Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids

The mTORC1 kinase promotes growth in response to growth factors, energy levels, and amino acids, and its activity is often deregulated in disease. The Rag GTPases interact with mTORC1 and are proposed to activate it in response to amino acids by promoting mTORC1 translocation to a membrane-bound compartment that contains the mTORC1 activator, Rheb. We show that amino acids induce the movement of mTORC1 to lysosomal membranes, where the Rag proteins reside. A complex encoded by the MAPKSP1, ROBLD3, and c11orf59 genes, which we term Ragulator, interacts with the Rag GTPases, recruits them to lysosomes, and is essential for mTORC1 activation. Constitutive targeting of mTORC1 to the lysosomal surface is sufficient to render the mTORC1 pathway amino acid insensitive and independent of Rag and Ragulator, but not Rheb, function. Thus, Rag-Ragulator-mediated translocation of mTORC1 to lysosomal membranes is the key event in amino acid signaling to mTORC1.National Institutes of Health (U.S.) (Grant CA103866)National Institutes of Health (U.S.) (Grant AI47389)United States. Dept. of Defense (W81XWH-07-0448)W. M. Keck FoundationJane Coffin Childs Memorial Fund for Medical ResearchLAM Foundation (Fellowship

The Folliculin Tumor Suppressor Is a GAP for the RagC/D GTPases That Signal Amino Acid Levels to mTORC1

The mTORC1 kinase is a master growth regulator that senses numerous environmental cues, including amino acids. The Rag GTPases interact with mTORC1 and signal amino acid sufficiency by promoting the translocation of mTORC1 to the lysosomal surface, its site of activation. The Rags are unusual GTPases in that they function as obligate heterodimers, which consist of RagA or B bound to RagC or D. While the loading of RagA/B with GTP initiates amino acid signaling to mTORC1, the role of RagC/D is unknown. Here, we show that RagC/D is a key regulator of the interaction of mTORC1 with the Rag heterodimer and that, unexpectedly, RagC/D must be GDP bound for the interaction to occur. We identify FLCN and its binding partners, FNIP1/2, as Rag-interacting proteins with GAP activity for RagC/D, but not RagA/B. Thus, we reveal a role for RagC/D in mTORC1 activation and a molecular function for the FLCN tumor suppressor.United States. National Institutes of Health (CA103866)United States. National Institutes of Health (AI47389)United States. Department of Defense (W81XWH-07-0448)National Cancer Institute (U.S.) (F30CA180754

mTORC1 Senses Lysosomal Amino Acids Through an Inside-Out Mechanism That Requires the Vacuolar H+-ATPase

The mTOR complex 1 (mTORC1) protein kinase is a master growth regulator that is stimulated by amino acids. Amino acids activate the Rag guanosine triphosphatases (GTPases), which promote the translocation of mTORC1 to the lysosomal surface, the site of mTORC1 activation. We found that the vacuolar H+–adenosine triphosphatase ATPase (v-ATPase) is necessary for amino acids to activate mTORC1. The v-ATPase engages in extensive amino acid–sensitive interactions with the Ragulator, a scaffolding complex that anchors the Rag GTPases to the lysosome. In a cell-free system, ATP hydrolysis by the v-ATPase was necessary for amino acids to regulate the v-ATPase-Ragulator interaction and promote mTORC1 translocation. Results obtained in vitro and in human cells suggest that amino acid signaling begins within the lysosomal lumen. These results identify the v-ATPase as a component of the mTOR pathway and delineate a lysosome-associated machinery for amino acid sensing.Damon Runyon Cancer Research FoundationMillennium Pharmaceuticals, Inc.American Lebanese Syrian Associated CharitiesHoward Hughes Medical Institut

C7orf59/LAMTOR4 phosphorylation and structural flexibility modulate ragulator assembly

Ragulator is a pentamer composed of p18, MP1, p14, C7orf59, and hepatitis B virus X-interacting protein (HBXIP; LAMTOR 1-5) which acts as a lysosomal scaffold of the Rag GTPases in the amino acid sensitive branch of TORC1 signaling. Here, we present the crystal structure of human HBXIP-C7orf59 dimer (LAMTOR 4/5) at 2.9 angstrom and identify a phosphorylation site on C7orf59 which modulates its interaction with p18. Additionally, we demonstrate the requirement of HBXIP-C7orf59 to stabilize p18 and allow further binding of MP1-p14. The structure of the dimer revealed an unfolded N terminus in C7orf59 (residues 1-15) which was shown to be essential for p18 binding. Full-length p18 does not interact stably with MP1-p14 in the absence of HBXIP-C7orf59, but deletion of p18 residues 108-161 rescues MP1-p14 binding. C7orf59 was phosphorylated by protein kinase A (PKA) in vitro and mutation of the conserved Ser67 residue to aspartate prevented phosphorylation and negatively affected the C7orf59 interaction with p18 both in cell culture and in vitro. C7orf59 Ser67 was phosphorylated in human embryonic kidney 293T cells. PKA activation with forskolin induced dissociation of p18 from C7orf59, which was prevented by the PKA inhibitor H-89. Our results highlight the essential role of HBXIP-C7orf59 dimer as a nucleator of pentameric Ragulator and support a sequential model of Ragulator assembly in which HBXIP-C7orf59 binds and stabilizes p18 which allows subsequent binding of MP1-p149915891602CNPQ - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPESP – Fundação de Amparo à Pesquisa Do Estado De São Paulo2014/12445-0; 2017/21455-7; 2014/17264-3190174/2012-

Amino acid regulation of rapamycin complex

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2013.Cataloged from PDF version of thesis. "September 2013."Includes bibliographical references.Mammalian target of rapamycin complex I (mTORC1) is an atypical Ser/Thr kinase that regulates cellular and organismal growth. Accordingly, mTORC1 has substantial roles in regulating insulin sensitivity and lifespan, and when deregulated, it is implicated in the pathogenesis of common cancers. mTORC1 responds to a diverse set of stimuli, including growth factors, oxygen availability, energy and amino acid levels in order to control essential anabolic and catabolic processes. Amino acids promote mTORC1 shuttling to the lysosomal surface, its site of activation. This translocation is mediated by a family of heterodimeric GTPases known as the Rags that reside on the lysosomal surface. Unique among the small GTPases, the Rags are obligate heterodimers: the highly related RagA and RagB are functionally redundant and bind to RagC or RagD, which are also very similar to each other. Amino acids regulate the binding of nucleotides to RagB, such that amino acid stimulation increases its GTP loading, leading to the recruitment and binding of mTORC1. In the work described here, we identify two Rag interacting complexes termed 'Ragulator' and 'GATOR' that form a lysosome based signaling platform that controls the activity of the Rags. We find that Ragulator is a pentameric complex that is both necessary and sufficient to determine the intracellular localization of Rags. Moreover, we find that Ragulator functions as a guanine nucleotide exchange factor (GEF) for RagA and RagB stimulating GTP-loading, a key event in the amino-acid dependent activation of mTORC1. Additionally, we describe the function of GATOR, an octomeric complex that is defined by two distinct subcomplexes termed GATOR1 and GATOR2. We find that GATOR2 functions as a positive regulator of mTORC1 whereas GATOR1 negatively controls this pathway. Epistasis analysis reveals GATOR2 functions upstream of GATOR1, which inhibits the Rags through its GTPase activating protein (GAP) activity towards RagA and RagB. GATOR1 components are mutated in gliolbastoma and ovarian tumors and GATOR1 deficient cancer cells are hypersensitive to the mTORC1 inhibitor rapamycin. Thus, we define the molecular mechanisms regulating the function of Rags and propose a model for the activation of the mTORC1 pathway by amino acids.by Liron Bar-Peled.Ph.D

Regulation of mTORC1 by amino acids

The mechanistic target of rapamycin complex I (mTORC1) is a central regulator of cellular and organismal growth, and hyperactivation of this pathway is implicated in the pathogenesis of many human diseases including cancer and diabetes. mTORC1 promotes growth in response to the availability of nutrients, such as amino acids, which drive mTORC1 to the lysosomal surface, its site of activation. How amino acid levels are communicated to mTORC1 is only recently coming to light by the discovery of a lysosome-based signaling system composed of Rags (Ras-related GTPases) and Ragulator v-ATPase, GATOR (GAP activity towards Rags), and folliculin (FLCN) complexes. Increased understanding of this pathway will not only provide insight into growth control but also into the human pathologies triggered by its deregulation.National Institutes of Health (U.S.) (CA103866)National Institutes of Health (U.S.) (AI473890United States. Department of Defense (W81XWH-07-0448

Disruption of the Rag-Ragulator Complex by c17orf59 Inhibits mTORC1

mTORC1 controls key processes that regulate cell growth, including mRNA translation, ribosome biogenesis, and autophagy. Environmental amino acids activate mTORC1 by promoting its recruitment to the cytosolic surface of the lysosome, where its kinase is activated downstream of growth factor signaling. mTORC1 is brought to the lysosome by the Rag GTPases, which are tethered to the lysosomal membrane by Ragulator, a lysosome-bound scaffold. Here, we identify c17orf59 as a Ragulator-interacting protein that regulates mTORC1 activity through its interaction with Ragulator at the lysosome. The binding of c17orf59 to Ragulator prevents Ragulator interaction with the Rag GTPases, both in cells and in vitro, and decreases Rag GTPase lysosomal localization. Disruption of the Rag-Ragulator interaction by c17orf59 impairs mTORC1 activation by amino acids by preventing mTOR from reaching the lysosome. By disrupting the Rag-Ragulator interaction to inhibit mTORC1, c17orf59 expression may represent another mechanism to modulate nutrient sensing by mTORC1

Pharmacological convergence reveals a lipid pathway that regulates C. elegans lifespan

Phenotypic screening has identified small-molecule modulators of aging, but the mechanism of compound action often remains opaque due to the complexities of mapping protein targets in whole organisms. Here, we combine a library of covalent inhibitors with activity-based protein profiling to coordinately discover bioactive compounds and protein targets that extend lifespan in Caenorhabditis elegans. We identify JZL184-an inhibitor of the mammalian endocannabinoid (eCB) hydrolase monoacylg-lycerol lipase (MAGL or MGLL)-as a potent inducer of longevity, a result that was initially perplexing as C. elegans does not possess an MAGL ortholog. We instead identify FAAH-4 as a principal target of JZL184 and show that this enzyme, despite lacking homology with MAGL, performs the equivalent metabolic function of degrading eCB-related monoacylglycerides in C. elegans. Small-molecule phenotypic screening thus illuminates pure pharmacological connections marking convergent metabolic functions in distantly related organisms, implicating the FAAH-4/monoacylglyceride pathway as a regulator of lifespan in C. elegans