Phosphatidic acid drives mTORC1 lysosomal translocation in the absence of amino acids.

Mammalian target of rapamycin complex 1 (mTORC1) promotes cell growth and proliferation in response to nutrients and growth factors. Amino acids induce lysosomal translocation of mTORC1 via the Rag GTPases. Growth factors activate Ras homolog enriched in brain (Rheb), which in turn activates mTORC1 at the lysosome. Amino acids and growth factors also induce the phospholipase D (PLD)-phosphatidic acid (PA) pathway, required for mTORC1 signaling through mechanisms that are not fully understood. Here, using human and murine cell lines, along with immunofluorescence, confocal microscopy, endocytosis, PLD activity, and cell viability assays, we show that exogenously supplied PA vesicles deliver mTORC1 to the lysosome in the absence of amino acids, Rag GTPases, growth factors, and Rheb. Of note, pharmacological or genetic inhibition of endogenous PLD prevented mTORC1 lysosomal translocation. We observed that precancerous cells with constitutive Rheb activation through loss of tuberous sclerosis complex subunit 2 (TSC2) exploit the PLD-PA pathway and thereby sustain mTORC1 activation at the lysosome in the absence of amino acids. Our findings indicate that sequential inputs from amino acids and growth factors trigger PA production required for mTORC1 translocation and activation at the lysosome.

Phospholipase D-dependent mTOR complex 1 (mTORC1) activation by glutamine.

Glutamine is a key nutrient required for sustaining cell proliferation, contributing to nucleotide, protein, and lipid synthesis. The mTOR complex 1 (mTORC1) is a highly conserved protein complex that acts as a sensor of nutrients, relaying signals for the shift from catabolic to anabolic metabolism. Although glutamine plays an important role in mTORC1 activation, the mechanism is not clear. Here we describe a leucine- and Rag-independent mechanism of mTORC1 activation by glutamine that depends on phospholipase D and the production of phosphatidic acid, which is required for the stability and activity of mTORC1. The phospholipase D-dependent activation of mTORC1 by glutamine depended on the GTPases ADP ribosylation factor 1 (Arf1), RalA, and Rheb. Glutamine deprivation could be rescued by α-ketoglutarate, a downstream metabolite of glutamine. This mechanism represents a distinct nutrient input to mTORC1 that is independent of Rag GTPases and leucine.

Lipid sensing by mTOR complexes via de novo synthesis of phosphatidic acid.

mTOR, the mammalian target of rapamycin, integrates growth factor and nutrient signals to promote a transformation from catabolic to anabolic metabolism, cell growth, and cell cycle progression. Phosphatidic acid (PA) interacts with the FK506-binding protein-12-rapamycin-binding (FRB) domain of mTOR, which stabilizes both mTOR complexes: mTORC1 and mTORC2. We report here that mTORC1 and mTORC2 are activated in response to exogenously supplied fatty acids via the de novo synthesis of PA, a central metabolite for membrane phospholipid biosynthesis. We examined the impact of exogenously supplied fatty acids on mTOR in KRas-driven cancer cells, which are programmed to utilize exogenous lipids. The induction of mTOR by oleic acid was dependent upon the enzymes responsible for de novo synthesis of PA. Suppression of the de novo synthesis of PA resulted in G1 cell cycle arrest. Although it has long been appreciated that mTOR is a sensor of amino acids and glucose, this study reveals that mTOR also senses the presence of lipids via production of PA.

Reciprocal regulation of AMP-activated protein kinase and phospholipase D.

AMP-activated protein kinase (AMPK), a critical sensor of energy sufficiency, acts as central metabolic switch in cell metabolism. Once activated by low energy status, AMPK phosphorylates key regulatory substrates and turns off anabolic biosynthetic pathways. In contrast, the mammalian/mechanistic target of rapamycin (mTOR) is active when there are sufficient nutrients for anabolic reactions. A critical factor regulating mTOR is phosphatidic acid (PA), a central metabolite of membrane lipid biosynthesis and the product of the phospholipase D (PLD)-catalyzed hydrolysis of phosphatidylcholine. PLD is a downstream target of the GTPase Rheb, which is turned off in response to AMPK via the tuberous sclerosis complex. Although many studies have linked AMPK with mTOR, very little is known about the connection between AMPK and PLD. In this report, we provide evidence for reciprocal regulation of PLD by AMPK and regulation of AMPK by PLD and PA. Suppression of AMPK activity led to an increase in PLD activity, and conversely, activation of AMPK suppressed PLD activity. Suppression of PLD activity resulted in elevated AMPK activity. Exogenously supplied PA abolished the inhibitory effects of elevated AMPK activity on mTOR signaling. In contrast, exogenously supplied PA could not overcome the effect AMPK activation if either mTOR or Raptor was suppressed, indicating that the inhibitory effects of PLD and PA on AMPK activity are mediated by mTOR. These data suggest a reciprocal feedback mechanism involving AMPK and the PLD/mTOR signaling node in cancer cells with therapeutic implications.

Phospholipase D and the maintenance of phosphatidic acid levels for regulation of mammalian target of rapamycin (mTOR).

Phosphatidic acid (PA) is a critical metabolite at the heart of membrane phospholipid biosynthesis. However, PA also serves as a critical lipid second messenger that regulates several proteins implicated in the control of cell cycle progression and cell growth. Three major metabolic pathways generate PA: phospholipase D (PLD), diacylglycerol kinase (DGK), and lysophosphatidic acid acyltransferase (LPAAT). The LPAAT pathway is integral to de novo membrane phospholipid biosynthesis, whereas the PLD and DGK pathways are activated in response to growth factors and stress. The PLD pathway is also responsive to nutrients. A key target for the lipid second messenger function of PA is mTOR, the mammalian/mechanistic target of rapamycin, which integrates both nutrient and growth factor signals to control cell growth and proliferation. Although PLD has been widely implicated in the generation of PA needed for mTOR activation, it is becoming clear that PA generated via the LPAAT and DGK pathways is also involved in the regulation of mTOR. In this minireview, we highlight the coordinated maintenance of intracellular PA levels that regulate mTOR signals stimulated by growth factors and nutrients, including amino acids, lipids, glucose, and Gln. Emerging evidence indicates compensatory increases in one source of PA when another source is compromised, highlighting the importance of being able to adapt to stressful conditions that interfere with PA production. The regulation of PA levels has important implications for cancer cells that depend on PA and mTOR activity for survival.

Regulation of mTOR by phosphatidic acid.

mTORC1, the mammalian target of rapamycin complex 1, is a key regulator of cellular physiology. The lipid metabolite phosphatidic acid (PA) binds to and activates mTORC1 in response to nutrients and growth factors. We review structural findings and propose a model for PA activation of mTORC1. PA binds to a highly conserved sequence in the α4 helix of the FK506 binding protein 12 (FKBP12)/rapamycin-binding (FRB) domain of mTOR. It is proposed that PA binding to two adjacent positively charged amino acids breaks and shortens the C-terminal region of helix α4. This has profound consequences for both substrate binding and the catalytic activity of mTORC1.

Inhibiting glutamine utilization creates a synthetic lethality for suppression of ATP citrate lyase in KRas-driven cancer cells.

Metabolic reprogramming is now considered a hallmark of cancer cells. KRas-driven cancer cells use glutaminolysis to generate the tricarboxylic acid cycle intermediate α-ketoglutarate via a transamination reaction between glutamate and oxaloacetate. We reported previously that exogenously supplied unsaturated fatty acids could be used to synthesize phosphatidic acid-a lipid second messenger that activates both mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and mTOR complex 2 (mTORC2). A key target of mTORC2 is Akt-a kinase that promotes survival and regulates cell metabolism. We report here that mono-unsaturated oleic acid stimulates the phosphorylation of ATP citrate lyase (ACLY) at the Akt phosphorylation site at S455 in an mTORC2 dependent manner. Inhibition of ACLY in KRas-driven cancer cells in the absence of serum resulted in loss of cell viability. We examined the impact of glutamine (Gln) deprivation in combination with inhibition of ACLY on the viability of KRas-driven cancer cells. While Gln deprivation was somewhat toxic to KRas-driven cancer cells by itself, addition of the ACLY inhibitor SB-204990 increased the loss of cell viability. However, the transaminase inhibitor aminooxyacetate was minimally toxic and the combination of SB-204990 and aminooxtacetate led to significant loss of cell viability and strong cleavage of poly-ADP ribose polymerase-indicating apoptotic cell death. This effect was not observed in MCF7 breast cancer cells that do not have a KRas mutation or in BJ-hTERT human fibroblasts which have no oncogenic mutation. These data reveal a synthetic lethality between inhibition of glutamate oxaloacetate transaminase and ACLY inhibition that is specific for KRas-driven cancer cells and the apparent metabolic reprogramming induced by activating mutations to KRas.

Surface Characterization of Lipid Biomimetic Systems.

Zeta potential and dipole potential measures are direct operational methodologies to determine the adsorption, insertion and penetration of ions, amphipathic and neutral compounds into the membranes of cells and model systems. From these results, the contribution of charged and dipole groups can be deduced. However, although each method may give apparent affinity or binding constants, care should be taken to interpret them in terms of physical meaning because they are not independent properties. On the base of a recent model in which the lipid bilayer is considered as composed by two interphase regions at each side of the hydrocarbon core, this review describes how dipole potential and zeta potential are correlated due to water reorganization. From this analysis, considering that in a cell the interphase region the membrane extends to the cell interior or overlaps with the interphase region of another supramolecular structure, the correlation of dipole and electrostatic forces can be taken as responsible of the propagation of perturbations between membrane and cytoplasm and vice versa. Thus, this picture gives the membrane a responsive character in addition to that of a selective permeability barrier when integrated to a complex system.

Cancer cells with defective RB and CDKN2A are resistant to the apoptotic effects of rapamycin.

Inhibition of mammalian target of rapamycin complex 1 (mTORC1) with rapamycin in the absence of transforming growth factor-ß (TGFß) signaling induces apoptosis in many cancer cell lines. In the presence of TGFß, rapamycin induces G1 cell cycle arrest; however, in the absence of TGFß, cells do not arrest in G1 and progress into S-phase where rapamycin is cytotoxic rather than cytostatic. However, we observed that DU145 prostate and NCI-H2228 lung cancer cells were resistant to the cytotoxic effect of rapamycin. Of interest, the rapamycin-resistant DU145 and NCI-H2228 cells have mutations in the RB and CDKN2A tumor suppressor genes. The gene products of RB and CDKN2A (pRb and p14ARF) suppress E2F family transcription factors that promote cell cycle progression from G1 into S. Restoration of wild type RB or inhibition of E2F activity in DU145 and NCI-H2228 cells led to rapamycin sensitivity. These data provide evidence that the combination of mutant RB and mutant CDKN2A in cancer cells leads to rapamycin resistance, which has implications for precision medicine approaches to anti-cancer therapies.

mSin1 is necessary for Akt/PKB phosphorylation, and its isoforms define three distinct mTORC2s.

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that participates in at least two distinct multiprotein complexes, mTORC1 and mTORC2 . These complexes play important roles in the regulation of cell growth, proliferation, survival, and metabolism. mTORC2 is a hydrophobic motif kinase for the cell-survival protein Akt/PKB and, here, we identify mSin1 as a component of mTORC2 but not mTORC1. mSin1 is necessary for the assembly of mTORC2 and for its capacity to phosphorylate Akt/PKB. Alternative splicing generates at least five isoforms of the mSin1 protein , three of which assemble into mTORC2 to generate three distinct mTORC2s. Even though all mTORC2s can phosphorylate Akt/PKB in vitro, insulin regulates the activity of only two of them. Thus, we propose that cells contain several mTORC2 flavors that may phosphorylate Akt/PKB in response to different signals.

Effects of Phenylalanine on the Liquid-Expanded and Liquid-Condensed States of Phosphatidylcholine Monolayers.

BACKGROUND: Phenylalanine (Phe) is involved in physiological and pathological processes in cell membranes in which expanded and condensed states coexist. In this direction, it was reported that surface hydration is important for the binding affinity of the amino acid which significantly perturbs 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayer structure and morphology. A deeper insight showed that Phe inserts in DPPC monolayer defects as a monomer at pH 5 and forms aggregates that adsorb to the membrane surface generating a reconfiguration of the lipid arrangement in areas of higher packing. This new arrangement in the monolayer causes the reorientation of dipoles of lipid and water molecules which is congruent with the dehydration and surface tension changes reported above. With this background, this article studies the affinity of Phe in liquid-expanded 1,2-dimyristoyl-sn-glycero-3 phosphocholine (LE DMPC) and liquid-condensed 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (LC DPPC) monolayers and their effects on membrane properties. RESULTS: The adsorption of Phe can be described by a cooperative process in non-independent sites suggesting that Phe/lipid systems reorganize to form new structures at a high degree of coverage. Compressibility modulus and Brewster angle microscopy (BAM) images allow to propose that Phe causes a new phase in 1,2-dimyristoyl-sn-glycero-3 phosphocholine (DMPC) and DPPC. CONCLUSIONS: Phe imposes new arrangements in the lipid phase to form new structures with different compressibility behavior than lipid binary mixtures of DMPC and DPPC. Phe interaction with the LC and LE phases gives place to a process in which a synergistic effect between non-independent sites can be produced. These features of Phe/lipid interaction would be of great importance to understand the multiple effects of Phe on cell membranes.

Correlation between the hydration of acyl chains and phosphate groups in lipid bilayers: Effect of phase state, head group, chain length, double bonds and carbonyl groups.

This paper demonstrates by means of FTIR/ATR analysis that water molecules intercalate at different extents in the acyl chain region of lipid membranes in correlation with the hydration of the phosphate groups. This correlation is sensible to the chain length, the presence of double bonds and the phase state of the lipid membrane. The presence of carbonyl groups CO modifies the profile of hydration of the two regions as observed from the comparison of DMPC and 14:0 Diether PC. The different water populations in lipid interphases would give arrangements with different free energy states that could drive the interaction of biological effectors with membranes.

The Enigma of Rapamycin Dosage.

The mTOR pathway is a critical regulator of cell growth, proliferation, metabolism, and survival. Dysregulation of mTOR signaling has been observed in most cancers and, thus, the mTOR pathway has been extensively studied for therapeutic intervention. Rapamycin is a natural product that inhibits mTOR with high specificity. However, its efficacy varies by dose in several contexts. First, different doses of rapamycin are needed to suppress mTOR in different cell lines; second, different doses of rapamycin are needed to suppress the phosphorylation of different mTOR substrates; and third, there is a differential sensitivity of the two mTOR complexes mTORC1 and mTORC2 to rapamycin. Intriguingly, the enigmatic properties of rapamycin dosage can be explained in large part by the competition between rapamycin and phosphatidic acid (PA) for mTOR. Rapamycin and PA have opposite effects on mTOR whereby rapamycin destabilizes and PA stabilizes both mTOR complexes. In this review, we discuss the properties of rapamycin dosage in the context of anticancer therapeutics.

Regulatory consequences of neuronal ELAV-like protein binding to coding and non-coding RNAs in human brain.

Neuronal ELAV-like (nELAVL) RNA binding proteins have been linked to numerous neurological disorders. We performed crosslinking-immunoprecipitation and RNAseq on human brain, and identified nELAVL binding sites on 8681 transcripts. Using knockout mice and RNAi in human neuroblastoma cells, we showed that nELAVL intronic and 3' UTR binding regulates human RNA splicing and abundance. We validated hundreds of nELAVL targets among which were important neuronal and disease-associated transcripts, including Alzheimer's disease (AD) transcripts. We therefore investigated RNA regulation in AD brain, and observed differential splicing of 150 transcripts, which in some cases correlated with differential nELAVL binding. Unexpectedly, the most significant change of nELAVL binding was evident on non-coding Y RNAs. nELAVL/Y RNA complexes were specifically remodeled in AD and after acute UV stress in neuroblastoma cells. We propose that the increased nELAVL/Y RNA association during stress may lead to nELAVL sequestration, redistribution of nELAVL target binding, and altered neuronal RNA splicing.

Coordination forces between lipid bilayers produced by ferricyanide and Ca2+.

Attractive forces usually invoked to take place in membrane-membrane contact in aggregation are hydrogen bonding cross-linkings and hydrophobic interactions between opposing surfaces. However, little is known in relation to the presence of coordination forces in the membrane-membrane interaction. These are understood as those that may be favoured by the formation or the participation of coordination complexes between surface specific groups. In this work, we have analyzed the formation of this type of aggregates between phosphatidylcholine vesicles mediated by a coadsorption of ferricyanide and Ca(2+) ions to the interface. The results obtained by surface potential measures, optical and electronic microscopy, FTIR and (1)H NMR spectroscopies indicate that ferricyanide [Fe(CN)(6)](3-) but not of ferrocyanide [Fe(CN)(6)](4-) can form the complex when Ca(2+) has been adsorbed previously to the membrane surface. In this condition, the anion is likely to act as a bridge between two opposing membranes causing a tight aggregation in which geometry and the polarizability of the ligands to Fe(3+) play a role.

Integrative modeling defines the Nova splicing-regulatory network and its combinatorial controls.

The control of RNA alternative splicing is critical for generating biological diversity. Despite emerging genome-wide technologies to study RNA complexity, reliable and comprehensive RNA-regulatory networks have not been defined. Here, we used Bayesian networks to probabilistically model diverse data sets and predict the target networks of specific regulators. We applied this strategy to identify approximately 700 alternative splicing events directly regulated by the neuron-specific factor Nova in the mouse brain, integrating RNA-binding data, splicing microarray data, Nova-binding motifs, and evolutionary signatures. The resulting integrative network revealed combinatorial regulation by Nova and the neuronal splicing factor Fox, interplay between phosphorylation and splicing, and potential links to neurologic disease. Thus, we have developed a general approach to understanding mammalian RNA regulation at the systems level.