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Phospho-S6 Ribosomal Protein (Ser240/244) (D68F8) XP Rabbit mAb antibody

RRID:AB_10694233

Antibody ID

AB_10694233

Target Antigen

Phospho-S6 Ribosomal Protein (Ser240/244) (D68F8) XP Rabbit mAb h, m, r, mk, mouse, human, non-human primate, rat

Proper Citation

(Cell Signaling Technology Cat# 5364, RRID:AB_10694233)

Clonality

monoclonal antibody

Comments

Applications: W, IHC-P, IF-IC, F. Consolidation on 9/2016: AB_10695727, AB_10698889.

Host Organism

rabbit

Vendor

Cell Signaling Technology

Brain Somatic Mutations in MTOR Disrupt Neuronal Ciliogenesis, Leading to Focal Cortical Dyslamination.

  • Park SM
  • Neuron
  • 2018 Jun 9

Literature context:


Abstract:

Focal malformations of cortical development (FMCDs), including focal cortical dysplasia (FCD) and hemimegalencephaly (HME), are major etiologies of pediatric intractable epilepsies exhibiting cortical dyslamination. Brain somatic mutations in MTOR have recently been identified as a major genetic cause of FMCDs. However, the molecular mechanism by which these mutations lead to cortical dyslamination remains poorly understood. Here, using patient tissue, genome-edited cells, and mouse models with brain somatic mutations in MTOR, we discovered that disruption of neuronal ciliogenesis by the mutations underlies cortical dyslamination in FMCDs. We found that abnormal accumulation of OFD1 at centriolar satellites due to perturbed autophagy was responsible for the defective neuronal ciliogenesis. Additionally, we found that disrupted neuronal ciliogenesis accounted for cortical dyslamination in FMCDs by compromising Wnt signals essential for neuronal polarization. Altogether, this study describes a molecular mechanism by which brain somatic mutations in MTOR contribute to the pathogenesis of cortical dyslamination in FMCDs.

Funding information:
  • NHLBI NIH HHS - HL073284(United States)

mTORC1 Promotes Metabolic Reprogramming by the Suppression of GSK3-Dependent Foxk1 Phosphorylation.

  • He L
  • Mol. Cell
  • 2018 Jun 7

Literature context:


Abstract:

The mammalian Target of Rapamycin Complex 1 (mTORC1)-signaling system plays a critical role in the maintenance of cellular homeostasis by sensing and integrating multiple extracellular and intracellular cues. Therefore, uncovering the effectors of mTORC1 signaling is pivotal to understanding its pathophysiological effects. Here we report that the transcription factor forkhead/winged helix family k1 (Foxk1) is a mediator of mTORC1-regulated gene expression. Surprisingly, Foxk1 phosphorylation is increased upon mTORC1 suppression, which elicits a 14-3-3 interaction, a reduction of DNA binding, and nuclear exclusion. Mechanistically, this occurs by mTORC1-dependent suppression of nuclear signaling by the Foxk1 kinase, Gsk3. This pathway then regulates the expression of multiple genes associated with glycolysis and downstream anabolic pathways directly modulated by Foxk1 and/or by Foxk1-regulated expression of Hif-1α. Thus, Foxk1 mediates mTORC1-driven metabolic rewiring, and it is likely to be critical for metabolic diseases where improper mTORC1 signaling plays an important role.

Funding information:
  • NHLBI NIH HHS - R01 HL121266()
  • NIGMS NIH HHS - P41 GM103533(United States)
  • NIGMS NIH HHS - R01 GM051405()

Acid Suspends the Circadian Clock in Hypoxia through Inhibition of mTOR.

  • Walton ZE
  • Cell
  • 2018 Jun 28

Literature context:


Abstract:

Recent reports indicate that hypoxia influences the circadian clock through the transcriptional activities of hypoxia-inducible factors (HIFs) at clock genes. Unexpectedly, we uncover a profound disruption of the circadian clock and diurnal transcriptome when hypoxic cells are permitted to acidify to recapitulate the tumor microenvironment. Buffering against acidification or inhibiting lactic acid production fully rescues circadian oscillation. Acidification of several human and murine cell lines, as well as primary murine T cells, suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling, a key regulator of translation in response to metabolic status. We find that acid drives peripheral redistribution of normally perinuclear lysosomes away from perinuclear RHEB, thereby inhibiting the activity of lysosome-bound mTOR. Restoring mTORC1 signaling and the translation it governs rescues clock oscillation. Our findings thus reveal a model in which acid produced during the cellular metabolic response to hypoxia suppresses the circadian clock through diminished translation of clock constituents.

Funding information:
  • NCRR NIH HHS - L30 RR020478(United States)

R-Ras1 and R-Ras2 Are Essential for Oligodendrocyte Differentiation and Survival for Correct Myelination in the Central Nervous System.

  • Sanz-Rodriguez M
  • J. Neurosci.
  • 2018 May 30

Literature context:


Abstract:

Rapid and effective neural transmission of information requires correct axonal myelination. Modifications in myelination alter axonal capacity to transmit electric impulses and enable pathological conditions. In the CNS, oligodendrocytes (OLs) myelinate axons, a complex process involving various cellular interactions. However, we know little about the mechanisms that orchestrate correct myelination. Here, we demonstrate that OLs express R-Ras1 and R-Ras2. Using female and male mutant mice to delete these proteins, we found that activation of the PI3K/Akt and Erk1/2-MAPK pathways was weaker in mice lacking one or both of these GTPases, suggesting that both proteins coordinate the activity of these two pathways. Loss of R-Ras1 and/or R-Ras2 diminishes the number of OLs in major myelinated CNS tracts and increases the proportion of immature OLs. In R-Ras1-/- and R-Ras2-/--null mice, OLs show aberrant morphologies and fail to differentiate correctly into myelin-forming phenotypes. The smaller OL population and abnormal OL maturation induce severe hypomyelination, with shorter nodes of Ranvier in R-Ras1-/- and/or R-Ras2-/- mice. These defects explain the slower conduction velocity of myelinated axons that we observed in the absence of R-Ras1 and R-Ras2. Together, these results suggest that R-Ras1 and R-Ras2 are upstream elements that regulate the survival and differentiation of progenitors into OLs through the PI3K/Akt and Erk1/2-MAPK pathways for proper myelination.SIGNIFICANCE STATEMENT In this study, we show that R-Ras1 and R-Ras2 play essential roles in regulating myelination in vivo and control fundamental aspects of oligodendrocyte (OL) survival and differentiation through synergistic activation of PI3K/Akt and Erk1/2-MAPK signaling. Mice lacking R-Ras1 and/or R-Ras2 show a diminished OL population with a higher proportion of immature OLs, explaining the observed hypomyelination in main CNS tracts. In vivo electrophysiology recordings demonstrate a slower conduction velocity of nerve impulses in the absence of R-Ras1 and R-Ras2. Therefore, R-Ras1 and R-Ras2 are essential for proper axonal myelination and accurate neural transmission.

Funding information:
  • Intramural NIH HHS - ZIA BC011010-06(United States)

Palladin Is a Neuron-Specific Translational Target of mTOR Signaling That Regulates Axon Morphogenesis.

  • Umegaki Y
  • J. Neurosci.
  • 2018 May 23

Literature context:


Abstract:

The mTOR signaling pathway regulates protein synthesis and diverse aspects of neuronal morphology that are important for brain development and function. To identify proteins controlled translationally by mTOR signaling, we performed ribosome profiling analyses in mouse cortical neurons and embryonic stem cells upon acute mTOR inhibition. Among proteins whose translation was significantly affected by mTOR inhibition selectively in neurons, we identified the cytoskeletal regulator protein palladin, which is localized within the cell body and axons in hippocampal neurons. Knockdown of palladin eliminated supernumerary axons induced by suppression of the tuberous sclerosis complex protein TSC1 in neurons, demonstrating that palladin regulates neuronal morphogenesis downstream of mTOR signaling. Our findings provide novel insights into an mTOR-dependent mechanism that controls neuronal morphogenesis through translational regulation.SIGNIFICANCE STATEMENT This study reports the discovery of neuron-specific protein translational responses to alterations of mTOR activity. By using ribosome profiling analysis, which can reveal the location and quantity of translating ribosomes on mRNAs, multiple aspects of protein translation were quantitatively analyzed in mouse embryonic stem cells and cortical neurons upon acute mTOR inhibition. Neurons displayed distinct patterns of ribosome occupancy for each codon and ribosome stalling during translation at specific positions of mRNAs. Importantly, the cytoskeletal regulator palladin was identified as a translational target protein of mTOR signaling in neurons. Palladin operates downstream of mTOR to modulate axon morphogenesis. This study identifies a novel mechanism of neuronal morphogenesis regulated by mTOR signaling through control of translation of the key protein palladin.

Funding information:
  • Medical Research Council - (United Kingdom)
  • NINDS NIH HHS - R01 NS051255()

Adipocyte p62/SQSTM1 Suppresses Tumorigenesis through Opposite Regulations of Metabolism in Adipose Tissue and Tumor.

  • Huang J
  • Cancer Cell
  • 2018 Apr 9

Literature context:


Abstract:

Obesity is a leading risk factor for cancer. However, understanding the crosstalk between adipocytes and tumor cells in vivo, independently of dietary contributions, is a major gap in the field. Here we used a prostate cancer (PCa) mouse model in which the signaling adaptor p62/Sqstm1 is selectively inactivated in adipocytes. p62 loss in adipocytes results in increased osteopontin secretion, which mediates tumor fatty acid oxidation and invasion, leading to aggressive metastatic PCa in vivo. Furthermore, p62 deficiency triggers in adipocytes a general shutdown of energy-utilizing pathways through mTORC1 inhibition, which supports nutrient availability for cancer cells. This reveals a central role of adipocyte's p62 in the symbiotic adipose tissue-tumor collaboration that enables cancer metabolic fitness.

Funding information:
  • Medical Research Council - 22358(United Kingdom)
  • NCI NIH HHS - R01 CA192642()
  • NCI NIH HHS - R01 CA211794()
  • NCI NIH HHS - R01 CA218254()
  • NIDDK NIH HHS - R01 DK108743()

mTOR-dependent alterations of Kv1.1 subunit expression in the neuronal subset-specific Pten knockout mouse model of cortical dysplasia with epilepsy.

  • Nguyen LH
  • Sci Rep
  • 2018 Feb 23

Literature context:


Abstract:

Cortical dysplasia (CD) is a common cause for intractable epilepsy. Hyperactivation of the mechanistic target of rapamycin (mTOR) pathway has been implicated in CD; however, the mechanisms by which mTOR hyperactivation contribute to the epilepsy phenotype remain elusive. Here, we investigated whether constitutive mTOR hyperactivation in the hippocampus is associated with altered voltage-gated ion channel expression in the neuronal subset-specific Pten knockout (NS-Pten KO) mouse model of CD with epilepsy. We found that the protein levels of Kv1.1, but not Kv1.2, Kv1.4, or Kvβ2, potassium channel subunits were increased, along with altered Kv1.1 distribution, within the hippocampus of NS-Pten KO mice. The aberrant Kv1.1 protein levels were present in young adult (≥postnatal week 6) but not juvenile (≤postnatal week 4) NS-Pten KO mice. No changes in hippocampal Kv1.1 mRNA levels were found between NS-Pten KO and WT mice. Interestingly, mTOR inhibition with rapamycin treatment at early and late stages of the pathology normalized Kv1.1 protein levels in NS-Pten KO mice to WT levels. Together, these studies demonstrate altered Kv1.1 protein expression in association with mTOR hyperactivation in NS-Pten KO mice and suggest a role for mTOR signaling in the modulation of voltage-gated ion channel expression in this model.

Funding information:
  • NIAID NIH HHS - R01AI067979(United States)
  • NICHD NIH HHS - U54 HD083092()
  • NINDS NIH HHS - R01 NS081053()

mTORC1/rpS6 regulates blood-testis barrier dynamics and spermatogenetic function in the testis in vivo.

  • Li SYT
  • Am. J. Physiol. Endocrinol. Metab.
  • 2018 Feb 1

Literature context:


Abstract:

The blood-testis barrier (BTB), conferred by Sertoli cells in the mammalian testis, is an important ultrastructure that supports spermatogenesis. Studies using animal models have shown that a disruption of the BTB leads to meiotic arrest, causing defects in spermatogenesis and male infertility. To better understand the regulation of BTB dynamics, we report findings herein to understand the role of ribosomal protein S6 (rpS6), a downstream signaling protein of mammalian target of rapamycin complex 1 (mTORC1), in promoting BTB disruption in the testis in vivo, making the barrier "leaky." Overexpression of wild-type rpS6 (rpS6-WT, the full-length cDNA cloned into the mammalian expression vector pCI-neo) and a constitutively active quadruple phosphomimetic mutant cloned into pCI-neo (p-rpS6-MT) vs. control (empty pCI-neo vector) was achieved by transfecting adult rat testes with the corresponding plasmid DNA using a Polyplus in vivo-jetPEI transfection reagent. On the basis of an in vivo functional BTB integrity assay, p-rpS6-MT was found to induce BTB disruption better than rpS6-WT did (and no effects in empty vector control), leading to defects in spermatogenesis, including loss of spermatid polarity and failure in the transport of cells (e.g., spermatids) and organelles (e.g., phagosomes), to be followed by germ exfoliation. More important, rpS6-WT and p-rpS6-MT exert their disruptive effects through changes in the organization of actin- and microtubule (MT)-based cytoskeletons, which are mediated by changes in the spatiotemporal expression of actin- and MT-based binding and regulatory proteins. In short, mTORC1/rpS6 signaling complex is a regulator of spermatogenesis and BTB by modulating the organization of the actin- and MT-based cytoskeletons.

Funding information:
  • Canadian Institutes of Health Research - (Canada)
  • NICHD NIH HHS - R01 HD056034()

RHOA G17V Induces T Follicular Helper Cell Specification and Promotes Lymphomagenesis.

  • Cortes JR
  • Cancer Cell
  • 2018 Feb 12

Literature context:


Abstract:

Angioimmunoblastic T cell lymphoma (AITL) is an aggressive tumor derived from malignant transformation of T follicular helper (Tfh) cells. AITL is characterized by loss-of-function mutations in Ten-Eleven Translocation 2 (TET2) epigenetic tumor suppressor and a highly recurrent mutation (p.Gly17Val) in the RHOA small GTPase. Yet, the specific role of RHOA G17V in AITL remains unknown. Expression of Rhoa G17V in CD4+ T cells induces Tfh cell specification; increased proliferation associated with inducible co-stimulator (ICOS) upregulation and increased phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase signaling. Moreover, RHOA G17V expression together with Tet2 loss resulted in development of AITL in mice. Importantly, Tet2-/-RHOA G17V tumor proliferation in vivo can be inhibited by ICOS/PI3K-specific blockade, supporting a driving role for ICOS signaling in Tfh cell transformation.

Funding information:
  • NCI NIH HHS - R01 CA197945()
  • NIDDK NIH HHS - R37 DK44746(United States)

Sensing and Transmitting Intracellular Amino Acid Signals through Reversible Lysine Aminoacylations.

  • He XD
  • Cell Metab.
  • 2018 Jan 9

Literature context:


Abstract:

Amino acids are known regulators of cellular signaling and physiology, but how they are sensed intracellularly is not fully understood. Herein, we report that each aminoacyl-tRNA synthetase (ARS) senses its cognate amino acid sufficiency through catalyzing the formation of lysine aminoacylation (K-AA) on its specific substrate proteins. At physiologic levels, amino acids promote ARSs bound to their substrates and form K-AAs on the ɛ-amine of lysines in their substrates by producing reactive aminoacyl adenylates. The K-AA marks can be removed by deacetylases, such as SIRT1 and SIRT3, employing the same mechanism as that involved in deacetylation. These dynamically regulated K-AAs transduce signals of their respective amino acids. Reversible leucylation on ras-related GTP-binding protein A/B regulates activity of the mammalian target of rapamycin complex 1. Glutaminylation on apoptosis signal-regulating kinase 1 suppresses apoptosis. We discovered non-canonical functions of ARSs and revealed systematic and functional amino acid sensing and signal transduction networks.

Funding information:
  • NIAID NIH HHS - P01AI045757(United States)

mTORC1 Activation during Repeated Regeneration Impairs Somatic Stem Cell Maintenance.

  • Haller S
  • Cell Stem Cell
  • 2017 Dec 7

Literature context:


Abstract:

The balance between self-renewal and differentiation ensures long-term maintenance of stem cell (SC) pools in regenerating epithelial tissues. This balance is challenged during periods of high regenerative pressure and is often compromised in aged animals. Here, we show that target of rapamycin (TOR) signaling is a key regulator of SC loss during repeated regenerative episodes. In response to regenerative stimuli, SCs in the intestinal epithelium of the fly and in the tracheal epithelium of mice exhibit transient activation of TOR signaling. Although this activation is required for SCs to rapidly proliferate in response to damage, repeated rounds of damage lead to SC loss. Consistently, age-related SC loss in the mouse trachea and in muscle can be prevented by pharmacologic or genetic inhibition, respectively, of mammalian target of rapamycin complex 1 (mTORC1) signaling. These findings highlight an evolutionarily conserved role of TOR signaling in SC function and identify repeated rounds of mTORC1 activation as a driver of age-related SC decline.

Funding information:
  • BLRD VA - I01 BX002324()
  • NCRR NIH HHS - UL1 RR024989(United States)
  • NHLBI NIH HHS - R01 HL132996()
  • NIA NIH HHS - K99 AG041764()
  • NIA NIH HHS - P01 AG036695()
  • NIA NIH HHS - R00 AG041764()
  • NIA NIH HHS - R01 AG047497()
  • NIA NIH HHS - R01 AG047820()
  • NIA NIH HHS - R37 AG023806()
  • NIDDK NIH HHS - R01 DK100342()
  • NIDDK NIH HHS - R01 DK113144()

Post-transcriptional Regulation of De Novo Lipogenesis by mTORC1-S6K1-SRPK2 Signaling.

  • Lee G
  • Cell
  • 2017 Dec 14

Literature context:


Abstract:

mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here, we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70K to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes, which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.

Funding information:
  • NHLBI NIH HHS - R01 HL074894(United States)

Oxysterol Restraint of Cholesterol Synthesis Prevents AIM2 Inflammasome Activation.

  • Dang EV
  • Cell
  • 2017 Nov 16

Literature context:


Abstract:

Type I interferon restrains interleukin-1β (IL-1β)-driven inflammation in macrophages by upregulating cholesterol-25-hydroxylase (Ch25h) and repressing SREBP transcription factors. However, the molecular links between lipid metabolism and IL-1β production remain obscure. Here, we demonstrate that production of 25-hydroxycholesterol (25-HC) by macrophages is required to prevent inflammasome activation by the DNA sensor protein absent in melanoma 2 (AIM2). We find that in response to bacterial infection or lipopolysaccharide (LPS) stimulation, macrophages upregulate Ch25h to maintain repression of SREBP2 activation and cholesterol synthesis. Increasing macrophage cholesterol content is sufficient to trigger IL-1β release in a crystal-independent but AIM2-dependent manner. Ch25h deficiency results in cholesterol-dependent reduced mitochondrial respiratory capacity and release of mitochondrial DNA into the cytosol. AIM2 deficiency rescues the increased inflammasome activity observed in Ch25h-/-. Therefore, activated macrophages utilize 25-HC in an anti-inflammatory circuit that maintains mitochondrial integrity and prevents spurious AIM2 inflammasome activation.

Funding information:
  • Howard Hughes Medical Institute - F30 AI120527()
  • NHLBI NIH HHS - P01 HL020948()
  • NIAID NIH HHS - R01 AI040098()
  • NIAID NIH HHS - R37 AI040098()
  • NIDCD NIH HHS - R01 DC014447(United States)
  • NIDDK NIH HHS - P30 DK063720()
  • NIGMS NIH HHS - T32 GM007618()

PTEN Loss Increases the Connectivity of Fast Synaptic Motifs and Functional Connectivity in a Developing Hippocampal Network.

  • Barrows CM
  • J. Neurosci.
  • 2017 Sep 6

Literature context:


Abstract:

Changes in synaptic strength and connectivity are thought to be a major mechanism through which many gene variants cause neurological disease. Hyperactivation of the PI3K-mTOR signaling network, via loss of function of repressors such as PTEN, causes epilepsy in humans and animal models, and altered mTOR signaling may contribute to a broad range of neurological diseases. Changes in synaptic transmission have been reported in animal models of PTEN loss; however, the full extent of these changes, and their effect on network function, is still unknown. To better understand the scope of these changes, we recorded from pairs of mouse hippocampal neurons cultured in a two-neuron microcircuit configuration that allowed us to characterize all four major connection types within the hippocampus. Loss of PTEN caused changes in excitatory and inhibitory connectivity, and these changes were postsynaptic, presynaptic, and transynaptic, suggesting that disruption of PTEN has the potential to affect most connection types in the hippocampal circuit. Given the complexity of the changes at the synaptic level, we measured changes in network behavior after deleting Pten from neurons in an organotypic hippocampal slice network. Slices containing Pten-deleted neurons showed increased recruitment of neurons into network bursts. Importantly, these changes were not confined to Pten-deleted neurons, but involved the entire network, suggesting that the extensive changes in synaptic connectivity rewire the entire network in such a way that promotes a widespread increase in functional connectivity.SIGNIFICANCE STATEMENT Homozygous deletion of the Pten gene in neuronal subpopulations in the mouse serves as a valuable model of epilepsy caused by mTOR hyperactivation. To better understand how gene deletions lead to altered neuronal activity, we investigated the synaptic and network effects that occur 1 week after Pten deletion. PTEN loss increased the connectivity of all four types of hippocampal synaptic connections, including two forms of increased inhibition of inhibition, and increased network functional connectivity. These data suggest that single gene mutations that cause neurological diseases such as epilepsy may affect a surprising range of connection types. Moreover, given the robustness of homeostatic plasticity, these diverse effects on connection types may be necessary to cause network phenotypes such as increased synchrony.

mTOR Inhibition Restores Amino Acid Balance in Cells Dependent on Catabolism of Extracellular Protein.

  • Nofal M
  • Mol. Cell
  • 2017 Sep 21

Literature context:


Abstract:

Scavenging of extracellular protein via macropinocytosis is an alternative to monomeric amino acid uptake. In pancreatic cancer, macropinocytosis is driven by oncogenic Ras signaling and contributes substantially to amino acid supply. While Ras signaling promotes scavenging, mTOR signaling suppresses it. Here, we present an integrated experimental-computational method that enables quantitative comparison of protein scavenging rates across cell lines and conditions. Using it, we find that, independently of mTORC1, amino acid scarcity induces protein scavenging and that under such conditions the impact of mTOR signaling on protein scavenging rate is minimal. Nevertheless, mTOR inhibition promotes growth of cells reliant on eating extracellular protein. This growth enhancement depends on mTORC1's canonical function in controlling translation rate: mTOR inhibition slows translation, thereby matching protein synthesis to the limited amino acid supply. Thus, paradoxically, in amino acid-poor conditions the pro-anabolic effects of mTORC1 are functionally opposed to growth.

Funding information:
  • NCI NIH HHS - F31 CA186513()
  • NCI NIH HHS - R01 CA163591()
  • NIDDK NIH HHS - DP1 DK113643()

A Ketogenic Diet Extends Longevity and Healthspan in Adult Mice.

  • Roberts MN
  • Cell Metab.
  • 2017 Sep 5

Literature context:


Abstract:

Calorie restriction, without malnutrition, has been shown to increase lifespan and is associated with a shift away from glycolysis toward beta-oxidation. The objective of this study was to mimic this metabolic shift using low-carbohydrate diets and to determine the influence of these diets on longevity and healthspan in mice. C57BL/6 mice were assigned to a ketogenic, low-carbohydrate, or control diet at 12 months of age and were either allowed to live their natural lifespan or tested for physiological function after 1 or 14 months of dietary intervention. The ketogenic diet (KD) significantly increased median lifespan and survival compared to controls. In aged mice, only those consuming a KD displayed preservation of physiological function. The KD increased protein acetylation levels and regulated mTORC1 signaling in a tissue-dependent manner. This study demonstrates that a KD extends longevity and healthspan in mice.

Funding information:
  • NIA NIH HHS - P01 AG025532()
  • NIDDK NIH HHS - U24 DK092993()

Spatial Activation of TORC1 Is Regulated by Hedgehog and E2F1 Signaling in the Drosophila Eye.

  • Kim W
  • Dev. Cell
  • 2017 Aug 21

Literature context:


Abstract:

Target of rapamycin complex 1 (TORC1) regulates cell growth in response to nutrients and growth factors. Although TORC1 signaling has been thoroughly studied at the cellular level, the regulation of TORC1 in multicellular tissues and organs has remained elusive. Here we found that TORC1 is selectively activated in the second mitotic wave (SMW), the terminal synchronous cell division, of the developing Drosophila eye. We demonstrated that Hedgehog (Hh) signaling regulates TORC1 through E2F1 and the cyclin D/Cdk4 complex in the SMW, and this regulation is independent from insulin and amino acid signaling pathways. TORC1 is necessary for the proper G1/S transition of the cells, and the activation of TORC1 rescues the cell-cycle defect of Hh signaling-deficient cells in the SMW. Based on this evolutionarily conserved regulation of TORC1 by Hh signaling, we propose that Hh-dependent developmental signaling pathways spatially regulate TORC1 activity in multicellular organisms.

Funding information:
  • NIMHD NIH HHS - G12 MD007592(United States)

Role of mTOR Inhibitors in Growth Hormone-Producing Pituitary Adenomas Harboring Different FGFR4 Genotypes.

  • Jalali S
  • Endocrinology
  • 2017 Jun 27

Literature context:


Abstract:

Pituitary adenomas (PAs) are common intracranial lesions. Available medical therapies are limited in PAs, and therefore, it is essential to identify treatments that control PA growth when surgery is not an option. Fibroblast growth factor 4 is implicated in PA pathogenesis; therefore, in this study, we used an isogenic mammosomatotroph cell line (GH4C1) harboring different fibroblast growth factor receptor (FGFR)-4 genotypes to establish and characterize intracranial xenograft mouse models that can be used for preclinical drug testing. We show that proliferating GH4C1 tumors have an average latency of 3 weeks to form. Histological analysis revealed that prototypic FGFR4 (G388) tumors express increased prolactin and less GH, whereas tumors possessing the polymorphic variant of FGFR4 (R388) express increased GH relative to prolactin. All tumors show abundant mammalian target of rapamycin (mTOR) signaling as confirmed using phosphorylated (p)-S6 and p-4E-binding protein 1 as downstream regulators of this pathway. We subsequently demonstrate that the mTOR inhibitor RAD001 decreases tumor growth rate and reduces p-S6 but not p-4E-binding protein 1 activation, regardless of FGFR4 status. More importantly, GH activity was significantly reduced after mTOR inhibition in the R388 polymorphic variant tumors. This reduction was also associated with a concomitant reduction in serum IGF-1 levels in the R388 group. In summary, we demonstrate that the GH4C1 FGFR polymorphic xenograft is a useful model for examining PAs. Furthermore, we show that RAD001 can efficiently reduce tumor growth rate by a reduction in mTOR signaling and more importantly results in control of GH expression and IGF-1 secretion, providing further support for using mTOR inhibitors in PA patients, in particular GH-producing adenomas.

Funding information:
  • Medical Research Council - MR/L011719/1(United Kingdom)
  • NHLBI NIH HHS - R37HL023081(United States)

LARP1 functions as a molecular switch for mTORC1-mediated translation of an essential class of mRNAs.

  • Hong S
  • Elife
  • 2017 Jun 26

Literature context:


Abstract:

The RNA binding protein, LARP1, has been proposed to function downstream of mTORC1 to regulate the translation of 5'TOP mRNAs such as those encoding ribosome proteins (RP). However, the roles of LARP1 in the translation of 5'TOP mRNAs are controversial and its regulatory roles in mTORC1-mediated translation remain unclear. Here we show that LARP1 is a direct substrate of mTORC1 and Akt/S6K1. Deep sequencing of LARP1-bound mRNAs reveal that non-phosphorylated LARP1 interacts with both 5' and 3'UTRs of RP mRNAs and inhibits their translation. Importantly, phosphorylation of LARP1 by mTORC1 and Akt/S6K1 dissociates it from 5'UTRs and relieves its inhibitory activity on RP mRNA translation. Concomitantly, phosphorylated LARP1 scaffolds mTORC1 on the 3'UTRs of translationally-competent RP mRNAs to facilitate mTORC1-dependent induction of translation initiation. Thus, in response to cellular mTOR activity, LARP1 serves as a phosphorylation-sensitive molecular switch for turning off or on RP mRNA translation and subsequent ribosome biogenesis.

Funding information:
  • NIDDK NIH HHS - R01 DK083491()
  • NIGMS NIH HHS - R01 GM088565()
  • NIGMS NIH HHS - R01 GM110019()

mTORC1 Signaling Contributes to Drinking But Not Blood Pressure Responses to Brain Angiotensin II.

  • Muta K
  • Endocrinology
  • 2017 May 31

Literature context:


Abstract:

Mechanistic target of rapamycin complex 1 (mTORC1) is a molecular node that couples extracellular cues to a wide range of cellular events controlling various physiological processes. Here, we identified mTORC1 signaling as a critical mediator of angiotensin II (Ang II) action in the brain. In neuronal GT1-7 cells, we show that Ang II stimulates neuronal mTORC1 signaling in an Ang II type 1 receptor-dependent manner. In mice, a single intracerebroventricular (ICV) injection or chronic sc infusion of Ang II activated mTORC1 signaling in the subfornical organ, a critical brain region in cardiovascular control and fluid balance. Moreover, transgenic sRA mice with brain-specific overproduction of Ang II displayed increased mTORC1 signaling in the subfornical organ. To test the functional role of brain mTORC1 in mediating the action of Ang II, we examined the consequence of mTORC1 inhibition with rapamycin on Ang II-induced increase in water intake and arterial pressure. ICV pretreatment with rapamycin blocked ICV Ang II-mediated increases in the frequency, duration, and amount of water intake but did not interfere with the pressor response evoked by Ang II. In addition, ICV delivery of rapamycin significantly reduced polydipsia, but not hypertension, of sRA mice. These results demonstrate that mTORC1 is a novel downstream pathway of Ang II type 1 receptor signaling in the brain and selectively mediates the effect of Ang II on drinking behavior.

Funding information:
  • NIGMS NIH HHS - R01 GM043154(United States)

rpS6 regulates blood-testis barrier dynamics through Arp3-mediated actin microfilament organization in rat sertoli cells. An in vitro study.

  • Mok KW
  • Endocrinology
  • 2015 May 18

Literature context:


Abstract:

In the seminiferous epithelium of rat testes, preleptotene spermatocytes residing in the basal compartment are transported across the blood-testis barrier (BTB) to enter the adluminal compartment at stage VIII of the epithelial cycle. This process involves redistribution of tight junction (TJ) proteins via reorganization of actin cytoskeleton in Sertoli cells that serves as attachment site for adhesion protein complexes. Ribosomal protein S6 (rpS6), a downstream molecule of mTORC1 (mammalian target of rapamycin complex 1), participates in this process via a yet-to-be defined mechanism. Here, we constructed an rpS6 quadruple phosphomimetic mutant by converting Ser residues at 235, 236, 240, and 244 to Glu via site-directed mutagenesis, making this mutant constitutively active. When this rpS6 mutant was overexpressed in Sertoli cells cultured in vitro with an established TJ barrier mimicking the BTB in vivo, it perturbed the TJ permeability by down-regulating and redistributing TJ proteins at the cell-cell interface. These changes are mediated by a reorganization of actin microfilaments, which was triggered by a redistribution of activated actin-related protein 3 (Arp3) as well as changes in Arp3-neuronal Wiskott-Aldrich Syndrome protein (N-WASP) interaction. This in turn induced reorganization of actin microfilaments, converting them from a "bundled" to an "unbundled/branched" configuration, concomitant with a reduced actin bundling activity, thereby destabilizing the TJ-barrier function. These changes were mediated by Akt (transforming oncogene of v-akt), because an Akt knockdown by RNA interference was able to mimic the phenotypes of rpS6 mutant overexpression at the Sertoli cell BTB. In summary, this study illustrates a mechanism by which mTORC1 signal complex regulates BTB function through rpS6 downstream by modulating actin organization via the Arp2/3 complex, which may be applicable to other tissue barriers.

Funding information:
  • NINDS NIH HHS - NS-23805(United States)

The role of hypothalamic mTORC1 signaling in insulin regulation of food intake, body weight, and sympathetic nerve activity in male mice.

  • Muta K
  • Endocrinology
  • 2015 Apr 21

Literature context:


Abstract:

Insulin action in the brain particularly the hypothalamus is critically involved in the regulation of several physiological processes, including energy homeostasis and sympathetic nerve activity, but the underlying mechanisms are poorly understood. The mechanistic target of rapamycin complex 1 (mTORC1) is implicated in the control of diverse cellular functions, including sensing nutrients and energy status. Here, we examined the role of hypothalamic mTORC1 in mediating the anorectic, weight-reducing, and sympathetic effects of central insulin action. In a mouse hypothalamic cell line (GT1-7), insulin treatment increased mTORC1 activity in a time-dependent manner. In addition, intracerebroventricular (ICV) administration of insulin to mice activated mTORC1 pathway in the hypothalamic arcuate nucleus, a key site of central action of insulin. Interestingly, inhibition of hypothalamic mTORC1 with rapamycin reversed the food intake- and body weight-lowering effects of ICV insulin. Rapamycin also abolished the ability of ICV insulin to cause lumbar sympathetic nerve activation. In GT1-7 cells, we found that insulin activation of mTORC1 pathway requires phosphatidylinositol 3-kinase (PI3K). Consistent with this, genetic disruption of PI3K in mice abolished insulin stimulation of hypothalamic mTORC1 signaling as well as the lumbar sympathetic nerve activation evoked by insulin. These results demonstrate the importance of mTORC1 pathway in the hypothalamus in mediating the action of insulin to regulate energy homeostasis and sympathetic nerve traffic. Our data also highlight the key role of PI3K as a link between insulin receptor and mTORC1 signaling in the hypothalamus.

Funding information:
  • NHLBI NIH HHS - R01 HL122494(United States)