Reactive nitrogen species regulate autophagy through ATM-AMPK-TSC2–mediated suppression of mTORC1

Abstract

Reactive intermediates such as reactive nitrogen species play essential roles in the cell as signaling molecules but, in excess, constitute a major source of cellular damage. We found that nitrosative stress induced by steady-state nitric oxide (NO) caused rapid activation of an ATM damage-response pathway leading to downstream signaling by this stress kinase to LKB1 and AMPK kinases, and activation of the TSC tumor suppressor. As a result, in an ATM-, LKB1-, TSC-dependent fashion, mTORC1 was repressed, as evidenced by decreased phosphorylation of S6K, 4E-BP1, and ULK1, direct targets of the mTORC1 kinase. Decreased ULK1 phosphorylation by mTORC1 at S757 and activation of AMPK to phosphorylate ULK1 at S317 in response to nitrosative stress resulted in increased autophagy: the LC3-II/LC3-I ratio increased as did GFP-LC3 puncta and acidic vesicles; p62 levels decreased in a lysosomedependent manner, confirming an NO-induced increase in autophagic flux. Induction of autophagy by NO correlated with loss of cell viability, suggesting that, in this setting, autophagy was functioning primarily as a cytotoxic response to excess nitrosative stress. These data identify a nitrosative-stress signaling pathway that engages ATM and the LKB1 and TSC2 tumor suppressors to repress mTORC1 and regulate autophagy. As cancer cells are particularly sensitive to nitrosative stress, these data open another path for therapies capitalizing on the ability of reactive nitrogen species to induce autophagy-mediated cell death. signal transduction | cancer therapyA utophagy is a self-digestion process by which a eukaryotic cell degrades and recycles aggregate-prone proteins, macromolecules, and organelles. During autophagy, cytoplasmic contents are sequestered in double-membrane bound vesicles called autophagosomes and delivered to lysosomes for degradation, thereby allowing cells to eliminate and recycle the contents (1-3). Autophagy participates in both prosurvival (recycling of cellular building blocks) and prodeath (excess catalysis) pathways. A comprehensive understanding of signaling pathways that regulate autophagy holds great promise for new therapeutic opportunities by opening the possibility to compromise prosurvival autophagic pathways that enable tumor cells to evade therapy, or by promoting prodeath autophagic pathways to kill cancer cells.The classical pathway regulating autophagy in mammalian cells involves the serine/threonine kinase, mammalian target of rapamycin (mTOR). Active mTOR kinase in the mTORC1 complex phosphorylates and inhibits ULK1, a key proautophagy adapter involved in nucleation of the autophagophore membrane. Inactivation of mTORC1, either pharmacologically with rapamycin or via activation of the tuberous sclerosis complex (TSC) tumor suppressor, leads to downstream dephosphorylation events, including loss of ULK1 phosphorylation at S757. The TSC1/2 heterodimer is itself regulated by upstream kinases, including the AMP-activated protein kinase (AMPK), which regulates several metabolic processes and activates t...

Organelle-Specific Initiation of Autophagy

“…ROS per se promote autophagy as they oxidize a specific cysteine residue located near the catalytic site of ATG4, a protease involved in the maturation of LC3 (Scherz-Shouval et al, 2007). Besides being genotoxic (which means they can engage nuclear systems of response to stress, see below), ROS as well as reactive nitrogen species (RNS) can be detected by a cytoplasmic pool of ATM (a key sensor of DNA damage), resulting in the activation of serine/ threonine kinase 11 (STK11, best known as LKB1), a positive regulator of AMPK signaling (Alexander et al, 2010;Tripathi et al, 2013). Finally, ROS alter the abundance of various Bcl-2 family proteins, and this favors the so-called mitochondrial permeability transition (MPT), entailing the dissipation of the Dc m (Galluzzi et al, 2012a).…”

“…Moreover, lysosomes play a key role in adaptive autophagy caused by an increased demand of metabolic intermediates. Chauhan et al, 2015;Knodler and Celli, 2011;Mandell et al, 2014 hypoxia mitophagy relies on the transactivation of BNIP3 and BNIP3L by HIF1, as well as on FUNDC1 Bellot et al, 2009;Chen et al, 2014Li et al, 2014Liu et al, 2014;Maxwell et al, 1999;Mazure and Pouyssé gur, 2009;Zhang et al, 2008 lipid droplets lipophagy contributes to hormone-and starvation-driven lipolysis Eid et al, 2013;Singh et al, 2009 protein aggregates aggrephagy relies on p62, OPTN, and ALFY Lamark and Johansen, 2012 ribosomes ribophagy not yet observed in mammalian cells Kraft et al, 2008 RNS ROS non-selective promote autophagy via an ATM/ LKB1 signaling pathway Alexander et al, 2010;Tripathi et al, 2013 Lysosomes amino acid deprivation non-selective stimulates autophagy via Ragulator/ MTORC1 signaling Bar-Peled et al, 2012;Rebsamen et al, 2015;Sancak et al, 2010;Wang et al, 2015;Zoncu et al, 2011 glucose deprivation non-selective stimulates autophagy via Ragulator/ LBK1/AMPK signaling Zhang et al, 2014 lysosomotropic agents lysophagy involves p62 Maejima et al, 2013;Hung et al, 2013 Peroxisomes responses to xenobiotic pexophagy involves PEX3, PEX14, NBR1, and ATG30 Burnett et al, 2015;Deosaran et al, 2013;Farré et al, 2008;Hara-Kuge and Fujiki, 2008;Yamashita et al, 2014 AKT1, v-akt murine thymoma viral oncogene homolog 1; ALFY (official name WDFY3), WD repeat and FYVE domain containing 3; AMPK (official name PRKA), protein kinase, AMP-activated; ATF, activating transcription factor; BECN1, beclin 1; BNIP3, BCL2/adenovirus E1B 19 kDa interacting protein 3; BNIP3L, BNIP3-like; CAMKKb (official name CAMKK2), calcium/calmodulin-dependent protein kinase kinase 2 beta; cAMP, cyclic AMP; CASP2, caspase 2; CCF, condensed chromatin fragment; CXCR4, chemokine (C-X-C motif) receptor 4; DAPK1, death-associated protein kina...…”

Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy

“…[30][31][32] Accumulating evidence has unveiled the complex crosstalk between DDR pathways and autophagy. Activation of ATM [33][34][35][36] by various insults induces autophagy, and TP53 [37][38][39] and MTOR 35,36,[39][40][41] are 2 representative downstream signaling nodes connecting the DDR pathways to autophagy. The descriptions of autophagy regulation by ATR are rather scarce, but there is strong evidence showing that ATR is a potential upstream regulator of autophagy.…”

Isogambogenic Acid Inhibits the Growth of Glioma Through Activation of the AMPK-mTOR Pathway

“…In addition, the activity of mTOR is usually up-regulated in cancers, including gliomas [14]. Moreover, activation of AMPK leads to the inhibition of mTOR activity and further regulates autophagy [15-17]. …”