Autophagy up and down by outsmarting the incredible ULK

Macroautophagy/autophagy initiation is finely regulated by post-translational modifications of key proteins, to comply with the fast kinetics of the cellular response to several stress stimuli. Phosphorylation and ubiquitination play a central role in controlling autophagy by influencing the activity, recruitment and turnover of autophagic components. Recently, we found that, upon autophagy progression, ULK1 kinase is specifically ubiquitinated by the E3 ligase NEDD4L and then degraded via the proteasome. However, during prolonged autophagy, while the ULK1 protein undergoes this inhibition, ULK1 mRNA is actively transcribed, translated and then inhibited again by MTOR-dependent inhibitory phosphorylation. This regulation is essential to promptly restore the ULK1 protein to its original levels to keep autophagy under a safe and physiological threshold.     

The E3 ubiquitin ligase NEDD4 is an LC3-interactive protein and regulates autophagy

The MAP1LC3/LC3 family plays an essential role in autophagosomal biogenesis and transport. In this report, we show that the HECT family E3 ubiquitin ligase NEDD4 interacts with LC3 and is involved in autophagosomal biogenesis. NEDD4 binds to LC3 through a conserved WXXL LC3-binding motif in a region between the C2 and the WW2 domains. Knockdown of NEDD4 impaired starvation- or rapamycin-induced activation of autophagy and autophagosomal biogenesis and caused aggregates of the LC3 puncta colocalized with endoplasmic reticulum membrane markers. Electron microscopy observed gigantic deformed mitochondria in NEDD4 knockdown cells, suggesting that NEDD4 might function in mitophagy. Furthermore, SQSTM1 is ubiquitinated by NEDD4 while LC3 functions as an activator of NEDD4 ligase activity. Taken together, our studies define an important role of NEDD4 in regulation of autophagy.     

The E3 ubiquitin ligase NEDD4 enhances killing of membrane-perturbing intracellular bacteria by promoting autophagy

The E3 ubiquitin ligase NEDD4 has been intensively studied in processes involved in viral infections, such as virus budding. However, little is known about its functions in bacterial infections. Our investigations into the role of NEDD4 in intracellular bacterial infections demonstrate that Mycobacterium tuberculosis and Listeria monocytogenes, but not Mycobacterium bovis BCG, replicate more efficiently in NEDD4 knockdown macrophages. In parallel, NEDD4 knockdown or knockout impaired basal macroautophagy/autophagy, as well as infection-induced autophagy. Conversely, NEDD4 expression promoted autophagy in an E3 catalytic activity-dependent manner, thereby restricting intracellular Listeria replication. Mechanistic studies uncovered that endogenous NEDD4 interacted with BECN1/Beclin 1 and this interaction increased during Listeria infection. Deficiency of NEDD4 resulted in elevated K48-linkage ubiquitination of endogenous BECN1. Further, NEDD4 mediated K6- and K27- linkage ubiquitination of BECN1, leading to elevated stability of BECN1 and increased autophagy. Thus, NEDD4 participates in killing of intracellular bacterial pathogens via autophagy by sustaining the stability of BECN1.     

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.     

KLHL20 links the ubiquitin-proteasome system to autophagy termination

Autophagy is a dynamic and self-limiting process. The amplitude and duration of this process need to be properly controlled to maintain cell homeostasis, and excessive or insufficient autophagy activity could each lead to disease states. Compared to our understanding of the molecular mechanisms of autophagy induction, little is known about how the autophagy process is turned off after its activation. We recently identified KLHL20 as a key regulator of autophagy termination. By functioning as a substrate-binding subunit of CUL3 ubiquitin ligase, KLHL20 targets the activated ULK1 and phagophore-residing PIK3C3/VPS34 and BECN1 for ubiquitination and proteasomal degradation, which in turn triggers a destabilization of their complex components ATG13 and ATG14. These hierarchical degradation events cause the exhaustion of the autophagic pool of ULK1 and PIK3C3/VPS34 complexes, thereby preventing persistent and excessive autophagy activity. Impairment of KLHL20-dependent feedback regulation of autophagy enhances cell death under prolonged starvation and aggravates muscle atrophy in diabetic mice, which highlights the pathophysiological significance of this autophagy termination mechanism in cell survival and tissue homeostasis. Modulation of this autophagy termination pathway may be effective for treating diseases associated with deregulation of autophagy activity.     

The deubiquitinating enzyme USP20 stabilizes ULK1 and promotes autophagy initiation

Autophagy begins with the formation of autophagosomes, a process that depends on the activity of the serine/threonine kinase ULK1 (hATG1). Although earlier studies indicated that ULK1 activity is regulated by dynamic polyubiquitination, the deubiquitinase involved in the regulation of ULK1 remained unknown. In this study, we demonstrate that ubiquitin‐specific protease 20 (USP20) acts as a positive regulator of autophagy initiation through stabilizing ULK1. At basal state, USP20 binds to and stabilizes ULK1 by removing the ubiquitin moiety, thereby interfering with the lysosomal degradation of ULK1. The stabilization of basal ULK1 protein levels is required for the initiation of starvation‐induced autophagy, since the depletion of USP20 by RNA interference inhibits LC3 puncta formation, a marker of autophagic flux. At later stages of autophagy, USP20 dissociates from ULK1, resulting in enhanced ULK1 degradation and apoptosis. Taken together, our findings provide the first evidence that USP20 plays a crucial role in autophagy initiation by maintaining the basal expression level of ULK1.     

AMPK-dependent phosphorylation of ULK1 regulates ATG9 localization

Autophagy is activated in response to a variety of cellular stresses including metabolic stress. While elegant genetic studies in yeast have identified the core autophagy machinery, the signaling pathways that regulate this process are less understood. AMPK is an energy sensing kinase and several studies have suggested that AMPK is required for autophagy. The biochemical connections between AMPK and autophagy, however, have not been elucidated. In this report, we identify a biochemical connection between a critical regulator of autophagy, ULK1, and the energy sensing kinase, AMPK. ULK1 forms a complex with AMPK, and AMPK activation results in ULK1 phosphorylation. Moreover, we demonstrate that the immediate effect of AMPK-dependent phosphorylation of ULK1 results in enhanced binding of the adaptor protein YWHAZ/14-3-3ζ; and this binding alters ULK1 phosphorylation in vitro. Finally, we provide evidence that both AMPK and ULK1 regulate localization of a critical component of the phagophore, ATG9, and that some of the AMPK phosphorylation sites on ULK1 are important for regulating ATG9 localization. Taken together these data identify an ULK1-AMPK signaling cassette involved in regulation of the autophagy machinery.     

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.     

NEDD8 Ultimate Buster 1 Long (NUB1L) Protein Suppresses Atypical Neddylation and Promotes the Proteasomal Degradation of Misfolded Proteins

Neddylation is a posttranslational modification that controls diverse biological processes by covalently conjugating the ubiquitin-like protein NEDD8 to specific targets. Neddylation is commonly mediated by NEDD8-specific enzymes (typical neddylation) and, sometimes, by ubiquitin enzymes (atypical neddylation). Although typical neddylation is known to regulate protein function in many ways, the regulatory mechanisms and biological consequence of atypical neddylation remain largely unexplored. Here we report that NEDD8 conjugates were accumulated in the diseased hearts from mouse models and human patients. Proteotoxic stresses induced typical and atypical neddylation in cardiomyocytes. Loss of NUB1L exaggerated atypical neddylation, whereas NUB1L overexpression repressed atypical neddylation through promoting the degradation of NEDD8. Activation of atypical neddylation accumulated a surrogate misfolded protein, GFPu. In contrast, suppression of atypical neddylation by NUB1L overexpression enhanced GFPu degradation. Moreover, NUB1L depletion accumulated a cardiomyopathy-linked misfolded protein, CryAB^R120G, whereas NUB1L overexpression promoted its degradation through suppressing neddylation of ubiquitinated proteins in cardiomyocytes. Consequently, NUB1L protected cells from proteotoxic stress-induced cell injury. In summary, these data indicate that NUB1L suppresses atypical neddylation and promotes the degradation of misfolded proteins by the proteasome. Our findings also suggest that induction of NUB1L could potentially become a novel therapeutic strategy for diseases with increased proteotoxic stress.     

NEDD4L binds the proteasome and promotes autophagy and bortezomib sensitivity in multiple myeloma

Multiple myeloma (MM) remains an incurable plasma cell cancer characterized by abnormal secretion of monoclonal immunoglobulins. The molecular mechanism that regulates the drug sensitivity of MM cells is being intensively studied. Here, we report an unexpected finding that the protein encoded by neural precursor cell-expressed developmentally downregulated gene 4L (NEDD4L), which is a HECT E3 ligase, binds the 19S proteasome, limiting its proteolytic function and enhancing autophagy. Suppression of NEDD4L expression reduced bortezomib (Bor) sensitivity in vitro and in vivo, mainly through autophagy inhibition mediated by low NEDD4L expression, which was rescued by an autophagy activator. Clinically, elevated expression of NEDD4L is associated with a considerably increased probability of responding to Bor, a prolonged response duration, and improved overall prognosis, supporting both the use of NEDD4L as a biomarker to identify patients most likely to benefit from Bor and the regulation of NEDD4L as a new approach in myeloma therapy.Subject terms: Myeloma, Protein quality control     

ULK-Atg13-FIP200 Complexes Mediate mTOR Signaling to the Autophagy Machinery

Autophagy, the starvation-induced degradation of bulky cytosolic components, is up-regulated in mammalian cells when nutrient supplies are limited. Although mammalian target of rapamycin (mTOR) is known as the key regulator of autophagy induction, the mechanism by which mTOR regulates autophagy has remained elusive. Here, we identify that mTOR phosphorylates a mammalian homologue of Atg13 and the mammalian Atg1 homologues ULK1 and ULK2. The mammalian Atg13 binds both ULK1 and ULK2 and mediates the interaction of the ULK proteins with FIP200. The binding of Atg13 stabilizes and activates ULK and facilitates the phosphorylation of FIP200 by ULK, whereas knockdown of Atg13 inhibits autophagosome formation. Inhibition of mTOR by rapamycin or leucine deprivation, the conditions that induce autophagy, leads to dephosphorylation of ULK1, ULK2, and Atg13 and activates ULK to phosphorylate FIP200. These findings demonstrate that the ULK-Atg13-FIP200 complexes are direct targets of mTOR and important regulators of autophagy in response to mTOR signaling.     

Role of ULK-FIP200 complex in mammalian autophagy: FIP200, a counterpart of yeast Atg17?

The yeast serine threonine kinase Atg1 appears to be a key regulator of autophagy and its kinase activity is crucial for autophagy induction. Recent reports have indicated that a mammalian Atg1 homolog, UNC-51-like kinase (ULK) 1, is required for autophagy. We found that ULK1 localizes to the autophagic isolation membrane and its kinase activity is important for autophagy induction. Furthermore, we identified a focal adhesion kinase (FAK) family interacting protein of 200 kD (FIP200) as a ULK-interacting protein. FIP200 also localizes to the isolation membrane together with ULK. Using FIP200-deficient cells, we found that FIP200 is essential for autophagosome formation and the proper function of ULK. Here, we discuss the role of the ULK-FIP200 complex in autophagy and the possibility that FIP200 functions as a mammalian counterpart of Atg17.     

Parkin-mediated K63-linked polyubiquitination: A signal for targeting misfolded proteins to the aggresome-autophagy pathway

The yeast serine threonine kinase Atg1 appears to be a key regulator of autophagy and its kinase activity is crucial for autophagy induction. Recent reports have indicated that a mammalian Atg1 homolog, UNC-51-like kinase (ULK) 1, is required for autophagy. We found that ULK1 localizes to the autophagic isolation membrane and its kinase activity is important for autophagy induction. Furthermore, we identified a focal adhesion kinase (FAK) family interacting protein of 200 kD (FIP200) as a ULK-interacting protein. FIP200 also localizes to the isolation membrane together with ULK. Using FIP200-deficient cells, we found that FIP200 is essential for autophagosome formation and the proper function of ULK. Here, we discuss the role of the ULK-FIP200 complex in autophagy and the possibility that FIP200 functions as a mammalian counterpart of Atg17.     

UNC51‐like kinase 1, autophagic regulator and cancer therapeutic target

Autophagy, the cell process of self‐digestion, plays a pivotal role in maintaining energy homoeostasis and protein synthesis. When required, it causes degradation of long‐lived proteins and damaged organelles, indicating that it may play a dual role in cancer, by both protecting against and promoting cell death. The autophagy‐related gene (Atg) family, with more than 35 members, regulates multiple stages of the process. Serine/threonine protein kinase Atg1 in yeast, for example, can interact with other ATG gene products, functioning in autophagosome formation. One mammalian homologue of Atg1, UNC‐51‐like kinase 1 (ULK1) and its related complex ULK1–mAtg13–FIP200 can mediate autophagy under nutrient‐deprived conditions, by protein–protein interactions and post‐translational modifications. Although specific mechanisms of how ULK1 and its complex transduces upstream signals to the downstream central autophagy pathways is not fully understood, past studies have indicated that ULK1 can both suppress and promote tumour growth under different conditions. Here, we summarize some properties of ULK1 which can regulate autophagy in cancer, which may shed new light on future cancer therapy strategies, utilizing ULK1 as a potential new target.     

NEDD8 Ultimate Buster-1 Long (NUB1L) Protein Promotes Transfer of NEDD8 to Proteasome for Degradation through the P97UFD1/NPL4 Complex

The NEDD8 protein and neddylation levels in cells are modulated by NUB1L or NUB1 through proteasomal degradation, but the underlying molecular mechanism is not well understood. Here, we report that NUB1L down-regulated the protein levels of NEDD8 and neddylation through specifically recognizing NEDD8 and P97/VCP. NUB1L directly interacted with NEDD8, but not with ubiquitin, on the key residue Asn-51 of NEDD8 and with P97/VCP on its positively charged VCP binding motif. In coordination with the P97-UFD1-NPL4 complex (P97^UFD1/NPL4), NUB1L promotes transfer of NEDD8 to proteasome for degradation. This mechanism is also exemplified by the canonical neddylation of cullin 1 for SCF (SKP1-cullin1-F-box) ubiquitin E3 ligases that is exquisitely regulated by the turnover of NEDD8.     

Downregulation of autophagy through CUL3-KLHL20-mediated turnover of the ULK1 and PIK3C3/VPS34 complexes

The molecular mechanism of macroautophagy/autophagy induction has been intensively studied, but little is known about downregulation of autophagy and how this process is restricted. In particular, how is autophagy maintained at an appropriate homeostatic level when cells are subjected to prolonged stress? In this study (see the related punctum in Autophagy 12–5), Liu et al. report a function of the CUL3-KLHL20 ubiquitin ligase in feedback regulation, leading to the downregulation of autophagy through the degradation of the ULK1 and PIK3C3/VPS34 complexes.     

Deubiquitinase inhibition by WP1130 leads to ULK1 aggregation and blockade of autophagy

Autophagy represents an intracellular degradation process which is involved in both regular cell homeostasis and disease settings. In recent years, the molecular machinery governing this process has been elucidated. The ULK1 kinase complex consisting of the serine/threonine protein kinase ULK1 and the adapter proteins ATG13, RB1CC1, and ATG101, is centrally involved in the regulation of autophagy initiation. This complex is in turn regulated by the activity of different nutrient- or energy-sensing kinases, including MTOR, AMPK, and AKT. However, next to phosphorylation processes it has been suggested that ubiquitination of ULK1 positively influences ULK1 function. Here we report that the inhibition of deubiquitinases by the compound WP1130 leads to increased ULK1 ubiquitination, the transfer of ULK1 to aggresomes, and the inhibition of ULK1 activity. Additionally, WP1130 can block the autophagic flux. Thus, treatment with WP1130 might represent an efficient tool to inhibit the autophagy-initiating ULK1 complex and autophagy.     

NEDD8 ultimate buster-1L interacts with the ubiquitin-like protein FAT10 and accelerates its degradation

FAT10 is an interferon-gamma-inducible ubiquitin-like protein that consists of two ubiquitin-like domains. FAT10 bears a diglycine motif at its C terminus that can form isopeptide bonds to so far unidentified target proteins. Recently we found that FAT10 and its conjugates are rapidly degraded by the proteasome and that the N-terminal fusion of FAT10 to a long lived protein markedly reduces its half-life. FAT10 may hence direct target proteins to the proteasome for degradation. In this study we report a new interaction partner of FAT10 that may link FAT10 to the proteasome. A yeast two-hybrid screen identified NEDD8 ultimate buster-1L (NUB1L) as a non-covalent binding partner of FAT10, and this interaction was confirmed by coimmunoprecipitation and glutathione S-transferase pull-down experiments. NUB1L is also an interferon-inducible protein that has been reported to interact with the ubiquitin-like protein NEDD8, thus leading to accelerated NEDD8 degradation. Here we show that NUB1L binds to FAT10 much stronger than to NEDD8 and that NEDD8 cannot compete with FAT10 for NUB1L binding. The interaction of FAT10 and NUB1L is specific as green fluorescent fusion proteins containing ubiquitin or SUMO-1 do not bind to NUB1L. The coexpression of NUB1L enhanced the degradation rate of FAT10 8-fold, whereas NEDD8 degradation was only accelerated 2-fold. Because NUB1 was shown to bind to the proteasome subunit RPN10 in vitro and to be contained in 26 S proteasome preparations, it may function as a linker that targets FAT10 for degradation by the proteasome.