ASCT2 silencing regulates mammalian target-of-rapamycin growth and survival signaling in human hepatoma cells

2007 ◽  
Vol 293 (1) ◽  
pp. C55-C63 ◽  
Author(s):  
Bryan C. Fuchs ◽  
Richard E. Finger ◽  
Marie C. Onan ◽  
Barrie P. Bode
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Ribosomal Protein ◽  
Binding Protein ◽  
Mammalian Target Of Rapamycin ◽  
Amino Acid Transporter ◽  
Human Hepatoma ◽  
Growth And Survival ◽  
Mtorc1 Signaling ◽  
Survival Signaling

System ASC amino acid transporter-2 (ASCT2) was previously demonstrated to be essential for human hepatoma cell growth and survival, as its silencing via inducible antisense RNA expression results in complete apoptosis within 48 h by a mechanism that transcends its role in amino acid delivery. To gain mechanistic insights into the reliance of cancerous liver cells on ASCT2, the aim of this study was to determine the early consequences of its silencing on the growth and survival signaling that presage apoptosis. Induced antisense ASCT2 RNA in SK-Hep1 cells led to >90% suppression of ASCT2 mRNA by 6 h and inhibition of mammalian target-of-rapamycin (mTOR)/raptor (mTOR complex-1; mTORC1) signaling by 8 h, as manifested by diminished p70 ribosomal protein S6 kinase-1 and eukaryotic initiation factor-4E (eIF4E) binding protein-1 phosphorylation, while protein synthesis rates declined by nearly 50% despite no measurable decreases in the cap binding protein eIF4G or cellular ribosomal protein content. Depressed mTORC1 signaling occurred before detectable reduction in ASCT2 activity but coincided with a 30% decline in total cellular ASCT2 protein. By 12 h after ASCT2 silencing, further decrements were observed in protein synthesis rates and ASCT2 protein and activity, each by ∼50%, while signaling from mTOR/rictor (mTOR complex-2; mTORC2) was stimulated as indexed by enhanced phosphorylation of the Akt/PKB kinase on serine-473 and of its proapoptotic substrate Bad on serine-136. These results suggest that ASCT2 silencing inhibits mTORC1 signaling to the translational machinery followed by an mTORC2-initiated survival response, establishing a link between amino acid transporter expression and mTOR function.

scholarly journals An increase in essential amino acid availability upregulates amino acid transporter expression in human skeletal muscle

2010 ◽  
Vol 298 (5) ◽  
pp. E1011-E1018 ◽  
Author(s):  
Micah J. Drummond ◽  
Erin L. Glynn ◽  
Christopher S. Fry ◽  
Kyle L. Timmerman ◽  
Elena Volpi ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Protein Expression ◽  
Human Skeletal Muscle ◽  
Amino Acid Transporter ◽  
Dependent Manner ◽  
Mtorc1 Signaling ◽  
Acid Transporter ◽  
Transporter Expression

Essential amino acids (EAA) stimulate skeletal muscle mammalian target of rapamycin complex 1 (mTORC1) signaling and protein synthesis. It has recently been reported that an increase in amino acid (AA) transporter expression during anabolic conditions is rapamycin-sensitive. The purpose of this study was to determine whether an increase in EAA availability increases AA transporter expression in human skeletal muscle. Muscle biopsies were obtained from the vastus lateralis of seven young adult subjects (3 male, 4 female) before and 1–3 h after EAA ingestion (10 g). Blood and muscle samples were analyzed for leucine kinetics using stable isotopic techniques. Quantitative RT-PCR, and immunoblotting were used to determine the mRNA and protein expression, respectively, of AA transporters and members of the general AA control pathway [general control nonrepressed (GCN2), activating transcription factor (ATF4), and eukaryotic initiation factor (eIF2) α-subunit (Ser52)]. EAA ingestion increased blood leucine concentration, delivery of leucine to muscle, transport of leucine from blood into muscle, intracellular muscle leucine concentration, ribosomal protein S6 (Ser240/244) phosphorylation, and muscle protein synthesis. This was followed with increased L-type AA transporter (LAT1), CD98, sodium-coupled neutral AA transporter (SNAT2), and proton-coupled amino acid transporter (PAT1) mRNA expression at 1 h ( P < 0.05) and modest increases in LAT1 protein expression (3 h post-EAA) and SNAT2 protein expression (2 and 3 h post-EAA, P < 0.05). Although there were no changes in GCN2 expression and eIF2α phosphorylation, ATF4 protein expression reached significance by 2 h post-EAA ( P < 0.05). We conclude that an increase in EAA availability upregulates human skeletal muscle AA transporter expression, perhaps in an mTORC1-dependent manner, which may be an adaptive response necessary for improved AA intracellular delivery.

scholarly journals Insulin Stimulates Human Skeletal Muscle Protein Synthesis via an Indirect Mechanism Involving Endothelial-Dependent Vasodilation and Mammalian Target of Rapamycin Complex 1 Signaling

2010 ◽  
Vol 24 (6) ◽  
pp. 1306-1306
Author(s):  
Kyle L. Timmerman ◽  
Jessica L. Lee ◽  
Hans C. Dreyer ◽  
Shaheen Dhanani ◽  
Erin L. Glynn ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Mammalian Target Of Rapamycin ◽  
Skeletal Muscle Protein ◽  
Capillary Recruitment ◽  
Isotope Tracers ◽  
Skeletal Muscle Protein Synthesis ◽  
Mtorc1 Signaling

Abstract Objective: Our objective was to determine whether endothelial-dependent vasodilation is an essential mechanism by which insulin stimulates human skeletal muscle protein synthesis and anabolism. Subjects: Subjects were healthy young adults (n = 14) aged 31 ± 2 yr. Design: Subjects were studied at baseline and during local leg infusion of insulin alone (control, n = 7) or insulin plus the nitric oxide synthase inhibitor NG-monomethyl-l-arginine (L-NMMA, n = 7) to prevent insulin-induced vasodilation. Methods: We measured skeletal muscle protein metabolism with stable isotope tracers, blood flow with indocyanine green, capillary recruitment with contrast enhanced ultrasound, glucose metabolism with stable isotope tracers, and phosphorylation of proteins associated with insulin (Akt) and amino acid-induced mammalian target of rapamycin(mTOR) complex 1 (mTORC1) signaling (mTOR, S6 kinase 1, and eukaryotic initiation factor 4Ebinding protein 1) with Western blot analysis. Results: No basal differences between groups were detected. During insulin infusion, blood flow and capillary recruitment increased in the control (P &lt; 0.05) group only; Akt phosphorylation and glucose uptake increased in both groups (P &lt; 0.05), with no group differences; and mTORC1 signaling increased more in control (P &lt; 0.05) than in l-NMMA. Phenylalanine net balance increased (P &lt; 0.05) in both groups, but with opposite mechanisms: increased protein synthesis (basal, 0.051 ± 0.006%/h; insulin, 0.077 ± 0.008%/h; P &lt; 0.05) with no change in proteolysis in control and decreased proteolysis (P &lt; 0.05) with no change in synthesis (basal, 0.061 ± 0.004%/h; insulin, 0.050 ± 0.006%/h; P value not significant) in l-NMMA. Conclusions: Endothelial-dependent vasodilation and the consequent increase in nutritive flow and mTORC1 signaling, rather than Akt signaling, are fundamental mechanisms by which insulin stimulates muscle protein synthesis in humans. Additionally, these data underscore that insulin modulates skeletal muscle proteolysis according to its effects on nutritive flow.

Leucine and mTORC1: a complex relationship

2012 ◽  
Vol 302 (11) ◽  
pp. E1329-E1342 ◽  
Author(s):  
Kayleigh M. Dodd ◽  
Andrew R. Tee
Keyword(s):  
Signal Transduction ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Protein Turnover ◽  
Muscle Growth ◽  
Protein Translation ◽  
Amino Acid Availability ◽  
Intracellular Amino Acid ◽  
System L ◽  
Mtorc1 Signaling

Amino acid availability is a rate-limiting factor in the regulation of protein synthesis. When amino acid supplies become restricted, mammalian cells employ homeostatic mechanisms to rapidly inhibit processes such as protein synthesis, which demands high levels of amino acids. Muscle cells in particular are subject to high protein turnover rates to maintain amino acid homeostasis. Mammalian target of rapamycin complex 1 (mTORC1) is an evolutionary conserved multiprotein complex that coordinates a network of signaling cascades and functions as a key mediator of protein translation, gene transcription, and autophagy. Signal transduction through mTORC1, which is centrally involved in muscle growth through enhanced protein translation, is governed by intracellular amino acid supply. The branched-chain amino acid leucine is critical for muscle growth and acts in part through activation of mTORC1. Recent research has revealed that mTORC1 signaling is coordinated primarily at the lysosomal membranes. This discovery has sparked a wealth of research in this field, revealing several different signaling molecules involved in transducing the amino acid signal to mTORC1, including the Rag GTPases, MAP4K3, and Vps34/ULK1. This review evaluates the current knowledge regarding cellular mechanisms that control and sense the intracellular amino acid pool. We discuss the role of leucine and mTORC1 in the regulation of amino acid transport via the system L and system A transporters such as LAT1 and SNAT2, as well as protein degradation via autophagic and proteasomal pathways. We also describe the complexities of energy homeostasis via AMPK and cell receptor-mediated growth signals that also converge on mTORC1. Leucine is a particularly potent regulator of protein turnover, to the extent where leucine stimulation alone is sufficient to stimulate mTORC1 signal transduction. The significance of leucine in this context is not yet known; however, recent advancements in this area will also be covered within this review.

scholarly journals The hnRNA-Binding Proteins hnRNP L and PTB Are Required for Efficient Translation of the Cat-1 Arginine/Lysine Transporter mRNA during Amino Acid Starvation

2009 ◽  
Vol 29 (10) ◽  
pp. 2899-2912 ◽  
Author(s):  
Mithu Majumder ◽  
Ibrahim Yaman ◽  
Francesca Gaccioli ◽  
Vladimir V. Zeenko ◽  
Chuanping Wang ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Binding Protein ◽  
Mrna Translation ◽  
Amino Acid Starvation ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Polypyrimidine Tract Binding Protein ◽  
Global Protein Synthesis

ABSTRACT The response to amino acid starvation involves the global decrease of protein synthesis and an increase in the translation of some mRNAs that contain an internal ribosome entry site (IRES). It was previously shown that translation of the mRNA for the arginine/lysine amino acid transporter Cat-1 increases during amino acid starvation via a mechanism that utilizes an IRES in the 5′ untranslated region of the Cat-1 mRNA. It is shown here that polypyrimidine tract binding protein (PTB) and an hnRNA binding protein, heterogeneous nuclear ribonucleoprotein L (hnRNP L), promote the efficient translation of Cat-1 mRNA during amino acid starvation. Association of both proteins with Cat-1 mRNA increased during starvation with kinetics that paralleled that of IRES activation, although the levels and subcellular distribution of the proteins were unchanged. The sequence CUUUCU within the Cat-1 IRES was important for PTB binding and for the induction of translation during amino acid starvation. Binding of hnRNP L to the IRES or the Cat-1 mRNA in vivo was independent of PTB binding but was not sufficient to increase IRES activity or Cat-1 mRNA translation during amino acid starvation. In contrast, binding of PTB to the Cat-1 mRNA in vivo required hnRNP L. A wider role of hnRNP L in mRNA translation was suggested by the decrease of global protein synthesis in cells with reduced hnRNP L levels. It is proposed that PTB and hnRNP L are positive regulators of Cat-1 mRNA translation via the IRES under stress conditions that cause a global decrease of protein synthesis.

Deletion of the amino acid transporter Slc6a14 suppresses tumour growth in spontaneous mouse models of breast cancer

2015 ◽  
Vol 469 (1) ◽  
pp. 17-23 ◽  
Author(s):  
Ellappan Babu ◽  
Yangzom D. Bhutia ◽  
Sabarish Ramachandran ◽  
Jaya P. Gnanaprakasam ◽  
Puttur D. Prasad ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Amino Acid ◽  
Mouse Models ◽  
Therapeutic Target ◽  
Tumour Growth ◽  
Mammalian Target Of Rapamycin ◽  
Amino Acid Transporter ◽  
Mtor Pathway ◽  
Target Of Rapamycin ◽  
Acid Transporter

Deletion of the amino acid transporter Slc6a14 in mice suppresses tumour growth in spontaneous models of breast cancer via interference with mammalian target of rapamycin (mTOR) pathway; this indicates an obligatory role for SLC6A14 in breast cancer, highlighting its potential as a therapeutic target.

scholarly journals Rag proteins regulate amino-acid-induced mTORC1 signalling

2009 ◽  
Vol 37 (1) ◽  
pp. 289-290 ◽  
Author(s):  
Yasemin Sancak ◽  
David M. Sabatini
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Cell Growth ◽  
Mammalian Target Of Rapamycin ◽  
Target Of Rapamycin ◽  
Master Regulator ◽  
Growth And Survival ◽  
Field Identification ◽  
Rag Proteins ◽  
Cell Growth And Survival

The serum- and nutrient-sensitive protein kinase mTOR (mammalian target of rapamycin) is a master regulator of cell growth and survival. The mechanisms through which nutrients regulate mTOR have been one of the major unanswered questions in the mTOR field. Identification of the Rag (Ras-related GTPase) family of GTPases as mediators of amino acid signalling to mTOR is an important step towards our understanding of this mechanism.

scholarly journals Activation of protein synthesis in cardiomyocytes by the hypertrophic agent phenylephrine requires the activation of ERK and involves phosphorylation of tuberous sclerosis complex 2 (TSC2)

2005 ◽  
Vol 388 (3) ◽  
pp. 973-984 ◽  
Author(s):  
Mark ROLFE ◽  
Laura E. McLEOD ◽  
Phillip F. PRATT ◽  
Christopher G. PROUD
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Tuberous Sclerosis ◽  
Tuberous Sclerosis Complex ◽  
Mammalian Target Of Rapamycin ◽  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Initiation Factor ◽  
Erk Activation ◽  
Mitogen Activated Protein

The hypertrophic Gq-protein-coupled receptor agonist PE (phenylephrine) activates protein synthesis. We showed previously that activation of protein synthesis by PE requires MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] and mTOR (mammalian target of rapamycin). However, it remained unclear whether ERK activation was required and which downstream components were involved in activating mTOR and protein synthesis. Using an adenovirus encoding the MKP3 (MAPK phosphatase 3) to inhibit ERK activity, we demonstrate that ERK is essential for the activation of protein synthesis by PE. Activation and phosphorylation of S6K1 (ribosomal protein S6 kinase 1) and phosphorylation of eIF4E (eukaryotic initiation factor 4E)-binding protein (both are mTOR targets) were also inhibited by MKP3, suggesting that ERK is also required for the activation of mTOR signalling. PE stimulation of cardiomyocytes induced the phosphorylation of TSC2 (tuberous sclerosis complex 2), a negative regulator of mTOR activity. TSC2 was phosphorylated only weakly at Thr1462, but phosphorylated at additional sites within the sequence RXRXX(S/T). This differs from the phosphorylation induced by insulin, indicating that MEK/ERK signalling targets distinct sites in TSC2. This phosphorylation may be mediated by p90RSK (90 kDa ribosomal protein S6K), which is activated by ERK, and appears to involve phosphorylation at Ser1798. Activation of protein synthesis by PE is partially insensitive to the mTOR inhibitor rapamycin. Inhibition of the MAPK-interacting kinases by CGP57380 decreases the phosphorylation of eIF4E and PE-induced protein synthesis. Moreover, CGP57380+rapamycin inhibited protein synthesis to the same extent as blocking ERK activation, suggesting that MAPK-interacting kinases and regulation of mTOR each contribute to the activation of protein synthesis by PE in cardiomyocytes.

scholarly journals Persistent mTORC1 signaling in cell senescence results from defects in amino acid and growth factor sensing

2017 ◽  
Vol 216 (7) ◽  
pp. 1949-1957 ◽  
Author(s):  
Bernadette Carroll ◽  
Glyn Nelson ◽  
Yoana Rabanal-Ruiz ◽  
Olena Kucheryavenko ◽  
Natasha A. Dunhill-Turner ◽  
...  
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Primary Cilia ◽  
Human Fibroblasts ◽  
Mammalian Target Of Rapamycin ◽  
Senescent Cell ◽  
Cell Senescence ◽  
Growth Factor Signaling ◽  
Mtorc1 Signaling ◽  
Underlying Mechanisms

Mammalian target of rapamycin complex 1 (mTORC1) and cell senescence are intimately linked to each other and to organismal aging. Inhibition of mTORC1 is the best-known intervention to extend lifespan, and recent evidence suggests that clearance of senescent cells can also improve health and lifespan. Enhanced mTORC1 activity drives characteristic phenotypes of senescence, although the underlying mechanisms responsible for increased activity are not well understood. We have identified that in human fibroblasts rendered senescent by stress, replicative exhaustion, or oncogene activation, mTORC1 is constitutively active and resistant to serum and amino acid starvation. This is driven in part by depolarization of senescent cell plasma membrane, which leads to primary cilia defects and a resultant failure to inhibit growth factor signaling. Further, increased autophagy and high levels of intracellular amino acids may act to support mTORC1 activity in starvation conditions. Interventions to correct these phenotypes restore sensitivity to the mTORC1 signaling pathway and cause death, indicating that persistent signaling supports senescent cell survival.

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