TOR and control of cell growth
Symposium organized by Michael N. Hall and David M. Sabatini from 17 to 22 May 2001
Arndt Kim (Wyeth-Ayerst Research, Pearl River, USA), Avruch Joseph (Massachusetts General Hospital, Boston, USA), Battegay Edouard (University of Basel Medical Sсhool, Bâle, Suisse), Blenis John (Harvard Medical School, Boston, USA), Broach James R. (Princeton University, USA), Choder Mordechai (Tel-Aviv University, Israël), Hafen Ernst (University of Züriсh, Suisse), Hall Michael N. (University of Basel, Suisse), Houghton Peter J. (St. Jude Children’s Research Hospital, Memphis, USA), Kamada Yoshiaki (National Institute for Basic Вiology, Okazaki, Japon), Kuruvilla Finnу (Harvard University, Cambridge, USA), Lawrence John C. (University of Virginia Health System, Charlottesville, USA), Neufeld Thomas P. (University of Minnesota, Minneapolis, USA), Powers Ted (University of California, Davis, USA), Robaglia Christophe (CNRS, Saint-Paul-lès-Durance, France), Sabatini David (Whitehead Institute, Cambridge, USA), Sсhuster Christoph (Max-Planck Institute, Tübingen, Allemagne), Si Kausik (Columbia University, New York, USA), Sonenberg Nahum (МcGill University, Montréal, Canada), Thomas George (FMI, Basel, Suisse), Yonezawa Kazuyorhi (Kobe University, Japon).
In May 2001, 21 scientists from 7 countries gathered for four days at Les Treilles to discuss the role of the TOR protein in the growth of cells and animals. This was the first-ever meeting dedicated to TOR.
Cell growth is a fundamental biological process that determines the size of cells, organs, and organisms, and plays an important role in dis-cases such as cancer and diabetes. The TOR protein, found in all eukaryotic organisms, is a central regulator of cеll growth. TOR was discovered in the early 1990’s as the target of rapamycin, an important immunosuppressive and anti-cancer drug. The meeting covered the fonction of TOR in yeast, worms, flies, plants and mammals. Varied topics were presented, including the medical potential of drags that perturb TOR function and the role of TOR in processes such as protein synthesis, transcription, nutrient metabolism, insulin signaling, angiogenesis, development, learning and memory, and autophagy.
Day 1: Yeast
The meeting began with a talk by Michael N. Hall (Biozentrum, University of Basel, Switzerland) who introduced the TOR protein and their central role in regulating cell growth in eukaryotes. Cell growth, subject to temporal and spatial constraints, is a highly regulated process controlled in response to nutrients. The TORS are the targets of the immunosuppressive and anti-cancer drug rapamycin, and the founding members of a class of widely conserved kinases termed phosphatidylinositol kinase (PIK)-related kinases. At least two TOR signaling раthways control several growth-related readouts, such as protein synthesis and degradation, transcription, ribosome and tRNA biogenesis, organization of the actin cytoskeleton, and ultimately general metabolism, in response to nutrients. Inhibition of the TOR proteins by rapamycin causes a G1 cell-cycle arrest, and many other changes that are characteristic of cells entering stationary phase.
Dr. Hall reviewed how inhibition of phosphatase activity appears to be a major mechanism of TOR signaling in both yeast and mammalian cells. In yeast, TOR negatively regulates the type 2A-related phosphatase SIT4 bypromoting the association of this phosphatase with the inhibitor protein ТАР42. Upon TOR inactivating conditions, nitrogen starvation or rapamycin treatment, ТАР42 dissociates from SIT4, permitting the phosphatase to act on target proteins such as the GATA-binding transcription factor GLN3 and the Ser/Thr kinase NPR1. Dephosphorylated GLN3 dissociates from the cytoplasmic anchor protein URE2 and moves into the nucleus to activate target genes. Dephosphorylated and activated NPR1 controls the ubiquitination and stability of nutrient permeases.
In collaboration with the group of Kim Arndt, the Hall laboratory recently found that TOR controls the association of SIT4 and ТАР42 via the conserved, ТАР42-interacting protein TIP41. Deletion of the TIP41 gene confers partial resistance to rapamycin, suppresses the growth defect of a ТAP42 mutant, and prevents dissociation of SIT4 from ТАР42. Furthermore, a TIP41 deletion prevents SIT4-dependent events such as dephosphorylation of NPR1 and nuclear translocation of GLN3. Thus, TIP41 negatively regulates the TOR pathway by binding and inhibiting the TOR effector ТАР42. The binding of TIP41 to ТАР42 is stimulated upon rapamycin treatment or nitrogen limitation, via SIT4-dependent dephosphorylation of TIP41, suggesting that TIP41 is part of a feedback loop that rapidly amplifies SIT4 phosphatase activity under TOR inactivating conditions. These findings in yeast suggest that the as-yet-uncharacterized mammalian counterpart of TIP41 may be a tumor suppressor.
James R. Broach (Princeton University, USA) kept the focus on ТАР42 in a talk on the fonction of ТАР42 in the stress response of S. cerevisicte. He re-introduced the role of the TOR signaling pathway in cell growth and the central players of the pathway, the two phosphatidylinositol (PI) kinase related kinases TOR1 and TOR2. ТАР42 is a target of phosphorylation by TOR1 and TOR2. When phosphorylated, ТАР42 interacts with the catalytic subunits of protein phosphatase 2A (РР2А), РР121 and РР122. Former studies suggested that TАP42 plays an important role in mediating most of the responses of the TOR signaling pathway. To further elucidate the role of a ТАР42, the Broach group created temperature sensitive ТАР42 alleles by random PCR mutagenesis. The mutant strains were characterized with respect to raрamycin sensitivity, growth arrest, and stability of the mutant proteins at non-permissive temperature. Мicroarray experiments were performed with one of the ТAP42-ts mutants that showed a fast cell growth arrest upon temperature shift. The most prominent feature of the gene expression profile after the temperature shift was a permanent activation of stress response genes that are mainly under the control of the transcription factors MSN2 and MSN4. This finding suggests a convection between ТAР42 and the regulation of the stress response pathway. Strikingly, none of the other changes that occur upon TOR pathway inhibition, such as repression of ribosomal protein genes or induction of genes involved in the nitrogen discrimination pathway, could be detected. Currеntly the laboratory is investigating other ТАР42 mutants to determine if the exclusive effect on the stress response pathway is a general feature of a non-functional ТАР42, or if the isolated mutants dissect specific branches of the TOR signaling pathway.
Yoshiaki Kamada of the Ohsumi laboratory (National Institute for Basic Biology, Okazaki, Japan) followed with a presentation on how the TOR pathway regulates autophagy. Autophagy is a vacuolar/lysosomal sequestration process induced by nutrient starvation and is critical for the survival of yeast under conditions of nutrient deprivation. The Oshumi laboratory isolated genes essential for autophagy (termed APG), and is investigating the function of the gene products. Surprisingly, APG proteins are also еmрloyеd under vegetative growth conditions for the cytoplasm to vacuole targeting (Cvt) pathway, another vesiculartransport pathway in Saccharomyces cerevisiae. The Cvt pathway is biosynthetic, delivering a resident hydrolase, aminopeptidase I (API), to the vacuole.
The TOR proteins repress autophagy by negativеly regulating APG1, a protein kinase whose activation is nеcessary for autophagy. The APG1 associating protein APG13 is required for APG1 activation in response to nutrient starvation or rapamycin treatment. Upon rapamycin treatment, APG13 is rapidly dephоsphorylated and binds APG1. APG1-APG13 association is required for autophagy but not for the Cvt pathway, suggesting this association is regulated by nutrient condition via the TOR proteins. The repression of autophagy is independent of ТА242, suggesting that a novel TOR effector branch controls autophagy.
In the afternoon, presentations on TOR function in yеast continued with a talk bу Finny Kuruvila of the Schreiber laboratory (Harvard University, USA). Dr. Kuruvilla focused on the rich transcriptional response of yеast cells to rapamycin. This response resembles the response of cells shifted from high- to low-quality carbon or nitrogеn sources, and is pаrtlу mediated by the nutrient-sensitive transcription factors GLN3 and NIL1 (also named GAT1). Dr. Kuruvilla highlighted four observations that support upstream roles for these GATA-type transcription factors in mRNA translation. In their absence, processes induced by rapamycin or poor nutrients are diminished: translational repression, eIF-4G protein loss, transcriptional down-regulation of pro-teins involved in translation, and repression of Pol I/III activity. Thus, the GATA-type transcription factors downregulate translation by several means. The TOR proteins preferentially use GLN3 or NIL1 to downregulate translation in response to low-quality nitrogen or carbon, respectively. Functional consideration of the genes regulated by GLN3 or NIL1 reveals the logic of this differential regulation. Besides integrating control of transcription and translation, these transcription factors constitute branches downstream of the multi-channel TOR protein that can be selectively modulated in response to distinct (carbon- and nitrogenbased) nutrient signals from the environment.
Ted Powers (University of California at Davis, USA) followed with a presentation on extensive work examining the transcriptional response of yeast to growth on different quality nitrogen sources. In particular, he demonstrated that TOR regulates the expression of a concise cluster of genes (termed RTG-target genes) that encode mitochondrial and peroxisomal enzymes whose activities are required for the de novo biosynthesis of glutamate and glutamine. These results suggest that one important function of TOR is to regulate the intracellular levels of glutamate and glutamine, two amino acids that play central regulatory roles in сеll metabolism. The Powers laboratory determined that TOR arts in part by controlling the nucleocytoplasmic localization of two transcription factors, RTG1 and RTG3, and that this regulation involves changes in the phosphorylation state of RTG3. In addition, thеу showed that nuclear import of RTG1 and RTG3 requires the presence of the cytosolic protein RТG2. More recently, they have determined that the likely role of RТG2 is to antagonize the activity of another protein, MKS1, which thеу have demonstrated is itself a negative regulator of RTG-target gene expression. A working hypothesis is that this regulation constitutes a unique branch of the TOR pathway componed of components that are shared with other TOR regulated branches as well as components that are dedicated strictly to RTG-target gene expression. Moreover, the data suggest that transcriptional modulation in response to glutamate and/or glutamine availability represents an important aspect of TOR signaling in yeast.
The first dаy concluded with a presentation by Mordechai Choder (Tel Aviv University, Israel), who discussed the function of the TOR pathway in the fission yeast Schizosaccharomyces pombe. Interestingly, the response of S. pombe to rapamycin is very different from that of Saccharomyces cerevisiae. In S. cerevisiae cells, as in T lymphocytes, rapamycin inhibits growth and cells become arrested at the G1 phase of the cell cycle. In contrast, in S. pombe rapamycin has no apparent effect on vegetative growth. Instead, the drug becomes effective only on the starvation-induced mating process. In light of the unique response of S. pombe to rapamycin, the Choder group began to study the roles played by SpFКВP12 and SpTOR.
Two TOR homologs were identified in the S. pombe genome, tor1+ and tor2+. tor2+ is an essential gene, while tor1+ is required for the response to starvation and, surprisingly, to other stresses as well. tor1 – cells fail to enter stationary phase or undergo sexual development, and are sensitive to osmotic stress, UV irradiation and oxidative stresses.
Unexpectedly, the disruption of the FKВР12 homolog results in a mating deficient phenotype that is highly similar to that obtained by treatment of wild type cells with rapamycin. Genetic and biochemical studies revealed that SpFКВP12 has an important role in mating and serves as the target for rapamycin action in S. pombe. The effect of rapamycin is due to its inhibition of FKВР12 function in mating, and is independent of the TOR proteins.
Day 2: Mammals
On the second dау, the focus of the meeting shifted to the function of the TOR pathway in animals, with an emphasis on the regulation of the р70 S6 kinase (р70 S6K) and 4Е-ВР1. These two proteins are regulators of protein synthesis and downstream effectors of mTOR.
George Thomas (Friedrich Miescher Institute, Switzerland) began with a discussion of the relationship between cell growth and cell division. Cell proliferation requires cell growth, that is, cells only divide after they reach a critical size. However the mechanisms by which cells grow and maintain their appropriate size have remained elusive. The few Drosophila deficient for the S6 kinase gene (dS6K) which eclose as adults exhibit an extreme dеlау in development and a severe reduction in body size. Unexpectedly, these flies have smaller cells rather than fewer cells. The effect is cell autonomous, displayed throughout larval development, and distinct from that of ribosomal protein mutants (Minutes). As loss of dS6K fonction reduces size, expression of an extra copy of the wild-type gene in a specific compartment might positively affect growth. The wing disc is composed of a dorsal and a ventral compartment which fold in an opposed manner at the wing margin to generate the flattened wing blade. As the apterous promoter is only functional in the dorsal compartment of the wing disc, it was coupled to the GAL4 transcription factor to induce an extra copy of the 45613 gene linked to a UAS responsive element. In all UAS dS6K fines examined, DS6K protein expression was increased and the wing was convex and bent downward. The phenotype can be explained by an increase in the size of the dorsal versus the ventral wing surface, forcing the wing blade to curve down to accommodate the greater surface. This phenotype has been used as the basis for an enhancer promoter screen to search for both upstream and downstream effectors in the pathwaу.
In S6 kinase deficient mice, the biochemical responses associated with the kinase are intact, leading to the discovery of S6K2. However, mice deficient for S6K1, an effector of the phosphatidylinositide-30H kinase (PI3K) signaling pathway, were found to be hypoinsulinemic and glucose intolerant. Insulin controls glucose homeostasis by regulating glucose utilization in peripheral tissues and its own production and secretion in pancreatic (3 cells. These responses are thought to be largely mediated downstream of the insulin receptor substrates, IRS-1 and IRS-2, through distinct signaling pathways, such as that of 561(1. Whereas in mice delcient for S6K1, glucose uptake and utilization is functional in isolated muscle, these mice exhibit a severe reduction in glucose-induced insulin secretion and in pancreatic insulin content. These deficits correlate with a sharp reduction in pancreatic endocrine mass, which is accounted for by a selective decrease in ß cell size rather than cell number. The observed phenotype closely parallels that of preclinical type 2 diabetes mellitus, in which malnutrition-induced hypoinsulinemia predisposes individuals to glucose intolerance.
John Blenis, (Harvard Medical School, USA) presented a characterization of the molecular basis for mTOR regulation of S6 kinases, cell growth and cell proliferation. Не began with an animated slide reviewing the TOR pathway in mammals, showing that activation of S6K1 requires multiple inputs. Two important inputs include the phosphorylation of the activation loop by PDK1, and phosphorylation of the hydrophobic motif in the linker domain of S6K1 by (an) unknown mechanism(s). Phosphorylation of this latter site is very sensitive to rapamycin and mTOR has been shown to directly phosphorylate this site in vitro. Evidence from the Bleuis laboratory suggests that phosphorylation of the hydrophobic motif site can be regulated by at least two mechanisms. The first mechanism is via autophosphorylation that requires PDK1 and PKC zeta inputs. The second mechanism requires mTOR function. However, it is difficult to determine whether mTOR has a direct positive input, or whether mTOR is inhibiting an S6K phosphatase. Indeed, evidence for both modes of mTOR action was presented. Structure/function analysis has revealed a small region within the N-terminal acidic domain of S6K that is critical for the mTOR input.
Rapamycin antagonizes both the increase in cell size (growth) and proliferation of U20S cells. Since mTOR regulates several downstream targets such as S6K1 and 4Е-ВР1, the Blenis group examined the contribution of S6K1 to the regulation of cell growth and proliferation. Using rapamycin-resistant alleles of mTOR and S6K1, plus overexpression of active S6K1, they found that expression of rapamycin-resistant S6K1 cannot aune rescue the inhibition of cell size increase or cell proliferation by rapamycin. S6K1 overexpression, however, is sufficient to increase cell size and provide a proliferative advantage to some сец types. Thus, S6K1, when other mTOR regulated processes are operating, does play an important role in the regulation of cell growth aid proliferation.
Joseph Avruch (Harvard Medical School, USA) kept the discussion focused on the р70 S6 kinase with a detailed presentation on the mechanisms regulating the phosphorylation and activation of р70 S6K. The р70 S6 kinase was one of the first insulin/mitogen activated protein ser/thr kinases to be discovered as well as perhaps the first molecular element in the TOR pathway of metazoans. Although the specific cellular roles of this kinase are stil incоmpletely understood, it appears to provide a point of convergence for the control of mRNA translation and cell growth in response to nutrients and mitogenic signals. The p70 S6 kinase is activated through a complex sequence of phosphоrylations catalyzed by several different kinases, and the sequential action of each is required to achieve significant activity. Inhibition of TOR with rapamyсin causes a very rapid reversal of p70 S6K activity by all stimuli, but this inhibition can be overcome by a recombinant TOR that does rot bind the rapamycin/FKBP complex. Rapаmycin causes the dephosphorylation of many p70 S6K sites, but dephosphorylation of a single site (Threonine 412 in p70 S6K alphal) corresponds most closely in time with the rapamyсin-induced deactivation of p70 S6K. Threonine 412 (Thr412) is phosphorylated in an insulin-sensitive, PI 3-kinase dependent manner in vivo. mTOR can directly phosphorylate this site in vitro, concomitant with p70 S6K activation. However mTOR does rot appear to be the insulin-directed kinase acting on this site in vivo, as a mutant of p70 S6K that is insensitive to inhibition by rapamycin continues to exhibit insulinstimulated, wortmannin sensitive phosphorylation of this site in vivo in the presence of high concentrations of rapamycin. This suggests that mTOR controls Thr412 phosphorylation indirеctly, through negative regulation of a protein phosphatase. However, direct evidence for TOR regulated phosphatases in mammalian cells is scant. The Avruch laboratory has recently identified a protein kinase capable of phosphorylating p70 S6K Thr 412 in vivo and in vitro; its physiologic relevance remains to be defined.
Dr. Avruch then turned to the regulation of mTOR kinase activity, a topic on which little information is currently available. mTOR kinase can be potently activated by CNTF (сiliary neurotrophic factor) in a marner that suggests covalent modification occurring in vivo; whether this is dependent on the elements of the PI 3 kinase pathway is unknown. The phosphorylation of p70 S6K and 4Е-BP are controlled by amino acid suшсiency and rapamycin in a parallel marner, suggesting that mTOR activity mаy be controlled by amino acid sufficiency; however, direct evidence in support of this conclusion is lacking.
Kazuyoshi Yonezawa (Kobe University, Japan) shifted the emphasis to a structure/fonction analysis of mTOR. Although mTOR is a member of a phosphatidylinositol-kinase (PIK)-related kinase family, kinase activities associated with mTOR are mainly serine-threonine kinase activities. Interestingly, these serine-threonine kinase activities associated with immunopurified mTOR appear to show different detergent sensitivity and include several activities, winch exhibit different substrate specificities. Those kinase activities are 1) autophosphorylation activity (site: HSF); 2) 4E-ВР kinase activity (sites: S/T-P, detergent-sensitive); 3) p70 S6K kinase activities (sites: FTY and S/T–P). Further study of mTORassociated proteins should clarify why immunopurified mTOR has multiple kinase activities.
Nahum Sonenberg (МcGill University, Quebec) opened his talk with a review on the initiation of translation and the role of the 4E-BPs. In most instances, translation is regulated at the initiation phase, when a ribosome is recruited to the 5′ end of an mRNA. The eIF-4E-binding proteins (4E-BPs) interdict translation initiation by binding to the translation factor eIF-4E, and preventing recruitment of the translation machinery to mRNA. The 4E-BPs inhibit translation in a reversible manier. Нypophosphorylated 4E-BPs interact avidly with eIF-4E, whereas 4E-BP hyperphosphorylation, elicited by stimulation of cells with hormones, cytokines, or growth factors, results in an abrogation of eIF-4E binding activity. The Sonenberg laboratory previously reported that phosphorylation of 4E-BP1 on Thr37 and Thr46 is relatively insensitive to serum deprivation and rapamycin treatment, and that phosphorylation of these residues is required for subsequent phosphorylation of a set of unidentified serum-responsive sites. Using mass spectrometry, thеу identify the serum-responsive, rapamycin-sensitive sites as Ser65 and Thr70. Utilizing a novel combination of two-dimensional isoelectric focusing/SDS-PAGE and Western blotting with phosphospecific antibodies, thеу also establish the order of 4E-ВР1 phosphorylation in vivo; phosphorylation of Thr37/Thr46 is followed by Thr70 phosphorylation, and Ser65 is phosphorylated last. Finally, thеу demonstrate that phosphorylation of Ser65 and Thr70 aloe is insufficient to mediate eIF-4E release, indicating that a combination of phosphorylation events is necessary to dissociate 4Е-ВР1 from eIF-4E.
Dr. Sonenberg followed the work on mammalian 4Е-BP1 with a presentation on a protein of similar function inDrosophila melanogaster. Не showed that the Drosophile 4Е-BP (д4Е-ВР) is a downstream target of the PI3Kinase (PI3K) signal transduction cascade, which affects d4Е-ВР interaction with eIF-4E. Ectopic expression of a highly active mutant д4Е-ВР protein in wing imaginal discs results in reduced size, brought about by a decrease in cell size and number. A marked reduction in cell size was also observed in post-mitotic cells. Expression of d4Е-BР in the еуе and wing together with PI3K or dAkt1 resulted in suppression of the growth phenotype elicited by these kinases. Taken together, their results support a role for д4Е-ВР as a regulator of cell growth.
David Sabatini (Whitehead Institute, USA) ended the dау with a talk discussing work on the mammalian TOR pathway. Dr. Sabatini is seeking to identify (1) the intracellular signals that normally regulate mTOR and (2) the critical function of the mTOR pathway in cell proliferation. Using oligonucleotide DNA microarrays that simultaneously detect the levels of thousands of different mRNAs, the Sabatini group identified the genes affected by rapamycin treatment of human cells. The set of rapamycin-sensitive genes shows remarkable overlap with the set of genes regulated by nutrient/energy withdrawal, particularly deprivation of glutamine. However, the microarray data suggest that mTOR is fit sensing glutamine per se but rather a metabolite that requires mitochon- drial action for its generation. Не proposed that the normal function of the mTOR pathway is coordinating cell growth (i.e. accumulation of cell mass) to available energy. Inhibition of the pathway by rapamycin leads to a significant decrease in cell size with a corresponding reduction in cell mass. Because most cellular growth occurs during the G1 phase of tEe cell cycle, this тау explain why rapamycin leads to a preferential dеlау in this phase of the cell cycle.
Day 3: Medical importance of the TOR pathway
John C. Lawrence, Jr. (University of Virginia, USA) spoke on the role of mTOR signaling in insulin action. Не reviewed how the identification of adipocyte proteins that are phosphorylated in response to insulin led to the discovery of at least two important downstream elements of the mTOR signaling pathway, PHAS-I (also known as 4Е-ВР) and lipin. PHAS-I is an important regulator of mRNA translation. Nonphosphorylated PHАS-I binds tightly to eIF-4E, the mRNA capbinding protein, and blicks translation. When phosphorylated in the appropriate sites, PHAS-Idissociates from eIF-4E, resulting in an increase in cap-dependent translation. The control of PHАS-I phosphorylation by insulin is complex, involving at least five sites and an ordered mechanism. Lipin is the protein whose mutation in the mouse results in the fatty liver dystrophy (fld) phenotype. Among the defects exhibited by fld mice are slow growth, abnormal аdiроcytе development, and hyperlipidemia. Interestingly, these defects resemble effects produced by treating animais with rapamycin. How mTOR signaling is coupled to PHAS- I and lipin phosphorylation has not been established, but it requires the kinase function of mTOR. PHАS-I mау be directly phosphorylated by mTOR, providing a useful biochemical assay for assessing changes in mTOR activity. Insulin treatment of cells increases the Ser/Thr kinase activity of mTOR assayed in vitro using recombinant PHAS-I as a sub-strate. The activation of mTOR is associated with the phosphorylation of Ser2448, which is located in a 9 amino acid insert not found in the yeast TOR proteins. Binding of the antibody mTAbl to this region of mTOR also increases mTOR activity several fold, and after activation with mTAbl, mTOR phosphorylates nit only Thr36 aid Thr45, but also Thr69 and Ser64 of PHAS-I (the numbering is different from that used by the Sonenberg laboratory because the rat protein, PHAS-I, has one less amino acid than the human protein, 4Е-ВР). Phosphorylation of these sites is markedly attenuated by rapamycin-FKBP12, but not by FK506-FKBP12. A mutant recombinant mTOR lacking the mTAbl epitope also exhibited increased activity toward these four phosphorylation sites. Thus, the mTAbl epitope appears to represent a regulatory domain whose function is potentially modulated by phosphorylation. It is well established that mutation of Ser2035 to I1e decreases the affinity of mTOR for rapamycin-FKBP12. Unexpectedly, this mutation also had a marked effect on the sites in PHAS-I phosphorylated by activated mTOR. Namely, the rate of phosphorylation of Thr36 and Thr45 was reduced, and the phosphorylation of Thr69 was increased. These results suggest that the rapamycin-binding domain influences substrate recognition. The results also cast a cautionary note on the use of this mutation in overexpression studies to investigate the effects of mTOR incells.
Edouard Battegay (University of Basel Medical School, Switzerland) followed with a presentation on the role of the mTOR pathway in angiogenesis and vascular wall proliferation. Angiogenesis, the formation of new vessels from existing ones, promotes wound healing, repair processes and diseases. Angiogenesis is associated with chronic inflammation or, in other instances, serves to preserve tissues that are vitally threatened by hypoxia. Hypoxia and other angiogenic stimuli induce outgrowth of new vessels via upregulation and release of a number of molecules including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF). PDGF is a hypoxia-driven gene that induces the formation of a vascularized connective tissue stroma in different diseases. The Battegay group used two- and three dimensional models to characterize the role of PDGF during angiogenesis in vitro; PDGF receptors have been found to be associated with an angiogenic phenotype of endothelial cells and to mediate endothelial proliferation, migration, and angiogenesis in vitro. Recently, thеу found that the proliferative response of endothelial cells, smooth muscle cells, and angiogenesis in vitro to PDGF is enhanced by hypoxia. Оnlу the enhanced proliferative response of the vascular wall cells and angiogenesis in vitro can be specifically inhibited by low concentrations of rapamycin. Thus, an mTOR driven signaling pathway additionally enhances the proliferative response of vascular wall cells (endothelial cells, smooth muscle cells) and angiogenesis in vitro to PDGF- and other angiogenic growth factors such as FGF during hypoxia.
Peter Houghton (St. Jude Children’s Research Hospital, USA) focused on the medical uses of rapamycin, particularly the consequences of inhibiting mTOR signaling in rhabdomyoblasts. Signaling through the insulin-like growth factor (IGF) axis becomes deregulated in many tumors. In some tumor cells the type I IGF receptor is overexpressed, whereas in other tumors IGF-II is secreted to yield autocrine growth. Rhabdomyosarcoma cells secrete IGF-II, and inhibition of the IGF-I receptor reduces growth and tumorgenicity. The macrocyclic lactone rapamycin potently inhibits IGF-I signaling, and suppresses growth of these cells. Interestingly, under conditions of autocrine growth (serumfree) rapamycin induces significant apoptosis rather than cytostasis in tumor cells lacking р53 tumor suppressor function. Expressing р53 or the cyclin-dependent kinase inhibitor р21сip1 arrests cell growth in the G1 phase of the cell cycle and completely protects cells from rapamycin-induced apoptosis. Rapamycin-induced apoptosis appears to be a consequence of incomplete G1 arrest in cells with mutant р53, raising the suggestion that normally р53 seises mTOR inhibition and reinforces G1 biock in rapamycin treated cells. Studies with murine еmbryо fibroblasts (MEFs) support this model. MEFs nullizygous for р53 or р21сiр1 progress through G1 in the presence of rapamycin and die. In contrast wild type cens arrest in G1 aid remain viable. These results suggest that loss of р53 mау result in a synthetic lethal interaction when mTOR is inhibited by rapamycin.
Paradoxically, rapamycin-induced apoptosis is completely inhibited by exogenous IGF-I or insulin. In rhabdomyosarcoma cells, IGF-I activation of MAP kinases (р44/42 Erk1/2) is inhibited by rapamycin. Current results indicate that rapamycin treatment prevents activation by inhibit- ing phosphorylation of Thr202 of р44/42. The rapamycin block is reversed by inhibitors of protein phosphatase 2A (РР2А). Data support a model in which IGF-I activates mTOR which then suppresses РР2А associated with p44/42. This allows phosphorylation of both Thr202 and Tyr204, facilitating full activation of these МАР kinases. The dornains of mTOR required to inhibit РР2А activity in these cells is being further investigated.
Kim Arndt (Wyeth-Ayerst Research, USA) presented the role of TAP42/alpha4 in the TOR pathway in both yeast and mammalian cells. In budding yeast, ТАР42 interacts with all type 2A (РР2А) and type 2A related (SIT4) protein phosphatases. For РР2А, ТА242 competes with the A and B subunits for binding to the РР2А catalytic C subunits РР121 and РР122. Likewise for SIТ4, TАР42 competes with the SAP subunits for binding to the catalytic subunit. TAP42/PPase complexes are regulated by the growth status of the cell; glucose imitation, nitrogen limitation, or addition of rapamycin result in dissociation of the complex. Genetic analysis has indicated that TAP42/PPxse complexes perform some fonction that is required, directly or indirесtly, for translation. Dr. Arndt proposed a model in which SIT4/SAP complexes have a unique late G1 fonction required for execution of Start, while PPH/A/B complexes have a unique function required for progression from G2 roto mitosis. Нowevеr, a strain temperature sensitive for SIT4 and РР2А catalytiс subunit fonction arrests in a nutrient limited GO-like state which suggests a shared GO function for SIT4 and РР2А. Because temperature sensitive ТАР42 mutants also arrest in a GO-like state, aid because ТАР42 forms complexes with SIT4 and all РР2А catalytic subunits, Dr. Arndt proposed that TAP42/РРasе complexes mediate some positive function that is required to process nutrient growth signals roto downstream events that are required for entry roto the cell division cyсlе. The requirement of TOR fonction in the formation of TAP42/PPases complexes suggests that TAP42/PPases complexes are downstream of TOR in tins process.
In mammalian cells, the TAP42-rеlated protein alpha4 also binds to type 2A and type 2A-related PPase catalytic subunits. In cells that are sensitive to rapamycin (such as Jurkat cells), alpha4/PPase complexes are completely dissociated by addition of rapamycin. However, while Тар42/PPase complexes in yeast dissociated in about 15 minutes after rapamycin addition, alpha4/PPase complexes take about 12 hours to dissociate after rapamycin addition. Ву contrast, the phosphorylation of р70 S6 kinase and 4Е-ВР is altered by rapamycin within 10 minutes. Currently, the Arndt group is comparing the mammalian alpha4/PPase systems to the yeast Тар42/PPase system and their regulation by TOR.
Day 4: Drosophila, plants and neuronal roles of the TOR pathway.
The next morning the emphasis of meeting shifted away from mammalian systems to the TOR pathway in Drosophile. Ernst Hafen (University of Zürich, Switzerland) opened with a talk on insulin signaling aid growth control in Drosophile. Loss-of-fonction mutations in genes coding for components of this insulin signaling pathway in Drosophile, including the insulin receptor homologue, Chiсo/IRS, P13 K, and PKB, affect growth by reducing cell size and сеll number without interfering with cell survival or differentiation. Flies mutant for dS6K also have smaller cens but the cell number appears constant. In a saturation screen for mutations affecting irgan growth using tissue specific recombination systems, the Hafen group identified a nimber of new genes in the Drosophile genome that are seemingly dedicated to cell growth. These genes include dPTEN, ТSC1 and TSC2, homologues of the mammalian tumor suppressor genes, as well as dTOR. Loss of dTOR fonction is lethal and causes a severe reduction in cell size. S6К is flot activated in dTOR mutant larvae. Partial lors-of-fonction mutations in dTOR elicit a phenotypе that it different from alterations in the РI3К/РКВ pathway. In dTOR mutant larvae, the endoreplicating tissue is severely reduced at the expense of the diploid adult tissue, while in РI3K mutants both tissues are reduced proportionally. This dTOR mutant phеnotypе resembles that of amino acid starved larvae and is thus consistent with a model that dTOR acts as an amino acid sensor also in Drosophile. Surprisingly, S6K activity in larval extracts depends on dTOR and dPDK1 activity but is independent of PI3K and Chico. These results suggest that S6K receives input from dTOR and dPDK1 even in the absence of stimulation of PI3K by the insulin pathway.
Thomas P. Neufeld (University of Minnesota, USA) followed with a talle on the roles of TOR in PI3K signaling and nutrient sensing during Drosophile development. Studies of mTOR/FRAP in higher eukaryotes have been limited by their reliance on rapamуcin, which inhibits TOR activity only partiаlly and by an unknown mechanism. Furthermore, such studies have not addressed the in vivo fonctions of TOR nor its roles during animal development. The Neufeld laboratory has generated complete loss-of-function mutations in the Drosophile TOR homologue, dTOR, and is using these mutants to understand the normal cellular fonctions of dTOR. The dTOR protein is 56% identical to human mTOR, with the highest levels of conservation in the kinase and FRB domains. The in vitro kinase activity of dTOR is inhibited by rapamyсin, and files heterozygous for dTOR mutations show hypersensitivity to rapamycin. Homozygous mutant Oies lacking ail dTOR protein suffer a growth arrest during еarly larval development. FACS analysis of dTOR mutant cells reveal a severe reduction in cell size, and an accumulation of cells in the G1 phase of the cell cycle. However, dTOR is not required for cell viability nor for differentiation, as mosaic clones of mutant cells can survive and generate normal (albeit small) adult structures.
To address the role of dTOR in relation to other growth pathways, the Neufeld group combined dTOR mutations with mutations affecting other signaling molecules. dTOR mutations completely suppress the overgrowth phenotype caused by loss of fonction mutations in dPTEN. In contrast, overexpression of dМyc or activated alleles of dS6K, but not PI3K or Ras, can bypass the growth arrest of dTOR mutants. Indeed, dTOR hypomorphic mutants can be rescued to viability by overexpression of dS6K oncles containing acidic residues at either Т389 (same as Т412 according to the numbering from the Avruch laboratory) or in the autoinhibitory domain. Thus, dS6K is a major, relevant target of dTOR activity. Finally, dTOR mutants display a number of phenotypes remarkаbly similar to those caused by starvation for amino acids. Both starvation and dTOR mutation cause a rapid сеll cycle arrest in endoreplicating cells, while diploid cells such as neuroblasts continue to cycle. Furthermore, both conditions result in a two-fold reduction in nucleolar size, and cause aggregation of lipid vesicles in the larval fat body. Together, these findings suggest dTOR functions at the interface of growth factor and nutrient signaling.
Christophe Robaglia (СЕA Centre de Cadarache, France) presented work on the fonction eIF-4E translational regulators and TOR in the plant Arаbidopsis thalianа. Completion of the A. thаliаnа genome sequence revealed four genes encoding eIF-4ES. Expression analysis of two of them, encoding eIF-4E1 and eIF(iso)4Е, showed that thеy are differentially expressed during plant development. However, transposon inactivation of the gene encoding eIF(iso)4Е did not lead to any discernible phenotypе, suggesting that this gene is dispensable for normal growth. A search for eIF-4E binding proteins identified a type 2 lipoxygenase that can bind both eIF-4E and eIF(iso)4E in vivo and in vitro.
The A. thaliana homolog of the mammalian and yeast TOR genes was identified and characterized as a single, large gene located in chromosome 1. Sinсe plants are insensitive to rapamyсin, the capacity of the encoded protein to bind FKBP proteins in the presence of rapamycin was investigated. This showed that the FRB domain of AtTOR can form a ternary complex with Saccharomyces cerevisiаe FKBP and rapamyсin, but not with a plant FKBP, demonstrating that AtTOR is structurally related to other eukaryotic TOR proteins for rapamyсin binding. Searches for AtTOR knock-out alleles was undertaken using the INRA T-DNA insertion library Two independent insertion events in AtTOR were identified, one of them bearing a translational fusion with the GUS reporter gene allowing visualization of the expression pattern of AtTOR. AtTOR is expressed in cens undergoing both cytoplasmic growth and division, but not in elongating mature cells of the leaf and root tissue. This contrasts with animals where TOR is expressed in all tissues. Plants homozygous for an AtTOR mutation arrest growth early during embryo development, without endosperm cellularization, at a stage where еmbryoniс cells undergo a transition toward an increase in cell mass. Cells in metaphases were still observed in the arrested embryos showing that AtTOR is flot required for division itself. In future studies, the Robaglia group will investigate links between AtTOR and other A. thаliаnа components of the eukаryоtiс TOR pathwаy, together with the plant specific signais acting upstream of TOR.
In the afternoon, the meeting concluded with two presentations on the role of TOR in the nervous system. Christoph M. Schuster (Friedrich-Miescher-Laboratorium der Max-Planck, Tübingen, Germany) presented a potential role of TOR in experience-dependent regulation of subsynaptic translation in long-term junctional plasticity of Drosophila. Hе showed that the development of neural communication involves genetically programmed processes that lead to the formation of a complex pattern of neuronal connections. In addition, neuronal firing activity appears to remodel these initial synaptic connections resulting in long-lasting alterations of neuronal connectivity and communication. As a model system to analyze the molecular mechanisms of such activity dependent plasticity phenomena, the Schuster laboratory uses the developing larval neuromuscular junction (NMJ). They initiated a systematic mutant screen to identify genes involved in aсtivity-dépendent long-term strengthening of junctional signal transmission. This screen identified several genes encoding components involved in the regulation of mRNA translation, such as eIF4e and PABP (polу(A)-binding protein). Subséquent experiments showed that locahzed subsynaptic translation of specifically stored mRNAs results in local signals at synapses that control glutamate receptor synthesis and trafficking, junctional physiology and morphological growth. Thus, the regulation of subsynaptic translation appears to be a critical parameter in the control of physiological and morphological alterations at developing NMJs. This concept of localized subsynaptic translation and its consequences is therefore well suited to mediate specific functional and structural changes in particular in such corplex environments as neurons within the central nervous system. Some of the current work in the Schuster laboratory focuses on the signaling pathways that lead to an increased occurrence of subsynaptic translation aggregates upon elevated synaptic stimulation. Dr. Schuster mentioned that preliminary evidence implicates both insulin signaling as well as TOR signaling in the regulation of subsynaptic translation.
Kausik Si from the Kandel Laboratory (Columbia University, USA) ended the meeting with a presentation on local protein synthesis and synapse-specific long-term facilitation in Aplysia. Synaptic plasticity, the ability of neurons to modulate their synaptic strength in response to extra- or intracellular ones, is thought to be a common mechanism for behavioral learning and memory storage. Like behavioral memory, synaptic plasticity has at least two temporally distinct forms, a short-term form lasting minutes, and a long-term form that lasts for days and weeks. This temporal distinction is reflected in the molecular requirement for the formation of these two forms of memories. The short-term form does not require protein synthesis, whereas the long-term form requires synthesis of new protein and mRNA, and establishment of new connections. The requirement of transcription for the long-term plasticity raises the question: is long-term memory cell-wide or is it synapse-specific?
Long-term facilitation (LTF) in the connections between Aplysiа sensory and motor neurons is synapse-specific and can be captured through the “marking” of the activated synapse. Synapse-specific long-term facilitation requires CREB-dependent transcription and local protein synthesis, leading to the growth and maintenance of stable new connections. At least two requirements of local protein synthesis have been observed: 1) a rapamycin-insensitivе component nеcessary for the retrograde signal to the nucleus and 2) a rapamyсin-sensitive component necessary to mark the activated synapse for growth. The Kandel laboratory asked: what is the nature of the rapamycin-sensitive local protein synthеsis-dependent “mark” necessary for stabilization of synaptic growth?
Work on early development has revealed that translation of some mRNAs can be activated by increasing the length of the polу(A) tait. This poly(A)-dependent translation is regulated by a cytoplasmic polyadenylation element (СРЕ) binding protein, CPEB. CPEB is an attractive candidate as a synapse-specific mark and a local regulator of translation because it 1) is activated through an extracellular signal, 2) activates translationally dormant mRNAs, 3) is spatially restricted, and 4) targets growth related mRNAs. Indeed, The Kandel laboratory has found that СРЕВ is present in the Aplysiа sensory neuron and that the protein is induced by serotonin following learning-related stimulation. This induction of СРЕВ is independent of transcription but requires new protein synthesis that is sensitive to rapamycin. The СРЕВ protein can be induced in isolated neurites. The induction of СРЕВ in turf results in the polyadenylation of neuronal aсtin, which is needed for synaptiс growth. Blocking CPEB blocks the growth-dependent late-phase of long-term plasticity. Thus СРЕВ is most likely one of the components of the rapamycin sensitive “mark”. These findings also suggest that rapamycin can influence neuronal plasticity and thus certain forms of mеmory.
The variety and richness of the topics touched on by the speakers over four days underscored the fundamentally important role of TOR in the biology of the cеll. However, manу basic questions concerning TOR structure, fonction and regulation remain to be answered. Given the high level of enthusiasm at the meeting and a growing interest in the field, more TOR meetings will be held to address these open questions. The organizers and speakers thank the Fondation des Treilles for supporting and hosting the seminal meeting of the TOR field.