Rag proteins regulate amino-acid-induced mTORC1 signalling

The serum- and nutrient-sensitive protein kinase mTOR (mammalian target of rapamycin) is a master regulator of cell growth and survival. The mechanisms through which nutrients regulate mTOR have been one of the major unanswered questions in the mTOR field. Identification of the Rag (Ras-related GTPase) family of GTPases as mediators of amino acid signalling to mTOR is an important step towards our understanding of this mechanism.National Institutes of Health (U.S.) (R01 CA103866)National Institutes of Health (U.S.) (AI47389)United States. Dept. of Defense (grant number W81XWH-07-0448)W. M. Keck Foundatio

Regulation of mTOR complex 1 in response to growth factors and nutrients

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections."June 2010." Cataloged from student submitted PDF version of thesis.Includes bibliographical references.In multicellular organisms, cells ensure the simultaneous availability of growth factors and nutrients before they invest in cellular processes that lead to growth. The TOR kinase is a master regulator of cellular growth and nucleates two distinct protein complexes known as TOR complex 1 and 2 (TORC1 and 2). The activity of TORC2 is mainly regulated by growth factors, whereas TORC1 activity is responsive to both growth factors and nutrients, and thus acts as a detector of favorable growth conditions. The consequence of TORC1 activation is increased protein translation through its substrates S6K and 4E-BP1. Many of the upstream signals that lead to activation of TOR pathway are tumor suppressors and deregulation of the pathway leads to disease. We aim to better understand the regulation of mammalian TORC1 (mTORC1) by upstream signals. In the work described here, we identified PRAS40 as a new component of mTORC1. PRAS40 is phosphorylated in response to growth factors and this phosphorylation event leads to mTORC1 activation. We also showed that the small GTPase Rheb, a major upstream activator of mTORC1, activates it by directly interacting with mTORC1. Both PRAS40 and Rheb relay information from growth factors to mTORC1, and do not seem to be regulated by nutrients, particularly amino acids. In this work, we also investigated how amino acids lead to mTORC1 activation. We showed that amino acids activate mTORC1 by recruiting the complex to lysosomal surface through the action of evolutionarily conserved Rag GTPases. We also indentified a complex of three proteins (p14, p18 and MP1), that we call the "Ragulator" complex, as Rag GTPase interacting proteins. The Ragulator itself localizes to lysosomes through lipid modifications on p18, and tethers the Rag GTPases on the lysosomal surface. We speculate that amino acid-induced lysosomal localization of mTORC1 enables its encounter with Rheb. Through this work, we propose a model for growth factor and amino acid-induced mTORC1 activation.by Yasemin S. Sancak.Ph.D

mTOR Signalling, Nutrients and Disease Rag proteins regulate amino-acid-induced mTORC1 signalling

Abstract The serum-and nutrient-sensitive protein kinase mTOR (mammalian target of rapamycin) is a master regulator of cell growth and survival. The mechanisms through which nutrients regulate mTOR have been one of the major unanswered questions in the mTOR field. Identification of the Rag (Ras-related GTPase) family of GTPases as mediators of amino acid signalling to mTOR is an important step towards our understanding of this mechanism

MCU encodes the pore conducting mitochondrial calcium currents

Mitochondrial calcium (Ca2+) import is a well-described phenomenon regulating cell survival and ATP production. Of multiple pathways allowing such entry, the mitochondrial Ca2+ uniporter is a highly Ca2+-selective channel complex encoded by several recently-discovered genes. However, the identity of the pore-forming subunit remains to be established, since knockdown of all the candidate uniporter genes inhibit Ca2+ uptake in imaging assays, and reconstitution experiments have been equivocal. To definitively identify the channel, we use whole-mitoplast voltage-clamping, the technique that originally established the uniporter as a Ca2+ channel. We show that RNAi-mediated knockdown of the mitochondrial calcium uniporter (MCU) gene reduces mitochondrial Ca2+ current (IMiCa), whereas overexpression increases it. Additionally, a classic feature of IMiCa, its sensitivity to ruthenium red inhibition, can be abolished by a point mutation in the putative pore domain without altering current magnitude. These analyses establish that MCU encodes the pore-forming subunit of the uniporter channel. DOI: http://dx.doi.org/10.7554/eLife.00704.00

Synthesis, characterization and cyclization reactions of some new bisthiosemicarbazones

The reaction of trans-1, 4-dichloro-2-butene 1 with selected phenols affords (E)-1, 4-bis(aryloxy)-2-butenes 2a-d which are converted into bis-thiosemicarbazones 3a-h via the reactions with thiosemicarbazide and 4-methyl thiosemicarbazide, respectively. Similarly, 4-methyl-5-ethoxycarbonyl- 2, 3-dihydro-1, 3-thiazoles 4a-h are synthesized via the reaction of bis-thiosemicarbazones 3a-h with ethyl 2-chloroacetoacetate. trans-1, 4-Dithiocyanato-2-butene 5 is obtained from the reaction of KSCN and trans-1, 4-dichloro-2-butene 1. Furthermore, the bis-2-amino-1, 3, 4-thiadiazoles 6k and l are obtained from the reaction of trans-1, 4-dithiocyanato-2-butene 5 with thiosemicarbazide and 4-methyl thiosemicarbazide, respectively. These compounds are characterized by elemental analyses, infrared, 1H-, 13C-NMR and mass spectrometry. Finally, the microbial features of all compounds are determined. Some of them exhibited microbial activities at low level, and the electronic absorption spectra of the compounds 3b, d, f and h are measured in organic solvents (MeOH, DMF, DMSO and 1, 4-dioxane) with various polarities

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

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

DEPTOR Is an mTOR Inhibitor Frequently Overexpressed in Multiple Myeloma Cells and Required for Their Survival

The mTORC1 and mTORC2 pathways regulate cell growth, proliferation, and survival. We identify DEPTOR as an mTOR-interacting protein whose expression is negatively regulated by mTORC1 and mTORC2. Loss of DEPTOR activates S6K1, Akt, and SGK1, promotes cell growth and survival, and activates mTORC1 and mTORC2 kinase activities. DEPTOR overexpression suppresses S6K1 but, by relieving feedback inhibition from mTORC1 to PI3K signaling, activates Akt. Consistent with many human cancers having activated mTORC1 and mTORC2 pathways, DEPTOR expression is low in most cancers. Surprisingly, DEPTOR is highly overexpressed in a subset of multiple myelomas harboring cyclin D1/D3 or c-MAF/MAFB translocations. In these cells, high DEPTOR expression is necessary to maintain PI3K and Akt activation and a reduction in DEPTOR levels leads to apoptosis. Thus, we identify a novel mTOR-interacting protein whose deregulated overexpression in multiple myeloma cells represents a mechanism for activating PI3K/Akt signaling and promoting cell survival.Howard Hughes Medical Institute (Investigator)Dana-Farber Cancer Institute (High-Tech Research Fund)National Cancer Institute (U.S.)National Institutes of Health (U.S.) (Intramural Research Program)American Cancer SocietyCanadian Institutes of Health Research (Fellowship)American Diabetes Association (Fellowship)W. M. Keck FoundationNational Institutes of Health (U.S.) (R01 CA103866)National Institutes of Health (U.S.) (NIH; R01 AI47389

Engineered ascorbate peroxidase as a genetically-encoded reporter for electron microscopy

Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments1 or require light and are difficult to use2. Here we report the development of a simple and robust EM genetic tag, called “APEX,” that is active in all cellular compartments and does not require light. APEX is a monomeric 28 kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins. We also fused APEX to the N- or C-terminus of the mitochondrial calcium uniporter (MCU), a newly identified channel whose topology is disputed3,4. MCU-APEX and APEX-MCU give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N-and C-termini of MCU face the matrix

Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy

Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments[superscript 1] or require light and can be difficult to use[superscript 2]. Here we report the development of 'APEX', a genetically encodable EM tag that is active in all cellular compartments and does not require light. APEX is a monomeric 28-kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins using a simple and robust labeling procedure. We also fused APEX to the N or C terminus of the mitochondrial calcium uniporter (MCU), a recently identified channel whose topology is disputed[superscript 3, 4]. These fusions give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N and C termini of MCU face the matrix. Because APEX staining is not dependent on light activation, APEX should make EM imaging of any cellular protein straightforward, regardless of the size or thickness of the specimen.National Institutes of Health (U.S.) (Grant DP1 OD003961)National Science Foundation (U.S.). Graduate Research Fellowship ProgramUnited States. Dept. of Defense (National Defense Science and Engineering Graduate (NDSEG) Fellowships