WIPI3 and WIPI4 β-propellers are scaffolds for LKB1-AMPK-TSC signalling circuits in the control of autophagy

Abstract

Autophagy is controlled by AMPK and mTOR, both of which associate with ULK1 and control the production of phosphatidylinositol 3-phosphate (PtdIns3P), a prerequisite for autophagosome formation. Here we report that WIPI3 and WIPI4 scaffold the signal control of autophagy upstream of PtdIns3P production and have a role in the PtdIns3P effector function of WIPI1-WIPI2 at nascent autophagosomes. In response to LKB1-mediated AMPK stimulation, WIPI4-ATG2 is released from a WIPI4-ATG2/AMPK-ULK1 complex and translocates to nascent autophagosomes, controlling their size, to which WIPI3, in complex with FIP200, also contributes. Upstream, WIPI3 associates with AMPK-activated TSC complex at lysosomes, regulating mTOR. Our WIPI interactome analysis reveals the scaffold functions of WIPI proteins interconnecting autophagy signal control and autophagosome formation. Our functional kinase screen uncovers a novel regulatory link between LKB1-mediated AMPK stimulation that produces a direct signal via WIPI4, and we show that the AMPK-related kinases NUAK2 and BRSK2 regulate autophagy through WIPI4.

A conserved ATG2‐GABARAP family interaction is critical for phagophore formation

“…ATG2A can simultaneously interact with both GABARAP and WIPI4 (Fig D–F). Interestingly, previous reports that identified the WIPI4 interaction region on ATG2 and the ATG2 interaction site of WIPI4 did not address the role of these interaction mutants during autophagy flux. Considering the close proximity of both GABARAP and WIPI4 interaction motifs on ATG2A, we wanted to dissect the individual roles of ATG2A‐GABARAP and ATG2A‐WIPI4 interactions during autophagy.…”

“…Mammalian homologues of yeast Atg18 are the WIPI (WD repeat domain phosphoinositide-interacting) proteins (WIPI1-4) that are involved in various stages of autophagosome formation [27][28][29]. ATG2A and ATG2B preferentially interact with WIPI4 (WDR45) through a conserved Y/HFS motif [29][30][31]. Simultaneous depletion of both ATG2A and ATG2B results in the accumulation of small, open immature phagophore structures [32,33].…”

“…Simultaneous depletion of both ATG2A and ATG2B results in the accumulation of small, open immature phagophore structures [32,33]. The depletion of WIPI4 also causes open phagophore structures, but they are morphologically dissimilar to those generated after ATG2A/B depletion [29]. Interestingly, previous studies have not, despite mapping the ATG2-WIPI4 interaction, shown whether this interaction is required for the restoration of autophagy flux in ATG2A/B-depleted cells [29][30][31].…”

Mammalian Atg8 proteins regulate lysosome and autolysosome biogenesis through SNARE s

“…Examples of selective degradative autophagy include autophagy of mitochondria (mitophagy), peroxisomes (pexophagy), intracellular microbes (xenophagy), ribosomes (ribophagy), protein aggregates (aggrephagy), and specific intracellular multiprotein complexes (precision autophagy) (Birgisdottir et al, 2013;Randow & Youle, 2014;Kimura et al, 2016;An & Harper, 2018). These include (i) formation of a complex between the ULK1 kinase, FIP200, ATG13, and ATG101, transducing mTOR inhibition (Ganley et al, 2009;Hosokawa et al, 2009;Jung et al, 2009), and AMPK activation (Kim et al, 2011) to induce autophagy; (ii) generation of PI3P by the ATG14-endowed class III PI3-kinase complex I (PI3KC3-CI) containing VPS34 (PI3KC3-C1) (Petiot et al, 2000;Baskaran et al, 2014;Chang et al, 2019) and Beclin 1 (He & Levine, 2010), which can also be modified by AMPK to specifically activate PI3KC3-C1 and not the other PI3KC3 forms (Kim et al, 2013) § ; (iii) the ubiquitin-like conjugation system with ATG5-ATG12/ATG16L1 (Mizushima et al, 1998a,b) acting as an E3 ligase to lipidate mammalian homologs of yeast Atg8 (mAtg8s), some of which like LC3B have become key markers for autophagosomal membranes (Kabeya et al, 2000); and (iv) the only integral membrane ATG protein, ATG9, and the ATG2-WIPI protein complexes, of still unknown but essential functions (Young et al, 2006;Velikkakath et al, 2012;Bakula et al, 2017). These include (i) formation of a complex between the ULK1 kinase, FIP200, ATG13, and ATG101, transducing mTOR inhibition (Ganley et al, 2009;Hosokawa et al, 2009;Jung et al, 2009), and AMPK activation (Kim et al, 2011) to induce autophagy; (ii) generation of PI3P by the ATG14-endowed class III PI3-kinase complex I (PI3KC3-CI) containing VPS34 (PI3KC3-C1) (Petiot et al, 2000;Baskaran et al, 2014;Chang et al, 2019) and Beclin 1 (He & Levine, 2010), which can also be modified by AMPK to specifically activate PI3KC3-C1 and not the other PI3KC3 forms (Kim et al, 2013) § ; (iii) the...…”

“…Given the diversity of cargo, complexity of the protein components and membrane compartments engaged, as well as the exquisite responsiveness to a variety of cargo triggers (e.g., damaged organelles, aggregates) and stress conditions (e.g., starvation, hypoxia), autophagy is controlled by a collection of subsystems that have to come together in a modular fashion and cooperate in the initiation and execution of autophagy (Mizushima et al, 2011). These modules are for the most part interconnected, with FIP200 physically bridging via ATG16L1 the ULK1/2 complex with the mAtg8 conjugation system Gammoh et al, 2013;Nishimura et al, 2013), ATG16L1 and WIPI directly interacting (Dooley et al, 2014), ATG13 connecting the ULK1/2 complex with PI3C3-C1 via ATG13's HORMA domain binding to ATG14 of PI3KC3-C1 (Jao et al, 2013;Park et al, 2016), and PI3P, the product of VPS34 (Petiot et al, 2000), being detected on membranes by WIPIs (Bakula et al, 2017). These modules are for the most part interconnected, with FIP200 physically bridging via ATG16L1 the ULK1/2 complex with the mAtg8 conjugation system Gammoh et al, 2013;Nishimura et al, 2013), ATG16L1 and WIPI directly interacting (Dooley et al, 2014), ATG13 connecting the ULK1/2 complex with PI3C3-C1 via ATG13's HORMA domain binding to ATG14 of PI3KC3-C1 (Jao et al, 2013;Park et al, 2016), and PI3P, the product of VPS34 (Petiot et al, 2000), being detected on membranes by WIPIs (Bakula et al, 2017).…”