Role of Membrane Association and Atg14-Dependent Phosphorylation in Beclin-1-Mediated Autophagy

Abstract

During autophagy, a double membrane envelops cellular material for trafficking to the lysosome. Human beclin-1 and its yeast homologue, Atg6/Vps30, are scaffold proteins bound in a lipid kinase complex with multiple cellular functions, including autophagy. Several different Atg6 complexes exist, with an autophagy-specific form containing Atg14. However, the roles of Atg14 and beclin-1 in the activation of this complex remain unclear. We here addressed the mechanism of beclin-1 complex activation and reveal two critical steps in this pathway. First, we identified a unique domain in beclin-1, conserved in the yeast homologue Atg6, which is involved in membrane association and, unexpectedly, controls autophagosome size and number in yeast. Second, we demonstrated that human Atg14 is critical in controlling an autophagy-dependent phosphorylation of beclin-1. We map these novel phosphorylation sites to serines 90 and 93 and demonstrate that phosphorylation at these sites is necessary for maximal autophagy. These results help clarify the mechanism of beclin-1 and Atg14 during autophagy.A utophagy is a catabolic membrane trafficking process that turns over cytosolic material following encapsulation by autophagosomes and subsequent degradation in the lysosome (in higher eukaryotes) or the vacuole (in plants and unicellular eukaryotes). Genetic studies in yeast have identified a battery of conserved proteins that are required for starvation-induced autophagy (1). In both higher eukaryotes and yeast, autophagy also occurs constitutively as a cargo-selective quality control process (2). Consistent with its dual role in metabolism and cellular quality control, autophagy has been shown to play a role in a variety of human pathologies, including cancer and neurodegeneration (3).Many of the proteins identified as essential for autophagy in yeast have homologues in higher eukaryotes that carry out conserved functions. Among these is the tumor suppressor beclin-1, discovered in a two-hybrid screen as a protein that interacts with the antiapoptotic protein Bcl-2 (4, 5). Beclin-1 is a core component of the phosphatidylinositol 3-kinase complex, along with the catalytic subunit Vps34 and the putative protein kinase Vps15 (6). Atg6/Vps30, the yeast homologue of beclin-1, shares 24.4% amino acid homology and functions in selective and nonselective autophagy (7), as well as endosomal trafficking. Similar to beclin-1, additional components bind the core Atg6-lipid kinase complex to direct functions in these different membrane trafficking pathways (8).Localization of the yeast Atg6/Vps15/Vps34 complex to the preautophagosomal structure is largely dictated by the binding of Atg6 to Atg14 (7). Recently, the human homologue of Atg14, hAtg14/Barkor/Atg14L (here referred to as hAtg14), has been identified by several groups. hAtg14 has been shown to be a member of a beclin-1 complex analogous to that in yeast, although the mechanism of action in autophagy may be distinct (6,9,10,11). hAtg14 interacts with beclin-1 through its coiled-coil doma...

Downregulation of ATG14 by EGR1-MIR152 sensitizes ovarian cancer cells to cisplatin-induced apoptosis by inhibiting cyto-protective autophagy

“…A recent study reveals a new function in which an autophagy-dependent phosphorylation of BECN1/Beclin 1 at serines 90 and 93 is controlled by the binding partner ATG14, and is required for full autophagic activity. 34 Our results also showed that knockdown of ATG14 reduced basal autophagy in both cisplatin-resistant ovarian cancer cell lines. It is reported ATG14 can be transcriptional regulated by FOXO (forkhead box O) transcription factors and has a role in hepatic autophagy and lipid metabolism.…”

IL13 activates autophagy to regulate secretion in airway epithelial cells

“…S3). 41 Similar to ATG5 deficiency, ATG14 depletion resulted in reduced MUC5AC secretion following stimulation with IL13 (Fig. 6C).…”

Structural biology of the macroautophagy machinery

“…In yeast, Vps15 is a myristoylated protein that contains both a kinase and a WD repeat domain required for recruitment and phosphorylation-dependent activation of Vps34 kinase activity (Panaretou et al, 1997;Heenan et al, 2009). Atg6 mediates interaction with Atg14 Fogel et al, 2013). Recently, Atg38 was shown to mediate complex integrity by bridging the interaction of Vps15-Vps34 with Atg14-Atg6 through a predicted C-terminal coiled-coil domain (Araki et al, 2013).…”

“…Membrane association of Beclin1 ECD may be strictly driven by direct interaction with membranes exhibiting high curvature, such as cardiolipin-containing membranes, which induce negative curvature in membranes (Renner and Weibel, 2011). Although the aromatic finger is absent in the yeast Beclin1 ortholog Atg6, a recent study found that the C-terminal domain of yeast Atg6 (Atg6-CTD) is required for its localization to the PAS (Fogel et al, 2013). Furthermore, domain deletion studies showed that the ECD of yeast Atg6 may have an additional role in regulating the size and number of autophagosomes (Fogel et al, 2013).…”

“…Although the aromatic finger is absent in the yeast Beclin1 ortholog Atg6, a recent study found that the C-terminal domain of yeast Atg6 (Atg6-CTD) is required for its localization to the PAS (Fogel et al, 2013). Furthermore, domain deletion studies showed that the ECD of yeast Atg6 may have an additional role in regulating the size and number of autophagosomes (Fogel et al, 2013).…”