Target Of Rapamycin Complex 1 Inhibition Research Articles

The PP242 Mammalian Target of Rapamycin (mTOR) Inhibitor Activates Extracellular Signal-regulated Kinase (ERK) in Multiple Myeloma Cells via a Target of Rapamycin Complex 1 (TORC1)/ Eukaryotic Translation Initiation Factor 4E (eIF-4E)/RAF Pathway and Activation Is a Mechanism of Resistance

Activation of PI3-K-AKT and ERK pathways is a complication of mTOR inhibitor therapy. Newer mTOR inhibitors (like pp242) can overcome feedback activation of AKT in multiple myeloma (MM) cells. We, thus, studied if feedback activation of ERK is still a complication of therapy with such drugs in this tumor model. PP242 induced ERK activation in MM cell lines as well as primary cells. Surprisingly, equimolar concentrations of rapamycin were relatively ineffective at ERK activation. Activation was not correlated with P70S6kinase inhibition nor was it prevented by PI3-kinase inhibition. ERK activation was prevented by MEK inhibitors and was associated with concurrent stimulation of RAF kinase activity but not RAS activation. RAF activation correlated with decreased phosphorylation of RAF at Ser-289, Ser-296, and Ser-301 inhibitory residues. Knockdown studies confirmed TORC1 inhibition was the key proximal event that resulted in ERK activation. Furthermore, ectopic expression of eIF-4E blunted pp242-induced ERK phosphorylation. Since pp242 was more potent than rapamycin in causing sequestering of eIF-4E, a TORC1/4E-BP1/eIF-4E-mediated mechanism of ERK activation could explain the greater effectiveness of pp242. Use of MEK inhibitors confirmed ERK activation served as a mechanism of resistance to the lethal effects of pp242. Thus, although active site mTOR inhibitors overcome AKT activation often seen with rapalog therapy, feedback ERK activation is still a problem of resistance, is more severe than that seen with use of first generation rapalogs and is mediated by a TORC1- and eIF-4E-dependent mechanism ultimately signaling to RAF.

The TEA Transcription Factor Tec1 Links TOR and MAPK Pathways to Coordinate Yeast Development

In Saccharomyces cerevisiae, the TEA transcription factor Tec1 controls several developmental programs in response to nutrients and pheromones. Tec1 is targeted by the pheromone-responsive Fus3/Kss1 mitogen-activated protein kinase (MAPK) cascade, which destabilizes the transcription factor to ensure efficient mating of sexual partner cells. The regulation of Tec1 by signaling pathways that control cell division and development in response to nutrients, however, is not known. Here, we show that Tec1 protein stability is under control of the nutrient-sensitive target of rapamycin complex 1 (TORC1) signaling pathway via the Tip41-Tap42-Sit4 branch. We further show that degradation of Tec1 upon inhibition of TORC1 by rapamycin does not involve polyubiquitylation and appears to be proteasome independent. However, rapamycin-induced Tec1 degradation depends on the HECT ubiquitin ligase Rsp5, which physically interacts with Tec1 via conserved PxY motives. We further demonstrate that rapamycin and mating pheromone control Tec1 protein stability through distinct mechanisms by targeting different domains of the transcription factor. Finally, we show that Tec1 is a positive regulator of yeast chronological lifespan (CLS), a known TORC1-regulated process. Our findings indicate that in yeast, Tec1 links TORC1 and MAPK signaling pathways to coordinate control of cellular development in response to different stimuli.

WIPI1 Coordinates Melanogenic Gene Transcription and Melanosome Formation via TORC1 Inhibition

Recent studies implicate a role for WD repeat domain, phosphoinositide-interacting 1 (WIPI1) in the biogenesis of melanosomes, cell type-specific lysosome-related organelles. In this study, we determined that WIPI1, an ATG18 homologue that is shown to localize to both autophagosomes and early endosomes, inhibited mammalian target of rapamycin (MTOR) signaling, leading to increased transcription of melanogenic enzymes and the formation of mature melanosomes. WIPI1 suppressed the target of rapamycin complex 1 (TORC1) activity, resulting in glycogen synthase kinase 3β inhibition, β-Catenin stabilization, and increased transcription of microphthalmia transcription factor and its target genes. WIPI1-depleted cells accumulated stage I melanosomes but lacked stage III-IV melanosomes. Inhibition of TORC1 by rapamycin treatment resulted in the accumulation of stage IV melanosomes but not autophagosomes, whereas starvation resulted in the formation of autophagosomes but not melanin accumulation. Taken together, our studies define a distinct role for WIPI1 and TORC1 signaling in controlling the transcription of melanogenic enzymes and melanosome maturation, a process that is distinct from starvation-induced autophagy.

Identification of Ypk1 as a Novel Selective Substrate for Nitrogen Starvation-triggered Proteolysis Requiring Autophagy System and Endosomal Sorting Complex Required for Transport (ESCRT) Machinery Components

Nitrogen starvation-mediated reduction of Ypk1 is suggested to suppress translational initiation, possibly in parallel with the target of rapamycin complex 1 (TORC1) signaling. However, the molecular mechanism that regulates Ypk1 in nitrogen-starved cells is poorly understood. Here we report that Ypk1 is a novel selective substrate for nitrogen starvation-triggered proteolysis requiring autophagy system. Among various nutrient starvation methods used to elicit autophagy, rapid Ypk1 degradation was specific to nitrogen starvation. In screening genes required for such nitrogen starvation-specific vacuolar proteolysis, we found that autophagy-related degradation of Ypk1 depended on the endosomal sorting complex required for transport (ESCRT) machinery, which is conventionally thought to function in endosomal trafficking. In microscopic analyses, the disruption of ESCRT subunits resulted in the accumulation of both Ypk1 and autophagosomal Atg8 at a perivacuolar site that was distinct from conventional endosomes. ESCRT machinery was not involved in autophagic flux induced by the TORC1 inhibitor rapamycin, thus suggesting that ESCRT represents an exclusive mechanism of nitrogen starvation-specific proteolysis of Ypk1. Overall, we propose a novel regulation of Ypk1 that is specific to nitrogen limitation.

Rapamycin increases rDNA stability by enhancing association of Sir2 with rDNA in Saccharomyces cerevisiae

The target of rapamycin (TOR) kinase is an evolutionarily conserved key regulator of eukaryotic cell growth and proliferation. Recently, it has been reported that inhibition of TOR signaling pathway can delay aging and extend lifespan in several eukaryotic organisms, but how lifespan extension is mediated by inhibition of TOR signaling is poorly understood. Here we report that rapamycin treatment and nitrogen starvation, both of which cause inactivation of TOR complex 1 (TORC1), lead to enhanced association of Sir2 with ribosomal DNA (rDNA) in Saccharomyces cerevisiae. TORC1 inhibition increases transcriptional silencing of RNA polymerase II-transcribed gene integrated at the rDNA locus and reduces homologous recombination between rDNA repeats that causes formation of toxic extrachromosomal rDNA circles. In addition, TORC1 inhibition induces deacetylation of histones at rDNA. We also found that Pnc1 and Net1 are required for enhancement of association of Sir2 with rDNA under TORC1 inhibition. Taken together, our findings suggest that inhibition of TORC1 signaling stabilizes the rDNA locus by enhancing association of Sir2 with rDNA, thereby leading to extension of replicative lifespan in S. cerevisiae.

Stressin' Sestrins take an aging fight

Sestrins (Sesns) are a family of highly conserved stress-responsive proteins, transcriptionally regulated by p53 and forkhead transcription factor that exhibit oxidoreductase activity in vitro and can protect cells from oxidative stress. However, their major biochemical and physiological function does not appear to depend on their redox (reduction and oxidation) activity. Sesns promote activation of adenosine-5′-monophosphate (AMP)-dependent protein kinase in both mammals and flies. Stress-induced Sesn expression results in inhibition of the target of rapamycin complex 1 (TORC1) and the physiological and pathological implications of disrupting the Sesns-TORC1 crosstalk are now being unravelled. Detailing their mechanism of action and exploring their roles in human physiology point to exciting new insights to topics as diverse as stress, cancer, metabolism and aging.

The Rapamycin-sensitive Phosphoproteome Reveals That TOR Controls Protein Kinase A Toward Some But Not All Substrates

Regulation of cell growth requires extensive coordination of several processes including transcription, ribosome biogenesis, translation, nutrient metabolism, and autophagy. In yeast, the protein kinases Target of Rapamycin (TOR) and protein kinase A (PKA) regulate these processes and are thereby the main activators of cell growth in response to nutrients. How TOR, PKA, and their corresponding signaling pathways are coordinated to control the same cellular processes is not understood. Quantitative analysis of the rapamycin-sensitive phosphoproteome combined with targeted analysis of PKA substrates suggests that TOR complex 1 (TORC1) activates PKA but only toward a subset of substrates. Furthermore, we show that TORC1 signaling impinges on BCY1, the negative regulatory subunit of PKA. Inhibition of TORC1 with rapamycin leads to BCY1 phosphorylation on several sites including T129. Phosphorylation of BCY1 T129 results in BCY1 activation and inhibition of PKA. TORC1 inhibits BCY1 T129 phosphorylation by phosphorylating and activating the S6K homolog SCH9 that in turn inhibits the MAP kinase MPK1. MPK1 phosphorylates BCY1 T129 directly. Thus, TORC1 activates PKA toward some substrates by preventing MPK1-mediated activation of BCY1.

Mammalian Target of Rapamycin Signaling and Autophagy: Roles in Lymphangioleiomyomatosis Therapy

The pace of progress in lymphangioleiomyomatosis (LAM) is remarkable. In the year 2000, TSC2 gene mutations were found in LAM cells; in 2001 the tuberous sclerosis complex (TSC) genes were discovered to regulate cell size in Drosophila via the kinase TOR (target of rapamycin); and in 2008 the results were published of a clinical trial of rapamycin, a specific inhibitor of TOR, in patients with TSC and LAM with renal angiomyolipomas. This interval of just 8 years between a genetic discovery for which the relevant signaling pathway was as yet unknown, to the initiation, completion, and publication of a clinical trial, is an almost unparalleled accomplishment in modern biomedical research. This robust foundation of basic, translational, and clinical research in TOR, TSC, and LAM is now poised to optimize and validate effective therapeutic strategies for LAM. An immediate challenge is to deduce the mechanisms underlying the partial response of renal angiomyolipomas to rapamycin, and thereby guide the design of combinatorial approaches. TOR complex 1 (TORC1), which is known to be active in LAM cells, is a key inhibitor of autophagy. One hypothesis, which will be explored here, is that low levels of autophagy in TSC2-null LAM cells limits their survival under conditions of bioenergetic stress. A corollary of this hypothesis is that rapamycin, by inducing autophagy, promotes the survival of LAM cells, while simultaneously arresting their growth. If this hypothesis proves to be correct, then combining TORC1 inhibition with autophagy inhibition may represent an effective clinical strategy for LAM.

Mammalian Target of Rapamycin Complex 1 Suppresses Lipolysis, Stimulates Lipogenesis, and Promotes Fat Storage

OBJECTIVEIn metazoans, target of rapamycin complex 1 (TORC1) plays the key role in nutrient- and hormone-dependent control of metabolism. However, the role of TORC1 in regulation of triglyceride storage and metabolism remains largely unknown.RESEARCH DESIGN AND METHODSIn this study, we analyzed the effect of activation and inhibition of the mammalian TORC1 (mTORC1) signaling pathway on the expression of adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), lipolysis, lipogenesis, and lipid storage in different mammalian cells.RESULTSActivation of mTORC1 signaling in 3T3-L1 adipocytes by ectopic expression of Rheb inhibits expression of ATGL and HSL at the level of transcription, suppresses lipolysis, increases de novo lipogenesis, and promotes intracellular accumulation of triglycerides. Inhibition of mTORC1 signaling by rapamycin or by knockdown of raptor stimulates lipolysis primarily via activation of ATGL expression. Analogous results have been obtained in C2C12 myoblasts and mouse embryonic fibroblasts with genetic ablation of tuberous sclerosis 2 (TSC2) gene. Overexpression of ATGL in these cells antagonized the lipogenic effect of TSC2 knockout.CONCLUSIONSOur findings demonstrate that mTORC1 promotes fat storage in mammalian cells by suppression of lipolysis and stimulation of de novo lipogenesis.

The Vam6 GEF Controls TORC1 by Activating the EGO Complex

The target of rapamycin complex 1 (TORC1) is a central regulator of eukaryotic cell growth that is activated by a variety of hormones (e.g., insulin) and nutrients (e.g., amino acids) and is deregulated in various cancers. Here, we report that the yeast Rag GTPase homolog Gtr1, a component of the vacuolar-membrane-associated EGO complex (EGOC), interacts with and activates TORC1 in an amino-acid-sensitive manner. Expression of a constitutively active (GTP-bound) Gtr1(GTP), which interacted strongly with TORC1, rendered TORC1 partially resistant to leucine deprivation, whereas expression of a growth inhibitory, GDP-bound Gtr1(GDP), caused constitutively low TORC1 activity. We also show that the nucleotide-binding status of Gtr1 is regulated by the conserved guanine nucleotide exchange factor (GEF) Vam6. Thus, in addition to its regulatory role in homotypic vacuolar fusion and vacuole protein sorting within the HOPS complex, Vam6 also controls TORC1 function by activating the Gtr1 subunit of the EGO complex.

Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1

We have found earlier that Tor1 binds to 5S rDNA chromatin but the functional significance has not been established. Here, we show that association with 5S rDNA chromatin is necessary for TOR complex 1 (TORC1) to regulate the synthesis of 5S ribosomal RNA and transfer RNAs (tRNAs) by RNA polymerase (Pol) III, as well as the phosphorylation and binding to Pol III-transcribed genes of the Pol III repressor Maf1. Interestingly, TORC1 does not bind to tRNA genes, suggesting that TORC1 modulates tRNA synthesis indirectly through Maf1 phosphorylation at the rDNA loci. We also find that Maf1 cytoplasmic localization is dependent on the SSD1-v allele. In W303 cells that carry the SSD1-d allele, Maf1 is constitutively nuclear but its nucleolar localization is inhibited by TORC1, indicating that TORC1 regulates nucleoplasm-to-nucleolus transport of Maf1. Finally, we show that TORC1 interacts with Maf1 in vivo and phosphorylates Maf1 in vitro, and regulates Maf1 nucleoplasm-to-nucleolus translocation. Together, these observations provide new insights into the chromatin-dependent mechanism by which TORC1 controls transcription by Pol III.

Caffeine extends yeast lifespan by targeting TORC1

Dietary nutrient limitation (dietary restriction) is known to increase lifespan in a variety of organisms. Although the molecular events that couple dietary restriction to increased lifespan are not clear, studies of the model eukaryote Saccharomyces cerevisiae have implicated several nutrient-sensitive kinases, including the target of rapamycin complex 1 (TORC1), Sch9, protein kinase A (PKA) and Rim15. We have recently demonstrated that TORC1 activates Sch9 by direct phosphorylation. We now show that Sch9 inhibits Rim15 also by direct phosphorylation. Treatment of yeast cells with the specific TORC1 inhibitor rapamycin or caffeine releases Rim15 from TORC1-Sch9-mediated inhibition and consequently increases lifespan. This kinase cascade appears to have been evolutionarily conserved, suggesting that caffeine may extend lifespan in other eukaryotes, including man.