Growth regulation by the TOR pathway
Séminaire FSER organisé par Michael Hall (Biozentrum, Bâle) et David M. Sabatini (Whitehead Institute, Cambridge, Massachussets) du 4 au 9 juin 2012
John Blenis, Anne Brunet, Claudio De Virgilio, Eyal Gottlieb, Kun-Liang Wang, Michael N. Hall, D. Grahame Hardie, Marc W. Kirschner, Pierre Léopold, Alison Lloyd, Brendan D. Manning, Christian Meyer, Linda Partridge, Mario Pende, L. Bryan Ray (editor), David M. Sabatini, Reuben Shaw, Aurelio A. Teleman, Jeremy Thorner, Anders Zetterberg.
The “Growth regulation by the TOR Pathway” meeting was held at Les Treilles from June 4 to 9, 2012 and focused on the regulation of mass accumulation by the master growth control pathway anchored by the TOR kinase. Participants discussed the function of this and related pathways in many organisms ranging from unicellular fungi to plants and mammals. The 19 talks disclosed largely unpublished data on a diversity of topics, including nutrient sensing, coordination between the cell cycle and growth control, aging, epigenetics, cancer and small molecule metabolism. Participants learned of new molecules that are part of the TOR pathway as well as new connections of TOR to important physiological processes, like the control of metabolism. In addition, novel techniques were disclosed at the meeting, such as the use of a very high throughput imaging method to study growth control. The talks were arranged by similarity of topic and each was followed by extensive discussions that spilled over into the coffee and lunch and dinner times. There was universal agreement that the meeting was amongst the best any participant had ever attended.
The meeting opened on the morning of June 4, 2012 with David Sabatini (Whitehead/MIT) giving a short introduction to the mTOR pathway and its role in the control of growth. He then went on to present three unpublished stories, two focused on amino acid sensing by mTORC1. He presented evidence that the Ragulator complex has GEF activity towards the Rag GTPases and an analysis of mice carrying a constitutively active version of RagA. These animals have severe defects in nutrient homeostasis and die perinataly. The third story was a model to explain why the sensitivity of mTORC1 substrates varies with respect to rapamycin and nutrient deprivation.
Jeremy Thorner (Berkeley) then spoke about the regulation and downstream targets of the yeast AGC family protein kinase Ypk1, which requires activation by the yeast TORC2 complex for its optimal function, especially in response to stresses that perturb the composition and structure of the plasma membrane. Activity of yeast Ypk1 requires phosphorylation in its activation loop (at Thr504) by the upstream protein kinases Pkh1 or Pkh2, which Thorner’s prior work has shown are localized at discrete sites on the plasma membrane (dubbed « eisosomes » by others) and are the sequence homologs and functional orthologues of mammalian 3-phosphoinositide-dependent protein kinase (PDK1). As Thorner discussed, the primary biological role of Ypk1 is to control plasma membrane glycerolipid and sphingolipid homeostasis.
Michael Hall (Biozentrum) then presented three separate, unpublished stories.The first, a follow-up of the presentation by Jeremy Thorner, described the role of yeast TORC1 in controlling the ER Orm proteins and sphingolipid biosynthesis.The second story was about activation of Rag-mTORC1 signaling by glutaminolysis. Glutaminolysis provides a mechanism for the sensing of leucine and glutamine upstream of Rag.The third story was on the mTORC1- and mTORC2-dependent phosphoproteomes.This last story focused on the regulation of the triple enzyme CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) and de novo pyrimidine synthesis by mTORC1.
After lunch, Claudio De Virgilio (University of Fribourg) discussed work from his lab characterizing the role of TORC1 in both entry into and exit from quiescence in Saccharomyces cerevisiaeǤSimilar to its orthologous counterpart in higher eukaryotes (i.e. the greatwall/MASTL kinase) Rim15 phosphorylates the small endosulfines (Igo1/2 in yeast), which serve to inhibit the typeʹprotein phosphatase (PP2A) module PP2ACdc55ǤThe second half of his presentation was dedicated to the description of new insights into the regulation of the EGO complex (EGOC), which is required for exit from rapamycin-induced growth arrest in yeast. Based on the latest findings from the De Virgilio lab, which indicate that the conserved SEA complex harbors a Gtr1-GAP activity, these latter two models can/will be tested in the near future.
Linda Partridge (UCL/Max Planck) then spoke about the role of nutrientsensing signaling in ageing. First discovered through genetic analysis in the nematode worm Caenorhabditis elegans, the insulin/Igf signaling pathway has proved to play a role in ageing that is conserved in evolution to the fruit fly Drosophila and the mouse. The closely interacting TOR pathway is also involved in ageing, in yeast, worms, flies and mice, and inhibition of TOR with rapamycin can extend lifespan in flies and mice. This nutrient-sensing network also mediates at least part of the increase in lifespan from dietary restriction, an intervention that has been shown to extend lifespan in diverse organisms including rhesus monkeys. In Drosophila, the responses of fecundity and lifespan to dietary restriction is attributable entirely to the essential amino acids in the diet, and imbalance in these limits both life history traits. The in silico translated proteome of Drosophila can be used to predict the theoretical requirements for the essential amino acids.
Anne Brunet (Stanford) then presented on the plasticity of aging in response to nutrient availability. The first part of her talk was on the energysensing protein kinase AMPK, which mediates longevity in response to certain forms of dietary restriction in the worm C. elegansǤAnne described a chemical genetic screen to identify novel substrates of AMPK in mammalian cells, including proteins involved in mitosis. This work provides an interesting link between energy-sensing and mitotic progression, which may help preserve genomic stability. The second part of Anne’s talk was related to the regulation of aging by chromatin changes. Anne reported that histone modifiers playedkey role in regulating lifespan in C. elegans. Surprisingly, changes in chromatin states in parents led to epigenetic inheritance of longevity in descendants.
Kun-Liang Guan (UCSD) began the second day of the meeting by discussing cross talk between mTOR and the Hippo pathway, which plays a key role in organ size regulation. He presented evidence for YAP and PTEN in mediating the crosstalk of these two signaling pathways.In addition, Dr. Guan discussed the regulation of the Hippo pathway by G-protein coupled receptor (GPCR) signaling.He showed that lysophosphatidic acid (LPA) inhibits the Hippo pathway by activating G12/13 and Rho GTPase.Moreover, many GPCRs are shown to either inhibit or activate the Hippo pathway inmanner depending on their coupled G-proteins.
Grahame Hardie (Dundee) then introduced the AMP-activated protein kinase (AMPK) signaling pathway, a system involved in the sensing of cellular energy nutrient status that inactivates the TORC1 complex. He discussed evidence that many widely used drugs and natural products (mostly derived from plants) activate AMPK, in most cases by an indirect mechanism involving inhibition of mitochondrial ATP synthesis with consequent rises in AMP and ADP. However, the natural plant hormone salicylate, which is the major breakdown product of aspirin in the human body, activates AMPK by binding directly to the
same site on AMPK as a synthetic activator derived fromhigh-throughput screen (A-769662).
Reuben Shaw (Salk Institute) also discussed the AMPK signaling pathway, first specifically focusing on the common thread of inherited hamartoma syndromes all sharing hyperactivation of mTORC1 signaling as a common feature. LKB1 is the major kinase which activates AMPK, which in turn inhibits mTORC1 signaling through dual phosphorylation of TSC2 and raptor, providing a direct molecular explanation for the elevating mTORC1 in LKB1-deficient tumors. In addition to these direct connections, subsequent studies from the Shaw lab and others have revealednumber of downstream substrates of AMPK are also directly regulated by mTORC1-dependent signals, including the ULK1/Atg1 autophagy kinase and the cytoskeletal regulator CLIP1.
After lunch Alison Lloyd (UCL) initiated a session discussing how and if cells sense and control their size. Using primary mammalian cells that can proliferate at steady-state at dramatically different sizes, she highlighted data that indicates that the size of a cell has no influence on cell-cycle proliferation rate and that the independent regulation of proliferation and growth pathways allows a flexible mechanism for the extracellular regulation of cell size. Whereas cell biogenesis is frequently thought of as a uniform process, she presented evidence that the generation of cell mass and cell volume are separable processes with volume addition requiring de novo lipid biogenesis which requires SREBP2.
Anders Zetterberg (Karolinska) presented data on cell growth and cell size control in mammalian cells. The studies were performed on single cells in culture followed by time lapse video-microscopy. Cell size was found to increase in an “exponential-like” fashion during interphase, strongly correlated to the increase in total ribosomal-RNA content in the cell. Clear evidence was presented for the existence of an actively operating size control mechanism, allowing bigger cells to initiate DNA-replication earlier in the G1 phase of the cell cycle than smaller cells. It was also found that cell growth could be dissociated from cell cycle progression under various experimental conditions, and that both of these processes were controlled by different growth factors. Finally, data was presented, suggesting the existence of a switch for growth control in early G1, operating stringently in normal cells but absent or relaxed in cancer cells.
Marc Kirschner (Harvard) presented some very quantitative data on cell growth using new mathematical and biophysical tools.Remarkably the work seemed to take off from Anders Zetterberg’s experiments of 50 years ago (also reporter at the meeting), as if the questions had been in hibernation during that period; there were many points of agreement and correspondence. The main conclusion is that cell size variation reachesminimum at G1/S.During this time the general size dependence is reversed.Large cells grow faster than small cells in early G1 but in late G1 small cells grow faster than larger ones.Thus cells know their size and have a way to control their growth.Experiments with rapamycin suggest that the TOR pathway plays a role in either the size sensing or in the cellular response.
Eyal Gottlieb (Beatson Institute) began the third day of the meeting by discussing the field of cancer metabolism in general, and the need to study specific metabolic alterations required for cancer cell growth and survival. He focused on recent studies of cells deficient in the mitochondrial tumour suppressor, fumarate hydratase (FH). FH is a tricarboxylic acid (TCA) cycle enzyme and a tumour suppressor which is lost in some severe cases of renal cell cancer. Using metabolomics and transcriptomics data collected from geneticallymodified primary mouse renal cells with a conditional loss of FH, he constructed a computational model, generated to study the unique metabolome and to identify metabolic pathways which are required for the survival of these cells. These include the heme biosynthesis and degradation pathway as well as mechanisms of alleviating TCA cycle carbon stress.
Brendan Manning (Harvard) continued with four unpublished stories, two about upstream regulation of mTORC1 and two about downstream functions of mTORC1.The first involved his laboratory’s recent characterization of a third stoichiometric component of the TSC1-TSC2 complex, which iscritical negative regulator of mTORC1 signaling.Like other components of the complex, loss of this protein (called TBC1D7), results in elevated mTORC1 signaling and cell growth under poor growth conditions.The second story was focused on the spatial regulation of this mTORC1 regulatory complex.The final two stories had to do with points of metabolic control downstream of mTORC1, with a focus on transcriptional control of lipid metabolism through SREBP andnovel posttranslational mechanism affecting nucleic acid metabolism.
John Blenis (Harvard) then discussed his lab’s recent discoveries revealing how activated mTORC1 and S6K increase glutamine uptake and metabolism by regulating two critical enzymes required for the conversion of glutamine to glutamate (glutaminase, GLS) and glutamate to a-ketoglutarate (glutamate dehydrogenase, GDH).Regulation of this anaplerotic pathway provides added carbon-building blocks and energy via oxidative phosphorylation, supporting several anabolic processes required for mTOR-modulated cell growth and survival.Dr. Blenis also provided evidence for an AMPK-independent mechanism for sensing cellular energy status to promote the proper assembly of a functional dimeric mTORC1 complex.
Christian Meyer (Institut Jean-Pierre BOURGIN, INRA-Versailles) began the last day of the meeting by summarizing the current knowledge on the TOR signaling pathway in photosynthetic organisms. It is now clear that a conserved TORC1 complex is present in higher and lower plants. Meyer then presented the consequences of mutating the components of the TORC1 complex in the model plant Arabidopsis on global gene expression and metabolite composition. Inactivation of either the Tor or Lst8 genes resulted in higher accumulation of starch, amino-acids and soluble sugars whereas the production of raffinose, a stress-related sugar, was highly impaired. Moreover silencing of the Tor gene produced aberrant leaf and root development with altered cell division.
Pierre Leopold (Université de Nice) then discussed the regulation of growth in fly larvae by pathways other than the TOR pathway. He presented work on new insulin like peptide, dILP8, that has an essential role in timing the growth program in response to injury of an imaginal disk. Gain and loss of function alleles of dIPL8 have concomitant effects on the timing of pupation. His second story was about the aversion of flies to food that is lacking an essential amino acid. Dr. Leopold showed that the GCN2 pathway plays an essential role and acts through ATF4 and the GABA receptor.
Aurelio Teleman (DKFZ, Heidelberg) presented two stories using Drosophila to study the insulin/TOR signaling pathway. The first story looked at the in vivo function of PRAS40, showing that PRAS40 connects TOR activation to PI3K activation in some tissues such as the germline, but not in most other tissues of the animal. The second story identified interconnections between the insulin and hippo/yorkie signaling pathways. This leads to concurrent activation of both pathways whenever one of the two is activated.
In the last talk of the meeting Mario Pende (INSERM, Paris) introduced rare human genetic diseases due to mutations in genes encoding signal transduction elements of the mTOR pathway. He then focussed on tuberous sclerosis, a rare genetic disease due to mutations in TSC1 and TSC2 leading to benign tumours such as angiomyolipomas of the kidney, lyphangiomyomatosis of the lung, subependymal nodules of the brain, angiofibromas of the skin. Mario Pende described an animal model of the disease in which the temporal deletion of TSC1 during embryonic development leads to the formation of mesenchymal lesions similar to human patients. This experimental approach was instrumental to reveal the overexpression of the transcriptional regulator Yap1 in the TSCnegative murine and human tissues, thus identifyingnovel cross-talk between mTOR and another major pathway regulating growth.