AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1

Autophagy is a process by which components of the cell are degraded to maintain essential activity and viability in response to nutrient limitation. Extensive genetic studies have shown that the yeast ATG1 kinase has an essential role in autophagy induction. Furthermore, autophagy is promoted by AMP activated protein kinase (AMPK), which is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by the mammalian target of rapamycin (mTOR), a central cell-growth regulator that integrates growth factor and nutrient signals. Here we demonstrate a molecular mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast ATG1. Under glucose starvation, AMPK promotes autophagy by directly activating Ulk1 through phosphorylation of Ser 317 and Ser 777. Under nutrient sufficiency, high mTOR activity prevents Ulk1 activation by phosphorylating Ulk1 Ser 757 and disrupting the interaction between Ulk1 and AMPK. This coordinated phosphorylation is important for Ulk1 in autophagy induction. Our study has revealed a signalling mechanism for Ulk1 regulation and autophagy induction in response to nutrient signalling.     

Regulation of nutrient-sensitive autophagy by uncoordinated 51-like kinases 1 and 2

Macroautophagy, commonly referred to as autophagy, is a protein degradation pathway that occurs constitutively in cells, but can also be induced by stressors such as nutrient starvation or protein aggregation. Autophagy has been implicated in multiple disease mechanisms including neurodegeneration and cancer, with both tumor suppressive and oncogenic roles. Uncoordinated 51-like kinase 1 (ULK1) is a critical autophagy protein near the apex of the hierarchal regulatory pathway that receives signals from the master nutrient sensors MTOR and AMP-activated protein kinase (AMPK). In mammals, ULK1 has a close homolog, ULK2, although their functional distinctions have been unclear. Here, we show that ULK1 and ULK2 both function to support autophagy activation following nutrient starvation. Increased autophagy following amino acid or glucose starvation was disrupted only upon combined loss of ULK1 and ULK2 in mouse embryonic fibroblasts. Generation of PtdIns3P and recruitment of WIPI2 or ZFYVE1/DFCP1 to the phagophore following amino acid starvation was blocked by combined Ulk1/2 double knockout. Autophagy activation following glucose starvation did not involve recruitment of either WIPI1 or WIPI2 to forming autophagosomes. Consistent with a PtdIns3P-independent mechanism, glucose-dependent autophagy was resistant to wortmannin. Our findings support functional redundancy between ULK1 and ULK2 for nutrient-dependent activation of autophagy and furthermore highlight the differential pathways that respond to amino acid and glucose deprivation.     

Withdrawal of Essential Amino Acids Increases Autophagy by a Pathway Involving Ca2+/Calmodulin-dependent Kinase Kinase-β (CaMKK-β)

Autophagy is the main lysosomal catabolic process that becomes activated under stress conditions, such as amino acid starvation and cytosolic Ca²⁺ upload. However, the molecular details on how both conditions control autophagy are still not fully understood. Here we link essential amino acid starvation and Ca²⁺ in a signaling pathway to activate autophagy. We show that withdrawal of essential amino acids leads to an increase in cytosolic Ca²⁺, arising from both extracellular medium and intracellular stores, which induces the activation of adenosine monophosphate-activated protein kinase (AMPK) via Ca²⁺/calmodulin-dependent kinase kinase-β (CaMKK-β). Furthermore, we show that autophagy induced by amino acid starvation requires AMPK, as this induction is attenuated in its absence. Subsequently, AMPK activates UNC-51-like kinase (ULK1), a mammalian autophagy-initiating kinase, through phosphorylation at Ser-555 in a process that requires CaMKK-β. Finally, the mammalian target of rapamycin complex C1 (mTORC1), a negative regulator of autophagy downstream of AMPK, is inhibited by amino acid starvation in a Ca²⁺-sensitive manner, and CaMKK-β appears to be important for mTORC1 inactivation, especially in the absence of extracellular Ca²⁺. All these results highlight that amino acid starvation regulates autophagy in part through an increase in cellular Ca²⁺ that activates a CaMKK-β-AMPK pathway and inhibits mTORC1, which results in ULK1 stimulation.     

ROS-induced DNA damage and PARP-1 are required for optimal induction of starvation-induced autophagy

In response to nutrient stress, cells start an autophagy program that can lead to adaptation or death. The mechanisms underlying the signaling from starvation to the initiation of autophagy are not fully understood. In the current study we show that the absence or inactivation of PARP-1 strongly delays starvation-induced autophagy. We have found that DNA damage is an early event of starvation-induced autophagy as measured by γ-H2AX accumulation and comet assay, with PARP-1 knockout cells displaying a reduction in both parameters. During starvation, ROS-induced DNA damage activates PARP-1, leading to ATP depletion (an early event after nutrient deprivation). The absence of PARP-1 blunted AMPK activation and prevented the complete loss of mTOR activity, leading to a delay in autophagy. PARP-1 depletion favors apoptosis in starved cells, suggesting a pro-survival role of autophagy and PARP-1 activation after nutrient deprivation. In vivo results show that neonates of PARP-1 mutant mice subjected to acute starvation, also display deficient liver autophagy, implying a physiological role for PARP-1 in starvation-induced autophagy. Thus, the PARP signaling pathway is a key regulator of the initial steps of autophagy commitment following starvation.     

The serine/threonine kinase ULK1 is a target of multiple phosphorylation events

Autophagy is a cellular degradation process that is up-regulated upon starvation. Nutrition-dependent regulation of mTOR (mammalian target of rapamycin) is a major determinant of autophagy. RTK (receptor tyrosine kinase) signalling and AMPK (AMP-activated protein kinase) converge upon mTOR to suppress or activate autophagy. Nutrition-dependent regulation of autophagy is mediated via mTOR phosphorylation of the serine/threonine kinase ULK1 (unc51-like kinase 1). In the present study, we also describe ULK1 as an mTOR-independent convergence point for AMPK and RTK signalling. We initially identified ULK1 as a 14-3-3-binding protein and this interaction was enhanced by treatment with AMPK agonists. AMPK interacted with ULK1 and phosphorylated ULK1 at Ser(555) in vitro. Mutation of this residue to alanine abrogated 14-3-3 binding to ULK1, and in vivo phosphorylation of ULK1 was blocked by a dominant-negative AMPK mutant. We next identified a high-stringency Akt site in ULK1 at Ser(774) and showed that phosphorylation at this site was increased by insulin. Finally, we found that the kinase-activation loop of ULK1 contains a consensus phosphorylation site at Thr(180) that is required for ULK1 autophosphorylation activity. Collectively, our results suggest that ULK1 may act as a major node for regulation by multiple kinases including AMPK and Akt that play both stimulatory and inhibitory roles in regulating autophagy.     

siRNA screening of the kinome identifies ULK1 as a multidomain modulator of autophagy

Autophagy is a vital response to nutrient starvation. Here, we screened a kinase-specific siRNA library using an autophagy assay in human embryonic kidney 293 cells that measures lipidation of the marker protein GFP-LC3 following amino acid starvation. This screen identified ULK1 in addition to other novel candidates that could be confirmed with multiple siRNAs. Knockdown of ULK1, but not the related kinase ULK2, inhibited the autophagic response. Also, ULK1 knockdown inhibited rapamycin-induced autophagy consistent with a role downstream of mTOR. Overexpression of ULK1 inhibited autophagy and this inhibition was independent of its kinase activity. Deletion of the PDZ domain-binding Val-Tyr-Ala motif at the ULK1 C terminus generated a more potent dominant-negative protein. Further deletions revealed that the minimal ULK1 dominant-negative region could be mapped to residues 1-351. Full-length ULK1 localized to cytoplasmic structures, some of which were GFP-LC3-positive, and this localization required the conserved C-terminal domain. In contrast, ULK1-(1-351) was diffuse in the cytoplasm. These experiments reveal at least two domains in ULK1 which likely function via unique sets of effectors to regulate autophagy.     

Role of an intrinsically disordered conformation in AMPK-mediated phosphorylation of ULK1 and regulation of autophagy

Recently, it has been established that there is a direct link between adenosine monophosphate activated protein kinase (AMPK), which is an energy sensor and is activated by glucose starvation, and Unc-51-like kinase 1 (ULK1) in triggering autophagy. Proper phosphorylation of ULK1 is crucial for ULK1/AMPK association and subsequent ULK1 functions in response to nutrient deprivation. Signaling modulated via phosphorylation often involves a flexible/unstructured or an intrinsically disordered (ID) region of proteins. Structural analyses of the ULK1 protein suggest that most of its functionally important phosphorylation sites are located in an ID region. We propose that this ID nature facilitates AMPK-mediated phosphorylation of ULK1, which may provide a mechanism for ULK1 functions in response to nutrient deprivation. Understanding how an ID region of ULK1 modulates its post-translational modifications through AMPK in regulating allosteric coupling will significantly help in defining the cellular and molecular mechanisms involved in ULK1/AMPK functions and in regulation of autophagy.     

Regulation of autophagosome formation by Rho kinase

Macroautophagy, commonly referred to as autophagy, is a protein degradation pathway that functions at a constitutive level in cells, which may become further activated by stressors such as nutrient starvation or protein aggregation. Autophagy has multiple beneficial roles for maintaining normal cellular homeostasis and these roles are related to the implications of autophagy in disease mechanisms including neurodegeneration and cancer. We previously searched for novel autophagy regulators and identified Rho-kinase 1 (ROCK1) as a candidate. Here, we show that activated ROCK1 inhibits autophagy in human embryonic kidney 293 cells. Conversely, ROCK inhibitory compounds enhanced the autophagy response to amino acid starvation or rapamycin treatment. Inhibition of ROCK during the starvation period led to a more rapid response with the production of larger early autophagosomes that matured into enlarged late degradative autolysosomes. Despite the production of enlarged LC3-positive early autophagosomes, membrane precursors containing WD-repeat protein interacting with phosphoinositides 1 (WIPI1) and mammalian Atg9 were not affected by ROCK inhibition, suggesting that phagophore elongation had been unusually extended. However, the enlarged autophagosomes were enriched in ULK1 which was essential to allow progression of autophagy flux. Our results demonstrate a novel role for ROCK in the control of autophagosome size and degradative capacity.     

Starvation-induced autophagy is regulated by mitochondrial reactive oxygen species leading to AMPK activation

Starvation is the most extensively studied condition that induces autophagy. Previous studies have demonstrated that starvation-induced autophagy is regulated by reactive oxygen species (ROS) such as superoxide (O₂^?) but the source for ROS under starvation conditions and the downstream signaling pathways regulating autophagy are unclear. In this study, a cervical cancer HeLa cell line was generated that was deficient in mitochondrial electron transport chain (mETC) (HeLa ρ° cells). This resulted in endogenous levels of O₂^? being significantly reduced and failed to be induced under starvation of glucose, L-glutamine, pyruvate, and serum (GP) or of amino acids and serum (AA) compared to wild type (wt) HeLa cells. In contrast, H₂O₂ production failed to increase under GP starvation in both wild type and ρ° cells whereas it increased in wt cells but not in ρ° cells under AA starvation. GP or AA starvation induced autophagy was blocked in ρ° cells as determined by the amount of autophagosomes and autolysosomes. Autophagy is regulated by 5′ adenosine monophosphate-activated protein kinase (AMPK) activation and AMPK is activated under starvation conditions. We demonstrate that ρ° cells and HeLa cells over expressing manganese-superoxide dismutase 2 (SOD2) cells fail to activate AMPK activation following starvation. This indicates that mitochondrial ROS might regulate AMPK activation. In addition, inhibiting AMPK activation either by siRNA or compound C resulted in reduced autophagy during starvation. Using a ROS scavenger NAC, AMPK activation is reduced under starvation condition and mTOR signaling is increased. Taken together, mitochondria-generated ROS induces autophagy mediated by the AMPK pathway under starvation conditions.     

Inhibition of CLIC4 enhances autophagy and triggers mitochondrial and ER stress-induced apoptosis in human glioma U251 cells under starvation

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to mitochondria, endoplasmic reticulum (ER), nucleus and cytoplasm, and participates in the apoptotic response to stress. Apoptosis and autophagy, the main types of the programmed cell death, seem interconnected under certain stress conditions. However, the role of CLIC4 in autophagy regulation has yet to be determined. In this study, we demonstrate upregulation and nuclear translocation of the CLIC4 protein following starvation in U251 cells. CLIC4 siRNA transfection enhanced autophagy with increased LC3-II protein and puncta accumulation in U251 cells under starvation conditions. In that condition, the interaction of the 14-3-3 epsilon isoform with CLIC4 was abolished and resulted in Beclin 1 overactivation, which further activated autophagy. Moreover, inhibiting the expression of CLIC4 triggered both mitochondrial apoptosis involved in Bax/Bcl-2 and cytochrome c release under starvation and endoplasmic reticulum stress-induced apoptosis with CHOP and caspase-4 upregulation. These results demonstrate that CLIC4 nuclear translocation is an integral part of the cellular response to starvation. Inhibiting the expression of CLIC4 enhances autophagy and contributes to mitochondrial and ER stress-induced apoptosis under starvation.     

FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells

Autophagy is a membrane-mediated intracellular degradation system. The serine/threonine kinase Atg1 plays an essential role in autophagosome formation. However, the role of the mammalian Atg1 homologues UNC-51-like kinase (ULK) 1 and 2 are not yet well understood. We found that murine ULK1 and 2 localized to autophagic isolation membrane under starvation conditions. Kinase-dead alleles of ULK1 and 2 exerted a dominant-negative effect on autophagosome formation, suggesting that ULK kinase activity is important for autophagy. We next screened for ULK binding proteins and identified the focal adhesion kinase family interacting protein of 200 kD (FIP200), which regulates diverse cellular functions such as cell size, proliferation, and migration. We found that FIP200 was redistributed from the cytoplasm to the isolation membrane under starvation conditions. In FIP200-deficient cells, autophagy induction by various treatments was abolished, and both stability and phosphorylation of ULK1 were impaired. These results suggest that FIP200 is a novel mammalian autophagy factor that functions together with ULKs.     

Knockdown of AMPKα decreases ATM expression and increases radiosensitivity under hypoxia and nutrient starvation in an SV40-transformed human fibroblast cell line,LM217

Background

Presence of unperfused regions containing cells under hypoxia and nutrient starvation contributes to radioresistance in solid human tumors. It is well known that hypoxia causes cellular radioresistance, but little is known about the effects of nutrient starvation on radiosensitivity. We have reported that nutrient starvation induced decrease of mTORC1 activity and decrease of radiosensitivity in an SV40-transformed human fibroblast cell line, LM217, and that nutrient starvation induced increase of mTORC1 activity and increase of radiosensitivity in human liver cancer cell lines, HepG2 and HuH6 (Murata et al., BBRC 2015). Knockdown of mTOR using small interfering RNA (siRNA) for mTOR suppressed radiosensitivity under nutrient starvation alone in HepG2 cells, which suggests that mTORC1 pathway regulates radiosensitivity under nutrient starvation alone. In the present study, effects of hypoxia and nutrient starvation on radiosensitivity were investigated using the same cell lines.

Linking tRNA localization with activation of nutritional stress responses

Cells respond to nutrient deprivation a variety of ways. In addition to global down regulation of cap-dependent protein synthesis mediated by the GCN2 and mTORC1 signaling pathways, a catabolic process autophagy is upregulated to provide internal building blocks and energy needed to sustain viability. It has recently been shown that during nutrient deprivation tRNAs accumulate in the nucleus, but the functional role of this accumulation remains unknown. This study investigates whether subcellular localization of tRNAs plays a role in signaling nutritional stress and autophagy. We report that human fibroblasts that accumulate tRNA in the nucleus due to downregulation of their transportin, Xpo-t, show reduced mTORC1 activity and upregulated autophagy. This suggests that subcellular localization of tRNAs may regulate an intracellular response to starvation independently of the cellular nutritional status.