Diacylglycerol lipase regulates lifespan and oxidative stress response by inversely modulating TOR signaling in Drosophila and C. elegans

Target of rapamycin (TOR) signaling is a nutrient‐sensing pathway controlling metabolism and lifespan. Although TOR signaling can be activated by a metabolite of diacylglycerol (DAG), phosphatidic acid (PA), the precise genetic mechanism through which DAG metabolism influences lifespan remains unknown. DAG is metabolized to either PA via the action of DAG kinase or 2‐arachidonoyl‐sn‐glycerol by diacylglycerol lipase (DAGL). Here, we report that in Drosophila and Caenorhabditis elegans, overexpression of diacylglycerol lipase (DAGL/inaE/dagl‐1) or knockdown of diacylglycerol kinase (DGK/rdgA/dgk‐5) extends lifespan and enhances response to oxidative stress. Phosphorylated S6 kinase (p‐S6K) levels are reduced following these manipulations, implying the involvement of TOR signaling. Conversely, DAGL/inaE/dagl‐1 mutants exhibit shortened lifespan, reduced tolerance to oxidative stress, and elevated levels of p‐S6K. Additional results from genetic interaction studies are consistent with the hypothesis that DAG metabolism interacts with TOR and S6K signaling to affect longevity and oxidative stress resistance. These findings highlight conserved metabolic and genetic pathways that regulate aging.     

A small G protein Rhb1 and a GTPase-activating protein Tsc2 involved in nitrogen starvation-induced morphogenesis and cell wall integrity of Candida albicans

Rheb is a new member of the small G proteins of the Ras superfamily in eukaryotic organisms and controls various physiological processes. Activity of Rheb is regulated by Tsc2, a GTPase-activating protein (GAP). In this study, we have identified Candida albicans homologs of Rheb (named as Rhb1) and Tsc2. Deletion of the RHB1 gene showed enhanced sensitivity to rapamycin (an inhibitor of TOR kinase), suggesting that Rhb1 is associated with the TOR signaling pathway in C. albicans. Further analysis indicated RHB1 and TSC2 are involved in nitrogen starvation-induced filamentation, likely by controlling the expression of MEP2 whose gene product is an ammonium permease and a sensor for the nitrogen signal. Moreover, we have demonstrated that Rhb1 is also involved in cell wall integrity pathway, by transferring signals through the TOR kinase and the Mkc1 MAP kinase pathway. Together, this study brings new insights into the complex interplay of signaling and regulatory pathways in C. albicans.     

TORC1 signaling regulates cytoplasmic pH through Sir2 in yeast

Glucose controls the phosphorylation of silent information regulator 2 (Sir2), a NAD⁺‐dependent protein deacetylase, which regulates the expression of the ATP‐dependent proton pump Pma1 and replicative lifespan (RLS) in yeast. TORC1 signaling, which is a central regulator of cell growth and lifespan, is regulated by glucose as well as nitrogen sources. In this study, we demonstrate that TORC1 signaling controls Sir2 phosphorylation through casein kinase 2 (CK2) to regulate PMA1 expression and cytoplasmic pH (pHc) in yeast. Inhibition of TORC1 signaling by either TOR1 deletion or rapamycin treatment decreased PMA1 expression, pHc, and vacuolar pH, whereas activation of TORC1 signaling by expressing constitutively active GTR1 (GTR1Q65L) resulted in the opposite phenotypes. Deletion of SIR2 or expression of a phospho‐mutant form of SIR2 increased PMA1 expression, pHc, and vacuolar pH in the tor1Δ mutant, suggesting a functional interaction between Sir2 and TORC1 signaling. Furthermore, deletion of TOR1 or KNS1 encoding a LAMMER kinase decreased the phosphorylation level of Sir2, suggesting that TORC1 signaling controls Sir2 phosphorylation. It was also found that Sit4, a protein phosphatase 2A (PP2A)‐like phosphatase, and Kns1 are required for TORC1 signaling to regulate PMA1 expression and that TORC1 signaling and the cyclic AMP (cAMP)/protein kinase A (PKA) pathway converge on CK2 to regulate PMA1 expression through Sir2. Taken together, these findings suggest that TORC1 signaling regulates PMA1 expression and pHc through the CK2–Sir2 axis, which is also controlled by cAMP/PKA signaling in yeast.     

Tor1, Sch9 and PKA downregulation in quiescence rely on Mtl1 to preserve mitochondrial integrity and cell survival

Here we show that Mtl1, member of the cell wall integrity pathway of Saccharomyces cerevisiae, plays a positive role in chronological life span (CLS). The absence of Mtl1 shortens CLS and causes impairment in the mitochondrial function. This is reflected in a descent in oxygen consumption during the postdiauxic state, an increase in the uncoupled respiration and mitochondrial membrane potential and also a descent in aconitase activity. We demonstrate that all these effects are a consequence of signalling defects suppressed by TOR1 (target of rapamycin) and SCH9 deletion and less efficiently by Protein kinase A (PKA) inactivation. Mtl1 also plays a role in the regulation of both Bcy1 stability and phosphorylation, mainly in response to glucose depletion. In postdiauxic phase and in conditions of glucose depletion, Mtl1 negatively regulates TOR1 function leading to Sch9 inactivation and Bcy1 phosphorylation converging in PKA inhibition. Slt2/Mpk1 kinase partially contributes to Bcy1 phosphorylation, although additional targets are not excluded. Mtl1 links mitochondrial dysfunction with TOR and PKA pathways in quiescence, glucose being the main signalling molecule.     

Growth control via TOR kinase signaling,an intracellular sensor of amino acid and energy availability,with crosstalk potential to proline metabolism

The TOR (Target of Rapamycin) protein kinase pathway plays a central role in sensing and responding to nutrients, stress, and intracellular energy state. TOR complex 1 (TORC1) is comprised of TOR, Raptor, and Lst8 and its activity is sensitive to inhibition by the macrolide antibiotic rapamycin. TORC1 regulates protein synthesis, ribosome biogenesis, autophagy, and ultimately cell growth through the phosphorylation of S6 K, 4E-BP, and other substrates. As TORC1 activity is positively or negatively modulated in response to upstream regulators, cellular growth rate is, respectively, enhanced or suppressed. A separate multiprotein TOR complex, TORC2, is insensitive to direct inhibition by rapamycin and does not regulate growth patterns directly; TORC2 can, however, impact certain aspects of TORC1 signaling and cell survival. TOR signaling is an ancient pathway, conserved among the yeasts, Dictyostelium, C. elegans, Drosophila, mammals, and Arabidopsis. This review will focus on the regulation of TORC1 in mammalian cells in the context of amino acid sensing/regulation and intracellular ATP homeostasis, but will also include comparisons among other organisms.     

The target of rapamycin kinase affects biomass accumulation and cell cycle progression by altering carbon/nitrogen balance in synchronized Chlamydomonas reinhardtii cells

Several metabolic processes tightly regulate growth and biomass accumulation. A highly conserved protein complex containing the target of rapamycin (TOR) kinase is known to integrate intra‐ and extracellular stimuli controlling nutrient allocation and hence cellular growth. Although several functions of TOR have been described in various heterotrophic eukaryotes, our understanding lags far behind in photosynthetic organisms. In the present investigation, we used the model alga Chlamydomonas reinhardtii to conduct a time‐resolved analysis of molecular and physiological features throughout the diurnal cycle after TOR inhibition. Detailed examination of the cell cycle phases revealed that growth is not only repressed by 50%, but also that significant, non‐linear delays in the progression can be observed. By using metabolomics analysis, we elucidated that the growth repression was mainly driven by differential carbon partitioning between anabolic and catabolic processes. Accordingly, the time‐resolved analysis illustrated that metabolic processes including amino acid‐, starch‐ and triacylglycerol synthesis, as well RNA degradation, were redirected within minutes of TOR inhibition. Here especially the high accumulation of nitrogen‐containing compounds indicated that an active TOR kinase controls the carbon to nitrogen balance of the cell, which is responsible for biomass accumulation, growth and cell cycle progression.     

Lifespan regulation in α/β posterior neurons of the fly mushroom bodies by Rab27

Brain function has been implicated to control the aging process and modulate lifespan. However, continuous efforts remain for the identification of the minimal sufficient brain region and the underlying mechanism for neuronal regulation of longevity. Here, we show that the Drosophila lifespan is modulated by rab27 functioning in a small subset of neurons of the mushroom bodies (MB), a brain structure that shares analogous functions with mammalian hippocampus and hypothalamus. Depleting rab27 in the α/βp neurons of the MB is sufficient to extend lifespan, enhance systemic stress responses, and alter energy homeostasis, all without trade‐offs in major life functions. Within the α/βp neurons, rab27KO causes the mislocalization of phosphorylated S6K thus attenuates TOR signaling, resulting in decreased protein synthesis and reduced neuronal activity. Consistently, expression of dominant‐negative S6K in the α/βp neurons increases lifespan. Furthermore, the expression of phospho‐mimetic S6 in α/βp neurons of rab27KO rescued local protein synthesis and reversed lifespan extension. These findings demonstrate that inhibiting TOR‐mediated protein synthesis in α/βp neurons is sufficient to promote longevity.     

The Arabidopsis THADA homologue modulates TOR activity and cold acclimation

• Low temperature is one of the most important environmental factors that affect global survival of humans and animals and equally importantly the distribution of plants and crop productivity. Survival of metazoan cells under cold stress requires regulation of the sensor‐kinase Target Of Rapamycin (TOR). TOR controls growth of eukaryotic cells by adjusting anabolic and catabolic metabolism. Previous studies identified the Thyroid Adenoma Associated (THADA) gene as the major effect locus by positive selection in the evolution of modern human adapted to cold. Here we investigate the role of THADA in TOR signaling and cold acclimation of plants.
• We applied BLAST searches and homology modeling to identify the AtTHADA (AT3G55160) in Arabidopsis thaliana as the highly probable orthologue protein. Reverse genetics approaches were combined with immunological detection of TOR activity and metabolite profiling to address the role of the TOR and THADA for growth regulation and cold acclimation.
• Depletion of the AtTHADA gene caused complete or partial loss of full‐length mRNA, respectively, and significant retardation of growth under non‐stressed conditions. Furthermore, depletion of AtTHADA caused hypersensitivity towards low‐temperatures. Atthada displayed a lowered energy charge. This went along with decreased TOR activity, which offers a molecular explanation for the slow growth phenotype of Atthada. Finally, we used TOR RNAi lines to identify the de‐regulation of TOR activity as one determinant for sensitivity towards low‐temperatures.
• Taken together our results provide evidence for a conserved function of THADA in cold acclimation of eukaryotes and suggest that cold acclimation in plants requires regulation of TOR.

     

A drug-resistant mutation in plant target of rapamycin validates the specificity of ATP-competitive TOR inhibitors in vivo

Kinases are major components of cellular signaling pathways, regulating key cellular activities through phosphorylation. Kinase inhibitors are efficient tools for studying kinase targets and functions, however assessing their kinase specificity in vivo is essential. The identification of resistant kinase mutants has been proposed to be the most convincing approach to achieve this goal. Here, we address this issue in plants via a pharmacogenetic screen for mutants resistant to the ATP-competitive TOR inhibitor AZD-8055. The eukaryotic TOR (Target of Rapamycin) kinase is emerging as a major hub controlling growth responses in plants largely thanks to the use of ATP-competitive inhibitors. We identified a dominant mutation in the DFG motif of the Arabidopsis TOR kinase domain that leads to very strong resistance to AZD-8055. This resistance was characterized by measuring root growth, photosystem II (PSII) activity in leaves and phosphorylation of YAK1 (Yet Another Kinase 1) and RPS6 (Ribosomal protein S6), a direct and an indirect target of TOR respectively. Using other ATP-competitive TOR inhibitors, we also show that the dominant mutation is particularly efficient for resistance to drugs structurally related to AZD-8055. Altogether, this proof-of-concept study demonstrates that a pharmacogenetic screen in Arabidopsis can be used to successfully identify the target of a kinase inhibitor in vivo and therefore to demonstrate inhibitor specificity. Thanks to the conservation of kinase families in eukaryotes, and the possibility of creating amino acid substitutions by genome editing, this work has great potential for extending studies on the evolution of signaling pathways in eukaryotes.     

Glucose Activates TORC2-Gad8 Protein via Positive Regulation of the cAMP/cAMP-dependent Protein Kinase A (PKA) Pathway and Negative Regulation of the Pmk1 Protein-Mitogen-activated Protein Kinase Pathway

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.     

Introducing fluorescence resonance energy transfer‐based biosensors for the analysis of cAMP‐PKA signalling in the fungal pathogen Candida glabrata

The cyclic adenosine monophosphate‐protein kinase A (cAMP‐PKA) pathway is central to signal transduction in many organisms. In pathogenic fungi such as Candida albicans, this signalling cascade has proven to be involved in several processes, such as virulence, indicating its potential importance in antifungal drug discovery. Candida glabrata is an upcoming pathogen of the same species, yet information regarding the role of cAMP‐PKA signalling in virulence is largely lacking. To enable efficient monitoring of cAMP‐PKA activity in this pathogen, we here present the usage of two FRET‐based biosensors. Both variations in the activity of PKA and the quantity of cAMP can be detected in a time‐resolved manner, as we exemplify by glucose‐induced activation of the pathway. We also present information on how to adequately process and analyse the data in a mathematically correct and physiologically relevant manner. These sensors will be of great benefit for scientists interested in linking the cAMP‐PKA signalling cascade to downstream processes, such as virulence, possibly in a host environment.