RESUMO
TOR protein kinases serve as the catalytic subunit of the TORC1 and TORC2 complexes, which regulate cellular growth, proliferation, and survival. In the fission yeast, Schizosaccharomyces pombe, cells lacking TORC2 or its downstream kinase Gad8 (AKT or SGK1 in human cells) exhibit sensitivity to a wide range of stress conditions, including DNA damage stress. One of the first responses to DNA damage is the phosphorylation of C-terminal serine residues within histone H2AX in human cells (γH2AX), or histone H2A in yeast cells (γH2A). The kinases responsible for γH2A in S. pombe are the two DNA damage checkpoint kinases Rad3 and Tel1 (ATR and ATM, respectively, in human cells). Here we report that TORC2-Gad8 signaling is required for accumulation of γH2A in response to DNA damage and during quiescence. Using the TOR-specific inhibitor, Torin1, we demonstrate that the effect of TORC2 on γH2A in response to DNA damage is immediate, rather than adaptive. The lack of γH2A is restored by deletion mutations of transcription and chromatin modification factors, including loss of components of Paf1C, SAGA, Mediator, and the bromo-domain proteins Bdf1/Bdf2. Thus, we suggest that TORC2-Gad8 may affect the accumulation of γH2A by regulating chromatin structure and function.
Assuntos
Dano ao DNA , Histonas , Alvo Mecanístico do Complexo 2 de Rapamicina , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Histonas/metabolismo , Histonas/genética , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/genética , Transdução de Sinais , Fosforilação , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Humanos , Proteínas Serina-Treonina QuinasesRESUMO
The fission yeast TOR complex 2 (TORC2) is required for gene silencing at subtelomeric regions and for the induction of gene transcription in response to DNA replication stress. Thus, TORC2 affects transcription regulation both negatively and positively. Whether these two TORC2-dependent functions share a common molecular mechanism is currently unknown. Here, we show that Gad8 physically interacts with proteins that regulate transcription, including subunits of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex and the BET bromodomain protein Bdf2. We demonstrate that in the absence of TORC2, Gcn5, the histone acetyltransferase subunit of SAGA, accumulates at subtelomeric genes and at non-induced promoters of DNA replication genes. Remarkably, the loss of Gcn5 in TORC2 mutant cells restores gene silencing as well as transcriptional induction in response to DNA replication stress. Loss of Bdf2 alleviates excess of Gcn5 binding in TORC2 mutant cells and also rescues the aberrant regulation of transcription in these cells. Furthermore, the loss of either SAGA or Bdf2 suppresses the sensitivity of TORC2 mutant cells to a variety of stresses, including DNA replication, DNA damage, temperature and nutrient stresses. We suggest a role of TORC2 in transcriptional regulation that is critical for gene silencing and gene induction in response to stress and involves the binding of Gcn5 to the chromatin.
Assuntos
Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Acetiltransferases/genética , Replicação do DNA/genética , Proteínas Fúngicas/genética , Heterocromatina/metabolismo , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
The Target of rapamycin (TOR) protein kinase forms part of TOR complex 1 (TORC1) and TOR complex 2 (TORC2), two multi-subunit protein complexes that regulate growth, proliferation, survival and developmental processes by phosphorylation and activation of AGC-family kinases. In the fission yeast, Schizosaccharomyces pombe, TORC2 and its target, the AGC kinase Gad8 (an orthologue of human AKT or SGK1) are required for viability under stress conditions and for developmental processes in response to starvation cues. In this study, we describe the isolation of gad8 mutant alleles that bypass the requirement for TORC2 and reveal a separation of function of TORC2 and Gad8 under stress conditions. In particular, osmotic and nutritional stress responses appear to form a separate branch from genotoxic stress responses downstream of TORC2-Gad8. Interestingly, TORC2-independent mutations map into the regulatory PIF pocket of Gad8, a highly conserved motif in AGC kinases that regulates substrate binding in PDK1 (phosphoinositide dependent kinase-1) and kinase activity in several AGC kinases. Gad8 activation is thought to require a two-step mechanism, in which phosphorylation by TORC2 allows further phosphorylation and activation by Ksg1 (an orthologue of PDK1). We focus on the Gad8-K263C mutation and demonstrate that it renders the Gad8 kinase activity independent of TORC2 in vitro and independent of the phosphorylation sites of TORC2 in vivo. Molecular dynamics simulations of Gad8-K263C revealed abnormal high flexibility at T387, the phosphorylation site for Ksg1, suggesting a mechanism for the TORC2-independent Gad8 activity. Significantly, the K263 residue is highly conserved in the family of AGC-kinases, which may suggest a general way of keeping their activity in check when acting downstream of TOR complexes.
Assuntos
Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Osmorregulação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiologia , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Sítios de Ligação/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Simulação de Dinâmica Molecular , Mutação , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
The target of rapamycin (TOR) protein kinase is at the core of growth factor- and nutrient-dependent signaling pathways that are well-known for their regulation of metabolism, growth, and proliferation. However, TOR is also involved in the regulation of gene expression, genomic and epigenomic stability. TOR affects nuclear functions indirectly through its activity in the cytoplasm, but also directly through active nuclear TOR pools. The mechanisms by which TOR regulates its nuclear functions are less well-understood compared with its cytoplasmic activities. TOR is an important pharmacological target for several diseases, including cancer, metabolic and neurological disorders. Thus, studies of the nuclear functions of TOR are important for our understanding of basic biological processes, as well as for clinical implications.
Assuntos
Epigenoma/genética , Serina-Treonina Quinases TOR/genética , Transcrição Gênica/genética , Núcleo Celular/genética , Humanos , Complexos Multiproteicos/genética , Transdução de Sinais/genéticaRESUMO
The evolutionarily conserved TOR complex 1 (TORC1) activates cell growth and proliferation in response to nutritional signals. In the fission yeast Schizosaccharomyces pombe, TORC1 is essential for vegetative growth, and its activity is regulated in response to nitrogen quantity and quality. Yet, how TORC1 senses nitrogen is poorly understood. Rapamycin, a specific TOR inhibitor, inhibits growth in S. pombe only under conditions in which the activity of TORC1 is compromised. In a genetic screen for rapamycin-sensitive mutations, we isolated caa1-1, a loss-of-function mutation of the cytosolic form of aspartate aminotransferase (Caa1). We demonstrate that loss of caa1+ partially mimics loss of TORC1 activity and that Caa1 is required for full TORC1 activity. Disruption of caa1+ resulted in aspartate auxotrophy, a finding that prompted us to assess the role of aspartate in TORC1 activation. We found that the amino acids glutamine, asparagine, arginine, aspartate, and serine activate TORC1 most efficiently following nitrogen starvation. The glutamine synthetase inhibitor l-methionine sulfoximine abolished the ability of asparagine, arginine, aspartate, or serine, but not that of glutamine, to induce TORC1 activity, consistent with a central role for glutamine in activating TORC1. Neither addition of aspartate nor addition of glutamine restored TORC1 activity in caa1-deleted cells or in cells carrying a Caa1 variant with a catalytic site substitution, suggesting that the catalytic activity of Caa1 is required for TORC1 activation. Taken together, our results reveal the contribution of the key metabolic enzyme Caa1 to TORC1 activity in S. pombe.
Assuntos
Aspartato Aminotransferases/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Mutação , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Arginina/farmacologia , Asparagina/farmacologia , Aspartato Aminotransferases/metabolismo , Ácido Aspártico/farmacologia , Citosol/enzimologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Isoenzimas/genética , Isoenzimas/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metionina Sulfoximina/farmacologia , Nitrogênio/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Sirolimo/farmacologiaRESUMO
BACKGROUND: Cellular quiescence is a reversible differentiation state during which cells modify their gene expression program to inhibit metabolic functions and adapt to a new cellular environment. The epigenetic changes accompanying these alterations are not well understood. We used fission yeast cells as a model to study the regulation of quiescence. When these cells are starved for nitrogen, the cell cycle is arrested in G1, and the cells enter quiescence (G0). A gene regulatory program is initiated, including downregulation of thousands of genes-for example, those related to cell proliferation-and upregulation of specific genes-for example, autophagy genes-needed to adapt to the physiological challenge. These changes in gene expression are accompanied by a marked alteration of nuclear organization and chromatin structure. RESULTS: Here, we investigated the role of Leo1, a subunit of the conserved RNA polymerase-associated factor 1 (Paf1) complex, in the quiescence process using fission yeast as the model organism. Heterochromatic regions became very dynamic in fission yeast in G0 during nitrogen starvation. The reduction of heterochromatin in early G0 was correlated with reduced target of rapamycin complex 2 (TORC2) signaling. We demonstrated that cells lacking Leo1 show reduced survival in G0. In these cells, heterochromatic regions, including subtelomeres, were stabilized, and the expression of many genes, including membrane transport genes, was abrogated. TOR inhibition mimics the effect of nitrogen starvation, leading to the expression of subtelomeric genes, and this effect was suppressed by genetic deletion of leo1. CONCLUSIONS: We identified a protein, Leo1, necessary for survival during quiescence. Leo1 is part of a conserved protein complex, Paf1C, linked to RNA polymerase II. We showed that Leo1, acting downstream of TOR, is crucial for the dynamic reorganization of chromosomes and the regulation of gene expression during cellular quiescence. Genes encoding membrane transporters are not expressed in quiescent leo1 mutant cells, and cells die after 2 weeks of nitrogen starvation. Taken together, our results suggest that Leo1 is essential for the dynamic regulation of heterochromatin and gene expression during cellular quiescence.
Assuntos
Heterocromatina/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fase de Repouso do Ciclo Celular/genética , Ciclo Celular/genética , Epigênese Genética , Regulação Fúngica da Expressão Gênica , Heterocromatina/genética , Histonas/metabolismo , Proteínas Nucleares/metabolismo , RNA Polimerase II/genética , Proteínas de Ligação a RNA/genética , Fase de Repouso do Ciclo Celular/fisiologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
The conserved serine/threonine protein kinase target of rapamycin (TOR) is a major regulator of eukaryotic cellular and organismal growth and a valuable target for drug therapy. TOR forms the core of two evolutionary conserved complexes, TOR complex 1 (TORC1) and TORC2. In the fission yeast Schizosaccharomyces pombe, TORC2 responds to glucose levels and, by activating the protein kinase Gad8 (an orthologue of human AKT), is required for well-regulated cell cycle progression, starvation responses, and cell survival. Here, we report that TORC2-Gad8 is also required for gene silencing and the formation of heterochromatin at the S. pombe mating-type locus and at subtelomeric regions. Deletion of TORC2-Gad8 resulted in loss of the heterochromatic modification of histone 3 lysine 9 dimethylation (H3K9me2) and an increase in euchromatic modifications, including histone 3 lysine 4 trimethylation (H3K4me3) and histone 4 lysine 16 acetylation (H4K16Ac). Accumulation of RNA polymerase II (Pol II) at subtelomeric genes in TORC2-Gad8 mutant cells indicated a defect in silencing at the transcriptional level. Moreover, a concurrent decrease in histone 4 lysine 20 dimethylation (H4K20me2) suggested elevated histone turnover. Loss of gene silencing in cells lacking TORC2-Gad8 is partially suppressed by loss of the anti-silencer Epe1 and fully suppressed by loss of the Pol II-associated Paf1 complex, two chromatin regulators that have been implicated in heterochromatin stability and spreading. Taken together, our findings suggest that TORC2-Gad8 signaling contributes to epigenetic stability at subtelomeric regions and the mating-type locus in S. pombe.
Assuntos
Cromatina/genética , Inativação Gênica , Heterocromatina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Telômero/genética , Cromatina/metabolismo , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Telômero/metabolismoRESUMO
Environmental changes, such as nutrient limitation or starvation induce different signal transducing pathways, which require coordinated cooperation of several genes. Our previous data revealed that the fhl1 fork-head type transcription factor of the fission yeast could be involved in sporulation, which was typically induced under poor conditions. Since the exact role of Fhl1 in this process was not known, we wanted to identify its downstream targets and to investigate its possible cooperation with another known regulator of sporulation. Gene expression and Northern blot analysis of the fhl1∆ mutant strain revealed the target genes involved in mating and sporulation. Our results also showed that Fhl1 could regulate nutrient sensing, the transporter and permease genes. Since the majority of these genes belonged to the nitrogen starvation response, the possible cooperation of fhl1 and tor2 was also investigated. Comparison of their microarray data and the expression of fhl1 + from a strong promoter in the tor2-ts mutant cells suggested that one part of the target genes are commonly regulated by Fhl1 and Tor2. Since the expression of fhl1 + from a strong promoter could rescue rapamycin and temperature sensitivity and suppressed the hyper-sporulation defect of the tor2-ts mutant cells, we believe that Fhl1 acts in TOR signaling, downstream of Tor2. Thus, this work shed light on certain novel details of the regulation of the sexual processes and a new member of the TOR pathway, but further experiments are needed to confirm the involvement of Fhl1 in nutrient sensing.
Assuntos
Fatores de Transcrição Forkhead/genética , Regulação Fúngica da Expressão Gênica , Meiose/genética , Complexos Multiproteicos/metabolismo , Nitrogênio/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Análise por Conglomerados , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Alvo Mecanístico do Complexo 1 de Rapamicina , Anotação de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Esporos Fúngicos/genéticaRESUMO
All organisms can respond to the availability of nutrients by regulating their metabolism, growth, and cell division. Central to the regulation of growth in response to nutrient availability is the target of rapamycin (TOR) signaling that is composed of two structurally distinct complexes: TOR complex 1 (TORC1) and TOR complex 2 (TORC2). The TOR genes were first identified in yeast as target of rapamycin, a natural product of a soil bacterium, which proved beneficial as an immunosuppressive and anticancer drug and is currently being tested for a handful of other pathological conditions including diabetes, neurodegeneration, and age-related diseases. Studies of the TOR pathway unraveled a complex growth-regulating network. TOR regulates nutrient uptake, transcription, protein synthesis and degradation, as well as metabolic pathways, in a coordinated manner that ensures that cells grow or cease growth in response to nutrient availability. The identification of specific signals and mechanisms that stimulate TOR signaling is an active and exciting field of research that has already identified nitrogen and amino acids as key regulators of TORC1 activity. The signals, as well as the cellular functions of TORC2, are far less well understood. Additional open questions in the field concern the relationships between TORC1 and TORC2, as well as the links with other nutrient-responsive pathways. Here I review the main features of TORC1 and TORC2, with a particular focus on yeasts as model organisms.
Assuntos
Sirolimo/metabolismo , Leveduras/crescimento & desenvolvimento , Leveduras/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
The target of rapamycin (TOR) kinase is found at the core of two evolutionarily conserved complexes known as TOR complexes 1 and 2 (TORC1 and TORC2). In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. We have previously reported that loss of function of TORC2 renders cells highly sensitive to DNA replication stress; however, the mechanism underlying this sensitivity is unknown. TORC2 has one known direct substrate, the kinase Gad8, which is related to AKT in human cells. Here we show that both TORC2 and its substrate Gad8 are found in the nucleus and are bound to the chromatin. We also demonstrate that Gad8 physically interacts with the MluI cell cycle box-binding factor (MBF) transcription complex that regulates the G1/S progression and the response to DNA stress. In mutant cells lacking TORC2 or Gad8, the binding of the MBF complex to its cognate promoters is compromised, and the induction of MBF target genes in response to DNA replication stress is reduced. Consistently, the protein levels of Cdt2 and Cig2, two MBF target genes, are reduced in the absence of TORC2-Gad8 signaling. Taken together, our findings highlight critical functions of TORC2 in the nucleus and suggest a role in surviving DNA replication stress via transcriptional regulation of MBF target genes.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Complexos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/genética , Ciclina B/genética , Ciclina B/metabolismo , Fase G1/genética , Alvo Mecanístico do Complexo 2 de Rapamicina , Complexos Multiproteicos/genética , Proteínas Serina-Treonina Quinases/genética , Fase S/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Fatores de Transcrição/genéticaRESUMO
UNLABELLED: The TOR (target of rapamycin [sirolimus]) is a universally conserved kinase that couples nutrient availability to cell growth. TOR complex 1 (TORC1) in Schizosaccharomyces pombe positively regulates growth in response to nitrogen availability while suppressing cellular responses to nitrogen stress. Here we report the identification of the GATA transcription factor Gaf1 as a positive regulator of the nitrogen stress-induced gene isp7(+), via three canonical GATA motifs. We show that under nitrogen-rich conditions, TORC1 positively regulates the phosphorylation and cytoplasmic retention of Gaf1 via the PP2A-like phosphatase Ppe1. Under nitrogen stress conditions when TORC1 is inactivated, Gaf1 becomes dephosphorylated and enters the nucleus. Gaf1 was recently shown to negatively regulate the transcription induction of ste11(+), a major regulator of sexual development. Our findings support a model of a two-faceted role of Gaf1 during nitrogen stress. Gaf1 positively regulates genes that are induced early in the response to nitrogen stress, while inhibiting later responses, such as sexual development. Taking these results together, we identify Gaf1 as a novel target for TORC1 signaling and a step-like mechanism to modulate the nitrogen stress response. IMPORTANCE: TOR complex 1 (TORC1) is an evolutionary conserved protein complex that positively regulates growth and proliferation, while inhibiting starvation responses. In fission yeast, the activity of TORC1 is downregulated in response to nitrogen starvation, and cells reprogram their transcriptional profile and prepare for sexual development. We identify Gaf1, a GATA-like transcription factor that regulates transcription and sexual development in response to starvation, as a downstream target for TORC1 signaling. Under nitrogen-rich conditions, TORC1 positively regulates the phosphorylation and cytoplasmic retention of Gaf1 via the PP2A-like phosphatase Ppe1. Under nitrogen stress conditions when TORC1 is inactivated, Gaf1 becomes dephosphorylated and enters the nucleus. Budding yeast TORC1 regulates GATA transcription factors via the phosphatase Sit4, a structural homologue of Ppe1. Thus, the TORC1-GATA transcription module appears to be conserved in evolution and may also be found in higher eukaryotes.
Assuntos
Regulação Fúngica da Expressão Gênica , Complexos Multiproteicos/metabolismo , Nitrogênio/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/genética , Serina-Treonina Quinases TOR/metabolismo , Transativadores/metabolismo , Proteínas de Ciclo Celular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Fosfoproteínas Fosfatases/metabolismo , Schizosaccharomyces/fisiologia , Proteínas de Schizosaccharomyces pombe/biossíntese , Estresse FisiológicoRESUMO
The inhibition of the central growth regulatory kinase TOR, which participates in two complexes, TORC1 and TORC2, has been a focus of metabolic and cancer studies for many years. Most studies have dealt with TORC1, the canonical target of rapamycin, and the role of this complex in autophagy, protein synthesis, and cell growth control. Recent work on TORC2 in budding and fission yeast species points to a conserved role of this lesser-known TOR complex in the survival of DNA damage. In budding yeast, TORC2 controls lipid biosynthesis and actin cytoskeleton through downstream AGC kinases, which are now, surprisingly, implicated in the survival of oxidative DNA damage. Preliminary data from mTORC2 modulation in cancer cells suggest that an extension to human chemotherapy is worth exploring.
Assuntos
Dano ao DNA , Instabilidade Genômica , Complexos Multiproteicos/metabolismo , Estresse Oxidativo , Saccharomycetales/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Actinas/metabolismo , Metabolismo dos Lipídeos , Alvo Mecanístico do Complexo 2 de RapamicinaRESUMO
The target of rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol 3-kinase-related kinases. TOR proteins are found at the core of two evolutionary conserved complexes, known as TORC1 and TORC2. In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. TORC2 has been implicated in a wide variety of functions; however, the signals that regulate TORC2 activity have so far remained obscure. TORC2 has one known direct substrate, the AGC kinase Gad8, which is related to AKT in human cells. Gad8 is phosphorylated by TORC2 at Ser-546 (equivalent to AKT Ser-473), leading to its activation. Here, we show that glucose is necessary and sufficient to induce Gad8 Ser-546 phosphorylation in vivo and Gad8 kinase activity in vitro. The glucose signal that activates TORC2-Gad8 is mediated via the cAMP/PKA pathway, a major glucose-sensing pathway. By contrast, Pmk1, similar to human extracellular signal-regulated kinases and a major stress-induced mitogen activated protein kinase (MAPK) in fission yeast, inhibits TORC2-dependent Gad8 phosphorylation and activation. Inhibition of TORC2-Gad8 also occurs in response to ionic or osmotic stress, in a manner dependent on the cAMP/PKA and Pmk1-MAPK signaling pathways. Our findings highlight the significance of glucose availability in regulation of TORC2-Gad8 and indicate a novel link between the cAMP/PKA, Pmk1/MAPK, and TORC2-Gad8 signaling.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Glucose/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Parede Celular/enzimologia , Parede Celular/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina , Fosforilação , Schizosaccharomyces/enzimologiaRESUMO
Genetic analysis of protein function requires a rapid means of inactivating the gene under study. Typically, this exploits temperature-sensitive mutations or promoter shut-off techniques. We report the adaptation to Schizosaccharomyces pombe of the anchor-away technique, originally designed in budding yeast by Laemmli lab. This method relies on a rapamycin-mediated interaction between the FRB- and FKBP12-binding domains to relocalize nuclear proteins of interest to the cytoplasm. We demonstrate a rapid nuclear depletion of abundant proteins as proof of principle.
Assuntos
Componente 4 do Complexo de Manutenção de Minicromossomo/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/fisiologia , Sirolimo/farmacologia , Proteína 1A de Ligação a Tacrolimo/fisiologia , Cruzamentos Genéticos , DNA Fúngico/química , DNA Fúngico/genética , Microscopia de Fluorescência , Plasmídeos , Reação em Cadeia da Polimerase , Transformação GenéticaRESUMO
TOR proteins reside in two distinct complexes, TOR complexes 1 and 2 (TORC1 and TORC2), that are central for the regulation of cellular growth, proliferation, and survival. TOR is also the target for the immunosuppressive and anticancer drug rapamycin. In Schizosaccharomyces pombe, disruption of the TSC complex, mutations in which can lead to the tuberous sclerosis syndrome in humans, results in a rapamycin-sensitive phenotype under poor nitrogen conditions. We show here that the sensitivity to rapamycin is mediated via inhibition of TORC1 and suppressed by overexpression of isp7(+), a member of the family of 2-oxoglutarate-Fe(II)-dependent oxygenase genes. The transcript level of isp7(+) is negatively regulated by TORC1 but positively regulated by TORC2. Yet we find extensive similarity between the transcriptome of cells disrupted for isp7(+) and cells mutated in the catalytic subunit of TORC1. Moreover, Isp7 regulates amino acid permease expression in a fashion similar to that of TORC1 and opposite that of TORC2. Overexpression of isp7(+) induces TORC1-dependent phosphorylation of ribosomal protein Rps6 while inhibiting TORC2-dependent phosphorylation and activation of the AGC-like kinase Gad8. Taken together, our findings suggest a central role for Isp7 in amino acid homeostasis and the presence of isp7(+)-dependent regulatory loops that affect both TORC1 and TORC2.
Assuntos
Aminoácidos/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Transdução de Sinais/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina , Complexos Multiproteicos/metabolismo , Mutação/efeitos dos fármacos , Mutação/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Transcriptoma/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
DNA damage can occur due to environmental insults or intrinsic metabolic processes and is a major threat to genome stability. The DNA damage response is composed of a series of well coordinated cellular processes that include activation of the DNA damage checkpoint, transient cell cycle arrest, DNA damage repair, and reentry into the cell cycle. Here we demonstrate that mutant cells defective for TOR complex 2 (TORC2) or the downstream AGC-like kinase, Gad8, are highly sensitive to chronic replication stress but are insensitive to ionizing radiation. We show that in response to replication stress, TORC2 is dispensable for Chk1-mediated cell cycle arrest but is required for the return to cell cycle progression. Rad52 is a DNA repair and recombination protein that forms foci at DNA damage sites and stalled replication forks. TORC2 mutant cells show increased spontaneous nuclear Rad52 foci, particularly during S phase, suggesting that TORC2 protects cells from DNA damage that occurs during normal DNA replication. Consistently, the viability of TORC2-Gad8 mutant cells is dependent on the presence of the homologous recombination pathway and other proteins that are required for replication restart following fork replication stalling. Our findings indicate that TORC2 is required for genome integrity. This may be relevant for the growing amount of evidence implicating TORC2 in cancer development.
Assuntos
Genoma Fúngico/fisiologia , Instabilidade Genômica/fisiologia , Complexos Multiproteicos/metabolismo , Fase S/fisiologia , Schizosaccharomyces/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina , Complexos Multiproteicos/genética , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Serina-Treonina Quinases TOR/genéticaRESUMO
Telomeres are nucleoprotein structures that protect the ends of eukaryotic chromosomes and play important roles in ensuring the genome's integrity. Telomere length is maintained by complex mechanisms that ensure length homeostasis. Recent work has linked telomere length maintenance to the Tor protein kinases, which are central regulators of cellular growth. Here we summarize these results, which suggest a link between nutrient availability, telomere length maintenance and chronological lifespan.
Assuntos
Serina-Treonina Quinases TOR/metabolismo , Telômero/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Homeostase do Telômero , Fatores de Transcrição/metabolismoRESUMO
The Target Of Rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol-3-kinase-related kinases (PIKKs). TOR proteins are found at the core of two distinct evolutionarily conserved complexes, TORC1 and TORC2. Disruption of TORC1 or TORC2 results in characteristically dissimilar phenotypes. TORC1 is a major cell growth regulator, while the cellular roles of TORC2 are not well understood. In the fission yeast Schizosaccharomyces pombe, Tor1 is a component of the TORC2 complex, which is particularly required during starvation and various stress conditions. Our genome-wide gene expression analysis of Deltator1 mutants indicates an extensive similarity with chromatin structure mutants. Consistently, TORC2 regulates several chromatin-mediated functions, including gene silencing, telomere length maintenance, and tolerance to DNA damage. These novel cellular roles of TORC2 are rapamycin insensitive. Cells lacking Tor1 are highly sensitive to the DNA-damaging drugs hydroxyurea (HU) and methyl methanesulfonate, similar to mutants of the checkpoint kinase Rad3 (ATR). Unlike Rad3, Tor1 is not required for the cell cycle arrest in the presence of damaged DNA. Instead, Tor1 becomes essential for dephosphorylation and reactivation of the cyclin-dependent kinase Cdc2, thus allowing reentry into mitosis following recovery from DNA replication arrest. Taken together, our data highlight critical roles for TORC2 in chromatin metabolism and in promoting mitotic entry, most notably after recovery from DNA-damaging conditions. These data place TOR proteins in line with other PIKK members, such as ATM and ATR, as guardians of genome stability.
Assuntos
Sobrevivência Celular/fisiologia , Dano ao DNA , Inativação Gênica , Proteínas Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Telômero/metabolismo , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Replicação do DNA , Ativação Enzimática , Regulação Fúngica da Expressão Gênica , Hidroxiureia/farmacologia , Metanossulfonato de Metila/farmacologia , Mitose/efeitos dos fármacos , Mitose/fisiologia , Complexos Multiproteicos/metabolismo , Mutagênicos/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Quinases/genética , Schizosaccharomyces/citologia , Schizosaccharomyces/efeitos dos fármacos , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
The TOR protein kinases exhibit a conserved role in regulating cellular growth and proliferation. In the fission yeast two TOR homologs are present. tor1(+) is required for starvation and stress responses, while tor2(+) is essential. We report here that Tor2 depleted cells show a phenotype very similar to that of wild-type cells starved for nitrogen, including arrest at the G(1) phase of the cell cycle, induction of nitrogen-starvation-specific genes, and entrance into the sexual development pathway. The phenotype of tor2 mutants is in a striking contrast to the failure of tor1 mutants to initiate sexual development or arrest in G(1) under nitrogen starvation conditions. Tsc1 and Tsc2, the genes mutated in the human tuberous sclerosis complex syndrome, negatively regulate the mammalian TOR via inactivation of the GTPase Rheb. We analyzed the genetic relationship between the two TOR genes and the Schizosaccharomyces pombe orthologs of TSC1, TSC2, and Rheb. Our data suggest that like in higher eukaryotes, the Tsc1-2 complex negatively regulates Tor2. In contrast, the Tsc1-2 complex and Tor1 appear to work in parallel, both positively regulating amino acid uptake through the control of expression of amino acid permeases. Additionally, either Tsc1/2 or Tor1 are required for growth on a poor nitrogen source such as proline. Mutants lacking Tsc1 or Tsc2 are highly sensitive to rapamycin under poor nitrogen conditions, suggesting that the function of Tor1 under such conditions is sensitive to rapamycin. We discuss the complex genetic interactions between tor1(+), tor2(+), and tsc1/2(+) and the implications for rapamycin sensitivity in tsc1 or tsc2 mutants.