Impaired autophagy due to constitutive mTOR activation sensitizes TSC2-null cells to cell death under stress

It has been well documented that cells deficient in either TSC1 or TSC2 are highly sensitive to various cell death stimuli. In this study, we utilized the TSC2 (-/-) mouse embryonic fibroblasts (MEFs) to study the involvement of autophagy in the enhanced susceptibility of TSC2-null cells to cell death. We first confirmed that both TSC1-null and TSC2-null MEFs are more sensitive to apoptosis in response to amino acid starvation (EBSS) and hypoxia. Second, we found that both the basal and inducible autophagy in TSC2 (-/-) MEFs is impaired, mainly due to constitutive activation of mTORC1. Third, suppression of autophagy by chloroquine and Atg7 knockdown sensitizes TSC2 (+/+) cells, but not TSC2 (-/-) cells, to EBSS-induced cell death. Conversely, the inhibition of mTORC1 by raptor knockdown and rapamycin activates autophagy and subsequently rescues TSC2 (-/-) cells. Finally, in starved cells, nutrient supplementations (insulin-like growth factor-1 (IGF-1) and leucine) enhanced cell death in TSC2 (-/-) cells, but reduced cell death in TSC2 (+/+) cells. Taken together, these data indicate that constitutive activation of mTORC1 in TSC2 (-/-) cells leads to suppression of autophagy and enhanced susceptibility to stress-mediated cell death. Our findings thus provide new insights into the complex relationships among mTOR, autophagy and cell death, and support the possible autophagy-targeted intervention strategies for the treatment of TSC-related pathologies.     

cAMP inhibits mammalian target of rapamycin complex-1 and -2 (mTORC1 and 2) by promoting complex dissociation and inhibiting mTOR kinase activity

cAMP and mTOR signalling pathways control a number of critical cellular processes including metabolism, protein synthesis, proliferation and cell survival and therefore understanding the signalling events which integrate these two signalling pathways is of particular interest. In this study, we show that the pharmacological elevation of [cAMP]_i in mouse embryonic fibroblasts (MEFs) and human embryonic kidney 293 (HEK293) cells inhibits mTORC1 activation via a PKA-dependent mechanism. Although the inhibitory effect of cAMP on mTOR could be mediated by impinging on signalling cascades (i.e. PKB, MAPK and AMPK) that inhibit TSC1/2, an upstream negative regulator of mTORC1, we show that cAMP inhibits mTORC1 in TSC2 knockout (TSC2^−/−) MEFs. We also show that cAMP inhibits insulin and amino acid-stimulated mTORC1 activation independently of Rheb, Rag GTPases, TSC2, PKB, MAPK and AMPK, indicating that cAMP may act independently of known regulatory inputs into mTOR. Moreover, we show that the prolonged elevation in [cAMP]_i can also inhibit mTORC2. We provide evidence that this cAMP-dependent inhibition of mTORC1/2 is caused by the dissociation of mTORC1 and 2 and a reduction in mTOR catalytic activity, as determined by its auto-phosphorylation on Ser2481. Taken together, these results provide an important insight into how cAMP signals to mTOR and down-regulates its activity, which may lead to the identification of novel drug targets to inhibit mTOR that could be used for the treatment and prevention of human diseases such as cancer.     

Impaired autophagy due to constitutive mTOR activation sensitizes TSC2-null cells to cell death under stress

It has been well documented that cells deficient in either TSC1 or TSC2 are highly sensitive to various cell death stimuli. In this study, we utilized the TSC2^-/- mouse embryonic fibroblasts (MEFs) to study the involvement of autophagy in the enhanced susceptibility of TSC2-null cells to cell death. We first confirmed that both TSC1-null and TSC2-null MEFs are more sensitive to apoptosis in response to amino acid starvation (EBSS) and hypoxia. Second, we found that both the basal and inducible autophagy in TSC2^-/- MEFs is impaired, mainly due to constitutive activation of mTORC1. Third, suppression of autophagy by chloroquine and Atg7 knockdown sensitizes TSC2^+/+ cells, but not TSC2^-/- cells, to EBSS-induced cell death. Conversely, the inhibition of mTORC1 by raptor knockdown and rapamycin activates autophagy and subsequently rescues TSC2^-/- cells. Finally, in starved cells, nutrient supplementations (insulin-like growth factor-1 (IGF-1) and leucine) enhanced cell death in TSC2^-/- cells, but reduced cell death in TSC2^+/+ cells. Taken together, these data indicate that constitutive activation of mTORC1 in TSC2^-/- cells leads to suppression of autophagy and enhanced susceptibility to stress-mediated cell death. Our findings thus provide new insights into the complex relationships among mTOR, autophagy and cell death, and support the possible autophagy-targeted intervention strategies for the treatment of TSC-related pathologies.     

Akt activates the mammalian target of rapamycin by regulating cellular ATP level and AMPK activity

The serine/threonine kinase Akt is an upstream positive regulator of the mammalian target of rapamycin (mTOR). However, the mechanism by which Akt activates mTOR is not fully understood. The known pathway by which Akt activates mTOR is via direct phosphorylation and inhibition of tuberous sclerosis complex 2 (TSC2), which is a negative regulator of mTOR. Here we establish an additional pathway by which Akt inhibits TSC2 and activates mTOR. We provide for the first time genetic evidence that Akt regulates intracellular ATP level and demonstrate that Akt is a negative regulator of the AMP-activated protein kinase (AMPK), which is an activator of TSC2. We show that in Akt1/Akt2 DKO cells AMP/ATP ratio is markedly elevated with concomitant increase in AMPK activity, whereas in cells expressing activated Akt there is a dramatic decrease in AMP/ATP ratio and a decline in AMPK activity. Currently, the Akt-mediated phosphorylation of TSC2 and the inhibition of AMPK-mediated phosphorylation of TSC2 are viewed as two separate pathways, which activate mTOR. Our results demonstrate that Akt lies upstream of these two pathways and induces full inhibition of TSC2 and activation of mTOR both through direct phosphorylation and by inhibition of AMPK-mediated phosphorylation of TSC2. We propose that the activation of mTOR by Akt-mediated cellular energy and inhibition of AMPK is the predominant pathway by which Akt activates mTOR in vivo.     

Multi-mechanisms are involved in reactive oxygen species regulation of mTORC1 signaling

The mammalian target of rapamycin complex 1(mTORC1) integrates diverse signals to control cell growth, proliferation, survival, and metabolism. Role of reactive oxygen species (ROS) on mTORC1 signaling remains obscure and mechanisms through which ROS modulate mTORC1 are not known. We demonstrate that low doses ROS exposure stimulate mTORC1 while high concentrations or long-term ROS treatment decrease mTORC1 activity in vivo and in a variety of cell lines. The dose/time needed for inhibition or activation are cell type-dependent. In HEK293 cells hydrogen peroxide (H₂O₂) stimulates phosphorylation of AMP-activated kinase (AMPK) (T172) and Raptor (S792), enhances association of activated AMPK with Raptor. Furthermore, AMPK inhibitor compound c inhibits H₂O₂-induced Raptor (S792) phosphorylation and reverses H₂O₂-induced de-phosphorylation of mTORC1 downstream targets p70-S6K1 (T389), S6 (S235/236) and 4E-BP1 (T37/46). H₂O₂ also stimulates association of endogenous protein phosphatase 2A catalytic subunit (PP2Ac) with p70-S6K1. Like compound c, inhibitor of PP2A, okadaic acid partially reverses inactivation of mTORC1 substrates induced by H₂O₂. Moreover, inhibition of PP2A and AMPK partially rescued cells from H₂O₂-induced cell death. High doses of H₂O₂ inhibit while low doses of H₂O₂ activate mTORC1 both in TSC2^?/? P53^?/? and TSC2^+/+ P53^?/? MEFs. These data suggest that PP2A and AMPK-mediated phosphorylation of Raptor mediate H₂O₂-induced inhibition of mTORC1 signaling.     

AMPK phosphorylation of raptor mediates a metabolic checkpoint

AMPK is a highly conserved sensor of cellular energy status that is activated under conditions of low intracellular ATP. AMPK responds to energy stress by suppressing cell growth and biosynthetic processes, in part through its inhibition of the rapamycin-sensitive mTOR (mTORC1) pathway. AMPK phosphorylation of the TSC2 tumor suppressor contributes to suppression of mTORC1; however, TSC2-deficient cells remain responsive to energy stress. Using a proteomic and bioinformatics approach, we sought to identify additional substrates of AMPK that mediate its effects on growth control. We report here that AMPK directly phosphorylates the mTOR binding partner raptor on two well-conserved serine residues, and this phosphorylation induces 14-3-3 binding to raptor. The phosphorylation of raptor by AMPK is required for the inhibition of mTORC1 and cell-cycle arrest induced by energy stress. These findings uncover a conserved effector of AMPK that mediates its role as a metabolic checkpoint coordinating cell growth with energy status.     

Tuberous sclerosis complex proteins 1 and 2 control serum-dependent translation in a TOP-dependent and -independent manner

The tuberous sclerosis complex (TSC) proteins TSC1 and TSC2 regulate protein translation by inhibiting the serine/threonine kinase mTORC1 (for mammalian target of rapamycin complex 1). However, how TSC1 and TSC2 control overall protein synthesis and the translation of specific mRNAs in response to different mitogenic and nutritional stimuli is largely unknown. We show here that serum withdrawal inhibits mTORC1 signaling, causes disassembly of translation initiation complexes, and causes mRNA redistribution from polysomes to subpolysomes in wild-type mouse embryo fibroblasts (MEFs). In contrast, these responses are defective in Tsc1(-/-) or Tsc2(-/-) MEFs. Microarray analysis of polysome- and subpolysome-associated mRNAs uncovered specific mRNAs that are translationally regulated by serum, 90% of which are TSC1 and TSC2 dependent. Surprisingly, the mTORC1 inhibitor, rapamycin, abolished mTORC1 activity but only affected approximately 40% of the serum-regulated mRNAs. Serum-dependent signaling through mTORC1 and polysome redistribution of global and individual mRNAs were restored upon re-expression of TSC1 and TSC2. Serum-responsive mRNAs that are sensitive to inhibition by rapamycin are highly enriched for terminal oligopyrimidine and for very short 5' and 3' untranslated regions. These data demonstrate that the TSC1/TSC2 complex regulates protein translation through mainly mTORC1-dependent mechanisms and implicates a discrete profile of deregulated mRNA translation in tuberous sclerosis pathology.     

Cell-type-dependent regulation of mTORC1 by REDD1 and the tumor suppressors TSC1/TSC2 and LKB1 in response to hypoxia

mTORC1 is a critical regulator of cell growth that integrates multiple signals and is deregulated in cancer. We previously reported that mTORC1 regulation by hypoxia involves Redd1 and the Tsc1/Tsc2 complex. Here we show that Redd1 induction by hypoxia is tissue dependent and that hypoxia signals are relayed to mTORC1 through different pathways in a tissue-specific manner. In the liver, Redd1 induction is restricted to the centrilobular area, and in primary hepatocytes, mTORC1 inhibition by hypoxia is independent of Redd1. Furthermore, Tsc1/Tsc2 and Arnt (Hif-1β) are similarly dispensable. Hypoxia signaling in hepatocytes involves Lkb1, AMP-activated protein kinase (AMPK), and raptor. Differences in signal relay extend beyond hypoxia and involve AMPK signaling. AMPK activation (using 5-aminoimidazole-4-carboxamide riboside [AICAR]) induces raptor phosphorylation and inhibits mTORC1 in both mouse embryo fibroblasts (MEFs) and hepatocytes, but whereas mTORC1 inhibition is Tsc1/Tsc2 dependent in MEFs, it is independent in hepatocytes. In liver cells, raptor phosphorylation is essential for both AMPK and hypoxia signaling. Thus, context-specific signals are required for raptor phosphorylation-induced mTORC1 inhibition. Our data illustrate a heretofore unappreciated topological complexity in mTORC1 regulation. Interestingly, topological differences in mTORC1 regulation by the tumor suppressor proteins Lkb1 and Tsc1/Tsc2 may underlie their tissue specificity of tumor suppressor action.     

Tumor necrosis factor-alpha (TNF-α)-mediated in vitro human retinal pigment epithelial (RPE) cell migration mainly requires Akt/mTOR complex 1 (mTORC1), but not mTOR complex 2 (mTORC2) signaling

When rhegmatogenous retinal detachment occurs, tumor necrosis factor-alpha (TNF-α) among other cytokines leaks into the subretinal space, induces resident retinal pigment epithelial (RPE) cells to migrate, which is the initial step of proliferative vitreoretinopathy (PVR). In the current study, we aim to understand how this is regulated by focusing the cellular mechanisms involved. Here we identified an Akt/Tuberous sclerosis protein 2 (TSC2)/mTOR complex1 (mTORC1) signaling pathway after TNF-α treatment to mediate RPE cell migration. Suppression of mTORC1 activation, either by its inhibitor rapamycin, or by activation of its suppressor AMP activated protein kinase (AMPK), inhibited TNF-α-mediated RPE cell migration, while RNA interference (RNAi)-mediated knocking-down of SIN1 or Rictor, two key components of mTOR complex 2 (mTORC2), had no significant effect on TNF-α-induced RPE cell migration. Our data provide initial evidence that TNF-α-mediated in vitro RPE cell migration mainly requires Akt/mTORC1, but not mTORC2 signaling. The results of this study may lead to indentify novel signaling targets against PVR.     

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.     

The insulin-like growth factor I receptor is required for Akt activation and suppression of anoikis in cells transformed by the ETV6-NTRK3 chimeric tyrosine kinase

Signaling through the insulin-like growth factor I receptor (IGF-IR) axis is essential for transformation by many dominantly acting oncoproteins. However, the mechanism by which IGF-IR contributes to oncogenesis remains unknown. To examine this, we compared transformation properties of the oncogenic ETV6-NTRK3 (EN) chimeric tyrosine kinase in IGF-IR-null R- mouse embryo fibroblasts with R- cells engineered to reexpress IGF-IR (R+ cells). We previously showed that R- cells expressing EN (R- EN cells) are resistant to transformation but that transformation is restored in R+ cells. We now show that while R- EN cells have intact Ras-extracellular signal-regulated kinase signaling and cell cycle progression, they are defective in phosphatidylinositol-3-kinase (PI3K)-Akt activation and undergo detachment-induced apoptosis (anoikis) under anchorage-independent conditions. In contrast, R+ cells expressing EN (R+ EN cells) suppress anoikis and are fully transformed. The requirement for IGF-IR in R- EN cells is overcome by ectopic expression of either activated Akt or a membrane-targeted form of EN. Moreover, compared to R- EN cells, R+ EN cells show a dramatic increase in membrane localization of insulin receptor substrate 1 (IRS-1) in association with EN. Since EN is known to bind IRS-1 as an adaptor protein, our findings suggest that IGF-IR may function to localize EN/IRS-1 complexes to cell membranes, in turn facilitating PI3K-Akt activation and suppression of anoikis.     

Chemical inhibition of RNA viruses reveals REDD1 as a host defense factor

A chemical genetics approach was taken to identify inhibitors of NS1, a major influenza A virus virulence factor that inhibits host gene expression. A high-throughput screen of 200,000 synthetic compounds identified small molecules that reversed NS1-mediated inhibition of host gene expression. A counterscreen for suppression of influenza virus cytotoxicity identified naphthalimides that inhibited replication of influenza virus and vesicular stomatitis virus (VSV). The mechanism of action occurs through activation of REDD1 expression and concomitant inhibition of mammalian target of rapamycin complex 1 (mTORC1) via TSC1-TSC2 complex. The antiviral activity of naphthalimides was abolished in REDD1(-/-) cells. Inhibition of REDD1 expression by viruses resulted in activation of the mTORC1 pathway. REDD1(-/-) cells prematurely upregulated viral proteins via mTORC1 activation and were permissive to virus replication. In contrast, cells conditionally expressing high concentrations of REDD1 downregulated the amount of viral protein. Thus, REDD1 is a new host defense factor, and chemical activation of REDD1 expression represents a potent antiviral intervention strategy.     

Inhibition of AMP-activated protein kinase α (AMPKα) by doxorubicin accentuates genotoxic stress and cell death in mouse embryonic fibroblasts and cardiomyocytes: role of p53 and SIRT1

Doxorubicin, an anthracycline antibiotic, is widely used in cancer treatment. Doxorubicin produces genotoxic stress and p53 activation in both carcinoma and non-carcinoma cells. Although its side effects in non-carcinoma cells, especially in heart tissue, are well known, the molecular targets of doxorubicin are poorly characterized. Here, we report that doxorubicin inhibits AMP-activated protein kinase (AMPK) resulting in SIRT1 dysfunction and p53 accumulation. Spontaneously immortalized mouse embryonic fibroblasts (MEFs) or H9C2 cardiomyocyte were exposed to doxorubicin at different doses and durations. Cell death and p53, SIRT1, and AMPK levels were examined by Western blot. In MEFs, doxorubicin inhibited AMPK activation, increased cell death, and induced robust p53 accumulation. Genetic deletion of AMPKα1 reduced NAD(+) levels and SIRT1 activity and significantly increased the levels of p53 and cell death. Pre-activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside or transfection with an adenovirus encoding a constitutively active AMPK (AMPK-CA) markedly reduced the effects of doxorubicin in MEFs from Ampkα1 knock-out mice. Conversely, pre-inhibition of Ampk further sensitized MEFs to doxorubicin-induced cell death. Genetic knockdown of p53 protected both wild-type and Ampkα1(-/-) MEFs from doxorubicin-induced cell death. p53 accumulation in Ampkα1(-/-) MEFs was reversed by SIRT1 activation by resveratrol. Taken together, these data suggest that AMPK inhibition by doxorubicin causes p53 accumulation and SIRT1 dysfunction in MEFs and further suggest that pharmacological activation of AMPK might alleviate the side effects of doxorubicin.     

CCCP induces autophagy in an AMPK-independent manner

AMP-activated protein kinase (AMPK) is an important sensor of cellular energy status, and is involved in cell growth and autophagy through mammalian target of rapamycin complex 1 (mTORC1). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, leads to AMPK activation and Parkin-dependent mitophagy, respectively. However, the detailed biochemical mechanism of how CCCP induces autophagy or mitophagy has not been investigated yet. Here, we showed that CCCP inhibits mTORC1 independently of AMPK, although CCCP induces AMPK activation. Using wild type (WT) and AMPKα1/α2 double knockout (DKO) MEFs, we observed that CCCP promotes endogenous LC3 lipidation and formation of RFP-LC3 puncta, indicating autophagosome or autolysosome, in an AMPK-independent manner. Finally, we also revealed that the percentage of CCCP-dependent colocalization between mitochondria and RFP-LC3 puncta is similar both in WT and AMPKα1/α2 DKO MEFs. Based on these data, we concluded that AMPK is not essential in regulation of CCCP-induced autopahgy including mitophagy.     

Cryptotanshinone induces G1 cell cycle arrest and autophagic cell death by activating the AMP-activated protein kinase signal pathway in HepG2 hepatoma

AMP-activated protein kinase (AMPK) performs a pivotal function in energy homeostasis via the monitoring of intracellular energy status. Once activated under the various metabolic stress conditions, AMPK regulates a multitude of metabolic pathways to balance cellular energy. In addition, AMPK also induces cell cycle arrest or apoptosis through several tumor suppressors including LKB1, TSC2, and p53. LKB1 is a direct upstream kinase of AMPK, while TSC2 and p53 are direct substrates of AMPK. Therefore, it is expected that activators of AMPK signal pathway might be useful for treatment or prevention of cancer. In the present study, we report that cryptotanshinone, a natural compound isolated from Salvia miltiorrhiza, robustly activated AMPK signaling pathway, including LKB1, p53, TSC2, thereby leading to suppression of mTORC1 in a number of LKB1-expressing cancer cells including HepG2 human hepatoma, but not in LKB1-deficient cancer cells. Cryptotanshinone induced HepG2 cell cycle arrest at the G1 phase in an AMPK-dependent manner, and a portion of cells underwent apoptosis as a result of long-term treatment. It also induced autophagic HepG2 cell death in an AMPK-dependent manner. Cryptotanshinone significantly attenuated tumor growth in an HCT116 cancer xenograft in vivo model, with a substantial activation of AMPK signal pathways. Collectively, we demonstrate for the first time that cryptotanshinone harbors the therapeutic potential for the treatment of cancer through AMPK activation.     

N-Formyl-3,4-methylenedioxy-benzylidene-γ-butyrolaetam, KNK437 induces caspase-3 activation through inhibition of mTORC1 activity in Cos-1 cells

The mammalian target of rapamycin complex 1 (mTORC1: mTOR-raptor interaction) and heat shock protein 70 (Hsp70) regulate various cellular processes and are crucial for the progression of many cancers and metabolic diseases. In the recent study, we reported that interaction of Hsp70 with tuberous sclerosis complex 1 (TSC1) regulated apoptosis. This study was designed to elucidate the underlying mechanism in Cos-1 cells. Here, we show that N-formyl-3,4-methylenedioxy-benzylidene-γ-butyrolaetam (KNK437), which inhibits the expression level of Hsp70, abrogated phosphorylation of mTOR and S6K in response to insulin, and inhibited mTORC1 activity via disruption of an interaction between mTOR and raptor. In addition, KNK437 did not alter TSC1/2 complex formation. Furthermore, KNK437 inhibited the mTOR-raptor interaction on the outer membrane of the mitochondria and triggered caspase-3 activation. A reduction in the level of Hsp70 could result in the inhibition of the mTORC1 signaling pathway, thereby inducing apoptosis.     

Inactivation of Rheb by PRAK-mediated phosphorylation is essential for energy-depletion-induced suppression of mTORC1

Cell growth can be suppressed by stressful environments, but the role of stress pathways in this process is largely unknown. Here we show that a cascade of p38β mitogen-activated protein kinase (MAPK) and p38-regulated/activated kinase (PRAK) plays a role in energy-starvation-induced suppression of mammalian target of rapamycin (mTOR), and that energy starvation activates the p38β-PRAK cascade. Depletion of p38β or PRAK diminishes the suppression of mTOR complex 1 (mTORC1) and reduction of cell size induced by energy starvation. We show that p38β-PRAK operates independently of the known mTORC1 inactivation pathways--phosphorylation of tuberous sclerosis protein 2 (TSC2) and Raptor by AMP-activated protein kinase (AMPK)--and surprisingly, that PRAK directly regulates Ras homologue enriched in brain (Rheb), a key component of the mTORC1 pathway, by phosphorylation. Phosphorylation of Rheb at Ser 130 by PRAK impairs the nucleotide-binding ability of Rheb and inhibits Rheb-mediated mTORC1 activation. The direct regulation of Rheb by PRAK integrates a stress pathway with the mTORC1 pathway in response to energy depletion.     

AMP-Activated Protein Kinase: A Universal Regulator of Autophagy?

Autophagy is a lysosomal pathway involved in the turnover of cellular macromolecules and organelles. Starvation and various other stresses increase autophagic activity above the low basal levels observed in unstressed cells, where it is kept down by mammalian target of rapamycin complex 1 (mTORC1). In starved cells, LKB1 activates AMP-activated protein kinase (AMPK) that inhibits mTORC1 activity via a pathway involving tuberous sclerosis complex 1 and 2 (TSC1/2) and its substrate Rheb. The present study suggests that AMPK inhibits mTORC1 and autophagy also in non-starved cells. Various Ca²⁺ mobilizing agents (vitamin D compounds, thapsigargin, ATP and ionomycin) activate AMPK via activation of Ca²⁺/calmodulin-dependent kinase kinase-β (CaMKK-β), and this pathway is required for Ca²⁺-induced mTORC1 inhibition and autophagy. Thus, we propose that an increase in free cytosolic Ca²⁺ ([Ca²⁺]c) induces autophagy via the CaMKK/β-AMPK-TSC1/2-Rheb-mTORC1 signaling pathway and that AMPK is a more general regulator of autophagy than previously expected.

Addendum to:

Control of Macroautophagy by Calcium, Calmodulin-Dependent Kinase Kinase-β and Bcl-2

M. Høyer-Hansen, L. Bastholm, P. Szyniarowski, M. Campanella, G. Szabadkai, T. Farkas, K. Bianchi, N. Fehrenbacher, F. Elling, R. Rizzuto, I.S. Mathiasen and M. Jäättelä

Mol Cell 2007; 25:193-205     

Peptide combinatorial libraries identify TSC2 as a death-associated protein kinase (DAPK) death domain-binding protein and reveal a stimulatory role for DAPK in mTORC1 signaling

Death-associated protein kinase (DAPK) is a multidomain enzyme that plays a central role in autophagic and apoptotic signaling, although the protein-protein interactions regulating DAPK functions are not well defined. Peptide aptamer libraries were used to identify the tumor suppressor protein tuberin (TSC2) as a novel DAPK death domain-binding protein, and we evaluated whether DAPK is a positive or negative effector of the TSC2-regulated mammalian target of rapamycin (mTORC1) signaling pathway. Binding studies using death domain miniproteins in vitro and deletion analysis in vivo determined that the death domain of DAPK is the major site for the interaction with TSC2. Recombinant DAPK phosphorylates TSC2 in vitro, and DAPK kinase activity is stimulated by growth factor signaling. Transfection of DAPK promotes phosphorylation of TSC2 in vivo, whereas short interfering RNA-mediated attenuation of DAPK reduces growth factor-stimulated phosphorylation of TSC2. DAPK-dependent phosphorylation leads to TSC1-TSC2 complex dissociation, and consequently manipulation of DAPK by transfection or short interfering RNA demonstrated that DAPK is a positive regulator of mTORC1 in response to growth factor activation. Epistatic studies suggest that DAPK functions downstream from the RAS-MEK-ERK and phosphatidylinositol 3-kinase-AKT growth factor signaling pathways. DAPK(+/-) mouse embryo fibroblasts have attenuated mTORC1 signaling compared with DAPK+/+ counterparts, and overexpression of DAPK in DAPK(+/-) MEFs stimulates mTORC1 activity. These data uncover a novel interaction between DAPK and TSC2 proteins that has revealed a positive link between growth factor stimulation of DAPK and mTORC1 signaling that may ultimately affect autophagy, cell survival, or apoptosis.