How the Atg1 complex assembles to initiate autophagy

The Atg1 complex, comprising Atg1, Atg13, Atg17, Atg29, and Atg31, is a key initiator of autophagy. The Atg17-Atg31-Atg29 subcomplex is constitutively present at the phagophore assembly site (PAS), while Atg1 and Atg13 join the complex when autophagy is triggered by starvation or other signals. We sought to understand the energetics and dynamics of assembly using isothermal titration calorimetry (ITC), sedimentation velocity analytical ultracentrifugation, and hydrogen-deuterium exchange (HDX). We showed that the membrane and Atg13-binding domain of Atg1, Atg1_EAT, is dynamic on its own, but is rigidified in its high-affinity (～100 nM) complex with Atg13. Atg1_EAT and Atg13 form a 2:2 dimeric assembly and together associate with lower affinity (～10 μM) with the 2:2:2 Atg17-Atg31-Atg29 complex. These results lead to an overall model for the assembly pathway of the Atg1 complex. The model highlights the Atg13-Atg17 binding event as the weakest link in the assembly process and thus as a natural regulatory checkpoint.     

How the Atg1 complex assembles to initiate autophagy

The Atg1 complex, comprising Atg1, Atg13, Atg17, Atg29, and Atg31, is a key initiator of autophagy. The Atg17-Atg31-Atg29 subcomplex is constitutively present at the phagophore assembly site (PAS), while Atg1 and Atg13 join the complex when autophagy is triggered by starvation or other signals. We sought to understand the energetics and dynamics of assembly using isothermal titration calorimetry (ITC), sedimentation velocity analytical ultracentrifugation, and hydrogen-deuterium exchange (HDX). We showed that the membrane and Atg13-binding domain of Atg1, Atg1_EAT, is dynamic on its own, but is rigidified in its high-affinity (∼100 nM) complex with Atg13. Atg1_EAT and Atg13 form a 2:2 dimeric assembly and together associate with lower affinity (∼10 μM) with the 2:2:2 Atg17-Atg31-Atg29 complex. These results lead to an overall model for the assembly pathway of the Atg1 complex. The model highlights the Atg13-Atg17 binding event as the weakest link in the assembly process and thus as a natural regulatory checkpoint.     

The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy

A GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). C9orf72 encodes two C9orf72 protein isoforms of unclear function. Reduced levels of C9orf72 expression have been reported in C9ALS/FTD patients, and although C9orf72 haploinsufficiency has been proposed to contribute to C9ALS/FTD, its significance is not yet clear. Here, we report that C9orf72 interacts with Rab1a and the Unc‐51‐like kinase 1 (ULK1) autophagy initiation complex. As a Rab1a effector, C9orf72 controls initiation of autophagy by regulating the Rab1a‐dependent trafficking of the ULK1 autophagy initiation complex to the phagophore. Accordingly, reduction of C9orf72 expression in cell lines and primary neurons attenuated autophagy and caused accumulation of p62‐positive puncta reminiscent of the p62 pathology observed in C9ALS/FTD patients. Finally, basal levels of autophagy were markedly reduced in C9ALS/FTD patient‐derived iNeurons. Thus, our data identify C9orf72 as a novel Rab1a effector in the regulation of autophagy and indicate that C9orf72 haploinsufficiency and associated reductions in autophagy might be the underlying cause of C9ALS/FTD‐associated p62 pathology.     

RPA70 depletion induces hSSB1/2-INTS3 complex to initiate ATR signaling

The primary eukaryotic single-stranded DNA-binding protein, Replication protein A (RPA), binds to single-stranded DNA at the sites of DNA damage and recruits the apical checkpoint kinase, ATR via its partner protein, ATRIP. It has been demonstrated that absence of RPA incapacitates the ATR-mediated checkpoint response. We report that in the absence of RPA, human single-stranded DNA-binding protein 1 (hSSB1) and its partner protein INTS3 form sub-nuclear foci, associate with the ATR-ATRIP complex and recruit it to the sites of genomic stress. The ATRIP foci formed after RPA depletion are abrogated in the absence of INTS3, establishing that hSSB-INTS3 complex recruits the ATR-ATRIP checkpoint complex to the sites of genomic stress. Depletion of homologs hSSB1/2 and INTS3 in RPA-deficient cells attenuates Chk1 phosphorylation, indicating that the cells are debilitated in responding to stress. We have identified that TopBP1 and the Rad9-Rad1-Hus1 complex are essential for the alternate mode of ATR activation. In summation, we report that the single-stranded DNA-binding protein complex, hSSB1/2-INTS3 can recruit the checkpoint complex to initiate ATR signaling.     

ATM engages the TSC2/mTORC1 signaling node to regulate autophagy

The link between reactive oxygen species (ROS) and induction of autophagy has been well documented, but the molecular mechanisms regulating this phenomenon are only beginning to be elucidated. Autophagy is now being appreciated as an integral part of the cellular response to many diverse types of cellular stresses including nutrient deprivation, hypoxia, oxidative stress, and DNA damage, and likely the mechanism(s) for each type of stress vary considerably. The cellular outcome of inducing autophagy in response to stress is also quite complex, and depends on many factors including cellular context, type and magnitude of stress.     

AMPK-dependent phosphorylation of ULK1 induces autophagy

AMP-activated protein kinase (AMPK) is a highly conserved cellular energy sensor that plays a central role in metabolic homeostasis. A recent study in Science (Egan et al., 2011) identifies ULK1 as a substrate for AMPK phosphorylation, a modification required for selective autophagy of mitochondria and cell survival during starvation.     

TFG binds LC3C to regulate ULK1 localization and autophagosome formation

The early secretory pathway and autophagy are two essential and evolutionarily conserved endomembrane processes that are finely interlinked. Although growing evidence suggests that intracellular trafficking is important for autophagosome biogenesis, the molecular regulatory network involved is still not fully defined. In this study, we demonstrate a crucial effect of the COPII vesicle‐related protein TFG (Trk‐fused gene) on ULK1 puncta number and localization during autophagy induction. This, in turn, affects formation of the isolation membrane, as well as the correct dynamics of association between LC3B and early ATG proteins, leading to the proper formation of both omegasomes and autophagosomes. Consistently, fibroblasts derived from a hereditary spastic paraparesis (HSP) patient carrying mutated TFG (R106C) show defects in both autophagy and ULK1 puncta accumulation. In addition, we demonstrate that TFG activity in autophagy depends on its interaction with the ATG8 protein LC3C through a canonical LIR motif, thereby favouring LC3C‐ULK1 binding. Altogether, our results uncover a link between TFG and autophagy and identify TFG as a molecular scaffold linking the early secretion pathway to autophagy.     

The requirement of uncoordinated 51-like kinase 1 (ULK1) and ULK2 in the regulation of autophagy

Autophagy is an evolutionarily conserved physiological process of self-digestion by a cell to adapt to various stresses, including starvation. Its molecular basis involves the concerted activation of proteins encoded by the family of autophagy-related (Atg) genes. The best characterized is the serine/threonine protein kinase Atg1 in yeast which appears to be essential at the early stage of autophagy. In mammals, five Atg1 homologues have been identified as uncoordinated (UNC) 51-like kinase 1 to 4 and STK36. ULK1 and ULK2 are the most closely related members of the family, sharing 78% homology within their protein kinase domains. However, the specific function of ULK1 and ULK2 in mammalian autophagy is not fully understood. Here, we demonstrate that ULK1 and ULK2 are functionally redundant protein kinases required to mediate autophagy under nutrient-deprived conditions in fibroblasts. In contrast, ULK1, but not ULK2, is critical to induce the autophagic response of cerebellar granule neurons (CGN) to low potassium concentration in serum-free conditions. Furthermore, we found that ULK1 has a cytoprotective function in neurons. Together, these results provide strong genetic evidence that ULK1 is an essential component of the autophagic signaling pathway. The ability of ULK2 to compensate for the loss of ULK1 function is cell-type specific.     

The requirement of uncoordinated 51-like kinase 1 (ULK1) and ULK2 in the regulation of autophagy

Autophagy is an evolutionarily conserved physiological process of self-digestion by a cell to adapt to various stresses, including starvation. Its molecular basis involves the concerted activation of proteins encoded by the family of autophagy-related (Atg) genes. The best characterized is the serine/threonine protein kinase Atg1 in yeast which appears to be essential at the early stage of autophagy. In mammals, five Atg1 homologues have been identified as uncoordinated (UNC) 51-like kinase 1 to 4 and STK36. ULK1 and ULK2 are the most closely related members of the family, sharing 78% homology within their protein kinase domains. However, the specific function of ULK1 and ULK2 in mammalian autophagy is not fully understood. Here, we demonstrate that ULK1 and ULK2 are functionally redundant protein kinases required to mediate autophagy under nutrient-deprived conditions in fibroblasts. In contrast, ULK1, but not ULK2, is critical to induce the autophagic response of cerebellar granule neurons (CGN) to low potassium concentration in serum-free conditions. Furthermore, we found that ULK1 has a cytoprotective function in neurons. Together, these results provide strong genetic evidence that ULK1 is an essential component of the autophagic signaling pathway. The ability of ULK2 to compensate for the loss of ULK1 function is cell-type specific.     

Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy

Autophagy is an intracellular trafficking pathway sequestering cytoplasm and delivering excess and damaged cargo to the vacuole for degradation. The Atg1/ULK1 kinase is an essential component of the core autophagy machinery possibly activated by binding to Atg13 upon starvation. Indeed, we found that Atg13 directly binds Atg1, and specific Atg13 mutations abolishing this interaction interfere with Atg1 function in vivo. Surprisingly, Atg13 binding to Atg1 is constitutive and not altered by nutrient conditions or treatment with the Target of rapamycin complex 1 (TORC1)-inhibitor rapamycin. We identify Atg8 as a novel regulator of Atg1/ULK1, which directly binds Atg1/ULK1 in a LC3-interaction region (LIR)-dependent manner. Molecular analysis revealed that Atg13 and Atg8 cooperate at different steps to regulate Atg1 function. Atg8 targets Atg1/ULK1 to autophagosomes, where it may promote autophagosome maturation and/or fusion with vacuoles/lysosomes. Moreover, Atg8 binding triggers vacuolar degradation of the Atg1-Atg13 complex in yeast, thereby coupling Atg1 activity to autophagic flux. Together, these findings define a conserved step in autophagy regulation in yeast and mammals and expand the known functions of LIR-dependent Atg8 targets to include spatial regulation of the Atg1/ULK1 kinase.     

ULK1 translocates to mitochondria and phosphorylates FUNDC1 to regulate mitophagy

Autophagy eliminates dysfunctional mitochondria in an intricate process known as mitophagy. ULK1 is critical for the induction of autophagy, but its substrate(s) and mechanism of action in mitophagy remain unclear. Here, we show that ULK1 is upregulated and translocates to fragmented mitochondria upon mitophagy induction by either hypoxia or mitochondrial uncouplers. At mitochondria, ULK1 interacts with FUNDC1, phosphorylating it at serine 17, which enhances FUNDC1 binding to LC3. A ULK1‐binding‐deficient mutant of FUNDC1 prevents ULK1 translocation to mitochondria and inhibits mitophagy. Finally, kinase‐active ULK1 and a phospho‐mimicking mutant of FUNDC1 rescue mitophagy in ULK1‐null cells. Thus, we conclude that FUNDC1 regulates ULK1 recruitment to damaged mitochondria, where FUNDC1 phosphorylation by ULK1 is crucial for mitophagy.     

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.     

WHAMM links actin assembly via the Arp2/3 complex to autophagy

Macroautophagy (hereafter autophagy) is the process by which cytosolic material destined for degradation is enclosed inside a double-membrane cisterna known as the autophagosome and processed for secretion and/or recycling. This process requires a large collection of proteins that converge on certain sites of the ER membrane to generate the autophagosome membrane. Recently, it was shown that actin accumulates around autophagosome precursors and could play a role in this process, but the mechanism and role of actin polymerization in autophagy were unknown. Here, we discuss our recent finding that the nucleation-promoting factor (NPF) WHAMM recruits and activates the Arp2/3 complex for actin assembly at sites of autophagosome formation on the ER. Using high-resolution, live-cell imaging, we showed that WHAMM forms dynamic puncta on the ER that comigrate with several autophagy markers, and propels the spiral movement of these puncta by an Arp2/3 complex-dependent actin comet tail mechanism. In starved cells, WHAMM accumulates at the interface between neighboring autophagosomes, whose number and size increases with WHAMM expression. Conversely, knocking down WHAMM, inhibiting the Arp2/3 complex or interfering with actin polymerization reduces the size and number of autophagosomes. These findings establish a link between Arp2/3 complex-mediated actin assembly and autophagy.     

DAPK3 inhibits gastric cancer progression via activation of ULK1-dependent autophagy

Dysregulation of the balance between cell proliferation and cell death is a central feature of malignances. Death-associated protein kinase 3 (DAPK3) regulates programmed cell death including apoptosis and autophagy. Our previous study showed that DAPK3 downregulation was detected in more than half of gastric cancers (GCs), which was related to tumor invasion, metastasis, and poor prognosis. However, the precise molecular mechanism underlying DAPK3-mediated tumor suppression remains unclear. Here, we showed that the tumor suppressive function of DAPK3 was dependent on autophagy process. Mass spectrometry, in vitro kinase assay, and immunoprecipitation revealed that DAPK3 increased ULK1 activity by direct ULK1 phosphorylation at Ser556. ULK1 phosphorylation by DAPK3 facilitates the ULK1 complex formation, the VPS34 complex activation, and autophagy induction upon starvation. The kinase activity of DAPK3 and ULK1 Ser556 phosphorylation were required for DAPK3-modulated tumor suppression. The coordinate expression of DAPK3 with ULK1 Ser556 phosphorylation was confirmed in clinical GC samples, and this co-expression was correlated with favorable survival outcomes in patients. Collectively, these findings indicate that the tumor-suppressor roles of DAPK3 in GC are associated with autophagy and that DAPK3 is a novel autophagy regulator, which can directly phosphorylate ULK1 and activate ULK1. Thus, DAPK3 might be a promising prognostic autophagy-associated marker.Subject terms: Tumour-suppressor proteins, Macroautophagy     

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.     

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.