[Show abstract][Hide abstract] ABSTRACT: Autophagy is a conserved catabolic process that utilizes a defined series of membrane trafficking events to generate a de
novo double-membrane vesicle termed the autophagosome, which matures by fusing to the lysosome. Subsequently, the lysosome
facilitates the degradation and recycling of the cytoplasmic cargo. In yeast, the upstream signals that regulate the induction
of starvation-induced autophagy are clearly defined. The nutrient-sensing kinase Tor inhibits the activation of autophagy
by regulating the formation of the Atg1-Atg13-Atg17 complex, through hyperphosphorylation of Atg13. However, in mammals, the
ortholog complex ULK1-ATG13-FIP200 is constitutively formed. As such, the molecular mechanism by which mTOR regulates mammalian
autophagy is unknown. Here we report the identification and characterization of novel nutrient-regulated phosphorylation sites
on ATG13: Ser224 and Ser258. mTOR directly phosphorylates ATG13 on Ser258 while Ser224 is a putative AMPK phosphorylation
site. In ATG13 knockout cells reconstituted with an unphosphorylatable mutant of ATG13, ULK1 kinase activity is more potent,
and amino acid starvation induced more rapid ULK1 translocation and autophagy. Therefore, ATG13 phosphorylation plays a crucial
role in autophagy regulation.
Preview · Article · Jan 2016 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: A signature event during the cell intrinsic apoptotic pathway is mitochondrial outer membrane permeabilization (MOMP), leading to formation of the apoptosome, a caspase activation complex. The cellular apoptosis susceptibility protein (CAS) can facilitate apoptosome assembly by stimulating nucleotide exchange on Apaf-1, following binding of cytochrome C. We report here that CAS expression itself is upregulated during TRAIL-induced apoptosis, and knockdown of CAS renders cells resistant to TRAIL. We find that TRAIL induces upregulation of CAS in a post-transcriptional, caspase-8 dependent manner, through degradation of cIAP1, an E3 ligase that targets CAS for ubiquitin-dependent proteasomal degradation. Thus, we have identified a novel signaling pathway whereby caspase-8 engages a feed forward cascade that leads to CAS upregulation and amplifies the apoptotic signal. Furthermore, in silico analysis revealed that expression of CAS is upregulated at both the mRNA and DNA level in human breast tumors,consistent with its role in promoting cell proliferation. Overexpression of various oncogenes led to CAS upregulation in nontransformed cells. Intriguingly, oncogene-induced CAS upregulation also resulted in greater susceptibility to TRAIL induced cell death, consistent with its pro-apoptotic function. These findings suggest that CAS plays contrasting roles in proliferation and apoptosis, and overexpression of CAS in tumors could serve as a potential biomarker to guide therapeutic choices.
Preview · Article · Dec 2015 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Autophagy is a cellular catabolic process critical for cell viability and homoeostasis. Inhibition of mammalian target of rapamycin (mTOR) complex-1 (mTORC1) activates autophagy. A puzzling observation is that amino acid starvation triggers more rapid autophagy than pharmacological inhibition of mTORC1, although they both block mTORC1 activity with similar kinetics. Here we find that in addition to mTORC1 inactivation, starvation also causes an increase in phosphatase activity towards ULK1, an mTORC1 substrate whose dephosphorylation is required for autophagy induction. We identify the starvation-stimulated phosphatase for ULK1 as the PP2A-B55α complex. Treatment of cells with starvation but not mTORC1 inhibitors triggers dissociation of PP2A from its inhibitor Alpha4. Furthermore, pancreatic ductal adenocarcinoma cells, whose growth depends on high basal autophagy, possess stronger basal phosphatase activity towards ULK1 and require ULK1 for sustained anchorage-independent growth. Taken together, concurrent mTORC1 inactivation and PP2A-B55α stimulation fuel ULK1-dependent autophagy.
Preview · Article · Aug 2015 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: Autophagy is a catabolic process mediated by incorporation of cellular material into cytosolic membrane vesicles for lysosomal degradation. It is crucial for maintaining cell viability and homeostasis in response to numerous stressful conditions. In this Review, the role of autophagy in both normal biology and disease is discussed. Emphasis is given to the interplay of autophagy with nutrient signaling through the ULK1 autophagy pre-initiation complex. Furthermore, related cellular processes utilizing components of the canonical autophagy pathway are discussed due to their potential roles in nutrient scavenging. Finally, the role of autophagy in cancer and its potential as a cancer therapeutic target are considered.
No preview · Article · Jan 2015 · Journal of Clinical Investigation
[Show abstract][Hide abstract] ABSTRACT: Recently a noncanonical activity of autophagy proteins has been discovered that targets lipidation of microtubule-associated protein 1 light chain 3 (LC3) onto macroendocytic vacuoles, including macropinosomes, phagosomes, and entotic vacuoles. While this pathway is distinct from canonical autophagy, the mechanism of how these nonautophagic membranes are targeted for LC3 lipidation remains unclear. Here we present evidence that this pathway requires activity of the vacuolar-type H(+)-ATPase (V-ATPase) and is induced by osmotic imbalances within endolysosomal compartments. LC3 lipidation by this mechanism is induced by treatment of cells with the lysosomotropic agent chloroquine, and through exposure to the Heliobacter pylori pore-forming toxin VacA. These data add novel mechanistic insights into the regulation of noncanonical LC3 lipidation and its associated processes, including LC3-associated phagocytosis (LAP), and demonstrate that the widely and therapeutically used drug chloroquine, which is conventionally used to inhibit autophagy flux, is an inducer of LC3 lipidation.
[Show abstract][Hide abstract] ABSTRACT: The mitochondria-mediated caspase activation pathway is a major apoptotic pathway characterized by mitochondrial outer membrane permeabilization (MOMP) and subsequent release of cytochrome c into the cytoplasm to activate caspases. MOMP is regulated by the Bcl-2 family of proteins. This pathway plays important roles not only in normal development, maintenance of tissue homeostasis and the regulation of immune system, but also in human diseases such as immune disorders, neurodegeneration and cancer. In the past decades the molecular basis of this pathway and the regulatory mechanism have been comprehensively studied, yet a great deal of new evidence indicates that cytochrome c release from mitochondria does not always lead to irreversible cell death, and that caspase activation can also have non-death functions. Thus, many unsolved questions and new challenges are still remaining. Furthermore, the dysfunction of this pathway involved in cancer development is obvious, and targeting the pathway as a therapeutic strategy has been extensively explored, but the efficacy of the targeted therapies is still under development. In this review we will discuss the mitochondria-mediated apoptosis pathway and its physiological roles and therapeutic implications.
[Show abstract][Hide abstract] ABSTRACT: The biological function of the PTEN tumor suppressor is mainly attributed to its lipid phosphatase activity. This study demonstrates that mammalian PTEN is a protein tyrosine phosphatase that selectively dephosphorylates insulin receptor substrate-1 (IRS1), a mediator of insulin and IGF signals. IGF signaling was defective in cells lacking NEDD4, a PTEN ubiquitin ligase, whereas AKT activation triggered by EGF or serum was unimpaired. Defective IGF signaling caused by NEDD4 deletion, including phosphorylation of IRS1 and AKT, was rescued by PTEN ablation. We demonstrate the nature of PTEN as an IRS1 phosphatase by direct biochemical analysis and cellular reconstitution, showing that NEDD4 supports insulin-mediated glucose metabolism and is required for the proliferation of IGF1 receptor-dependent but not EGF receptor-dependent tumor cells. Thus, PTEN is a protein phosphatase for IRS1, and its antagonism by NEDD4 promotes signaling by IGF and insulin.
No preview · Article · May 2014 · Nature Structural & Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: The effects of selective PI3K and AKT inhibitors were compared in human tumor cell lines in which the pathway is dysregulated. Both caused inhibition of AKT, relief of feedback inhibition of RTKs, and growth arrest. However, only the PI3K inhibitors caused rapid induction of cell death. In seeking a mechanism for this phenomenon, we found that PI3K inhibition, but not AKT inhibition, causes rapid inhibition of wild type RAS and of RAF/MEK/ERK signaling. Inhibition of RAS-ERK signaling is transient, rebounding a few hours after drug addition, and is required for rapid induction of apoptosis. Combined MEK and AKT inhibition also promotes cell death and in murine models of HER2+ cancer, either pulsatile PI3K inhibition or combined MEK and AKT inhibition causes tumor regressions. We conclude that PI3K is upstream of RAS and AKT and that pulsatile inhibition of both pathways is sufficient for effective antitumor activity.
No preview · Article · Jan 2014 · Cancer Discovery
[Show abstract][Hide abstract] ABSTRACT: Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
[Show abstract][Hide abstract] ABSTRACT: We investigated the mechanism of Src homology and collagen homology (Shc) in autophagy caused by troglitazone (TZ).
To reveal the regulatory role of p52Shc in autophagy, we used confocal microscopy and immunoblotting to examine autophagy induced by TZ. Then we used small RNA interference (siRNA) to deplete Shc and plasmids transfection to overexpress wtShc as well as 3mShc in PAE cells. Finally, we reached conclusion by detecting autophagic status following the deprivation of UNC-51-like kinase 1 (Ulk1) by siRNA.
We found that the deprivation of Shc showed to enhance autophagy, whereas p52Shc over expression suppressed TZ-depended autophagy concurring with an attenuated AMP-activated protein kinase (AMPK) and Ulk1 signaling. Besides, it demonstrated that p52Shc tyrosine sites of 239, 240 and 317 implemented a critical role in the process.
Collectively, Shc adaptor protein was involved in TZ-inducing autophagy likely via affecting AMPK and Ulk1 signaling.
No preview · Article · Oct 2013 · ACTA MICROBIOLOGICA SINICA
[Show abstract][Hide abstract] ABSTRACT: Previously, it has been shown that GPI proteins are required for cell wall synthesis and organization in Aspergillus fumigatus, a human opportunistic pathogen causing life-threatening invasive aspergillosis (IA) in immunocompromised patients. Blocking GPI anchor synthesis leads to severe phenotypes such as cell wall defects, increased cell death, and attenuated virulence. However, the mechanism by which these phenotypes are induced is unclear. To gain insight into global effects of GPI anchoring in A. fumigatus, in this study a conditional expression mutant was constructed and a genome wide transcriptome analysis was carried out. Our results suggested that suppression of GPI anchor synthesis mainly led to activation of phosphatidylinositol (PtdIns) signaling and ER stress. Biochemical and morphological evidence showed that autophagy was induced in response to suppression of the GPI anchor synthesis, and also an increased necroptosis was observed. Based on our results, we propose that activation of PtdIns3K and increased cytosolic Ca(2+), which was induced by both ER stress and PtdIns signaling, acted as the main effectors to induce autophagy and possible necroptosis.
[Show abstract][Hide abstract] ABSTRACT: The Atg1/ULK1 complex plays a central role in starvation-induced autophagy, integrating signals from upstream sensors such as MTOR and AMPK and transducing them to the downstream autophagy pathway. Much progress has been made in the last few years in understanding the mechanisms by which the complex is regulated through protein-protein interactions and post-translational modifications, providing insights into how the cell modulates autophagy, particularly in response to nutrient status. However, how the ULK1 complex transduces upstream signals to the downstream central autophagy pathway is still unclear. Although the protein kinase activity of ULK1 is required for its autophagic function, its protein substrate(s) responsible for autophagy activation has not been identified. Furthermore, examples of potential ULK1-independent autophagy have emerged, indicating that under certain specific contexts, the ULK1 complex might be dispensable for autophagy activation. This raises the question of how the autophagic machinery is activated independent of the ULK1 complex and what are the biological functions of such noncanonical autophagy pathways.
[Show abstract][Hide abstract] ABSTRACT: As a major stress-responsive catabolic pathway, autophagy communicates with a variety of signal transduction pathways ranging from proliferative signaling, metabolic pathways, cell death pathways, and multiple cellular stresses. As such, autophagy needs to be able to integrate diverse signaling events and respond to various complex biological conditions in a highly orchestrated manner. Mechanistically, while the molecular basis underlying the function of the core autophagy machinery has been relatively well established, how does autophagy pathway sense diverse upstream signaling pathways? How does autophagy contribute to the eventual biological outcomes of these signaling pathways? Is there any feedback regulation from autophagy to the upstream signaling pathways? And what governs signal transduction within the core autophagy pathway? This chapter discusses the current understanding of these questions. Particularly, an emphasis is placed on the role of the Atg1/ULK1 complex in sensing the upstream nutrient/energy signaling and relaying the upstream signaling to downstream autophagy machinery.
[Show abstract][Hide abstract] ABSTRACT: Autophagy is a finely orchestrated cellular catabolic process that requires multiple autophagy-related gene products (ATG proteins). The ULK1 complex functions to integrate upstream signals to downstream ATG proteins through an unknown mechanism. Here we have identified an interaction between mammalian FIP200 and ATG16L1, essential components of the ULK1 and ATG5 complexes, respectively. Further analyses show this is a direct interaction mediated by a short domain of ATG16L1 that we term the FIP200-binding domain (FBD). The FBD is not required for ATG16L1 self-dimerization or interaction with ATG5. Notably, an FBD-deleted ATG16L1 mutant is defective in mediating amino acid starvation-induced autophagy, which requires the ULK1 complex. However, this mutant retains its function in supporting glucose deprivation-induced autophagy, a ULK1 complex-independent process. This study therefore identifies a previously uncharacterized interaction between the ULK1 and ATG5 complexes that can distinguish ULK1-dependent and -independent autophagy processes.
[Show abstract][Hide abstract] ABSTRACT: Cells respond to cytotoxicity by activating a variety of signal transduction pathways. One pathway frequently upregulated during cytotoxic response is macroautophagy (hereafter referred to as autophagy). Previously, we demonstrated that pan-histone deacetylase (HDAC) inhibitors, such as the anticancer agent suberoylanilide hydroxamic acid (SAHA, Vorinostat), can induce autophagy. In this study, we show that HDAC inhibition triggers autophagy by suppressing MTOR and activating the autophagic kinase ULK1. Furthermore, autophagy inhibition can sensitize cells to both apoptotic and nonapoptotic cell death induced by SAHA, suggesting the therapeutic potential of autophagy targeting in combination with SAHA therapy. This study also raised a series of questions: What is the role of HDACs in regulating autophagy? Do individual HDACs have distinct functions in autophagy? How do HDACs regulate the nutrient-sensing kinase MTOR? Since SAHA-induced nonapoptotic cell death is not driven by autophagy, what then is the mechanism underlying the apoptosis-independent death? Tackling these questions should lead to a better understanding of autophagy and HDAC biology and contribute to the development of novel therapeutic strategies.