PURPL represses autophagic cell death to promote cutaneous melanoma by modulating ULK1 phosphorylation

Uncontrolled overactivation of autophagy may lead to autophagic cell death, suppression of which is a pro-survival strategy for tumors. However, mechanisms involving key regulators in modulating autophagic cell death remain poorly defined. Here, we report a novel long noncoding RNA, p53 upregulated regulator of p53 levels (PURPL), functions as an oncogene to promote cell proliferation, colony formation, migration, invasiveness, and inhibits cell death in melanoma cells. Mechanistic studies showed that PURPL promoted mTOR-mediated ULK1 phosphorylation at Ser757 by physical interacting with mTOR and ULK1 to constrain autophagic response to avoid cell death. Loss of PURPL led to AMPK-mediated phosphorylation of ULK1 at Ser555 and Ser317 to over-activate autophagy and induce autophagic cell death. Our results identify PURPL as a key regulator to modulate the activity of autophagy initiation factor ULK1 to repress autophagic cell death in melanoma and may represent a potential intervention target for melanoma therapy.Subject terms: Oncogenes, Macroautophagy, Melanoma     

The tumor suppressor DAP kinase is a target of RSK-mediated survival signaling

The viability of vertebrate cells depends on a complex signaling interplay between survival factors and cell-death effectors. Subtle changes in the equilibrium between these regulators can result in abnormal cell proliferation or cell death, leading to various pathological manifestations. Death-associated protein kinase (DAPK) is a multidomain calcium/calmodulin (CaM)-dependent Ser/Thr protein kinase with an important role in apoptosis regulation and tumor suppression. The molecular signaling mechanisms regulating this kinase, however, remain unclear. Here, we show that DAPK is phosphorylated upon activation of the Ras-extracellular signal-regulated kinase (ERK) pathway. This correlates with the suppression of the apoptotic activity of DAPK. We demonstrate that DAPK is a novel target of p90 ribosomal S6 kinases (RSK) 1 and 2, downstream effectors of ERK1/2. Using mass spectrometry, we identified Ser-289 as a novel phosphorylation site in DAPK, which is regulated by RSK. Mutation of Ser-289 to alanine results in a DAPK mutant with enhanced apoptotic activity, whereas the phosphomimetic mutation (Ser289Glu) attenuates its apoptotic activity. Our results suggest that RSK-mediated phosphorylation of DAPK is a unique mechanism for suppressing the proapoptotic function of this death kinase in healthy cells as well as Ras/Raf-transformed cells.     

Regulating RIPK1: another way in which ULK1 contributes to survival

ABSTRACT

The mammalian ULK1 is the central initiating kinase of bulk and selective macroautophagy/autophagy processes. In the past, both autophagy-relevant and non-autophagy-relevant substrates of this Ser/Thr kinase have been reported. Here, we describe our recent finding that ULK1 also regulates TNF signaling pathways. We find that inhibition of autophagy or specifically ULK1 increases TNF-induced cell death. This autophagy-independent pro-survival function of ULK1 is mediated via the phosphorylation of RIPK1 at Ser357. RIPK1 is the central mediator of pro-inflammatory or pro-death signaling pathways induced by TNF, and ULK1-dependent phosphorylation regulates RIPK1 activation and distribution to different intracellular signaling complexes. Our results indicate that ULK1 exerts a cyto-protective function not only by initiating autophagy, but also by controlling RIPK1-mediated cell death.     

ULK1 phosphorylates Ser30 of BECN1 in association with ATG14 to stimulate autophagy induction

ULK1 (unc51-like autophagy activating kinase 1) is a serine/threonine kinase that plays a key role in regulating macroautophagy/autophagy induction in response to amino acid starvation. Despite the recent progress in understanding ULK1 functions, the molecular mechanism by which ULK1 regulates the induction of autophagy remains elusive. In this study, we determined that ULK1 phosphorylates Ser30 of BECN1 (Beclin 1) in association with ATG14 (autophagy-related 14) but not with UVRAG (UV radiation resistance associated). The Ser30 phosphorylation was induced by deprivation of amino acids or treatments with Torin 1 or rapamycin, the conditions that inhibit MTORC1 (mechanistic target of rapamycin complex 1), and requires ATG13 and RB1CC1 (RB1 inducible coiled-coil 1), proteins that interact with ULK1. Hypoxia or glutamine deprivation, which inhibit MTORC1, was also able to increase the phosphorylation in a manner dependent upon ULK1 and ULK2. Blocking the BECN1 phosphorylation by replacing Ser30 with alanine suppressed the amino acid starvation-induced activation of the ATG14-containing PIK3C3/VPS34 (phosphatidylinositol 3-kinase catalytic subunit type 3) kinase, and reduced autophagy flux and the formation of phagophores and autophagosomes. The Ser30-to-Ala mutation did not affect the ULK1-mediated phosphorylations of BECN1 Ser15 or ATG14 Ser29, indicating that the BECN1 Ser30 phosphorylation might regulate autophagy independently of those 2 sites. Taken together, these results demonstrate that BECN1 Ser30 is a ULK1 target site whose phosphorylation activates the ATG14-containing PIK3C3 complex and stimulates autophagosome formation in response to amino acid starvation, hypoxia, and MTORC1 inhibition.     

ULK1 inhibits mTORC1 signaling,promotes multisite Raptor phosphorylation and hinders substrate binding

Protein synthesis and autophagy work as two opposing processes to control cell growth in response to nutrient supply. The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway, which acts as a master regulator to control protein synthesis, has recently been shown to inhibit autophagy by phosphorylating and inactivating ULK1, an autophagy regulatory protein. ULK1 also inhibits phosphorylation of a mTORC1 substrate, S6K1, indicating that a complex signaling interplay exists between mTORC1 and ULK1. Here, we demonstrate that ULK1 induces multisite phosphorylation of Raptor in vivo and in vitro. Using phospho-specific antibodies we identify Ser855 and Ser859 as being strongly phosphorylated by ULK1, with moderate phosphorylation of Ser792 also observed. Interestingly, ULK1 overexpression also increases phosphorylation of Raptor Ser863 and the mTOR autophosphorylation site, Ser2481 in a mTORC1-dependent manner. Despite this evidence for heightened mTORC1 kinase activity following ULK1 overexpresssion, mTORC1-mediated phosphorylation of S6K1 and 4E-BP1 is significantly inhibited. ULK1 expression has no effect on protein-protein interactions between the components of mTORC1, but does reduce the ability of Raptor to bind to the substrate 4E-BP1. Furthermore, shRNA knockdown of ULK1 leads to increased phosphorylation of mTORC1 substrates and decreased phosphorylation of Raptor at Ser859 and Ser792. We propose a new mechanism whereby ULK1 contributes to mTORC1 inhibition through hindrance of substrate docking to Raptor. This is a novel negative feedback loop that occurs upon activation of autophagy to maintain mTORC1 inhibition when nutrient supplies are limiting.     

ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and hinders substrate binding

Protein synthesis and autophagy work as two opposing processes to control cell growth in response to nutrient supply. The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway, which acts as a master regulator to control protein synthesis, has recently been shown to inhibit autophagy by phosphorylating and inactivating ULK1, an autophagy regulatory protein. ULK1 also inhibits phosphorylation of a mTORC1 substrate, S6K1, indicating that a complex signaling interplay exists between mTORC1 and ULK1. Here, we demonstrate that ULK1 induces multisite phosphorylation of Raptor in vivo and in vitro. Using phospho-specific antibodies we identify Ser855 and Ser859 as being strongly phosphorylated by ULK1, with moderate phosphorylation of Ser792 also observed. Interestingly, ULK1 overexpression also increases phosphorylation of Raptor Ser863 and the mTOR autophosphorylation site, Ser2481 in a mTORC1-dependent manner. Despite this evidence for heightened mTORC1 kinase activity following ULK1 overexpresssion, mTORC1-mediated phosphorylation of S6K1 and 4E-BP1 is significantly inhibited. ULK1 expression has no effect on protein-protein interactions between the components of mTORC1, but does reduce the ability of Raptor to bind to the substrate 4E-BP1. Furthermore, shRNA knockdown of ULK1 leads to increased phosphorylation of mTORC1 substrates and decreased phosphorylation of Raptor at Ser859 and Ser792. We propose a new mechanism whereby ULK1 contributes to mTORC1 inhibition through hindrance of substrate docking to Raptor. This is a novel negative feedback loop that occurs upon activation of autophagy to maintain mTORC1 inhibition when nutrient supplies are limiting.     

DAPK2 is a novel regulator of mTORC1 activity and autophagy

Autophagy is a tightly regulated catabolic process, which is upregulated in cells in response to many different stress signals. Inhibition of mammalian target of rapmaycin complex 1 (mTORC1) is a crucial step in induction of autophagy, yet the mechanisms regulating the fine tuning of its activity are not fully understood. Here we show that death-associated protein kinase 2 (DAPK2), a Ca²⁺-regulated serine/threonine kinase, directly interacts with and phosphorylates mTORC1, and has a part in suppressing mTOR activity to promote autophagy induction. DAPK2 knockdown reduced autophagy triggered either by amino acid deprivation or by increases in intracellular Ca²⁺ levels. At the molecular level, DAPK2 depletion interfered with mTORC1 inhibition caused by these two stresses, as reflected by the phosphorylation status of mTORC1 substrates, ULK1 (unc-51-like kinase 1), p70 ribosomal S6 kinase and eukaryotic initiation factor 4E-binding protein 1. An increase in mTORC1 kinase activity was also apparent in unstressed cells that were depleted of DAPK2. Immunoprecipitated mTORC1 from DAPK2-depleted cells showed increased kinase activity in vitro, an indication that DAPK2 regulation of mTORC1 is inherent to the complex itself. Indeed, we found that DAPK2 associates with components of mTORC1, as demonstrated by co-immunoprecipitation with mTOR and its complex partners, raptor (regulatory-associated protein of mTOR) and ULK1. DAPK2 was also able to interact directly with raptor, as shown by recombinant protein-binding assay. Finally, DAPK2 was shown to phosphorylate raptor in vitro. This phosphorylation was mapped to Ser721, a site located within a highly phosphorylated region of raptor that has previously been shown to regulate mTORC1 activity. Thus, DAPK2 is a novel kinase of mTORC1 and is a potential new member of this multiprotein complex, modulating mTORC1 activity and autophagy levels under stress and steady-state conditions.Macroautophagy (hereafter referred to as autophagy) is a highly regulated intracellular bulk degradation process found ubiquitously in eukaryotes. During autophagy a double-membrane vesicle, termed an autophagosome, engulfs cytoplasmic materials, including whole organelles. The autophagosome is later fused with the lysosome and its content degraded by hydrolases.¹ Basal levels of autophagy are maintained within the cell during steady state, and are involved in cell homeostasis activities such as turnover of long-lived proteins, preventing accumulation of protein aggregates, and removal of damaged cellular structures.² Beyond this homeostatic function, autophagy is stimulated during various stress conditions, such as nutrient deprivation, intracellular Ca²⁺ increase, hypoxia, ER stress and oxidative stress, to ensure continuous cell survival under stress.³A critical step in the induction of autophagy comprises the inactivation of a key negative regulator of the process, the mammalian target of rapamycin (mTOR).⁴ mTOR is a conserved serine/threonine protein kinase that acts as a master regulator in the cell. mTOR forms a rapamycin-sensitive complex named mTORC1 with its binding partner raptor (regulatory-associated protein of mTOR), which mediates mTOR''s substrate presentation.⁵ mTORC1 senses nutrient availability, growth factors and energy levels, and, in response, regulates cell growth, metabolism and protein synthesis, mainly by phosphorylation of substrates involved in protein translation: the p70 ribosomal S6 kinase (p70S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Under nutrient-rich conditions, mTORC1 suppresses autophagy to basal levels by phosphorylating and inhibiting the autophagy proteins ULK1 (unc-51-like kinase 1) and Atg13. Upon autophagic stimulus, mTORC1 activity is inhibited and the ULK1 complex is activated, leading to autophagy induction.⁶ The activity levels of mTORC1 are regulated by several mechanisms, such as interacting proteins, cellular localization and phosphorylation events. Raptor phosphorylation has been suggested as a mechanism by which upstream kinases such as AMPK,⁷ RSK⁸ and ULK1⁹ can regulate mTORC1 activity.Death-associated protein kinase 2 (DAPK2; also named DRP-1) is a 42-kDa Ca²⁺/calmodulin (CaM)-regulated serine/threonine kinase,¹⁰ and a closely related homolog of DAPK, a gene originally discovered in an attempt to find positive regulators of cell death.¹¹ DAPK2 was identified based on homology to the catalytic domain of DAPK. DAPK2 is a soluble cytoplasmatic protein, which triggers massive membrane blebbing and appearance of double-membrane autophagic vesicles upon its overexpression (for a review see Shiloh et al.¹²). DAPK2''s substrates and interacting proteins are mostly unknown, with the exception of the myosin II regulatory light chain, which has been shown to be an in vitro and in vivo substrate.¹³ Although many publications have studied DAPK, its substrates and its role in cell death and autophagy,^{14, 15} very little is known about DAPK2 substrates, cellular functions or the molecular pathways that it regulates.In this work, we studied the involvement of DAPK2 in the autophagic module. We identified DAPK2 as a novel interacting protein of mTORC1, and as a negative regulator of the complex both during steady-state growth conditions and in response to different stress autophagic signals. We identified mTOR''s binding partner, raptor, as a substrate of DAPK2, and found Ser721 as its phosphorylation site.     

ULK1 inhibits the kinase activity of mTORC1 and cell proliferation

ULK1 (Unc51-like kinase, hATG1) is a Ser/Thr kinase that plays a key role in inducing autophagy in response to starvation. ULK1 is phosphorylated and negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1). Previous studies have shown that ULK1 is not only a downstream effector of mTORC1 but also a negative regulator of mTORC1 signaling.^1-3 Here, we investigated how ULK1 regulates mTORC1 signaling, and found that ULK1 inhibits the kinase activity of mTORC1 and cell proliferation. Deficiency or knockdown of ULK1 or its homolog ULK2 enhanced mTORC1 signaling, cell proliferation rates and accumulation of cell mass, whereas overexpression of ULK1 had the opposite effect. Knockdown of Atg13, the binding partner of ULK1 and ULK2, mimicked the effects of ULK1 or ULK2 deficiency or knockdown. Both insulin and leucine stimulated mTORC1 signaling to a greater extent when ULK1 or ULK2 was deficient or knocked down. In contrast, Atg5 deficiency did not have a significant effect on mTORC1 signaling and cell proliferation. The stimulatory effect of ULK1 knockdown on mTORC1 signaling occurred even in the absence of tuberous sclerosis complex 2 (TSC2), the negative regulator of mTORC1 signaling. In addition, ULK1 was found to bind raptor, induce its phosphorylation, and inhibit the kinase activity of mTORC1. These results demonstrate that ULK1 negatively regulates the kinase activity of mTORC1 and cell proliferation in a manner independent of Atg5 and TSC2. The inhibition of mTORC1 by ULK1 may be important to coordinately regulate cell growth and autophagy with optimized utilization of cellular energy.     

The association of AMPK with ULK1 regulates autophagy

Autophagy is a highly orchestrated intracellular bulk degradation process that is activated by various environmental stresses. The serine/threonine kinase ULK1, like its yeast homologue Atg1, is a key initiator of autophagy that is negatively regulated by the mTOR kinase. However, the molecular mechanism that controls the inhibitory effect of mTOR on ULK1-mediated autophagy is not fully understood. Here we identified AMPK, a central energy sensor, as a new ULK1-binding partner. We found that AMPK binds to the PS domain of ULK1 and this interaction is required for ULK1-mediated autophagy. Interestingly, activation of AMPK by AICAR induces 14-3-3 binding to the AMPK-ULK1-mTORC1 complex, which coincides with raptor Ser792 phosphorylation and mTOR inactivation. Consistently, AICAR induces autophagy in TSC2-deficient cells expressing wild-type raptor but not the mutant raptor that lacks the AMPK phosphorylation sites (Ser722 and Ser792). Taken together, these results suggest that AMPK association with ULK1 plays an important role in autophagy induction, at least in part, by phosphorylation of raptor to lift the inhibitory effect of mTOR on the ULK1 autophagic complex.     

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.     

Ser289 phosphorylation activates both DAPK1 and DAPK2 but in response to different intracellular signaling pathways

DAPK1 and DAPK2 are calmodulin (CaM)-regulated protein kinases that share a high degree of homology in their catalytic and CaM regulatory domains. Both kinases function as tumor suppressors, and both have been implicated in autophagy regulation. Over the years, common regulatory mechanisms for the two kinases as well as kinase-specific ones have been identified. In a recent work, we revealed that DAPK2 is phosphorylated on Ser289 by the metabolic sensor AMPK, and that this phosphorylation enhances DAPK2 catalytic activity. Notably, Ser289 is conserved between DAPK1 and DAPK2, and was previously found to be phosphorylated in DAPK1 by RSK. Intriguingly, Ser289 phosphorylation was conversely reported to inhibit the pro-apoptotic activity of DAPK1 in cells. However, as the direct effect of this phosphorylation on DAPK1 catalytic activity was not tested, indirect effects were not excluded. Here, we compared Ser289 phosphorylation of the two kinases in the same cells and found that the intracellular signaling pathways that lead to Ser289 phosphorylation are mutually-exclusive and different for each kinase. In addition, we found that Ser289 phosphorylation in fact enhances DAPK1 catalytic activity, similar to the effect on DAPK2. Thus, Ser289 phosphorylation activates both DAPK1 and DAPK2, but in response to different intracellular signaling pathways.     

ULK1 inhibits the kinase activity of mTORC1 and cell proliferation

ULK1 (Unc51-like kinase, hATG1) is a Ser/Thr kinase that plays a key role in inducing autophagy in response to starvation. ULK1 is phosphorylated and negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1). Previous studies have shown that ULK1 is not only a downstream effector of mTORC1 but also a negative regulator of mTORC1 signaling. ( 1-3) Here, we investigated how ULK1 regulates mTORC1 signaling, and found that ULK1 inhibits the kinase activity of mTORC1 and cell proliferation. Deficiency or knockdown of ULK1 or its homolog ULK2 enhanced mTORC1 signaling, cell proliferation rates and accumulation of cell mass, whereas overexpression of ULK1 had the opposite effect. Knockdown of Atg13, the binding partner of ULK1 and ULK2, mimicked the effects of ULK1 or ULK2 deficiency or knockdown. Both insulin and leucine stimulated mTORC1 signaling to a greater extent when ULK1 or ULK2 was deficient or knocked down. In contrast, Atg5 deficiency did not have a significant effect on mTORC1 signaling and cell proliferation. The stimulatory effect of ULK1 knockdown on mTORC1 signaling occurred even in the absence of tuberous sclerosis complex 2 (TSC2), the negative regulator of mTORC1 signaling. In addition, ULK1 was found to bind raptor, induce its phosphorylation, and inhibit the kinase activity of mTORC1. These results demonstrate that ULK1 negatively regulates the kinase activity of mTORC1 and cell proliferation in a manner independent of Atg5 and TSC2. The inhibition of mTORC1 by ULK1 may be important to coordinately regulate cell growth and autophagy with optimized utilization of cellular energy.     

Involvement of phosphorylation of ULK1 in alternative autophagy

ABSTRACT

Alternative autophagy is an ATG5 (autophagy related 5)-independent, Golgi membrane-derived form of macroautophagy. ULK1 (unc-51 like kinase 1) is an essential initiator not only for canonical autophagy but also for alternative autophagy. However, the mechanism as to how ULK1 differentially regulates both types of autophagy has remained unclear. Recently, we identified a novel phosphorylation site of ULK1 at Ser746, which is required for alternative autophagy, but not canonical autophagy. We also identify RIPK3 (receptor-interacting serine-threonine kinase 3) as the kinase responsible for genotoxic stress-induced ULK1 S746 phosphorylation. These findings indicate that RIPK3-dependent ULK1 S746 phosphorylation plays a pivotal role in genotoxic stress-induced alternative autophagy.     

Inhibition of mTOR kinase by AZD8055 can antagonize chemotherapy-induced cell death through autophagy induction and down-regulation of p62/sequestosome 1

AZD8055 is an ATP-competitive inhibitor of mammalian target of rapamycin (mTOR) that forms two multiprotein complexes, mTORC1 and mTORC2, and negatively regulates autophagy. We demonstrate that AZD8055 stimulates and potentiates chemotherapy-mediated autophagy, as shown by LC3I-II conversion and down-regulation of the ubiquitin-binding protein p62/sequestosome 1. AZD8055-induced autophagy was pro-survival as shown by its ability to attenuate cell death and DNA damage (p-H2AX), and to enhance clonogenic survival by cytotoxic chemotherapy. Autophagy inhibition by siRNA against Beclin 1 or LC3B, or by chloroquine, partially reversed the cytoprotective effect of AZD8055 that was independent of cell cycle inhibition. The pro-survival role of autophagy was confirmed using ectopic expression of Beclin 1 that conferred cytoprotection. To determine whether autophagy-mediated down-regulation of p62/sequestosome 1 contributes to its pro-survival role, we generated p62 knockdown cells using shRNA that showed protection from chemotherapy-induced cell death and DNA damage. We also overexpressed wild-type (wt) p62 that promoted chemotherapy-induced cell death, whereas mutated p62 at functional domains (PB1, UBA) failed to do so. The ability of ectopic wt p62 to promote cell death was blocked by AZD8055. AZD8055 was shown to inhibit phosphorylation of the autophagy-initiating kinase ULK1 at Ser(757) and inhibited known targets of mTORC1 (p-mTOR Ser(2448), p70S6K, p-S6, p4EBP1) and mTORC2 (p-mTOR Ser(2481), p-AKT Ser(473)). Knockdown of mTOR, but not Raptor or Rictor, reduced p-ULK1 at Ser(757) and enhanced chemotherapy-induced autophagy that resulted in a similar cytoprotective effect as shown for AZD8055. In conclusion, AZD8055 inhibits mTOR kinase and ULK1 phosphorylation to induce autophagy whose pro-survival effect is due, in part, to down-regulation of p62.     

The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG14

ULK1 (unc-51 like autophagy activating kinase 1), the key mediator of MTORC1 signaling to autophagy, regulates early stages of autophagosome formation in response to starvation or MTORC1 inhibition. How ULK1 regulates the autophagy induction process remains elusive. Here, we identify that ATG13, a binding partner of ULK1, mediates interaction of ULK1 with the ATG14-containing PIK3C3/VPS34 complex, the key machinery for initiation of autophagosome formation. The interaction enables ULK1 to phosphorylate ATG14 in a manner dependent upon autophagy inducing conditions, such as nutrient starvation or MTORC1 inhibition. The ATG14 phosphorylation mimics nutrient deprivation through stimulating the kinase activity of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex and facilitates phagophore and autophagosome formation. By monitoring the ATG14 phosphorylation, we determined that the ULK1 activity requires BECN1/Beclin 1 but not the phosphatidylethanolamine (PE)-conjugation machinery and the PIK3C3 kinase activity. Monitoring the phosphorylation also allowed us to identify that ATG9A is required to suppress the ULK1 activity under nutrient-enriched conditions. Furthermore, we determined that ATG14 phosphorylation depends on ULK1 and dietary conditions in vivo. These results define a key molecular event for the starvation-induced activation of the ATG14-containing PtdIns3K complex by ULK1, and demonstrate hierarchical relations between the ULK1 activation and other autophagy proteins involved in phagophore formation.     

Phosphorylation of ULK1 serine 746 dictates ATG5-independent autophagy

ABSTRACT

There is a type of noncanonical autophagy, which is independent of ATG5 (autophagy related 5), also referred to as alternative autophagy. Both canonical and ATG5-independent alternative autophagy require the initiator ULK1 (unc-51 like kinase 1), but how ULK1 regulates these two types of autophagy differently remains unclear. A recent paper from Torii et al. demonstrates that phosphorylation of ULK1 at Ser746 by RIPK3 (receptor interacting serine/threonine kinase 3) is the key difference between these two types of autophagy; this phosphorylation is exclusively found during alternative autophagy.     

FLCN,a novel autophagy component,interacts with GABARAP and is regulated by ULK1 phosphorylation

Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterized by benign hair follicle tumors, pneumothorax, and renal cancer. Folliculin (FLCN), the protein product of the FLCN gene, is a poorly characterized tumor suppressor protein, currently linked to multiple cellular pathways. Autophagy maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase ULK1 drives autophagy, the underlying mechanisms are still being unraveled and few ULK1 substrates have been identified to date. Here, we identify that loss of FLCN moderately impairs basal autophagic flux, while re-expression of FLCN rescues autophagy. We reveal that the FLCN complex is regulated by ULK1 and elucidate 3 novel phosphorylation sites (Ser406, Ser537, and Ser542) within FLCN, which are induced by ULK1 overexpression. In addition, our findings demonstrate that FLCN interacts with a second integral component of the autophagy machinery, GABA(A) receptor-associated protein (GABARAP). The FLCN-GABARAP association is modulated by the presence of either folliculin-interacting protein (FNIP)-1 or FNIP2 and further regulated by ULK1. As observed by elevation of GABARAP, sequestome 1 (SQSTM1) and microtubule-associated protein 1 light chain 3 (MAP1LC3B) in chromophobe and clear cell tumors from a BHD patient, we found that autophagy is impaired in BHD-associated renal tumors. Consequently, this work reveals a novel facet of autophagy regulation by ULK1 and substantially contributes to our understanding of FLCN function by linking it directly to autophagy through GABARAP and ULK1.     

FLCN,a novel autophagy component,interacts with GABARAP and is regulated by ULK1 phosphorylation

Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterized by benign hair follicle tumors, pneumothorax, and renal cancer. Folliculin (FLCN), the protein product of the FLCN gene, is a poorly characterized tumor suppressor protein, currently linked to multiple cellular pathways. Autophagy maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase ULK1 drives autophagy, the underlying mechanisms are still being unraveled and few ULK1 substrates have been identified to date. Here, we identify that loss of FLCN moderately impairs basal autophagic flux, while re-expression of FLCN rescues autophagy. We reveal that the FLCN complex is regulated by ULK1 and elucidate 3 novel phosphorylation sites (Ser406, Ser537, and Ser542) within FLCN, which are induced by ULK1 overexpression. In addition, our findings demonstrate that FLCN interacts with a second integral component of the autophagy machinery, GABA(A) receptor-associated protein (GABARAP). The FLCN-GABARAP association is modulated by the presence of either folliculin-interacting protein (FNIP)-1 or FNIP2 and further regulated by ULK1. As observed by elevation of GABARAP, sequestome 1 (SQSTM1) and microtubule-associated protein 1 light chain 3 (MAP1LC3B) in chromophobe and clear cell tumors from a BHD patient, we found that autophagy is impaired in BHD-associated renal tumors. Consequently, this work reveals a novel facet of autophagy regulation by ULK1 and substantially contributes to our understanding of FLCN function by linking it directly to autophagy through GABARAP and ULK1.     

Autophagy gets a brake: DAP1, a novel mTOR substrate,is activated to suppress the autophagic process

Autophagy, a highly regulated catabolic process, is controlled by the action of positive and negative regulators. While many of the positive mediators of autophagy have been identified, very little is known about negative regulators that might counterbalance the process. We recently identified death-associated protein 1 (DAP1) as a suppressor of autophagy and as a novel direct substrate of mammalian target of rapamycin (mTOR). We found that DAP1 is functionally silent in cells growing under rich nutrient supplies through mTOR-dependent inhibitory phosphorylation on two sites, which were mapped to Ser3 and Ser51. During amino acid starvation, mTOR activity is turned off resulting in a rapid reduction in the phosphorylation of DAP1. This caused the conversion of the protein into a suppressor of autophagy, thus providing a buffering mechanism that counterbalances the autophagic flux and prevents its overactivation under conditions of nutrient deprivation. Based on these studies we propose the “gas and brake” concept in which mTOR, the main sensor that regulates autophagy in response to amino acid deprivation, also controls the activity of a specific balancing brake to prevent the overactivation of autophagy.Key words: DAP1, mTOR, autophagy, amino acid starvation, phosphorylationIn recent years, many of the genes controlling and executing the autophagic process have been identified. Most of these genes act as positive mediators of the various steps of the process, including the ULK1 complex, which regulates the induction step, the Vps34-Beclin 1 complex that participates in the vesicle nucleation step and two ubiquitin-like pathways, the Atg12-Atg5 and the LC3-phosphatidylethanolamine (PE) conjugation steps, which play a central role in the vesicle elongation process. To date, only a few negative regulators of autophagy have been identified, including mTOR and the anti-apoptotic Bcl-2 family members. mTOR Ser/Thr kinase is a central suppressor of autophagy acting at the initiating regulatory steps of the process. Many signaling pathways act to inhibit mTOR activity, thus relieving its inhibitory effects on autophagy. The anti-apoptotic Bcl-2 and Bcl-X_L proteins, on the other hand, act at the nucleation step, by directly binding to Beclin 1''s BH3 domain, thus reducing the activation of Vps34 and subsequent autophagy. This inhibition can be relieved through dissociation of the complex, following either JNK-1 mediated phosphorylation of Bcl-2 or DAP kinase-mediated phosphorylation of the BH3 domain of Beclin 1.DAP1 is a small (∼15 kDa), ubiquitously expressed protein, rich in prolines and lacking known functional motifs. DAP1 was isolated more than a decade ago in our laboratory using a functional approach to gene cloning aimed at identifying novel mediators of IFNγ-induced cell death in mammalian cell cultures. Until recently, very little was known about the cellular and molecular functions of DAP1, mainly due to the lack of homology to other known proteins and the lack of functional motifs that could indicate a possible cellular function and studies in mammalian systems were missing.Recently, we discovered that DAP1 is another negative regulator of autophagy; yet, interestingly, its suppressive activity is selectively turned on during the autophagic process. Moreover, we found that DAP1 suppressive activity is tightly linked to the status of mTOR kinase activity. Under nutrient-rich culture conditions, DAP1 is phosphorylated by mTOR on two sites, Ser3 and Ser51, resulting in its inactivation. In response to nutrient deprivation, mTOR is inhibited and DAP1 undergoes rapid dephosphorylation. By knocking down the endogenous DAP1 and introducing either the phosphomimetic or the nonphosphorylatible DAP1 mutants, we found that the dephosphorylation leads to activation of the autophagic suppressive function of DAP1, whereas the phophorylated form is inactive. These results led to a “gas and brake” model, in which at the same time that autophagy is induced, some brakes such as DAP1 are also activated to provide a buffering mechanism that counterbalances the autophagic flux and prevents its overactivation under nutrient-deprivation conditions (Fig. 1). Notably, balancing autophagy is extremely important, since deregulated or excessive autophagy has been implicated in the pathogenesis of diverse diseases, such as certain types of neuronal degeneration and cancer and also in cellular aging.Open in a separate windowFigure 1“Gas and brake” model. During nutrient-rich conditions, active mTORC1 phosphorylates and inactivates the components of the ULK1 complex, ULK1 and Atg13, thus preventing the induction of autophagy. DAP1 is also inactivated simultaneously by mTORC1-mediated phosphorylation on Ser3 and Ser51. In addition, mTORC1 phosphorylates and activates p70S6K and 4E-BP1, which mediate the protein translation and cell growth activities of mTOR. Upon nutrient starvation, mTORC1 activity is attenuated, leading to dephosphorylation and activation of ULK1. ULK1, in turn, undergoes autophosphorylation and phosphorylates Atg13 and FIP200 resulting in ULK1 complex activation and induction of autophagy. On the other hand, activation of DAP1 by dephosphorylation, results in suppression of autophagy, thus inserting a brake into the process of autophagy. Note that the inactive proteins/complexes are faded out.The current challenge is to identify the molecular basis of the suppressive functions of DAP1 on autophagy. We have recently shown that DAP1 knockdown enhances LC3 lipidation and autophagosome accumulation both during amino acid starvation and rapamycin treatment. In addition, preliminary data indicate that the knockdown of DAP1 has no effect on mTOR complex 1 (mTORC1) activity in cells, at least during the first hours of starvation. Accordingly, DAP1 may function between the mTORC1 and the LC3 conjugation systems. The potential targets may fall into one of the multiprotein complexes functioning downstream of mTOR such as the ULK1 complex, the Vps34-Beclin 1 complex and more. Future studies will be performed to identify the molecular mechanism by which DAP1 suppresses autophagy. The lack of known functional motifs in the DAP1 protein sequence suggests that this small proline-rich protein may function as an adaptor blocking autophagy by binding to critical protein partners that still await identification.Although autophagy is primarily a protective process for the cell, it can also play a role in cell death. In response to prolonged starvation, autophagy can act either as a cell survival mechanism or be recruited as a cell death executer. In the future it would be interesting to examine whether the autophagy enhancement resulting from DAP1 knockdown contributes to increased cell death in our system or even may convert the survival properties of autophagy into death induction. This will fit the “gas and brake” model, in which autophagy, which is initially recruited as a cell survival mechanism, is converted into cell death machinery when a certain threshold is crossed due to the loss of the “brake” by the knockdown of DAP1.To date, very little is known about the putative mechanisms that restrict the intensity of the autophagic flux to maintain the continuous benefits of this process under stress. Therefore, the ability of DAP1 to counterbalance and buffer the process in a manner that is tightly linked to the status of a central player in autophagy (i.e., mTOR) is an important discovery in this field and provides a target for future drug design.