K, Akt, Ras homolog enriched in the brain (Rheb) (a small GTPase), and tuberous sclerosis complex (a GTPase-activating protein for Rheb) (Fig. 1A). mTORC1 signaling in turn drives anabolic (e.g., protein synthesis) and suppresses catabolic (e.g., autophagy) cellular processes. Evolutionarily conserved Rag GTPases play a critical role in amino acid sensing by mTORC1 (3, 4). Despite advances in understanding Rag structure and function, important mechanistic questions remain regarding how dynamic structural states of Rag proteins controlled by guanine nucleotide binding confer amino acid sensing by mTORC1. Egri and Shen used elegant kinetic and cell-based methods to quantitatively dissect dynamic structural elements within Rag subunits that enable mTORC1 to respond to fluctuating levels of amino acids appropriately and rapidly (5).Open in a separate windowFigure 1mTORC1 activation by growth factors (GFs) requires sufficient levels of amino acids (AAs). GFs and hormones (e.g., IGF-1; insulin) signal through PI3K, Akt, and TSC and activate Rheb through increased GTP loading (A). AAs drive Rag heterodimers toward a RagA/B^GTP–RagC/D^GDP “on” state; conversely, AA deprivation induces a switch toward a RagA/B^GDP–RagC/D^GTP “off” state. In the “on” state, Rag heterodimers bind to and recruit mTORC1 to the surface of lysosomes, where Rheb resides. Therefore, AAs and GFs activate mTORC1 cooperatively because of an induced proximity mechanism mediated by Rags and Rheb. A critical hydrogen bond (blue bar) between the NBD and CRD of RagA or RagC plays a critical role in maintaining the two stable “on” and “off” states (B). CRD, C-terminal roadblock domain; mTORC1, mechanistic target of rapamycin complex 1; NBD, nucleotide-binding domain; Rheb, Ras homolog enriched in the brain; TSC, tuberous sclerosis complex.Rag proteins function as obligate heterodimers, whereby mammalian RagA or RagB dimerizes with RagC or RagD. Rag proteins localize to lysosomal membranes by tethering to the LAMTOR/Ragulator complex (Fig. 1A) (6). In the active RagA/B^GTP–RagC/D^GDP state formed in amino acid–replete conditions, the Rag heterodimer recruits mTORC1 to the lysosomal surface through direct binding (6). Such recruitment enables Rheb to associate with and activate mTORC1 by an induced proximity mechanism (7). Upon amino acid withdrawal, GTP on RagA/B hydrolyzes to GDP, and GTP exchanges for GDP on RagC/D. This inactive RagA/B^GDP–RagC/D^GTP heterodimer releases mTORC1 into the cytosol. Thus, Rags function as dynamic molecular switches that control mTORC1 signaling in accordance with amino acid levels.Prior work (8) demonstrated that the two GTPase subunits of the Rag heterodimer (RagA/B and RagC/D) communicate with each other. GTP binding to one subunit limits binding of GTP to the other subunit and increases GTP hydrolysis if binding were to occur, and vice versa. Such intersubunit crosstalk prevents dual GTP loading, thus maintaining an opposite guanine nucleotide–loaded state and driving Rag heterodimers into two stable “on” or “off” states. The crystal structure of Rag heterodimers from budding yeast bound to GDP or GTP provided important structural information regarding how guanine nucleotide binding controls Rag architecture (9, 10). An individual Rag subunit consists of a nucleotide-binding domain (NBD) and a C-terminal roadblock domain (CRD) that mediates heterodimerization. In the GDP-bound state, the switch I domain within the NBD forms an alpha helix that orients toward the CRD; in the GTP-bound state, the switch I domain swings upward to the top of the nucleotide-binding pocket, away from the CRD. From the yeast Rag crystal structures (9, 10), Egri and Shen predicted that in the GDP- but not GTP-bound state, the hydroxyl group of Ser266 in the RagC CRB forms hydrogen bonds with Lys84 in the switch I alpha helix of the RagC NBD. As RagA Thr210 is analogous to RagC Ser266, they also predicted that Thr210 in the RagA CRB forms hydrogen bonds with Asn30 in the NBD. In the GTP-bound state, the switch I domain swings up and away from the CRD, preventing formation of these hydrogen bonds (Fig. 1B).Egri and Shen coupled these predictions with elegant quantitative kinetic in vitro assays of guanine nucleotide loading and GTP hydrolysis to demonstrate that a critical interdomain interaction in RagA and RagC maintains an opposite nucleotide-loading state in heterodimers and regulates mTORC1 activity (5). They first mutated RagA Thr210 and RagC Ser266 to Ala to abrogate the hydrogen bond and then biochemically purified WT and mutant Rag heterodimers. Ablation of the hydrogen bond had no effect on guanine nucleotide binding. When only one GTP was bound to the heterodimer, rates of GTP hydrolysis were similar on WT and mutant Rag heterodimers. When both Rag subunits of the heterodimer were forced to bind GTP, WT heterodimers displayed an increased rate of GTP hydrolysis compared with those loaded with a single GTP, indicating that the heterodimer actively resolves the dual GTP problem by hydrolyzing GTP on one subunit, consistent with prior work (8). GTP hydrolysis was increased even more for the RagA(T210A)–RagC and RagA–RagC(S266A) mutant heterodimers, suggesting that the mutations mimic a constitutive GTP-loaded conformation, driving faster GTP hydrolysis on the other subunit. In WT heterodimers, preloading the first subunit with GTP increased GTP hydrolysis on the other subunit relative to preloading with GDP. Interestingly, radioactive GTP hydrolysis in mutant heterodimers was strikingly faster than that of the WT when preloaded with either GTP or GDP, indicating that the RagA(T210) and RagC(S266A) mutations shift the heterodimer toward the GTP-loaded conformation. These results suggest that the hydrogen bond stabilizes the GDP-loaded state, and in its absence, Rag proteins tend to adopt a GTP-bound conformation even when bound to GDP, which accelerates GTP hydrolysis on the other subunit.Egri and Shen also investigated the functional significance of the RagA and RagC hydrogen bond in the control of mTORC1 signaling. Coimmunoprecipitation experiments and analysis of mTORC1 signaling to its well-established substrate S6K1 in intact cells demonstrated that the RagA(T210A)–RagC mutant associated with and activated mTORC1 inappropriately in the absence of amino acids. Upon amino acid stimulation, the RagA–RagC(S266A) mutant displayed reduced mTORC1 binding and failed to activate mTORC1 signaling. These results are consistent with RagA(T210A) mimicking a RagA^GTP “on” state and RagC(S266A) mimicking a RagC^GTP “off” state. Taken together, these results reveal the functional significance of the RagA and RagC interdomain hydrogen bond, demonstrating that it plays a critical role in regulation of mTORC1 signaling in accordance with amino acid levels.Mechanistic understanding of Rag heterodimer asanas (i.e., postures and poses) will improve our understanding of the role of mTORC1 in tumorigenesis and metabolism. For example, cancer-associated mutations have been identified in RagC, which increase mTORC1 binding (2). In addition, the physiologic importance of Rag proteins in metabolic control was demonstrated in mice engineered with an active RagA knock-in allele conferring constitutive GTP loading. These mice die perinatally, as they are unable to suppress mTORC1 signaling appropriately upon severance of the placental nutrient supply at birth. These mice fail to suppress energy expenditure, fail to induce autophagy and liberate amino acids as substrates for gluconeogenesis, and consequently fail to upregulate hepatic glucose production, responses essential for survival during fasting, unlike WT neonates (2). Thus, Rag GTPases play critical roles in cell and organismal physiology. Moving forward, deeper mechanistic insight into the yoga of Rag GTPases will improve our understanding of nutrient sensing, how its aberrant regulation contributes to a host of diseases such as cancer, obesity, and type II diabetes, and how its therapeutic targeting could treat these disorders. Namaste.     

Amino acid regulation of TOR complex 1

TOR complex 1 (TORC1), an oligomer of the mTOR (mammalian target of rapamycin) protein kinase, its substrate binding subunit raptor, and the polypeptide Lst8/GbetaL, controls cell growth in all eukaryotes in response to nutrient availability and in metazoans to insulin and growth factors, energy status, and stress conditions. This review focuses on the biochemical mechanisms that regulate mTORC1 kinase activity, with special emphasis on mTORC1 regulation by amino acids. The dominant positive regulator of mTORC1 is the GTP-charged form of the ras-like GTPase Rheb. Insulin, growth factors, and a variety of cellular stressors regulate mTORC1 by controlling Rheb GTP charging through modulating the activity of the tuberous sclerosis complex, the Rheb GTPase activating protein. In contrast, amino acids, especially leucine, regulate mTORC1 by controlling the ability of Rheb-GTP to activate mTORC1. Rheb binds directly to mTOR, an interaction that appears to be essential for mTORC1 activation. In addition, Rheb-GTP stimulates phospholipase D1 to generate phosphatidic acid, a positive effector of mTORC1 activation, and binds to the mTOR inhibitor FKBP38, to displace it from mTOR. The contribution of Rheb's regulation of PL-D1 and FKBP38 to mTORC1 activation, relative to Rheb's direct binding to mTOR, remains to be fully defined. The rag GTPases, functioning as obligatory heterodimers, are also required for amino acid regulation of mTORC1. As with amino acid deficiency, however, the inhibitory effect of rag depletion on mTORC1 can be overcome by Rheb overexpression, whereas Rheb depletion obviates rag's ability to activate mTORC1. The rag heterodimer interacts directly with mTORC1 and may direct mTORC1 to the Rheb-containing vesicular compartment in response to amino acid sufficiency, enabling Rheb-GTP activation of mTORC1. The type III phosphatidylinositol kinase also participates in amino acid-dependent mTORC1 activation, although the site of action of its product, 3'OH-phosphatidylinositol, in this process is unclear.     

Mechanisms involved in the coordinate regulation of mTORC1 by insulin and amino acids

In this study, we explored the coordinate regulation of mTORC1 by insulin and amino acids. Rat livers were perfused with medium containing various concentrations of insulin and/or amino acids. At fasting (1×) or 2× (2×AA) concentrations of amino acids, insulin maximally stimulated Akt phosphorylation but had no effect on global rates of protein synthesis. In the absence of insulin, 4×AA produced a moderate stimulation of protein synthesis and activation of mTORC1. The combination of 4×AA and insulin produced a maximal stimulation of protein synthesis and activation of mTORC1. These effects were accompanied by decreases in raptor and PRAS40 and an increase in RagC associated with mTOR (mammalian target of rapamycin). The studies were extended to a cell culture model in which mTORC1 activity was repressed by deprivation of leucine and serum, and resupplementation with the amino acid and insulin acted in an additive manner to restore mTORC1 activation. In deprived cells, mTORC1 was activated by expressing either constitutively active (ca) Rheb or a caRagB·caRagC complex, and coexpression of the constructs had an additive effect. Notably, resupplementation with leucine in cells expressing caRheb or with insulin in cells expressing the caRagB·caRagC complex was as effective as resupplementation with both leucine and insulin in non-transfected cells. Moreover, changes in mTORC1 activity correlated directly with altered association of mTOR with RagB/RagC, Rheb, raptor, and PRAS40. Overall, the results suggest that amino acids signal through the Rag complex and insulin through Rheb to achieve coordinate activation of mTORC1.     

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.     

SH3BP4 is a negative regulator of amino acid-Rag GTPase-mTORC1 signaling

Amino acids stimulate cell growth and suppress autophagy through activation of mTORC1. The activation of mTORC1 by amino acids is mediated by Rag guanosine triphosphatase (GTPase) heterodimers on the lysosome. The molecular mechanism by which amino acids regulate the Rag GTPase heterodimers remains to be elucidated. Here, we identify SH3 domain-binding protein 4 (SH3BP4) as a binding protein and a negative regulator of Rag GTPase complex. SH3BP4 binds to the inactive Rag GTPase complex through its Src homology 3 (SH3) domain under conditions of amino acid starvation and inhibits the formation of active Rag GTPase complex. As a consequence, the binding abrogates the interaction of mTORC1 with Rag GTPase complex and the recruitment of mTORC1 to the lysosome, thus inhibiting amino acid-induced mTORC1 activation and cell growth and promoting autophagy. These results demonstrate that SH3BP4 is a negative regulator of the Rag GTPase complex and amino acid-dependent mTORC1 signaling.     

Rheb binds and regulates the mTOR kinase

BACKGROUND: The target of rapamycin (TOR), in complex with the proteins raptor and LST8 (TOR complex 1), phosphorylates the p70S6K and 4E-BP1 to promote mRNA translation. Genetic evidence establishes that TOR complex activity in vivo requires the small GTPase Rheb, and overexpression of Rheb can rescue TOR from inactivation in vivo by amino-acid withdrawal. The Tuberous Sclerosis heterodimer (TSC1/TSC2) functions as a Rheb GTPase activator and inhibits TOR signaling in vivo. RESULTS: Here, we show that Rheb binds to the TOR complex specifically, independently of its ability to bind TSC2, through separate interactions with the mTOR catalytic domain and with LST8. Rheb binding to the TOR complex in vivo and in vitro does not require Rheb guanyl nucleotide charging but is modulated by GTP and impaired by certain mutations (Ile39Lys) in the switch 1 loop. Nucleotide-deficient Rheb mutants, although capable of binding mTOR in vivo and in vitro, are inhibitory in vivo, and the mTOR polypeptides that associate with nucleotide-deficient Rheb in vivo lack kinase activity in vitro. Reciprocally, mTOR polypeptides bound to Rheb(Gln64Leu), a mutant that is nearly 90% GTP charged, exhibit substantially higher protein kinase specific activity than mTOR bound to wild-type Rheb. CONCLUSIONS: The TOR complex 1 is a direct target of Rheb-GTP, whose binding enables activation of the TOR kinase.     

Regulation of mTOR Complex 1 (mTORC1) by Raptor Ser863 and Multisite Phosphorylation

The rapamycin-sensitive mTOR complex 1 (mTORC1) promotes protein synthesis, cell growth, and cell proliferation in response to growth factors and nutritional cues. To elucidate the poorly defined mechanisms underlying mTORC1 regulation, we have studied the phosphorylation of raptor, an mTOR-interacting partner. We have identified six raptor phosphorylation sites that lie in two centrally localized clusters (cluster 1, Ser⁶⁹⁶/Thr⁷⁰⁶ and cluster 2, Ser⁸⁵⁵/Ser⁸⁵⁹/Ser⁸⁶³/Ser⁸⁷⁷) using tandem mass spectrometry and generated phosphospecific antibodies for each of these sites. Here we focus primarily although not exclusively on raptor Ser⁸⁶³ phosphorylation. We report that insulin promotes mTORC1-associated phosphorylation of raptor Ser⁸⁶³ via the canonical PI3K/TSC/Rheb pathway in a rapamycin-sensitive manner. mTORC1 activation by other stimuli (e.g. amino acids, epidermal growth factor/MAPK signaling, and cellular energy) also promote raptor Ser⁸⁶³ phosphorylation. Rheb overexpression increases phosphorylation on raptor Ser⁸⁶³ as well as on the five other identified sites (e.g. Ser⁸⁵⁹, Ser⁸⁵⁵, Ser⁸⁷⁷, Ser⁶⁹⁶, and Thr⁷⁰⁶). Strikingly, raptor Ser⁸⁶³ phosphorylation is absolutely required for raptor Ser⁸⁵⁹ and Ser⁸⁵⁵ phosphorylation. These data suggest that mTORC1 activation leads to raptor multisite phosphorylation and that raptor Ser⁸⁶³ phosphorylation functions as a master biochemical switch that modulates hierarchical raptor phosphorylation (e.g. on Ser⁸⁵⁹ and Ser⁸⁵⁵). Importantly, mTORC1 containing phosphorylation site-defective raptor exhibits reduced in vitro kinase activity toward the substrate 4EBP1, with a multisite raptor 6A mutant more strongly defective that single-site raptor S863A. Taken together, these data suggest that complex raptor phosphorylation functions as a biochemical rheostat that modulates mTORC1 signaling in accordance with environmental cues.     

Human cytomegalovirus infection maintains mTOR activity and its perinuclear localization during amino acid deprivation

The mammalian target of rapamycin (mTOR) kinase is present in 2 functionally distinct complexes, mTOR complex 1 (mTORC1) and complex 2 (mTORC2). Active mTORC1 mediates phosphorylation of eIF4E-binding protein (4E-BP) and p70 S6 kinase (S6K), which is important for maintaining translation. During human cytomegalovirus (HCMV) infection, cellular stress responses are activated that normally inhibit mTORC1; however, previous data show that HCMV infection circumvents stress responses and maintains mTOR kinase activity. Amino acid deprivation is a stress response that normally inhibits mTORC1 activity. Amino acids can signal to mTORC1 through the Rag proteins, which promote the colocalization of mTORC1 with its activator Rheb-GTP in a perinuclear region, thereby inducing 4E-BP and S6K phosphorylation. As expected, our results show that amino acid depletion in mock-infected cells caused loss of mTORC1 activity and loss of the perinuclear localization; however, there was no loss of activity or perinuclear localization in HCMV-infected cells where the perinuclear localization of Rheb-GTP and mTOR coincided with the perinuclear assembly compartment (AC). This suggested that HCMV infection bypasses normal Rag-dependent amino acid signaling. This was demonstrated by short hairpin RNA (shRNA) depletion of Rag proteins, which had little effect on mTORC1 activity in infected cells but inhibited activity in mock-infected cells. Our data show that HCMV maintains mTORC1 activity in an amino acid- and Rag-independent manner through the colocalization of mTOR and Rheb-GTP, which occurs in association with the formation of the AC, thus bypassing inhibition that may result from lowered amino acid levels.     

Rheb binding to mammalian target of rapamycin (mTOR) is regulated by amino acid sufficiency

The removal of extracellular amino acids or leucine alone inhibits the ability of the mammalian target of rapamycin (mTOR) to signal to the raptor-dependent substrates, p70 S6 kinase and 4E-BP. This inhibition can be overcome by overexpression of the Rheb GTPase. Rheb binds directly to the amino-terminal lobe of the mTOR catalytic domain, and activates mTOR kinase in a GTP-dependent manner. Herein we show that the binding of Rheb to endogenous and recombinant mTOR is reversibly inhibited by withdrawal of all extracellular amino acids or just leucine. The effect of amino acid withdrawal is not attributable to changes in Rheb-GTP charging; amino acid withdrawal does not alter the GTP charging of recombinant Rheb. Moreover, the binding of mTOR to Rheb mutants that are unable to bind guanyl nucleotide in vivo is also inhibited by amino withdrawal. The inhibitory effect of amino acid withdrawal is exerted through an action on mTOR, at a site largely distinct from that responsible for the binding of Rheb; deletion of the larger, carboxyl-terminal lobe of the mTOR catalytic domain eliminates the inhibitory effect of amino acid withdrawal on Rheb binding, without altering Rheb binding per se. The lesser ability of the mTOR catalytic domain to bind Rheb after amino acid withdrawal does not persist after extraction and purification of the mTOR polypeptide. Amino acid withdrawal may generate an inhibitor of the Rheb-mTOR interaction that interferes with the signaling function of TOR complex 1.     

De novo <Emphasis Type="Italic">RRAGC</Emphasis> mutation activates mTORC1 signaling in syndromic fetal dilated cardiomyopathy

Idiopathic dilated cardiomyopathy (DCM) is a heritable, genetically heterogeneous disorder with variable age-dependent penetrance. We sought to identify the genetic underpinnings of syndromic, sporadic DCM in a newborn female diagnosed in utero. Postnatal evaluation revealed ventricular dilation and systolic dysfunction, bilateral cataracts, and mild facial dysmorphisms. Comprehensive metabolic and genetic testing, including chromosomal microarray, mitochondrial DNA and targeted RASopathy gene sequencing, and clinical whole exome sequencing for known cardiomyopathy genes was non-diagnostic. Following exclusion of asymptomatic DCM in the parents, trio-based whole exome sequencing was carried out on a research basis, filtering for rare, predicted deleterious de novo and recessive variants. An unreported de novo S75Y mutation was discovered in RRAGC, encoding Ras-related GTP binding C, an essential GTPase in nutrient-activated mechanistic target of rapamycin complex 1 (mTORC1) signaling. In silico protein modeling and molecular dynamics simulation predicted the mutation to disrupt ligand interactions and increase the GDP-bound state. Overexpression of RagC^S75Y rendered AD293 cells partially insensitive to amino acid deprivation, resulting in increased mTORC1 signaling compared to wild-type RagC. These findings implicate mTORC1 dysregulation through a gain-of-function mutation in RagC as a novel molecular basis for syndromic forms of pediatric heart failure, and expand genotype–phenotype correlation in RASopathy-related syndromes.     

Nuclear FAK: a new mode of gene regulation from cellular adhesions

Mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and autophagy. Its activity is regulated by the availability of amino acids and growth factors. The activation of mTORC1 by growth factors, such as insulin and insulin-like growth factor-1 (IGF-1), is mediated by tuberous sclerosis complex (TSC) 1 and 2 and Rheb GTPase. Relative to the growth factor-regulated mTORC1 pathway, the evolutionarily ancient amino acid-mTORC1 pathway remains not yet clearly defined. The amino acid-mTORC1 pathway is mediated by Rag GTPase heterodimers. Several binding proteins of Rag GTPases were discovered in recent studies. Here, we discuss the functions and mechanisms of the newly-identified binders of Rag GTPases. In particular, this review focuses on SH3 binding protein 4 (SH3BP4), the protein recently identifed as a negative regulator of Rag GTPases.     

p62 is a key regulator of nutrient sensing in the mTORC1 pathway

The signaling adaptor p62 is a critical mediator of important cellular functions, owing to its ability to establish interactions with various signaling intermediaries. Here, we identify raptor as an interacting partner of p62. Thus, p62 is an integral part of the mTORC1 complex and is necessary to mediate amino acid signaling for the activation of S6K1 and 4EBP1. p62 interacts in an amino acid-dependent manner with mTOR and raptor. In addition, p62 binds the Rags proteins and favors formation of the active Rag heterodimer that is further stabilized by raptor. Interestingly, p62 colocalizes with Rags at the lysosomal compartment and is required for the interaction of mTOR with Rag GTPases in vivo and for translocation of the mTORC1 complex to the lysosome, a crucial step for mTOR activation.     

Crystal Structure of the Gtr1pGTP-Gtr2pGDP Protein Complex Reveals Large Structural Rearrangements Triggered by GTP-to-GDP Conversion

The heterodimeric Rag GTPases consisting of RagA (or RagB) and RagC (or RagD) are the key regulator activating the target of rapamycin complex 1 (TORC1) in response to the level of amino acids. The heterodimer between GTP-loaded RagA/B and GDP-loaded RagC/D is the most active form that binds Raptor and leads to the activation of TORC1. Here, we present the crystal structure of Gtr1p(GTP)-Gtr2p(GDP), the active yeast Rag GTPase heterodimer. The structure reveals that GTP-to-GDP conversion on Gtr2p results in a large conformational transition of this subunit, including a large scale rearrangement of a long segment whose corresponding region in RagA is involved in binding to Raptor. In addition, the two GTPase domains of the heterodimer are brought to contact with each other, but without causing any conformational change of the Gtr1p subunit. These features explain how the nucleotide-bound statuses of the two GTPases subunits switch the Raptor binding affinity on and off.     

Redox regulates mammalian target of rapamycin complex 1 (mTORC1) activity by modulating the TSC1/TSC2-Rheb GTPase pathway

Mammalian target of rapamycin (mTOR) is a kinase that plays a key role in a wide array of cellular processes and exists in two distinct functional complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Although mTORC2 is primarily activated by growth factors, mTORC1 is regulated by numerous extracellular and intracellular signals such as nutrients, growth factors, and cellular redox. Previous study has shown that cysteine oxidants sufficiently activate mTORC1 activity under amino acid-depleted conditions and that a reducing agent effectively suppresses amino acid-induced mTORC1 activity, thereby raising the possibility that redox-sensitive mechanisms underlie amino acid-dependent mTORC1 regulation. However, the molecular mechanism by which redox regulates mTORC1 activity is not well understood. In this study, we show that the redox-sensitive regulation of mTORC1 occurs via Rheb but not the Rag small GTPase. Enhancing cellular redox potential with cysteine oxidants significantly increases Rheb GTP levels. Importantly, modulation of the cellular redox potential with a cysteine oxidant or reducing agent failed to alter mTORC1 activity in TSC1(-/-) or TSC2(-/-) mouse embryonic fibroblast cells. Furthermore, a cysteine oxidant has little effect on mTOR localization but sufficiently activates mTORC1 activity in both p18(-/-) and control mouse embryonic fibroblast cells, suggesting that the redox-sensitive regulation of mTORC1 occurs independent of the Ragulator·Rag complex. Taken together, our results suggest that the TSC complex plays an important role in redox-sensitive mTORC1 regulation and argues for the activation of mTORC1 in places other than the lysosome upon inhibition of the TSC complex.     

DAPK2 is a novel regulator of mTORC1 activity and autophagy

Autophagy is a tightly regulated catabolic process, which is upregulated in cells in response to many different stress signals. Inhibition of mammalian target of rapmaycin complex 1 (mTORC1) is a crucial step in induction of autophagy, yet the mechanisms regulating the fine tuning of its activity are not fully understood. Here we show that death-associated protein kinase 2 (DAPK2), a Ca²⁺-regulated serine/threonine kinase, directly interacts with and phosphorylates mTORC1, and has a part in suppressing mTOR activity to promote autophagy induction. DAPK2 knockdown reduced autophagy triggered either by amino acid deprivation or by increases in intracellular Ca²⁺ levels. At the molecular level, DAPK2 depletion interfered with mTORC1 inhibition caused by these two stresses, as reflected by the phosphorylation status of mTORC1 substrates, ULK1 (unc-51-like kinase 1), p70 ribosomal S6 kinase and eukaryotic initiation factor 4E-binding protein 1. An increase in mTORC1 kinase activity was also apparent in unstressed cells that were depleted of DAPK2. Immunoprecipitated mTORC1 from DAPK2-depleted cells showed increased kinase activity in vitro, an indication that DAPK2 regulation of mTORC1 is inherent to the complex itself. Indeed, we found that DAPK2 associates with components of mTORC1, as demonstrated by co-immunoprecipitation with mTOR and its complex partners, raptor (regulatory-associated protein of mTOR) and ULK1. DAPK2 was also able to interact directly with raptor, as shown by recombinant protein-binding assay. Finally, DAPK2 was shown to phosphorylate raptor in vitro. This phosphorylation was mapped to Ser721, a site located within a highly phosphorylated region of raptor that has previously been shown to regulate mTORC1 activity. Thus, DAPK2 is a novel kinase of mTORC1 and is a potential new member of this multiprotein complex, modulating mTORC1 activity and autophagy levels under stress and steady-state conditions.Macroautophagy (hereafter referred to as autophagy) is a highly regulated intracellular bulk degradation process found ubiquitously in eukaryotes. During autophagy a double-membrane vesicle, termed an autophagosome, engulfs cytoplasmic materials, including whole organelles. The autophagosome is later fused with the lysosome and its content degraded by hydrolases.¹ Basal levels of autophagy are maintained within the cell during steady state, and are involved in cell homeostasis activities such as turnover of long-lived proteins, preventing accumulation of protein aggregates, and removal of damaged cellular structures.² Beyond this homeostatic function, autophagy is stimulated during various stress conditions, such as nutrient deprivation, intracellular Ca²⁺ increase, hypoxia, ER stress and oxidative stress, to ensure continuous cell survival under stress.³A critical step in the induction of autophagy comprises the inactivation of a key negative regulator of the process, the mammalian target of rapamycin (mTOR).⁴ mTOR is a conserved serine/threonine protein kinase that acts as a master regulator in the cell. mTOR forms a rapamycin-sensitive complex named mTORC1 with its binding partner raptor (regulatory-associated protein of mTOR), which mediates mTOR''s substrate presentation.⁵ mTORC1 senses nutrient availability, growth factors and energy levels, and, in response, regulates cell growth, metabolism and protein synthesis, mainly by phosphorylation of substrates involved in protein translation: the p70 ribosomal S6 kinase (p70S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Under nutrient-rich conditions, mTORC1 suppresses autophagy to basal levels by phosphorylating and inhibiting the autophagy proteins ULK1 (unc-51-like kinase 1) and Atg13. Upon autophagic stimulus, mTORC1 activity is inhibited and the ULK1 complex is activated, leading to autophagy induction.⁶ The activity levels of mTORC1 are regulated by several mechanisms, such as interacting proteins, cellular localization and phosphorylation events. Raptor phosphorylation has been suggested as a mechanism by which upstream kinases such as AMPK,⁷ RSK⁸ and ULK1⁹ can regulate mTORC1 activity.Death-associated protein kinase 2 (DAPK2; also named DRP-1) is a 42-kDa Ca²⁺/calmodulin (CaM)-regulated serine/threonine kinase,¹⁰ and a closely related homolog of DAPK, a gene originally discovered in an attempt to find positive regulators of cell death.¹¹ DAPK2 was identified based on homology to the catalytic domain of DAPK. DAPK2 is a soluble cytoplasmatic protein, which triggers massive membrane blebbing and appearance of double-membrane autophagic vesicles upon its overexpression (for a review see Shiloh et al.¹²). DAPK2''s substrates and interacting proteins are mostly unknown, with the exception of the myosin II regulatory light chain, which has been shown to be an in vitro and in vivo substrate.¹³ Although many publications have studied DAPK, its substrates and its role in cell death and autophagy,^{14, 15} very little is known about DAPK2 substrates, cellular functions or the molecular pathways that it regulates.In this work, we studied the involvement of DAPK2 in the autophagic module. We identified DAPK2 as a novel interacting protein of mTORC1, and as a negative regulator of the complex both during steady-state growth conditions and in response to different stress autophagic signals. We identified mTOR''s binding partner, raptor, as a substrate of DAPK2, and found Ser721 as its phosphorylation site.     

Studies on the assay and activities of guanyl and adenyl cyclase of rat liver

In applying recently developed methods for measuring adenyl and guanyl cyclase activities, we found that some modifications produced much better cyclic nucleotide recovery, lower assay backgrounds, and greater reliability than previously reported. The reliability and specificity of the assay methods were confirmed by substrate and product analysis. Kinetic analysis of rat liver guanyl and adenyl cyclase was subsequently performed to investigate regulatory properties of both enzymes. The Michaelis-Menton constant of guanyl cyclase activity of a 30,000g supernatant fraction of rat liver for guanosine 5′-triphosphate (GTP) was 0.04 mm. This enzyme was competitively inhibited by adenosine 5′-triphosphate (ATP) (K_i = 0.011 mM). Guanyl cyclase was activated in vitro by secretin but unaffected by carbamylcholine, hist-amine, methoxamirie, serotonin, glucagon, and pancreozymin. Liver homogenate adenyl cyclase had a Michaelis-Menten constant for ATP of 0.2 mm. This enzyme was activated by secretin, pancreozymin, glucagon, sodium fluoride, and isoproterenol. GTP (0.005 mm) enhanced the activation by both isoproterenol and glucagon. Methoxamine had no effect on adenyl cyclase activity in the presence or absence of GTP. These results suggest that both guanyl cyclase and adenyl cyclase may be mediators of hormone action in the liver.     

Identification of residues in the human guanylate-binding protein 1 critical for nucleotide binding and cooperative GTP hydrolysis

The guanylate-binding proteins (GBPs) form a group of interferon-gamma inducible GTP-binding proteins which belong to the family of dynamin-related proteins. Like other members of this family, human guanylate-binding protein 1 (hGBP1) shows nucleotide-dependent oligomerisation that stimulates the GTPase activity of the protein. A unique feature of the GBPs is their ability to hydrolyse GTP to GDP and GMP. In order to elucidate the relationship between these findings, we designed point mutants in the phosphate-binding loop (P-loop) as well as in the switch I and switch II regions of the protein based on the crystal structure of hGBP1. These mutant proteins were analysed for their interaction with guanine nucleotides labeled with a fluorescence dye and for their ability to hydrolyse GTP in a cooperative manner. We identified mutations of amino acid residues that decrease GTPase activity by orders of magnitude a part of which are conserved in GTP-binding proteins. In addition, mutants in the P-loop were characterized that strongly impair binding of nucleotide. In consequence, together with altered GTPase activity and given cellular nucleotide concentrations this results in hGBP1 mutants prevailingly resting in the nucleotide-free (K51A and S52N) or the GTP bound form (R48A), respectively. Using size-exclusion chromatography and analytical ultracentrifugation we addressed the impact on protein oligomerisation. In summary, mutants of hGBP1 were identified and biochemically characterized providing hGBP1 locked in defined states in order to investigate their functional role in future cell biology studies.     

Amino Acid-Dependent mTORC1 Regulation by the Lysosomal Membrane Protein SLC38A9

The serine/threonine kinase mTORC1 regulates cellular homeostasis in response to many cues, such as nutrient status and energy level. Amino acids induce mTORC1 activation on lysosomes via the small Rag GTPases and the Ragulator complex, thereby controlling protein translation and cell growth. Here, we identify the human 11-pass transmembrane protein SLC38A9 as a novel component of the Rag-Ragulator complex. SLC38A9 localizes with Rag-Ragulator complex components on lysosomes and associates with Rag GTPases in an amino acid-sensitive and nucleotide binding state-dependent manner. Depletion of SLC38A9 inhibits mTORC1 activity in the presence of amino acids and in response to amino acid replenishment following starvation. Conversely, SLC38A9 overexpression causes RHEB (Ras homolog enriched in brain) GTPase-dependent hyperactivation of mTORC1 and partly sustains mTORC1 activity upon amino acid deprivation. Intriguingly, during amino acid starvation mTOR is retained at the lysosome upon SLC38A9 depletion but fails to be activated. Together, the findings of our study reveal SLC38A9 as a Rag-Ragulator complex member transducing amino acid availability to mTORC1 activity.     

The Legionella pneumophila GTPase Activating Protein LepB Accelerates Rab1 Deactivation by a Non-canonical Hydrolytic Mechanism

GTPase activating proteins (GAPs) from pathogenic bacteria and eukaryotic host organisms deactivate Rab GTPases by supplying catalytic arginine and glutamine fingers in trans and utilizing the cis-glutamine in the DXXGQ motif of the GTPase for binding rather than catalysis. Here, we report the transition state mimetic structure of the Legionella pneumophila GAP LepB in complex with Rab1 and describe a comprehensive structure-based mutational analysis of potential catalytic and recognition determinants. The results demonstrate that LepB does not simply mimic other GAPs but instead deploys an expected arginine finger in conjunction with a novel glutamic acid finger, which forms a salt bridge with an indispensible switch II arginine that effectively locks the cis-glutamine in the DXXGQ motif of Rab1 in a catalytically competent though unprecedented transition state configuration. Surprisingly, a heretofore universal transition state interaction with the cis-glutamine is supplanted by an elaborate polar network involving critical P-loop and switch I serines. LepB further employs an unusual tandem domain architecture to clamp a switch I tyrosine in an open conformation that facilitates access of the arginine finger to the hydrolytic site. Intriguingly, the critical P-loop serine corresponds to an oncogenic substitution in Ras and replaces a conserved glycine essential for the canonical transition state stereochemistry. In addition to expanding GTP hydrolytic paradigms, these observations reveal the unconventional dual finger and non-canonical catalytic network mechanisms of Rab GAPs as necessary alternative solutions to a major impediment imposed by substitution of the conserved P-loop glycine.