Autophagy suppression sensitizes glioma cells to IMP dehydrogenase inhibition-induced apoptotic death

We investigated the role of autophagy, a process of controlled self-digestion, in the in vitro anticancer action of the inosine monophosphate dehydrogenase (IMPDH) inhibitor ribavirin. Ribavirin-triggered oxidative stress, caspase activation, and apoptotic death in U251 human glioma cells were associated with the induction of autophagy, as confirmed by intracellular acidification, appearance of autophagic vesicles, conversion of microtubule associated protein 1 light chain 3 (LC3)-I to autophagosome-associated LC3-II, and degradation of autophagic target p62/sequestosome 1. Ribavirin downregulated the activity of autophagy-inhibiting mammalian target of rapamycin complex 1 (mTORC1), as indicated by a decrease in phosphorylation of the mTORC1 substrate ribosomal p70S6 kinase and reduction of the mTORC1-activating Src/Akt signaling. Guanosine supplementation inhibited, while IMPDH inhibitor tiazofurin mimicked ribavirin-mediated autophagy induction, suggesting the involvement of IMPDH blockade in the observed effect. Autophagy suppression by ammonium chloride, bafilomycin A1, or RNA interference-mediated knockdown of LC3 sensitized glioma cells to ribavirin-induced apoptosis. Ribavirin also induced cytoprotective autophagy associated with Akt/mTORC1 inhibition in C6 rat glioma cells. Our data demonstrate that ribavirin-triggered Akt/mTORC1-dependent autophagy counteracts apoptotic death of glioma cells, indicating autophagy suppression as a plausible therapeutic strategy for sensitization of cancer cells to IMPDH inhibition.     

Autophagy-dependent and -independent involvement of AMP-activated protein kinase in 6-hydroxydopamine toxicity to SH-SY5Y neuroblastoma cells

The role of the main intracellular energy sensor adenosine monophosphate (AMP)-activated protein kinase (AMPK) in the induction of autophagic response and cell death was investigated in SH-SY5Y human neuroblastoma cells exposed to the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). The induction of autophagy in SH-SY5Y cells was demonstrated by acridine orange staining of intracellular acidic vesicles, the presence of autophagosome- and autophagolysosome-like vesicles confirmed by transmission electron microscopy, as well as by microtubule-associated protein 1 light-chain 3 (LC3) conversion and p62 degradation detected by immunoblotting. 6-OHDA induced phosphorylation of AMPK and its target Raptor, followed by the dephosphorylation of the major autophagy inhibitor mammalian target of rapamycin (mTOR) and its substrate p70S6 kinase (S6K). 6-OHDA treatment failed to suppress mTOR/S6K phosphorylation and to increase LC3 conversion, p62 degradation and cytoplasmatic acidification in neuroblastoma cells in which AMPK expression was downregulated by RNA interference. Transfection of SH-SY5Y cells with AMPK or LC3β shRNA, as well as treatment with pharmacological autophagy inhibitors suppressed, while mTOR inhibitor rapamycin potentiated 6-OHDA-induced oxidative stress and apoptotic cell death. 6-OHDA induced phosphorylation of p38 mitogen-activated protein (MAP) kinase in an AMPK-dependent manner, and pharmacological inhibition of p38 MAP kinase reduced neurotoxicity, but not AMPK activation and autophagy triggered by 6-OHDA. Finally, the antioxidant N-acetyl cysteine antagonized 6-OHDA-induced activation of AMPK, p38 and autophagy. These data suggest that oxidative stress-mediated AMPK/mTOR-dependent autophagy and AMPK/p38-dependent apoptosis could be valid therapeutic targets for neuroprotection.     

Signal transduction pathways in FSH regulation of rat Sertoli cell proliferation

The final number of Sertoli cells reached during the proliferative periods determines sperm production capacity in adulthood. It is well known that FSH is the major Sertoli cell mitogen; however, little is known about the signal transduction pathways that regulate the proliferation of Sertoli cells. The hypothesis of this investigation was that FSH regulates proliferation through a PI3K/Akt/mTORC1 pathway, and additionally, AMPK-dependent mechanisms counteract FSH proliferative effects. The present study was performed in 8-day-old rat Sertoli cell cultures. The results presented herein show that FSH, in addition to increasing p-Akt, p-mTOR, and p-p70S6K levels, increases p-PRAS40 levels, probably contributing to improving mTORC1 signaling. Furthermore, the decrease in FSH-stimulated p-Akt, p-mTOR, p-p70S6K, and p-PRAS40 levels in the presence of wortmannin emphasizes the participation of PI3K in FSH signaling. Additionally, the inhibition of FSH-stimulated Sertoli cell proliferation by the effect of wortmannin and rapamycin point to the relevance of the PI3K/Akt/mTORC1 signaling pathway in the mitotic activity of FSH. On the other hand, by activating AMPK, several interesting observations were made. Activation of AMPK produced an increase in Raptor phosphorylation, a decrease in p70S6K phosphorylation, and a decrease in FSH-stimulated Sertoli cell proliferation. The decrease in FSH-stimulated cell proliferation was accompanied by an increased expression of the cyclin-dependent kinase inhibitors (CDKIs) p19INK4d, p21Cip1, and p27Kip1. In summary, it is concluded that FSH regulates Sertoli cell proliferation with the participation of a PI3K/Akt/mTORC1 pathway and that AMPK activation may be involved in the detention of proliferation by, at least in part, a decrease in mTORC1 signaling and an increase in CDKI expression.     

Compound C induces protective autophagy in cancer cells through AMPK inhibition-independent blockade of Akt/mTOR pathway

In the present study, we report that compound C, an inhibitor of a key intracellular energy sensor AMP-activated protein kinase (AMPK), can induce autophagy in cancer cells. The induction of autophagy in U251 human glioma cell line was demonstrated by acridine orange staining of intracellular acidic vesicles, Beclin 1 induction, p62 decrease and conversion of LC3-I to autophagosome-associated LC3-II in the presence of proteolysis inhibitors. The presence of autophagosome-like vesicles was confirmed by transmission electron microscopy. Compound C-mediated inhibition of AMPK and raptor in U251 cells was associated with paradoxical decrease in phosphorylation of AMPK/raptor-repressed mTOR, a major negative regulator of autophagy, and its downstream target p70S6K. The phosphorylation of an mTOR activator Akt and the PI3K-activating kinase Src was also impaired in compound C-treated cells. The siRNA-mediated AMPK silencing did not reduce the activity of the Akt/mTOR/p70S6K pathway and AMPK activators metformin and AIC AR failed to block compound C-induced autophagy. Autophagy inhibitors bafilomycin and chloroquine significantly increased the cytotoxicity of compound C towards U251 cells, as confirmed by increase in lactate dehydrogenase release, DNA fragmentation and caspase-3 activation. Similar effects of compound C were also observed in C6 rat glioma, L929 mouse fibrosarcoma and B16 mouse melanoma cell lines. Since compound C has previously been reported to suppress AMPK-dependent autophagy in different cell types, our findings suggest that the effects of compound C on autophagy might be dose-, cell type- and/or context-dependent. By demonstrating the ability of compound C to induce autophagic response in cancer cells via AMPK inhibition-independent downregulation of Akt/mTOR pathway, our results warrant caution when using compound C to inhibit AMPK-dependent cellular responses, but also support further exploration of compound C and related molecules as potential anticancer agents.     

Compound C induces protective autophagy in cancer cells through AMPK inhibition-independent blockade of Akt/mTOR pathway

In the present study, we report that compound C, an inhibitor of a key intracellular energy sensor AMP-activated protein kinase (AMPK), can induce autophagy in cancer cells. The induction of autophagy in U251 human glioma cell line was demonstrated by acridine orange staining of intracellular acidic vesicles, Beclin 1 induction, p62 decrease and conversion of LC3-I to autophagosome-associated LC3-II in the presence of proteolysis inhibitors. The presence of autophagosome-like vesicles was confirmed by transmission electron microscopy. Compound C-mediated inhibition of AMPK and raptor in U251 cells was associated with paradoxical decrease in phosphorylation of AMPK/raptor-repressed mTOR, a major negative regulator of autophagy, and its downstream target p70S6K. The phosphorylation of an mTOR activator Akt and the PI3K-activating kinase Src was also impaired in compound C-treated cells. The siRNA-mediated AMPK silencing did not reduce the activity of the Akt/mTOR/p70S6K pathway and AMPK activators metformin and AIC AR failed to block compound C-induced autophagy. Autophagy inhibitors bafilomycin and chloroquine significantly increased the cytotoxicity of compound C towards U251 cells, as confirmed by increase in lactate dehydrogenase release, DNA fragmentation and caspase-3 activation. Similar effects of compound C were also observed in C6 rat glioma, L929 mouse fibrosarcoma and B16 mouse melanoma cell lines. Since compound C has previously been reported to suppress AMPK-dependent autophagy in different cell types, our findings suggest that the effects of compound C on autophagy might be dose-, cell type- and/or context-dependent. By demonstrating the ability of compound C to induce autophagic response in cancer cells via AMPK inhibition-independent downregulation of Akt/mTOR pathway, our results warrant caution when using compound C to inhibit AMPK-dependent cellular responses, but also support further exploration of compound C and related molecules as potential anticancer agents.     

PF-4708671 Activates AMPK Independently of p70S6K1 Inhibition

The P70 ribosomal protein S6 kinase 1 (P70S6K1) is activated by the mammalian target of rapamycin (mTORC1) and regulates proliferation, growth, and metabolism. PF-4708671 is a novel, cell-permeable, has been proposed to be a highly specific inhibitor of p70S6K1. It is used in micromolar concentration range to dissect signaling pathways downstream of mTORC1 and to study the function of p70S6K1. Here we show that PF-4708671 induces AMP-activated protein kinase (AMPK) phosphorylation and activation in immortalized mouse embryonic fibroblasts (MEF) independently of p70S6K1, due to specific inhibition of mitochondrial respiratory chain Complex I.     

Curcumin represses mTORC1 signaling in Caco-2 cells by a two-sided mechanism involving the loss of IRS-1 and activation of AMPK

The mechanistic target of rapamycin complex 1 (mTORC1) is a central modulator of inflammation and tumorigenesis in the gastrointestinal tract. Growth factors upregulate mTORC1 via the PI3K/AKT and/or Ras/MAPK signal pathways. Curcumin (CUR), a polyphenol found in turmeric roots (Curcuma longa) can repress mTORC1 kinase activity in colon cancer cell lines; however, key aspects of CUR mechanism of action remain to be elucidated including its primary cellular target. We investigated the molecular effects of physiologically attainable concentration of CUR (20 μM) in the intestinal lumen on mTORC1 signaling in Caco-2 cells. CUR markedly inhibited mTORC1 kinase activity as determined by the decreased phosphorylation of p70S6K (Thr389, −99%, P < 0.0001) and S6 (Ser235/236, −92%, P < 0.0001). Mechanistically, CUR decreased IRS-1 protein abundance (−80%, P < 0.0001) thereby downregulating AKT phosphorylation (Ser473, −94%, P < 0.0001) and in turn PRAS40 phosphorylation (Thr246, −99%, P < 0.0001) while total PRAS40 abundance was unchanged. The use of proteasome inhibitor MG132 showed that CUR-mediated loss of IRS-1 involved proteasomal degradation. CUR lowered Raptor protein abundance, which combined with PRAS40 hypophosphorylation, suggests CUR repressed mTORC1 activity by inducing compositional changes that hinder the complex assembly. In addition, CUR activated AMPK (Thr172 phosphorylation, P < 0.0001), a recognized repressor of mTORC1, and AMPK upstream regulator LKB1. Although cargo adapter protein p62 was decreased by CUR (−49%, P < 0.004), CUR did not significantly induce autophagy. Inhibition of AKT/mTORC1 signaling by CUR may have lifted the cross-inhibition onto MAPK signaling, which became induced; p-ERK1/2 (+670%, P < 0.0001), p-p38 (+1433%, P < 0.0001). By concomitantly targeting IRS-1 and AMPK, CUR's mechanism of mTORC1 inhibition is distinct from that of rapamycin.     

Osteoprotegerin inhibit osteoclast differentiation and bone resorption by enhancing autophagy via AMPK/mTOR/p70S6K signaling pathway in vitro

Osteoclasts are highly differentiated terminal cells formed by fusion of hematopoietic stem cells. Previously, osteoprotegerin (OPG) inhibit osteoclast differentiation and bone resorption by blocking receptor activator of nuclear factor-κB ligand (RANKL) binding to RANK indirect mechanism. Furthermore, autophagy plays an important role during osteoclast differentiation and function. However, whether autophagy is involved in OPG-inhibited osteoclast formation and bone resorption is not known. To elucidate the role of autophagy in OPG-inhibited osteoclast differentiation and bone resorption, we used primary osteoclast derived from mice bone marrow monocytes/macrophages (BMM) by induced M-CSF and RANKL. The results showed that autophagy-related proteins expression were upregulated; tartrate-resistant acid phosphatase-positive osteoclast number and bone resorption activity were decreased; LC3 puncta and autophagosomes number were increased and activated AMPK/mTOR/p70S6K signaling pathway. In addition, chloroquine (as the autophagy/lysosome inhibitor, CQ) or rapamycin (as the autophagy/lysosome inhibitor, Rap) attenuated osteoclast differentiation and bone resorption activity by OPG treatment via AMPK/mTOR/p70S6K signaling pathway. Our data demonstrated that autophagy plays a critical role in OPG inhibiting osteoclast differentiation and bone resorption via AMPK/mTOR/p70S6K signaling pathway in vitro.     

Autophagy Is a Protective Mechanism for Human Melanoma Cells under Acidic Stress

Cyclic hypoxia and alterations in oncogenic signaling contribute to switch cancer cell metabolism from oxidative phosphorylation to aerobic glycolysis. A major consequence of up-regulated glycolysis is the increased production of metabolic acids responsible for the presence of acidic areas within solid tumors. Tumor acidosis is an important determinant of tumor progression and tumor pH regulation is being investigated as a therapeutic target. Autophagy is a cellular catabolic pathway leading to lysosomal degradation and recycling of proteins and organelles, currently considered an important survival mechanism in cancer cells under metabolic stress or subjected to chemotherapy. We investigated the response of human melanoma cells cultured in acidic conditions in terms of survival and autophagy regulation. Melanoma cells exposed to acidic culture conditions (7.0 < pH < 6.2) promptly accumulated LC3+ autophagic vesicles. Immunoblot analysis showed a consistent increase of LC3-II in acidic culture conditions as compared with cells at normal pH. Inhibition of lysosomal acidification by bafilomycin A1 further increased LC3-II accumulation, suggesting an active autophagic flux in cells under acidic stress. Acute exposure to acidic stress induced rapid inhibition of the mammalian target of rapamycin signaling pathway detected by decreased phosphorylation of p70S6K and increased phosphorylation of AMP-activated protein kinase, associated with decreased ATP content and reduced glucose and leucine uptake. Inhibition of autophagy by knockdown of the autophagic gene ATG5 consistently reduced melanoma cell survival in low pH conditions. These observations indicate that induction of autophagy may represent an adaptation mechanism for cancer cells exposed to an acidic environment. Our data strengthen the validity of therapeutic strategies targeting tumor pH regulation and autophagy in progressive malignancies.     

In vitro antiglioma action of indomethacin is mediated via AMP-activated protein kinase/mTOR complex 1 signalling pathway

We investigated the role of the intracellular energy-sensing AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in the in vitro antiglioma effect of the cyclooxygenase (COX) inhibitor indomethacin. Indomethacin was more potent than COX inhibitors diclofenac, naproxen, and ketoprofen in reducing the viability of U251 human glioma cells. Antiglioma effect of the drug was associated with p21 increase and G₂M cell cycle arrest, as well as with oxidative stress, mitochondrial depolarization, caspase activation, and the induction of apoptosis. Indomethacin increased the phosphorylation of AMPK and its targets Raptor and acetyl-CoA carboxylase (ACC), and reduced the phosphorylation of mTOR and mTOR complex 1 (mTORC1) substrates p70S6 kinase and PRAS40 (Ser183). AMPK knockdown by RNA interference, as well as the treatment with the mTORC1 activator leucine, prevented indomethacin-mediated mTORC1 inhibition and cytotoxic action, while AMPK activators metformin and AICAR mimicked the effects of the drug. AMPK activation by indomethacin correlated with intracellular ATP depletion and increase in AMP/ATP ratio, and was apparently independent of COX inhibition or the increase in intracellular calcium. Finally, the toxicity of indomethacin towards primary human glioma cells was associated with the activation of AMPK/Raptor/ACC and subsequent suppression of mTORC1/S6K. By demonstrating the involvement of AMPK/mTORC1 pathway in the antiglioma action of indomethacin, our results support its further exploration in glioma therapy.     

PFKP facilitates ATG4B phosphorylation during amino acid deprivation-induced autophagy

ATG4B facilitates autophagy by promoting autophagosome maturation through the reversible lipidation and delipidation of LC3. Recent reports have shown that phosphorylation of ATG4B regulates its activity and LC3 processing, leading to modulate autophagy activity. However, the mechanism about how ATG4B phosphorylation is involved in amino acid deprivation-induced autophagy is unclear. Here, we combined the tandem affinity purification with mass spectrometry (MS) and identified the ATG4B-interacting proteins including its well-known partner gamma-aminobutyric acid receptor-associated protein (GABARAP, a homolog of LC3) and phosphofructokinase 1 platelet isoform (PFKP). Further immunoprecipitation assays showed that amino acid deprivation strengthened the interaction between ATG4B and PFKP. By genetic depletion of PFKP using CRISPR/Cas9, we uncovered that PFKP loss reduced the degradation of LC3-II and p62 due to a partial block in autophagic flux. Furthermore, MS analysis of Flag-tagged ATG4B immunoprecipitates identified phosphorylation of ATG4B serine 34 residue (S34) and PFKP serine 386 residue (S386) under amino acid deprivation condition. In vitro kinase assay validated that PFKP functioning as a protein kinase phosphorylated ATG4B at S34. This phosphorylation could enhance ATG4B activity and p62 degradation. In addition, PFKP S386 phosphorylation was important to ATG4B S34 phosphorylation and autophagy in HEK293T cells. In brief, our findings describe that PFKP, a rate-limiting enzyme in the glycolytic pathway, functions as a protein kinase for ATG4B to regulate ATG4B activity and autophagy under amino acid deprivation condition.     

Perlecan inhibits autophagy to maintain muscle homeostasis in mouse soleus muscle

The autophagy–lysosome system is essential for muscle protein synthesis and degradation equilibrium, and its dysfunction has been linked to various muscle disorders. It has been reported that a diverse collection of extracellular matrix constituents, including decorin, collagen VI, laminin α2, endorepellin, and endostatin, can modulate autophagic signaling pathways. However, the association between autophagy and perlecan in muscle homeostasis remains unclear. The mechanical unloading of perlecan-deficient soleus muscles resulted in significantly decreased wet weights and cross-section fiber area compared with those of control mice. We found that perlecan deficiency in slow-twitch soleus muscles enhanced autophagic activity. This was accompanied by a decrease in autophagic substrates, such as p62, and an increase in LC3II levels. Furthermore, perlecan deficiency caused a reduction in the phosphorylation levels of p70S6k and Akt and increased the phosphorylation of AMPKα. Our findings suggested that perlecan inhibits the autophagic process through the activation of the mTORC1 pathway. This autophagic response may be a novel target for enhancing the efficacy of skeletal muscle atrophy treatment.     

AMP‐activated protein kinase counteracts brain‐derived neurotrophic factor‐induced mammalian target of rapamycin complex 1 signaling in neurons

Growth factors and nutrients, such as amino acids and glucose, regulate mammalian target of rapamycin complex 1 (mTORC1) signaling and subsequent translational control in a coordinated manner. Brain‐derived neurotrophic factor (BDNF), the most prominent neurotrophic factor in the brain, activates mTORC1 and induces phosphorylation of its target, p70S6 kinase (p70S6K), at Thr389 in neurons. BDNF also increases mammalian target of rapamycin‐dependent novel protein synthesis in neurons. Here, we report that BDNF‐induced p70S6K activation is dependent on glucose, but not amino acids, sufficiency in cultured cortical neurons. AMP‐activated protein kinase (AMPK) is the molecular background to this specific nutrient dependency. Activation of AMPK, which is induced by glucose deprivation, treatment with pharmacological agents such as 2‐Deoxy‐d ‐glucose, metformin, and 5‐aminoimidazole‐4‐carboxamide ribonucleoside or forced expression of a constitutively active AMPKα subunit, counteracts BDNF‐induced phosphorylation of p70S6K and enhanced protein synthesis in cortical neurons. These results indicate that AMPK inhibits the effects of BDNF on mTORC1‐mediated translation in neurons.

     

Phospholipase D regulates myogenic differentiation through the activation of both mTORC1 and mTORC2 complexes

How phospholipase D (PLD) is involved in myogenesis remains unclear. At the onset of myogenic differentiation of L6 cells induced by the PLD agonist vasopressin in the absence of serum, mTORC1 complex was rapidly activated, as reflected by phosphorylation of S6 kinase1 (S6K1). Both the long (p85) and short (p70) S6K1 isoforms were phosphorylated in a PLD1-dependent way. Short rapamycin treatment specifically inhibiting mTORC1 suppressed p70 but not p85 phosphorylation, suggesting that p85 might be directly activated by phosphatidic acid. Vasopressin stimulation also induced phosphorylation of Akt on Ser-473 through PLD1-dependent activation of mTORC2 complex. In this model of myogenesis, mTORC2 had a positive role mostly unrelated to Akt activation, whereas mTORC1 had a negative role, associated with S6K1-induced Rictor phosphorylation. The PLD requirement for differentiation can thus be attributed to its ability to trigger via mTORC2 activation the phosphorylation of an effector that could be PKCα. Moreover, PLD is involved in a counter-regulation loop expected to limit the response. This study thus brings new insights in the intricate way PLD and mTOR cooperate to control myogenesis.     

AMPK represses TOP mRNA translation but not global protein synthesis in liver

The AMP-activated protein kinase (AMPK) represses signaling through the mammalian target of rapamycin complex 1 (mTORC1). In muscle, repression of mTORC1 leads to a reduction in global protein synthesis. In contrast, repression of mTORC1 in the liver has no immediate effect on global protein synthesis. In the present study, signaling through mTORC1 and translation of specific mRNAs such as those bearing a 5′-terminal oligopyrimidine (TOP) tract and were examined in rat liver following activation of AMPK after treadmill running. Activation of AMPK repressed translation of the TOP mRNAs encoding rpS6, rpS8, and eEF1α. In contrast, neither global protein synthesis nor translation of mRNAs encoding GAPDH or β-actin was changed. Basal phosphorylation of the mTORC1 target 4E-BP1, but not S6K1 or rpS6, was reduced following activation of AMPK. Thus, in liver, AMPK activation repressed translation of TOP mRNAs through a mechanism distinct from downregulated phosphorylation of S6K1 or rpS6.     

CPTP: A sphingolipid transfer protein that regulates autophagy and inflammasome activation†

The macroautophagy/autophagy and inflammasome pathways are linked through their roles in innate immunity and chronic inflammatory disease. Ceramide-1-phosphate (C1P) is a bioactive sphingolipid that regulates pro-inflammatory eicosanoid production. Whether C1P also regulates autophagy and inflammasome assembly/activation is not known. Here we show that CPTP (a protein that traffics C1P from its site of phosphorylation in the trans-Golgi to target membranes) regulates both autophagy and inflammasome activation. In human epithelial cells, knockdown of CPTP (but not GLTP [glycolipid transfer protein]) or expression of C1P binding-site point mutants, stimulated an 8- to 10-fold increase in autophagosomes and altered endogenous LC3-II and SQSTM1/p62 protein expression levels. CPTP depletion-induced autophagy elevated early markers of autophagosome formation (Golgi-derived ATG9A-vesicles, WIPI1), required key phagophore assembly and elongation factors (ATG5, ATG7, ULK1), and suppressed MTOR phosphorylation and that of its downstream target, RPS6KB1/p70S6K. Wild-type CPTP overexpression exerted a protective effect against starvation-induced autophagy. In THP-1 macrophage-like surveillance cells, CPTP knockdown induced not only autophagy but also elevated CASP1/caspase-1 levels, and strongly increased IL1B/interleukin-1β and IL18 release via a NLRP3 (but not NLRC4) inflammasome-based mechanism, while only moderately increasing inflammatory (pyroptotic) cell death. Inflammasome assembly and activation stimulated by CPTP depletion were autophagy dependent. Elevation of intracellular C1P by exogenous C1P treatment (instead of CPTP inhibition) also induced autophagy and IL1B release. Our findings identify human CPTP as an endogenous regulator of early-stage autophagosome assembly and inflammasome-driven, pro-inflammatory cytokine generation and release.     

p53/TAp63 and AKT Regulate Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling through Two Independent Parallel Pathways in the Presence of DNA Damage

Under conditions of DNA damage, the mammalian target of rapamycin complex 1 (mTORC1) is inhibited, preventing cell cycle progression and conserving cellular energy by suppressing translation. We show that suppression of mTORC1 signaling to 4E-BP1 requires the coordinated activity of two tumor suppressors, p53 and p63. In contrast, suppression of S6K1 and ribosomal protein S6 phosphorylation by DNA damage is Akt-dependent. We find that loss of either p53, required for the induction of Sestrin 1/2, or p63, required for the induction of REDD1 and activation of the tuberous sclerosis complex, prevents the DNA damage-induced suppression of mTORC1 signaling. These data indicate that the negative regulation of cap-dependent translation by mTORC1 inhibition subsequent to DNA damage is abrogated in most human cancers.     

FMRP S499 Is Phosphorylated Independent of mTORC1-S6K1 Activity

Hyperactive mammalian target of rapamycin (mTOR) is associated with cognitive deficits in several neurological disorders including tuberous sclerosis complex (TSC). The phosphorylation of the mRNA-binding protein FMRP reportedly depends on mTOR complex 1 (mTORC1) activity via p70 S6 kinase 1 (S6K1). Because this phosphorylation is thought to regulate the translation of messages important for synaptic plasticity, we explored whether FMRP phosphorylation of the S6K1-dependent residue (S499) is altered in TSC and states of dysregulated TSC-mTORC1 signaling. Surprisingly, we found that FMRP S499 phosphorylation was unchanged in heterozygous and conditional Tsc1 knockout mice despite significantly elevated mTORC1-S6K1 activity. Neither up- nor down-regulation of the mTORC1-S6K1 axis in vivo or in vitro had any effect on phospho-FMRP S499 levels. In addition, FMRP S499 phosphorylation was unaltered in S6K1-knockout mice. Collectively, these data strongly suggest that FMRP S499 phosphorylation is independent of mTORC1-S6K1 activity and is not altered in TSC.     

The role of AMPK/mTOR/S6K1 signaling axis in mediating the physiological process of exercise-induced insulin sensitization in skeletal muscle of C57BL/6 mice

The crosstalk between mTORC1/S6K1 signaling and AMPK is emerging as a powerful and highly regulated way to gauge cellular energy and nutrient content. The aim of the current study was to determine the mechanism by which exercise training reverses lipid-induced insulin resistance and the role of AMPK/mTOR/S6K1 signaling axis in mediating this response in skeletal muscle. Our results showed that high-fat feeding resulted in decreased glucose tolerance, which was associated with decreased Akt expression and increased intramuscular triglyceride deposition in the skeletal muscle of C57BL/6 mice. Impairments in lipid metabolism were accompanied by increased total protein and phosphorylation of S6K1, SREBP-1c cleavage, and decreased AMPK phosphorylation. Exercise training reversed these impairments, resulting in improved serum lipid profiles and glucose tolerance. C2C12 myotubes were exposed to palmitate, resulting in an increased insulin-dependent Akt Ser473 phosphorylation, associated with a significant increase in the level of phosphorylation of S6K1 on T389. All these changes were reversed by activation of AMPK. Consistent with this, inhibition of AMPK by compound C induced an enhanced phosphorylation of both S6K1 and Akt, and silencing of S6K1 with siRNA showed no effect on Akt phosphorylation in both the absence and presence of palmitate cultured myotubes. In addition, compound C led to an elevated SREBP-1c cleavage but was blocked by S6K1 siRNA. In summary, exercise training inhibits SREBP-1c cleavage through AMPK/mTOR/S6K1 signaling, resulting in decreased intramyocellular lipid accumulation. Our results provide new insights into the mechanism by which AMPK/mTOR/S6K1 signaling axis mediates the physiological process of exercise-induced insulin sensitization.     

mTOR phosphorylated at S2448 binds to raptor and rictor

In mammalian cells, the mammalian target of rapamycin (mTOR) forms an enzyme complex with raptor (together with other proteins) named mTOR complex 1 (mTORC1), of which a major target is the p70 ribosomal protein S6 kinase (p70S6K). A second enzyme complex, mTOR complex 2 (mTORC2), contains mTOR and rictor and regulates the Akt kinase. Both mTORC1 and mTORC2 are regulated by phosphorylation, complex formation and localization. So far, the role of p70S6K-mediated mTOR S2448 phosphorylation has not been investigated in detail. Here, we report that endogenous mTOR phosphorylated at S2448 binds to both, raptor and rictor. Experiments with chemical inhibitors of the mTOR kinase and of the phosphatidylinositol-3-kinase revealed that downregulation of mTOR S2448 phosphorylation correlates with decreased mTORC1 activity but can occur decoupled of effects on mTORC2 activity. In addition, we found that the correlation of the mTOR S2448 phosphorylation status with mTORC1 activity is not a consequence of effects on the assembly of mTOR protein and raptor. Our data allow new insights into the role of mTOR phosphorylation for the regulation of its kinase activity.