PKC? replaces AMPK to regulate mitophagy: Another PEDF role on ischaemic cardioprotection.
ABSTRACT: Both decreased autophagy positive regulator AMP activated protein kinase (AMPK) level and promoted mitophagy are observed in oxygen-glucose deprivation (OGD) cardiomyocytes treated with pigment epithelium-derived factor (PEDF). This contradictory phenomenon and its underlying mechanisms have not been thoroughly elucidated. Our previous study reveals that PEDF increases the protein kinase C? (PKC?) and phospho-PKC? (p-PKC?) contents to promote mitophagy. Thus, we investigated the association between PKC? and mitophagy. Here we identify an interaction between PKC? and Unc-51-like kinase 1 (ULK1), essential component of mitophagy. Further analyses show this is a direct interaction within a domain of ULK1 that termed the serine/threonine-rich domain (S/T domain). Notably, a deletion mutant ULK1 that lacks the binding domain is defective in mediating PEDF-induced mitophagy. Furthermore, we demonstrate that ULK1 is phosphorylated at Ser317/555/777 and Raptor is also phosphorylated by phospho-PKC?. Phospho-ULK1 (p-ULK1) at these sites are all essential for PEDF-induced mitophagy and reduce the release of mitochondrial ROS and DNA. This study therefore identifies a previously uncharacterized interaction between the ULK1 and PKC? that can replace the AMPK-dependent mitophagy processes.
Project description:Mitochondrial health is critical for skeletal muscle function and is improved by exercise training through both mitochondrial biogenesis and removal of damaged/dysfunctional mitochondria via mitophagy. The mechanisms underlying exercise-induced mitophagy have not been fully elucidated. Here, we show that acute treadmill running in mice causes mitochondrial oxidative stress at 3-12?h and mitophagy at 6?h post-exercise in skeletal muscle. These changes were monitored using a novel fluorescent reporter gene, pMitoTimer, that allows assessment of mitochondrial oxidative stress and mitophagy in vivo, and were preceded by increased phosphorylation of AMP activated protein kinase (Ampk) at tyrosine 172 and of unc-51 like autophagy activating kinase 1 (Ulk1) at serine 555. Using mice expressing dominant negative and constitutively active Ampk in skeletal muscle, we demonstrate that Ulk1 activation is dependent on Ampk. Furthermore, exercise-induced metabolic adaptation requires Ulk1. These findings provide direct evidence of exercise-induced mitophagy and demonstrate the importance of Ampk-Ulk1 signaling in skeletal muscle.Exercise is associated with biogenesis and removal of dysfunctional mitochondria. Here the authors use a mitochondrial reporter gene to demonstrate the occurrence of mitophagy following exercise in mice, and show this is dependent on AMPK and ULK1 signaling.
Project description:Host-directed therapeutics for human cytomegalovirus (HCMV) requires elucidation of cellular mechanisms that inhibit HCMV. We report a novel pathway used by cardiac glycosides to inhibit HCMV replication: induction of AMP-activated protein kinase (AMPK) activity and autophagy flux through the Na+,K+/ATPase ?1 subunit. Our data illustrate an intricate balance between the autophagy regulators AMPK, mammalian target of rapamycin (mTOR), and ULK1 during infection and treatment with the cardiac glycoside digitoxin. Both infection and digitoxin induced AMPK phosphorylation, but ULK1 was differentially phosphorylated at unique sites leading to opposing effects on autophagy. Suppression of autophagy during infection occurred via ULK1 phosphorylation at Ser757 by enhanced mTOR activity. Digitoxin continuously phosphorylated AMPK, leading to ULK1 phosphorylation at Ser317, and suppressed mTOR, resulting in increased autophagy flux and HCMV inhibition. In ATG5-deficient human fibroblasts, digitoxin did not inhibit HCMV, supporting autophagy induction as a mechanism for virus inhibition. Drug combination studies with digitoxin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) further confirmed the role of autophagy activation in HCMV inhibition. Individually, each compound phosphorylated AMPK, but their combination reduced autophagy rather than inducing it and was antagonistic against HCMV, resulting in virus replication. The initial ULK1 activation by digitoxin was counteracted by AICAR, which prevented the downstream interaction of Beclin1 and phosphatidylinositol 3-kinase class III (PI3K-CIII), further supporting digitoxin-mediated HCMV inhibition through autophagy. Finally, the ?1 subunit was required for autophagy induction, since in ?1-deficient cells neither AMPK nor autophagy was activated and HCMV was not inhibited by digitoxin. In summary, induction of a novel pathway (?1-AMPK-ULK1) induces autophagy as a host-directed strategy for HCMV inhibition.IMPORTANCE Infection with human cytomegalovirus (HCMV) creates therapeutic challenges in congenitally infected children and transplant recipients. Side effects and selection of resistant mutants with the limited drugs available prompted evaluation of host-directed therapeutics. We report a novel mechanism of HCMV inhibition by the cardiac glycoside digitoxin. At low concentrations that inhibit HCMV, digitoxin induced signaling through the ?1 subunit of the Na+,K+/ATPase pump and the cellular kinase AMPK, resulting in binding and phosphorylation of ULK1 (Ser317) and autophagy activation. HCMV suppressed autophagy through ULK1 phosphorylation (Ser757) by activating the mTOR kinase. The pump-autophagy pathway was required for HCMV inhibition, since in ?1- or ATG5-deficient cells the virus was not inhibited. Furthermore, the AMPK activator AICAR antagonized digitoxin activity against HCMV, a phenomenon resulting from opposing effects downstream in the autophagy pathway, at the Beclin1 stage. In summary, autophagy may provide a strategy for harnessing HCMV replication.
Project description: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.
Project description:Currently, there is limited understanding about hormonal regulation of mitochondrial turnover. Thyroid hormone (T3) increases oxidative phosphorylation (OXPHOS), which generates reactive oxygen species (ROS) that damage mitochondria. However, the mechanism for maintenance of mitochondrial activity and quality control by this hormone is not known. Here, we used both in vitro and in vivo hepatic cell models to demonstrate that induction of mitophagy by T3 is coupled to oxidative phosphorylation and ROS production. We show that T3 induction of ROS activates CAMKK2 (calcium/calmodulin-dependent protein kinase kinase 2, ?) mediated phosphorylation of PRKAA1/AMPK (5' AMP-activated protein kinase), which in turn phosphorylates ULK1 (unc-51 like autophagy activating kinase 1) leading to its mitochondrial recruitment and initiation of mitophagy. Furthermore, loss of ULK1 in T3-treated cells impairs both mitophagy as well as OXPHOS without affecting T3 induced general autophagy/lipophagy. These findings demonstrate a novel ROS-AMPK-ULK1 mechanism that couples T3-induced mitochondrial turnover with activity, wherein mitophagy is necessary not only for removing damaged mitochondria but also for sustaining efficient OXPHOS.
Project description:Adenosine monophosphate-activated protein kinase (AMPK) is a conserved sensor of intracellular energy activated in response to low nutrient availability and environmental stress. In a screen for conserved substrates of AMPK, we identified ULK1 and ULK2, mammalian orthologs of the yeast protein kinase Atg1, which is required for autophagy. Genetic analysis of AMPK or ULK1 in mammalian liver and Caenorhabditis elegans revealed a requirement for these kinases in autophagy. In mammals, loss of AMPK or ULK1 resulted in aberrant accumulation of the autophagy adaptor p62 and defective mitophagy. Reconstitution of ULK1-deficient cells with a mutant ULK1 that cannot be phosphorylated by AMPK revealed that such phosphorylation is required for mitochondrial homeostasis and cell survival during starvation. These findings uncover a conserved biochemical mechanism coupling nutrient status with autophagy and cell survival.
Project description:The mitochondrial receptor protein FUN14 domain-containing-1 (FUNDC1) can induce mitophagy under hypoxic conditions, as well as playing important roles in normal metabolism and intracellular homeostasis. Exercise not only elevates mitochondrial biosynthesis, but also exerts a significant impact on mitochondrial fission, integration and mitophagy. However, it is still not clear whether FUNDC1 plays a regulatory role in this context. Electrical pulse stimulation (EPS) of cultured myotubes is widely used as an in vitro model of muscle contraction. We simulated the contraction of C2C12 myotubes by EPS (15 V, 1 Hz, 2 ms, 1 h) to examine the role of FUNDC1 in mitophagy. EPS was found to induce mitophagy by activating the AMPK-ULK1 pathway to an even greater extent than AICAR and FUNDC1 is involved in the associated mitophagy. However, when AMPK is inhibited, other pathways may regulate mitophagy. Our findings indicate that mitophagy helps maintain the normal functions of mitochondria. EPS of C2C12 myotubes results in contraction, induction of mitophagy and potential activation of the AMPK-ULK1 pathway that promotes the expression of FUNDC1.
Project description:Irbesartan has shown significant therapeutic effects in hypertensive patients with non-alcoholic fatty liver disease (NAFLD). To determine the underlying mechanisms of its action, we established an in vitro model of NAFLD by treating human and mouse hepatocytes with free fatty acids (FFAs) and angiotensin (Ang) II. Irbesartan significantly reversed AngII/FFA-induced lipid deposition and mitochondrial dysfunction by restoring ATP production and the mitochondrial membrane potential (MMP), and decreasing the levels of reactive oxygen species (ROS) and inflammatory markers. In addition, irbesartan also increased the autophagy flux, in terms of increased numbers of autolysosomes and autophagosomes, and the upregulation and mitochondrial localization of the autophagic proteins Atg5 and LC3BII/I. Activation of protein kinase C (PKC) and inhibition of the autophagic flux exacerbated mitochondrial dysfunction in the steatotic hepatocytes. Furthermore, AngII upregulated PKC which inhibited AMPK phosphorylation via direct interaction with the AngII receptor AT1-R. Irbesartan inhibited PKC and activated AMPK and its downstream effector ULK1, thereby inducing autophagy, decreasing lipid deposition, and restoring mitochondrial function. Taken together, irbesartan triggers autophagy via the PKC/AMPK/ULK1 axis to ameliorate the pathological changes in the steatotic hepatocytes.
Project description:Selective autophagy recycles damaged organelles?and clears intracellular pathogens to prevent their aberrant accumulation. How ULK1 kinase is targeted and activated during selective autophagic events remains to be elucidated. In this study, we used chemically inducible dimerization?(CID) assays in tandem with CRISPR KO lines to systematically analyze the molecular basis of selective autophagosome biogenesis. We demonstrate that ectopic placement of NDP52 on mitochondria or peroxisomes is sufficient to initiate selective autophagy by focally localizing and activating the ULK1 complex. The capability of NDP52 to induce mitophagy is dependent on its interaction with the FIP200/ULK1 complex, which is facilitated by TBK1. Ectopically tethering ULK1 to cargo bypasses the requirement for autophagy receptors and TBK1. Focal activation of ULK1 occurs independently of AMPK and mTOR. Our findings provide a parsimonious model of selective autophagy, which highlights the coordination of ULK1 complex localization by autophagy receptors and TBK1 as principal drivers of targeted autophagosome biogenesis.
Project description:Unc-51-like autophagy activating kinase 1 (ULK1)-autophagy-related 13 (ATG13) is the most upstream autophagy initiation complex that is phosphorylated by mammalian target-of-rapamycin complex 1 (mTORC1) and AMP-activated protein kinase (AMPK) to induce autophagy in asynchronous conditions. However, their phospho-regulation and functions in mitosis and cell cycle remain unknown. Here we show that ULK1-ATG13 complex is differentially regulated throughout the cell cycle, especially in mitosis, in which both ULK1 and ATG13 are highly phosphorylated by the key cell cycle machinery cyclin-dependent kinase 1 (CDK1)/cyclin B. Combining mass spectrometry and site-directed mutagenesis, we found that CDK1-induced ULK1-ATG13 phosphorylation promotes mitotic autophagy and cell cycle progression. Moreover, double knockout (DKO) of ULK1 and ATG13 could block cell cycle progression and significantly decrease cancer cell proliferation in cell line and mouse models. Our results not only bridge the mutual regulation between the core machinery of autophagy and mitosis but also illustrate the positive function of ULK1-ATG13 and their phosphorylation by CDK1 in mitotic autophagy regulation.
Project description:Miltefosine is an FDA approved oral drug for treating cutaneous and visceral leishmaniasis. Leishmania is a flagellated protozoa, which infects and differentiates in macrophages. Here, we studied the effects of Miltefosine on macrophage's lipid homeostasis, autophagy, and NLRP3 inflammasome assembly/activity. Miltefosine treatment conferred multiple effects on macrophage lipid homeostasis leading to increased cholesterol release from cells, increased lipid-raft disruption, decreased phosphatidylserine (PS) flip from the cell-surface, and redistribution of phosphatidylinositol 4,5-bisphosphate (PIP2) from the plasma membrane to actin rich regions in the cells. Enhanced basal autophagy, lipophagy and mitophagy was observed in cells treated with Miltefosine vs. control. Miltefosine treated cells showed marked increased in phosphorylation of kinases involved in autophagy induction such as; Adenosine monophosphate-activated protein kinase (AMPK) and Unc-51 like autophagy activating kinase (ULK1). The Toll like receptor (TLR) signaling pathway was blunted by Miltefosine treatment, resulting in decreased TLR4 recruitment to cell-surface and ~75% reduction in LPS induced pro-IL-1? mRNA levels. Miltefosine reduced endotoxin-mediated mitochondrial reactive oxygen species and protected the mitochondrial membrane potential. Miltefosine treatment induced mitophagy and dampened NLRP3 inflammasome assembly. Collectively, our data shows that Miltefosine induced ABCA1 mediated cholesterol release, induced AMPK phosphorylation and mitophagy, while dampening NLRP3 inflammasome assembly and IL-1? release.