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Regulation of Autophagy By Signaling Through the Atg1/ULK1 Complex

Daniel Papinski and Claudine Kraft

Max F. Perutz Laboratories, Vienna Biocenter, University of Vienna, A-1030 Vienna, Austria

Correspondence to Claudine Kraft: [email protected] http://dx.doi.org/10.1016/j.jmb.2016.03.030 Edited by James H. Hurley

Abstract

Autophagy is an intracellular degradation pathway highly conserved in eukaryotic species. It is characterized by selective or bulk trafficking of cytosolic structures—ranging from single proteins to cell organelles—to the vacuole or a lysosome, in which the autophagic cargo is degraded. Autophagy fulfils a large set of roles, including nutrient mobilization in starvation conditions, clearance of protein aggregates and host defence against intracellular pathogens. Not surprisingly, autophagy has been linked to several human diseases, among them neurodegenerative disorders and cancer. Autophagy is coordinated by the action of the Atg1/ ULK1 kinase, which is the target of several important stress signaling cascades. In this review, we will discuss the available information on both upstream regulation and downstream effectors of Atg1/ULK1, with special focus on reported and proposed kinase substrates. © 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Introduction tion of the core macroautophagy machinery, which is visible as a dot at the vacuole in fluorescence Autophagy is defined as an intracellular mechanism microscopy [2,3]. Autophagosome formation occurs for the turnover of cytosolic material dependent on a in distinct steps. At the PAS, vesicles nucleate a lytic compartment, that is, a lysosome or the vacuole double-sheeted isolation membrane. The isolation [1]. It is conserved in eukaryotic species from simple membrane expands in a cup-shaped fashion around single-cellular fungi like Saccharomyces cerevisiae to autophagic cargo before closing to form a mature complex organisms of the Animalia kingdom. Autoph- autophagosome. The outer membrane of the autop- agy is capable of rapidly turning over large amounts of hagosome then fuses with the vacuolar membrane, cytosolic material and organelles, ensuring cellular releasing the inner vesicle into the vacuolar lumen, homeostasis and cell survival under starvation condi- where its contents are degraded (reviewed in Ref. [4]). tions. There are several different types of autophagy These processes and many of the involved proteins pathways, including macroautophagy, microauto- are conserved in other eukaryotes, including mam- phagy and—in mammals—chaperone-mediated mals. One notable difference in mammals is that autophagy (CMA). They share as a common feature isolation membranes are formed not only at one site in the delivery of cargo material to and its degradation in each cell but at several sites in parallel [5].Thisislikely the vacuole or a lysosome, but differ in the way by due to the large size of mammalian cells compared to which the cargo is delivered to the lytic compartment yeast and the availability of numerous lysosomes as (Fig. 1). target compartments distributed in the cell. Macro- In macroautophagy, cytosolic material is delivered to autophagy exists in selective and non-selective forms the lytic compartment in double-membrane vesicles [6]. In non-selective, “bulk” macroautophagy, a random termed autophagosomes. In S. cerevisiae, autophago- part of the cytosol is engulfed by the isolation somes are formed at a single, perivacuolar site termed membrane and degraded in the vacuole. This general pre-autophagosomal structure or phagophore assem- degradation pathway is induced in starved cells to bly site (PAS). The PAS is defined by the co-localiza- mobilize resources and energy bound in potentially

0022-2836/© 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). J Mol Biol (2016) 428, 1725–1741 1726 Regulation of autophagy by Atg1/ULK1 signaling

Chaperone-mediated autophagy (CMA) KFERQ

HSP7C

Microautophagy

HSP7C

HSP7C

Lysosome / Vacuole

Macroautophagy

Fig. 1. Autophagy pathways. Different autophagy pathways deliver cargo to the lytic compartment by different means. In macroautophagy, a cup-shaped isolation membrane is formed de novo, engulfing cytosolic material and closing in order to form a double-membrane autophagosome. The outer membrane of the autophagosome fuses with the lysosomal or vacuolar membrane and releases the inner vesicle into the lumen, where it is degraded. In microautophagy, cargo is taken up directly by the lytic compartment. The lysosomal or vacuolar membrane invaginates, engulfs the cargo and buds off into the lumen as a vesicle, which is degraded together with its content. In CMA, an HSP7C chaperone complex recognizes target proteins by a KFERQ-like sequence and mediates their transport to the lysosomal membrane. There, the protein is unfolded and translocated into the lysosome through a protein pore by the concerted action of cytosolic and lysosomal HSP7C. superfluous proteins and organelles. Selective macro- phagy also targets intracellular pathogens [17,18] autophagy targets surplus or damaged organelles and and ferritin [19–21]. In all these pathways, selectivity intracellular structures, such as mitochondria [7], is conferred by specialized cargo receptor proteins, peroxisomes [8], the endoplasmic reticulum [9] and which link their specific cargo to the autophagy ribosomes [10], limits the activity of retrotransposons machinery [6]. [11] and removes ubiquitylated protein aggregates, In microautophagy, the lytic compartment directly which cannot be dealt with by proteasomal degrada- engulfs cytosolic material with its own membrane. The tion [12,13].InS. cerevisiae, the selective macro- lysosomal or vacuolar membrane invaginates, en- autophagy machinery delivers several enzymes, wraps cytoplasmic material in a random or selective including aminopeptidase 1 (Ape1), to the vacuole manner, and buds as a vesicle into the lumen of the lytic via the cytosol-to-vacuole targeting (Cvt) pathway compartment (reviewed in Ref. [22]). Selective target- [14–16]. In mammalian cells, selective macroauto- ing of mitochondria (micromitophagy), peroxisomes Regulation of autophagy by Atg1/ULK1 signaling 1727

(micropexophagy) and parts of the nucleus (piecemeal membranes of different sizes, proportional to their microautophagy of the nucleus) have been described kinase activity [32]. Starvation and TORC1 inhibition [23–25]. The molecular machineries of microautophagy also allow for efficient binding of Atg13 to Atg17, and macroautophagy overlap, as several core macro- another member of the core autophagy machinery. autophagy factors have been reported to play a role Atg17 is required for PAS organization and Atg1 also in microautophagy, including Atg1 [26–29]. kinase activity in starvation-induced autophagy [3,33]. In CMA, proteins harboring a KFERQ-like motif It forms a stable sub-complex with Atg29 and Atg31, are unfolded and translocated across the lysosomal which are required for proper Atg17 localization and membrane directly as single molecules by an HSP7C function [40–42]. Atg17 has been reported to form (heat shock cognate 71 kDa protein) chaperone homodimers with an S-shaped structure, reminiscent complex through a pore formed by LAMP-2A (lyso- of two BAR (Bin-Amphiphysin-Rvs) domain dimers some-associated membrane glycoprotein 2A) binding to each other [42]. BAR domains directly bind (reviewed in Ref. [30]). curved membranes [43], leading to the speculation that As this review focuses on macroautophagy, for Atg17, together with Atg1, might be involved in binding reasons of simplicity macroautophagy will be referred Atg9-positive vesicles at the PAS [42]. Atg17 has to as autophagy hereafter. recently been reported to bind to Atg9 directly and to tether Atg9 proteoliposomes in vitro [44].However,the nature of this interaction is controversial, as the The Atg1/ULK1 Kinase Core Complex N-terminal Hop1, Rev7 and Mad2 (HORMA) domain of Atg13, but not Atg17, has been found to bind Atg9 in Atg1 is a conserved serine/threonine kinase [31] vivo [45]. In selective autophagy, Atg17 is dispensable and, to date, the only kinase specific for autophagy and the PAS is organized by the scaffold protein Atg11. described in S. cerevisiae. Its deletion, or expression Atg11 binds to, among other proteins, Atg1, Atg9, of a kinase-dead mutant, leads to the accumulation Atg29 and the Cvt cargo receptor Atg19, and recruits of most other members of the autophagy machinery the autophagy machinery to the autophagic cargo at the PAS [3], and to the abrogation of autophagy [33,46–49]. progression at an early stage [31,32]. Thus, Atg1 is The mammalian homologues of Atg1 are the considered as pivotal to the regulation of autophagy. uncoordinated-51-like kinases 1 and 2 (ULK1 and The Atg1 core complex consists of Atg1, Atg13, ULK2). ULK1 and ULK2 likely can act redundantly, as Atg17–Atg29–Atg31 and Atg11 (Fig. 2a and b). ULK1 deficiency causes only a mild phenotype in mice Atg13 is required for Atg1 kinase activity and [50]. Their respective importance for autophagy, autophagy progression in both nutrient-rich and however, seems to vary depending on the cell line, starvation conditions [33]. Atg13 is strongly phos- as silencing of ULK1 alone is sufficient to inhibit phorylated in nutrient-rich conditions, but becomes autophagy in HEK293 cells [51].AsAtg1,ULK1 rapidly dephosphorylated upon nutrient deprivation harbors an autophosphorylation site, Thr180, in the or inhibition of target of rapamycin complex 1 kinase activation loop [52].ULK1ispartofamultimeric (TORC1) by treatment with rapamycin [33]. This protein complex, composed of mammalian Atg13, coincides with increased Atg1 kinase activity and the RB1CC1/FIP200 (RB1-inducible coiled-coil protein 1, induction of bulk autophagy [33,34]. In an early in autophagy research commonly referred to as FAK study, dephosphorylation of Atg13 was reported to family kinase-interacting protein of 200 kDa) and be required for its interaction with Atg1, leading to a Atg101 (Fig. 2a). In contrast to yeast, the binding model in which Atg13 binds to Atg1 only in starvation affinities within mammalian ULK1 complex seem not conditions [33]. This model has since been revised, to be regulated by nutrient signaling [53–57]. However, as Atg1 function requires Atg13 binding also in fed subtle differences in affinity might have been masked cells [35], and only the binding affinity is regulated by by protein overexpression in these studies. Small nutrient signaling [36]. The finding that increased affinity changes might also have been missed binding of Atg13 to Atg1 coincides with the upreg- because they seemed insignificant compared to the ulation of Atg1 kinase activity has led to the predominant model of yeast autophagy at the time, in assumption that Atg13—under the control of TOR- which Atg13 was thought not to interact with Atg1 in C1—acts as a direct activator of Atg1 kinase activity nutrient-rich conditions. This holds true for autophagy and bulk autophagy, possibly by promoting self-- in Drosophila melanogaster, where the interaction of activation of Atg1 in trans [37]. In agreement with Atg1 and Atg13 was found to increase approximately this, the phosphorylation of several sites on Atg1 is fourfold upon starvation [58]. Nevertheless, it was upregulated during bulk macroautophagy induction. reported to not be regulated by nutrient signaling, even Two of these sites, Thr226 and Ser230, are located in though this affinity difference closely resembles the the kinase activation loop and required for kinase one recently found in yeast [36]. Mammalian Atg13 was function [38,39]. The kinase activity of Atg1 directly independently identified and characterized by several regulates isolation membrane expansion, as different groups in parallel [54,56,57,59]. As yeast Atg13, it Atg1 mutants have been shown to form isolation features an N-terminal HORMA domain (Fig. 2b), but 1728 Regulation of autophagy by Atg1/ULK1 signaling possesses only weak sequence homology to the yeast tion, and for ULK1 stability and kinase activity. Similar to protein [60]. Mammalian Atg13 is required for targeting yeast Atg13, mammalian Atg13 is strongly phosphor- the ULK1 complex to sites of autophagosome forma- ylated under nutrient-rich conditions, but is only partially

(a) Yeast Mammals

Atg29 Atg31

FIP200 Atg11 Atg17

ULK1 Atg13 Atg101 Atg1 Atg13

(b) Atg13, Atg11 Raptor FIP200* Atg8 Atg13 GABARAP AMPK 877 1 332 897 1051 428 433 587 278 1 356 361 711 827

Atg1 Kinase tMIT ULK1 Kinase S34 T180 S474 S467 S637 S317 S555, T574 T226 , S230 S508, S515 S757, S777

Atg17 ULK1, FIP200 Atg9 Atg1 Atg101 LC3 268 424 436 521 738 1 190 384 441 447 516 1 460 12

Atg13 HORMA Atg13 HORMA MIM S355 S389 S344 S581 S515, S517 S428, S429, S437 S484, S494, S496

Atg13 Atg13, ULK1* Atg31 Atg17 GABARAP 243 388 208 223 333 417 1 699 704 1456 1584 1588 1 254

Atg17 FIP200 11h S968 S943 S1323 Atg29 Atg1, Atg11 Atg9 Atg19 Atg13 218 576 627 536 858 1178 198 1 1 4

Atg11 Atg101 HORMA S11 S203 Atg29 Atg31 Atg11 Atg17 15 100 1 213 1 52 92 175 196 Atg29 Atg31

Fig. 2. (legend on next page). Regulation of autophagy by Atg1/ULK1 signaling 1729 dephosphorylated upon starvation. FIP200 has no specific phosphatidylinositol 3-kinase class 3 (PI3KC3) clear sequence homologue in yeast, but contains an complex 1 remains tobeelucidated. Atg11 homology region and is considered the function- Several members of the Atg1/ULK1 core complex al mammalian counterpart of S. cerevisiae Atg17 and in yeast and mammals have been shown to interact Atg11 (Fig. 2aandb)[61–63]. Like Atg13, FIP200 is with Atg8-like proteins via LIR (LC3 interacting required for proper localization, stability and kinase region) motifs (Fig. 2B) [35,70–72]. By tethering the activity of ULK1 [53,54,56,57]. FIP200-deficient mouse Atg1/ULK1 complex to autophagic membranes, the embryonic fibroblasts have been found unable to form LIR motifs provide a means to both concentrate the isolation membranes [20]. There are conflicting data kinase complex on expanding autophagosomes and regarding the binding partners of FIP200 within the to target it to the lytic compartment for degradation. ULK1 complex: Ganley et al. reported that recombinant This may constitute a feedback mechanism regulat- FIP200 and ULK1 directly interact in vitro [56], whereas ing autophagic flux [35]. two other groups found this interaction to be in large part dependent on Atg13 in vitro and in vivo [54,57]. Interestingly, the C-terminal region of ULK1 is required Regulation of Autophagy by Phosphor- for its interaction with both FIP200 [53] and Atg13 ylation of the Atg1/ULK1 Complex [54,57]. This indicates that either the binding sites for Atg13 and FIP200 in ULK1 are in close proximity or that Autophagy activity is finely tuned to ensure cellular the interaction of ULK1 and FIP200 is bridged by Atg13, homeostasis. Belated induction of autophagy in similar to the yeast Atg1–Atg13–Atg17 complex. response to cellular stress like starvation may be The final member of the ULK1 complex, Atg101, has lethal to the cell. At the same time, failure to properly no homologue in S. cerevisiae, but is conserved in limit autophagy levels may lead to unchecked, Schizosaccharomyces pombe [64]. Atg101 binds to the detrimental cellular self-digestion. The Atg1/ULK1 ULK1 complex via the Atg13 N-terminus and is complex is pivotal in the induction of autophagy and required for autophagy function, the stability of Atg13 thus a prime target of regulatory pathways. Indeed, and ULK1, and the basal autophosphorylation of ULK1 several signaling kinases have been found to [55,65]. D. melanogaster Atg101 was predicted to fold phosphorylate Atg1 and ULK1, and their complex into a HORMA domain [66], similar to the N-terminus of partners, and will be discussed in the following yeast Atg13 [67]. Very recently, this was confirmed for sections. S. pombe and human Atg101 independently by three different groups (Fig. 2b) [60,68,69]. The structures Regulation by TORC1 determined by Suzuki et al. and Qi et al. show Atg101 in complex with the HORMA domain of Atg13, forming a Yeast Tor1 and Tor2 are conserved serine/ C-Mad2/O-Mad2-like heterodimer. A conserved loop in threonine kinases that act as major controllers of Atg101, termed WF finger, was found to be dispens- cell growth and metabolism. Tor kinases form two able for Atg13 binding, but required for the recruitment complexes of different composition, TORC1 (Tor of the phosphatidylinositol 3-phosphate (PI3P) binding complex 1, containing either Tor1 or Tor2) and proteins DFCP1/ZFYV1 (double FYVE-containing TORC2 (Tor complex 2, exclusively containing protein 1, also known as Zinc finger FYVE domain- Tor2). TORC1 activity is regulated by nutrient cues containing protein 1) and WIPI1 (WD repeat domain and can be inhibited by rapamycin treatment, phosphoinositide-interacting protein 1) to isolation whereas TORC2 is resistant to rapamycin treatment membranes [68]. Whether Atg101 acts directly in the and its regulation is not well understood [73,74]. recruitment of these proteins or plays a more upstream TORC1, but not TORC2, is also a key repressor of role in the recruitment or regulation of the autophagy- autophagy under nutrient-rich conditions, and

Fig. 2. Atg1/ULK1 core complexes. (a) Schematic representation of the S. cerevisiae Atg1 (left) and mammalian ULK1 (right) core complexes. Proteins are colored according to functional homology between yeast and mammals. The yeast core complex contains two scaffolding proteins, Atg11 and Atg17, which are required for selective and bulk autophagy, respectively. In the mammalian complex, FIP200 has been proposed to combine their functions. There are no known homologues of Atg29 and Atg31 in mammals or of Atg101 in S. cerevisiae. (b) Schematic representation of the domain structure of yeast Atg1 (left) and mammalian ULK1 (right) core complex members. Selected interaction partners and their binding regions, as well as critical phosphorylation sites discussed in the text are shown. Dashed lines indicate that no data on the binding region were available when preparing this review. Asterisks indicate conflicting data on the direct nature of the interaction—see text for details. Sites phosphorylated by Atg1/ULK1 are bold and underlined. 11h: Atg11 homology. Residue numbering refers to S. cerevisiae (yeast) or M. musculus (mammalian) proteins. When region boundaries were determined in other organisms, the corresponding S. cerevisiae or mouse residues were determined by sequence alignment. All proteins, domains and binding regions are drawn to scale. Please note that in Atg17 no defined binding region for Atg31 is shown because the interacting region is split between three Atg17 helices [42]. 1730 Regulation of autophagy by Atg1/ULK1 signaling inhibition of TORC1 leads to the rapid induction of under starvation. A serine-to-alanine substitution autophagy [34].InS. cerevisiae,Atg1complex also showed a mild defect in autophagy and Atg17 formation and activity are regulated by the cellular binding, consistent with a functional role of the nutrient state. TORC1 inhibition strengthens the hydrogen bond formed by Ser429. Taken together, interaction of Atg1, Atg13 and Atg17 [33,75]; strongly the data provide a mechanistic model for the enhances Atg1 kinase activity [33]; and therefore is formation of the yeast Atg1 core complex and its required for the efficient formation of the PAS in bulk regulation by phosphorylation. While the involved autophagy [40]. serine residues are phosphorylated in a TORC1- The Atg1 core complex member Atg13 is hyper- dependent manner, it remains to be determined phosphorylated under nutrient-rich conditions, but whether they are direct or indirect targets of TORC1. becomes quickly de-phosphorylated upon TORC1 As TORC1 activity inhibits Atg1 kinase activity, inactivation [33], making it a likely direct target of Atg1 might also be a substrate of TORC1. Several TORC1. Indeed, TORC1 can phosphorylate Atg13 in studies have identified phosphorylation sites on Atg1 vitro [76]. Eight serine residues in Atg13 were [38,39,77–79], but assignment of kinases has identified as potential TORC1 phosphorylation remained sparse. This may in part be due to Atg1 sites, either by mass spectrometry or by sequence autophosphorylation [31], which adds a level of similarity to known TORC1 substrates. Alanine complexity to phosphoproteomic studies. Five phos- substitution of these residues blocked Atg13 phos- phorylation sites in Atg1 have been found to be phorylation as indicated by an increased mobility in significantly regulated by TORC1 activity, of which SDS-PAGE. Overexpression of Atg13 constructs only one, Ser474, is downregulated upon rapamycin harboring different combinations of serine-to-alanine treatment [38]. This residue, however, is not con- mutations, which mimic the non-phosphorylated served in other yeast species. The other four state, was reported to induce bulk autophagy residues are conserved and their phosphorylation irrespective of TORC1 activity [76]. Whether these is upregulated upon TORC1 inhibition, indicative of mutations also induce bulk autophagy at endoge- Atg1-activating or autophosphorylation events. In- nous expression levels needs to be clarified. It is deed, Thr226 and an additional phosphorylation site, interesting to note that the overexpression of Atg13 Ser230, are located in the kinase activation loop and constructs without a Ser496Ala mutation did not required for Atg1 activity. Thr226 phosphorylation induce autophagy when TORC1 was active, even in was identified and shown to depend on autophos- the context of the seven other serine-to-alanine phorylation and the core complex members Atg13 substitutions. The relevance of Atg13 Ser496 has and Atg17 in an independent study [39] . Phosphor- been confirmed by work determining the crystal ylation of Ser34 in the Atg1 N-terminus was structure of the Atg13 regions interacting with Atg1 proposed to repress Atg1 activity, as serine-to- and Atg17 in complex with their respective binding aspartate or glutamate, but not serine-to-alanine partner [36]. The interaction of Atg1 with Atg13 was substitutions at this position abrogated Atg1 kinase found to be mediated by tandem microtubule activity [77]. However, no kinase mediating this interacting and transport (tMIT) domains in the phosphorylation was identified, and no further C-terminal domain of Atg1, which bind an MIT- investigation has followed up this finding. In sum- interacting motif (MIM) in the central region of Atg13 mary, the potential direct action of TORC1 on Atg1 (Fig. 2B). Intriguingly, the C-terminal half of the and its importance in autophagy regulation needs Atg13 MIM contains five serine residues found further investigation. It is conceivable that Atg1 phosphorylated in mass spectrometry, among them activity is more indirectly regulated by TORC1- Ser496 discussed above. Mimicking phosphoryla- mediated changes in the binding affinity of other tion of these residues by mutation to aspartate complex members toward Atg1. In line with this severely reduced binding of Atg13 to Atg1, explain- notion, autophosphorylation of Thr226 is thought to ing the striking effect of Ser496 observed previously occur in trans, perhaps by dimerization of Atg1 [36,76]. This leads to a model in which the N-terminal mediated by Atg13 [37,38]. part of the Atg13 MIM mediates the constitutive In higher eukaryotes, the Tor homologue mTOR binding of Atg1 and Atg13 reported previously [35], (mechanistic target of rapamycin) in TORC1 also while phosphorylation of the C-terminal half regu- negatively regulates autophagy [80], and inhibition of lates the binding affinity in response to the cellular TORC1 by starvation or rapamycin increases ULK1 nutrient state. kinase activity in mammalian cells [53,57].BothULK1 The same study [36] found the interaction of Atg13 and Atg13 are phosphorylated in nutrient-rich condi- and Atg17 to depend on hydrogen bonds between tions in vivo, but become de-phosphorylated upon two serine residues in Atg13 (Ser428 and Ser429) starvation [54,56,57]. There is good evidence for a and an aspartate residue in Atg17 (Asp427). direct action of TORC1 on both proteins: TORC1 has Replacing Atg13 Ser429 with aspartate strongly been shown to phosphorylate both ULK1 and Atg13 impaired starvation-induced autophagy and the in vitro [54,57]; in vivo, TORC1 overexpression increase in Atg13–Atg17 binding usually observed increases Atg13 phosphorylation [56] and TORC1 Regulation of autophagy by Atg1/ULK1 signaling 1731 activity is required for incorporation of radioactive 32P Regulation by PKA into Atg13 [57]; and ULK1 interacts with the TORC1 subunit Raptor (regulatory-associated protein of Akin to TORC1, the cAMP-dependent protein mTOR; Fig. 2b) [54,81]. However, similar to yeast, kinase A (PKA) is involved in the regulation of cellular the identification of specific sites phosphorylated by metabolism and proliferation. In S. cerevisiae, several TORC1 and their role in autophagy has remained a studies have found PKA to be a negative regulator of weak point. No data on TORC1-dependent phosphor- autophagy. PKA is activated by Ras proteins, and ylation sites are available for Atg13, and only two the expression of a constitutively active Ras2 serine residues in ULK1 have been described as mutant blocks autophagy induction by rapamycin TORC1 targets, even though a multitude of phos- treatment, while the expression of inactive Ras2 phorylation sites in ULK1 have been found in mass impedes proliferation and leads to autophagy spectrometry studies [82,83].ULK1Ser757(residue induction [89]. Also, chemical inhibition of an ATP numbering refers to mouse proteins unless stated analogue-sensitive PKA mutant has been shown to otherwise) phosphorylation by TORC1 has been induce autophagy [90]. In an evolutionary proteo- reported to regulate the interaction of ULK1 with mics approach Atg1, Atg13 and Atg18 have been AMP-activated protein kinase (AMPK) in two inde- identified as potential PKA substrates [79]. PKA pendent studies (Fig. 2b) [84,85].Inbothstudies, phosphorylates Atg1 at Ser508 and Ser515, and mutations of Ser757 to alanine and aspartate abro- phosphorylation of these residues inhibits recruit- gated ULK1–AMPK1 binding, but the authors inter- ment of Atg1 to the PAS, reproducing the phenotype preted the data differently. Shang et al. concluded that of the constitutively active Ras2Val19 mutant. In- TORC1-dependent phosphorylation was required for triguingly, serine-to-alanine substitutions of the Atg1 AMPK binding (implying that aspartate could not PKAsitesrescueAtg1PASlocalizationinthe mimic phosphorylation), while Kim et al. argued that Ras2Val19 mutant, but not the induction of autoph- the serine residue itself was required for the interac- agy [79]. This implies the existence of other PKA tion and phosphorylation would negatively regulate targets in autophagy inhibition. In line with this, a AMPK binding. Shang et al. also identified Ser637 as later study by the same group reported phosphor- both a TORC1- and an AMPK-dependent phosphor- ylation of Atg13 by PKA on Ser344, Ser437 and ylation site without a clear function in autophagy Ser581 [91]. Similar to Atg1, Atg13 serine-to-alanine regulation. mutants showed increased localization to the PAS, While the exact role of TORC1 in ULK1 complex even in the constitutively active Ras2Val19 back- regulation remains to be elucidated, the results of ground. Additionally, these mutations were found to several studies imply that TORC1 acts mechanistically increase the Atg13–Atg17 interaction. Ser437 lies different in mammals compared to yeast. As outlined adjacent to the Atg17-binding region in Atg13 above, yeast TORC1 regulates binding affinities within (residues 424–436, Fig. 2b), but was not included the Atg1 core complex in response to nutrient cues. In in the Atg13 fragment crystallized with Atg17 [36]. contrast, the mammalian ULK1 core complex has Thus, a possible direct role of Atg13 Ser437 been reported to change neither in composition nor in phosphorylation by PKA in Atg17 binding remains the binding affinities between its members when cells to be elucidated. It has been reported that the effects were starved or treated with rapamycin [53–57],and of TORC1 and PKA inhibition on autophagy induc- the Atg13–Atg101 and Atg13–ULK1 interactions tion are additive, suggesting that the two signaling within purified complexes persisted after treatment cascades act independently [91]. with phosphatase [65]. While subtle changes in In mammals, only one study has reported a link complex affinities might have been masked by protein between PKA and autophagy [92]. Here, the overexpression or overlooked, these findings indicate regulatory PKA subunit RIα associates with struc- that TORC1 likely either regulates ULK1 kinase tures positive for the isolation membrane marker activity directly by phosphorylating yet to be identified LC3 (Microtubule-associated proteins 1 A/1B light ULK1 residues, or does so indirectly via effector chain 3) and positively regulates autophagosome proteins like AMBRA1 (activating molecule in BECN1- formation. As the same study found the PKA subunit regulated autophagy protein 1). AMBRA1 has been RIα to also associate with mTOR and to negatively shown to facilitate ULK1 ubiquitylation, increasing regulate mTOR activity, mammalian PKA likely acts ULK1 stability and kinase activity. This is negatively indirectly in autophagy induction. regulated by TORC1-dependent phosphorylation of AMBRA1 Ser52 [86]. It is conceivable that TORC1 Regulation by Snf1/AMPK also regulates autophagy via routes not passing through the Atg1/ULK1 complexes in both yeast and AMPK is an important cellular energy sensor and higher eukaryotes. For instance, TORC1 regulates regulates the transition from anabolic to catabolic transcription in both yeast and mammals [74,87] metabolism upon energy stress. This role presents and has been shown to regulate Atg14 expression in an obvious link between AMPK and autophagy. yeast [88]. Interestingly and in contrast to PKA, more data are 1732 Regulation of autophagy by Atg1/ULK1 signaling available on the role of AMPK in autophagy in higher availability within a cell or mimic this by directly eukaryotes than on the yeast AMPK homologue, activating or inhibiting central signaling hubs like sucrose non-fermenting 1 (Snf1). TORC1. While these approaches are well suited to Snf1 has been reported as a positive regulator of study events during general upregulation of autopha- autophagy as Snf1 deficiency completely blocked gy, they fail to capture regulatory events in basal or autophagy induction [93]. In addition, the cyclin- locally induced selective autophagy. In mammals, a dependent kinase Pho85 (phosphate metabolism basal level of autophagy occurs also under non- 85) has been identified as a negative regulator of stimulated conditions, highlighted by the presence of autophagy in the same study, but the mechanism of LC3-positive structures in fed cells (e.g., reported in both Snf1 and Pho85 action in autophagy regulation Ref. [101]). This may be due to local activation of remains to be elucidated. autophagy in a global inhibiting environment or due to In mammalian cells, AMPK has been found to incomplete basal inhibition of autophagy. In yeast, transduce cytosolic calcium signals to induce au- non-selective autophagy is completely repressed in tophagy [94] and to negatively regulate TORC1 nutrient-rich conditions [7,34], but the constitutively activity by phosphorylation of TSC2 (tuberous active Cvt pathway also requires Atg1 kinase activity sclerosis complex 2) and Raptor [95,96]. A direct [16,102]. First evidence on how Atg1 is activated link of AMPK and the ULK1 complex has been during selective autophagy in the presence of global reported by several independent studies. Phosphor- inhibitory signaling has recently been reported [103]. ylation of Ser467, Ser555, Thr574 and Ser637 in Atg1 activity in nutrient-rich conditions was found to ULK1 by AMPK was found to be required for p62/ depend on Ape1 complexes bound to the Cvt cargo SQSTM1 (sequestosome-1) degradation during receptor Atg19 and the selective autophagy scaffold amino acid starvation, and for the autophagic protein Atg11. Approaches reconstituting Atg1 activity degradation of mitochondria (mitophagy) [97].In in vitro led to the conclusion that Atg19 and Atg11 only line with this, phosphorylation of ULK1 Ser555 by activated Atg1 in the presence of cargo complexes. AMPK has recently been shown to be required for Similar to the role of Atg19 and Atg11 in the Cvt the translocation of ULK1 to mitochondria in hypox- pathway, Atg11 and the pexophagy cargo receptor ia-induced mitophagy [98]. ULK1 translocation and Atg36 promoted Atg1 activity when peroxisomal mitophagy were found abrogated in AMPK-depleted damage was induced under nutrient-rich conditions. cells, but could be restored by expression of a A model based on these findings suggests that Atg1 is phosphorylation-mimetic ULK1 Ser555Asp mutant. activated when binding to selective autophagy cargo Ser637 was reported as a major AMPK phosphoryla- by clustering, inducing activation loop autophosphor- tion site on ULK1 in vitro [83], but the potential function ylation in trans. An interesting question arising from this of Ser637 phosphorylation in vivo has remained work is whether Atg1 binds to and is activated on cargo unclear. Two other sites on ULK1, Ser317 and complexes only once they reach the PAS, or already in Ser777, were also reported as direct AMPK targets the cytosol. As Atg1 is not required for PAS recruitment [85]. Phosphorylation of these residues was found to of Ape1 complexes [104], the former is more likely, be required for the induction of ULK1 activity and which would also help to restrict Atg1 activity to its site autophagy upon glucose withdrawal, but to be of action in a globally inhibitory setting. The study by dispensable during amino acid deprivation. Interest- Kamber et al. does not preclude additional regulatory ingly, AMPK has been reported to interact with ULK1 steps and factors for two reasons. First, an Atg13 (Fig. 2B) in several studies [83–85,99], but the data are mutant specifically designed to mimic starvation ambiguous regarding the regulation of this interaction. conditions [76] wasusedinthein vitro experiments, The ULK1–AMPK1 interaction has been found to be which might obscure the requirement for regulatory negatively [84] or positively [85] regulated by TORC1 elements by predisposing Atg1 for activation. Second, activity and TORC1-dependent phosphorylation of the experiments were carried out in yeast lysate, which ULK1 Ser757. This may have been caused by different may contain additional regulatory factors. For a experimental conditions or because the ULK1– profound understanding of Atg1 activation in selective AMPK1 interaction changes over time during nutrient autophagy, future studies need to address these deprivation [83]. AMPK-dependent phosphorylation possibilities. Another interesting direction for addition- has been found to induce binding of 14–3–3proteinsto al research is to elucidate whether similar mecha- ULK1 [83,97,100], but the mechanistic significance of nisms might govern selective autophagy in higher this interaction remains to be addressed. eukaryotes.

Regulation of Atg1/ULK1 in selective autophagy Autophagy-Related Substrates of It is worth noting that studies aiming to elucidate the Atg1/ULK1 regulation of autophagy and Atg1/ULK1 kinase activity commonly have used global stimuli to induce autoph- Even though the pivotal role of Atg1/ULK1 in agy, most of which drastically alter energy and nutrient autophagy had long been established, the identification Regulation of autophagy by Atg1/ULK1 signaling 1733 of Atg1/ULK1 substrates has been slow until recently. residues at the positions −3, +1 and +2 relative to the This was likely caused by the lack of a published, phosphorylation acceptor (Fig. 3a). As the Atg1 well-defined consensus phosphorylation sequence of consensus motif matched well with a reported ULK1 Atg1 and its homologues. We have recently deciphered target site in Beclin-1 [106], we reasoned that the Atg1 the consensus motif of yeast Atg1 in its native complex consensus motif needed to be highly conserved in [105]. We found Atg1 to have a unique phosphorylation ULK1. Indeed, the recently reported human ULK1 motif, with strong selection for aromatic or aliphatic consensus [107] closely matches the yeast Atg1

(a) (b)

(c)

(d) (e)

Fig. 3. Atg1/ULK1 targets. (a) The consensus phosphorylation motif of S. cerevisiae Atg1.The logo was created using WebLogo 3.0 by aligning sites phosphorylated by Atg1 [105,127]. (b) The consensus phosphorylation motif of H. sapiens ULK1. The logo was created using WebLogo 3.0 from positional scanning peptide library data [107,127]. (c) Alignment of Atg1 and ULK1 phosphorylation sites in yeast, fly and mammals. Underscore: phenotype associated with this site reported; gray text: low-matching sequence and/or conflicting data—see text for details; italic: unreported target site with matching sequence and/or homology to a reported target. The defining −3, +1 and +2 positions of the Atg1/ULK1 consensus are highlighted in gray, and preferred residues are color coded at these positions. Red: Met, Leu, preferred at −3; green: Ile, Val, Phe, Tyr, Trp preferred at +1 and +2; blue: Ser preferred at +1 and +2 (ULK1 only). (d) Alignment of the potential Atg1/ULK1 target site in fly (D.m.) Paxillin with its zebrafish (D.r.) and mouse (M.m.) homologues. Framed: consensus motif; color code as in (c). (e) Alignment of the reported ULK1 target sites in rat (R.n.) AMPKγ1 with its human (H.s.) and yeast (S.c.) homologues. Framed: consensus motif; color code as in (C). 1734 Regulation of autophagy by Atg1/ULK1 signaling consensus in its selection for aromatic or aliphatic Sqa phosphorylation in vitro and myosin II activation residues at positions −3, +1 and +2 from the and GFP-Atg8a puncta formation in vivo. While the phosphorylation acceptor site, but differs in some data clearly link Atg1 to Sqa and myosin II activation, details: compared to Atg1, ULK1 has stronger speci- Thr279 seems to be an unlikely Atg1 target site for ficity for Met at position −3, prefers aromatic over several reasons. First, the sequence flanking Thr279 aliphatic residues at position +1 and does not select does not conform well to the Atg1 and ULK1 against Ser at position +2 (Fig. 3b). Interestingly, in consensus motifs (Fig. 3c). Second, D. melanoga- contrast to the ULK1 peptide consensus, ULK1 in vivo ster Atg1, like its yeast and mammalian homologues, targets are not enriched in hydrophobic residues at features a phenylalanine residue at the DFG +1 position +2 [107]. We found no such striking differences position. This confers specificity for serine as a between the Atg1 peptide consensus and in vivo phosphorylation acceptor [108], making threonine an phosphorylation sites in Atg9 [105]. Both Atg1 and improbable substrate residue. Third, the correspond- ULK1 strongly prefer serine over threonine as phos- ing residue in the mammalian homologue DAPK3/ phorylation acceptor. This is in line with a conserved ZIPK (death-associated protein kinase 3/zipper-in- Phe residue immediately following the Asp-Phe-Gly teracting protein kinase), Thr265, has been reported (DFG) motif in the kinase domain, which has been as a major autophosphorylation site [112,113]. Thus, shown to confer serine specificity in serine/threonine phosphorylation by Atg1 may trigger Sqa autophos- kinases [108]. The deciphering of the Atg1 and ULK1 phorylation in D. melanogaster, but Sqa Thr279 consensus motifs has already led to the identification of does not seem to be a likely target residue of Atg1. novel substrates of these kinases and will greatly facilitate future screens for putative targets of Atg1/ Atg1 substrates in S. cerevisiae ULK1. Such screens will have to be followed up by thorough characterization of the identified potential Even though the yeast S. cerevisiae has been a target sites in vivo, as phosphorylation events do highly prominent model organism in autophagy depend not only on consensus motifs but also on the research, Atg1 substrates have remained elusive accessibility of the phosphorylation site and the for more than a decade. We recently reported the localization of the substrate and the kinase in the cell. first Atg1 substrates in budding yeast, Atg2 and Atg9 Nevertheless, knowledge of the Atg1/ULK1 phosphor- [105]. Phosphorylation of both proteins by Atg1 has ylation motifs allows for critical evaluation of potential been confirmed in a recent study [103]. We found Atg1 and published Atg1/ULK1 substrates, which we will to phosphorylate Atg2 at Ser249 and Atg9 at six endeavor in the following sections. residues, Ser19, Ser657, Ser802, Ser831, Ser948 and Ser969 (Fig. 3c). Atg2 mutants harboring serine-to- Atg1 substrates in D. melanogaster alanine substitutions at Ser249 and Ser1086, which also matches the Atg1 consensus motif, did not elicit a In D. melanogaster, Atg1 has been reported to phenotype in starvation-induced autophagy or the Cvt phosphorylate paxillin (dPax) [109] and spaghetti-s- pathway, indicating either functional redundancy or a quash activator (Sqa) [110]. A genetic interaction of specialized role in another type of selective autophagy. the cytoskeletal scaffolding protein paxillin and Atg1 In contrast, our data show phosphorylation of Atg9 to governs wing morphology and arista patterning, and be a crucial requirement for both Cvt pathway and bulk fly Atg1 directly phosphorylates an N-terminal frag- autophagy progression. In line with findings reported ment of dPax comprising residues 1–346 in vitro [109]. previously [114], we found that Atg9 phosphorylation Similar to fly Atg1, mouse ULK2 phosphorylates an facilitates binding of Atg18 to Atg9. Atg9 phosphory- N-terminal fragment of murine paxillin. Paxillin has lation is essential at an early stage of autophagosome been found to be required for autophagy in fly and formation: it is required for the stable recruitment of mammalian cells [109], but neither the role of paxillin Atg18 to the PAS, and the formation and expansion of phosphorylation nor the precise site of phosphoryla- the isolation membrane [105]. Interestingly, the ser- tion has been elucidated to date. Using ScanSite [111] ine-to-alanine substitutions at the Atg1 phosphoryla- and the yeast Atg1 consensus motif [105],wefound tion sites did not change Atg9 localization, while a dPax Ser260 to be a well-scoring Atg1 consensus site deletion of Atg1 leads to Atg9 accumulation at the PAS (Fig. 3c). The consensus motif is highly conserved in [114]. This indicates that Atg1 regulates Atg9 shuttling mouse and zebrafish paxillin (Ser272 and Ser248, by other means than Atg9 phosphorylation. In line with respectively, Fig. 3D), implying functional importance a slightly different role of mammalian Atg9 in autoph- and making this site an interesting target for future agy [115], sequence homology of yeast and mamma- studies. lian Atg9 is low in the cytosolic termini, and the yeast The kinase domain of the D. melanogaster myosin phosphorylation sites are not conserved in mammals. light chain kinase Sqa is phosphorylated by Atg1 in Whether ULK1 phosphorylates mammalian Atg9 is to vitro, and Thr279 has been reported as the main be determined in future studies. phosphorylation site [110]. A threonine-to-alanine Very recently, yeast Atg6 has been reported to be substitution at this position has been found to impair phosphorylated by Atg1 [103], similar to its mammalian Regulation of autophagy by Atg1/ULK1 signaling 1735 homologue Beclin-1, which is a substrate of ULK1 that Ser943 and Ser986 phosphorylation might rather [106,107]. However, no specific phosphorylation sites play a species-specific role in Homo sapiens.Two have been identified. The major ULK1 phosphorylation ULK1 phosphorylation sites were identified in human site in mouse Beclin-1, Ser14 [106,107], seems not to Atg101: Ser11 and Ser203 [107]. Both sites match the be conserved in Atg6. Human Beclin-1 Ser279 and ULK1 consensus well (Fig. 3c) and are conserved in Ser337, reported ULK1 target sites [107],arehomol- mouse and zebrafish. Similar to FIP200, evidence for a ogous to Atg6 Ser332 and Ser390, respectively. Both role of Atg101 phosphorylation in autophagy is lacking. sites fit the Atg1 consensus moderately well (Fig. 3c). ULK1 has also been reported to phosphorylate Two serine residues on yeast Atg6, Ser85 and Ser540, several members of the autophagy-specific PI3KC3 are exceptionally good matches for the Atg1 phos- complex 1: Beclin-1, Vps34/PIK3C3 (vacuolar pro- phorylation motif (Fig. 3c). Atg6 Ser85 is conserved in tein sorting 34/phosphatidylinositol 3-kinase catalyt- S. pombe, but not in Candida albicans. S. cerevisiae ic subunit type 3), and AMBRA1 [106,107,117]. The Atg6 Ser540 is conserved in S. pombe Atg6 Ser462 PI3KC3 complex 1 plays a central role in autophagy and corresponds to the negatively charged Glu514 by phosphorylating phosphatidylinositol to generate in C. albicans Atg6, hinting at a potential role of a PI3P, allowing for the recruitment of PI3P-binding negative charge at this position. However, Ser462 is proteins like DFCP1 and members of the WIPI family the antepenultimate residue in S. pombe Atg6; [118]. ULK1 has been reported to phosphorylate whether Atg1 can phosphorylate residues this close AMBRA1 in vivo and in vitro, and to be required for to the C-terminus has not been determined yet. the abrogation of PI3KC3 complex 1 binding to the dynein motor complex upon starvation [117]. This Mammalian ULK1 substrates binding is mediated by AMBRA1, and the overex- pression of AMBRA1 mutants unable to bind to the In mammals, ULK1 has been found to phosphor- dynein motor complex upregulates autophagy in fed ylate its binding partners Atg13, FIP200 and Atg101 cells. This implies that ULK1-mediated phosphory- [107,116]. In a SILAC (stable isotope labelling by lation of AMBRA1 is a key step in autophagy amino acids in cell culture) mass spectrometry induction. Two ULK1 phosphorylation sites in experiment, ULK1 was found to phosphorylate human AMBRA1 have been reported, but not human Atg13 isoform 2 at Ser318 in a manner characterized further: Ser465 and Ser635 (Fig. 3c) dependent on the Hsp90–Cdc37 (heat shock protein [107]. Both sites are conserved in mouse AMBRA1, 90–cell division cycle 37) chaperone complex [116]. and Ser465 also in the zebrafish homologue. In This residue corresponds to Ser354 and Ser355 in mouse, ULK1 has been found to activate the PI3KC3 the canonical murine and human isoforms, respec- complex 1 by directly phosphorylating Beclin-1 tively. Atg13 Ser355 phosphorylation is required for Ser14 (Fig. 3c), and this phosphorylation event is targeting Atg13 to depolarised mitochondria and for required for autophagy induction upon nutrient mitophagy, but not for starvation-induced autopha- deprivation [106]. Phosphorylation of this residue gy. This indicates that ULK1 might target different has been confirmed in human Beclin-1 [107], among substrates and phosphorylation sites depending on several other ULK1-dependent phosphorylation the cue that triggers autophagy activation. Human sites. These sites include Ser279 and Ser337 Atg13 S355 passably conforms to the ULK1 con- (Fig. 3c), both of which are conserved in yeast. sensus motif (Fig. 3c), but could not be confirmed as Human Vps34, the catalytic subunit of the PI3KC3 an ULK1 phosphorylation site in another study by complex 1, is also phosphorylated by ULK1 at Ser249 Egan et al. [107], possibly due to weak coverage (Fig. 3c) [107]. While this residue is conserved in of this region in mass spectrometry. Egan et al. yeast, a serine-to-alanine substitution did not elicit a reported human Atg13 Ser389 to be a target of ULK1 phenotype in starvation-induced autophagy. As in (Fig. 3c), but did not characterize it regarding a yeast Atg2 [105], this might be due to a redundant or potential function in autophagy. Given the close highly specialized function of Vps34 Ser249 phos- interaction of ULK1 and Atg13, and the fact that phorylation. In summary, there is striking evidence for Atg13 has been suggested to be a substrate of ULK1 the regulation of the PI3KC3 complex 1 by ULK1, but already when it was identified [54,57–59], clarifica- future studies will need to determine the functional role tion of Atg13 phosphorylation by ULK1 will be an of several reported phosphorylation events. interesting subject for future studies. The same holds FUNDC1 is an outer-membrane protein that acts as true for FIP200, which has been found to be a cargo receptor protein in hypoxia-induced mito- phosphorylated depending on ULK1 activity already phagy [119]. Phosphorylation of mouse FUNDC1 in an early study [57]. Evidence for phosphorylation Ser17 by ULK1 is required for efficient binding of LC3 of three FIP200 sites by ULK1 has been provided to FUNDC1 and mitophagy progression [120].Mouse [107], but their functional significance has not been FUNDC1 Ser17 matches the ULK1 consensus assessed. The reported sites in human FIP200 are (Fig. 3c) and is conserved in human and zebrafish, Ser943, Ser986 and Ser1323, of which only Ser1323 lending additional support to its role as a bona isconservedinmurineFIP200(Fig. 3c). This indicates fide ULK1 target site. Strikingly, expression of a 1736 Regulation of autophagy by Atg1/ULK1 signaling serine-to-aspartate mutant mimicking phosphorylation Conclusion was sufficient to drive mitophagy even in the absence of ULK1 [120]. This might hint that only ULK1, but not Since the identification of TORC1 as a major ULK2 phosphorylates FUNDC1, while the two kinases regulator of yeast autophagy in 1998, our understand- might act redundantly in other steps of mitophagy. ing of the regulatory network governing autophagy repression and induction has greatly advanced, both Atg1/ULK1-driven feedback loops in terms of underlying signaling cascades and their targets (Fig. 4). TORC1 and PKA are the major In several studies in D. melanogaster and mamma- repressors of autophagy in non-challenged cells, lian cells, Atg1 and ULK1 have been implicated in whereas AMPK triggers autophagy induction in negative regulation of TORC1 activity toward two response to several cues, including nutrient stress. major substrates, S6K (ribosomal protein S6 kinase) These pathways have been shown to regulate, often and 4E-BP1 (Eukaryotic translation initiation factor directly, Atg1/ULK1 activity, making Atg1/ULK1 the 4E-binding protein 1) [57,58,81,121,122].Itistempt- pivot of autophagy signaling. Recently, an important ing to think of TORC1 as a direct target of Atg1 or piece to this puzzle has been added by discovering a ULK1, forming a positive feedback loop in autophagy potential mechanism for yeast Atg1 activation in induction. Indeed, ULK1 has been found to phosphor- selective autophagy, when global signaling inhibits ylate the TORC1-specific subunit Raptor [81], and five the induction of non-selective autophagy. It will be human Raptor residues have been reported as ULK1 interesting to see whether a similar mechanism exists targets: Ser792, Ser855, Ser859, Ser863 and Ser877 in higher eukaryotes. While the big picture of (Fig. 3C) [122]. Ser792 matches the ULK1 phosphor- autophagy regulation has advanced in great steps, ylation motif nicely, but has previously been reported the understanding of the mechanistic events is largely as an AMPK target site [95]. Ser855 and Ser859 lacking. Future studies should aim to elucidate the conform to the ULK1 consensus motif reasonably well mechanisms by which phosphorylation events or and are the most likely targets of ULK1 among the other post-translational modifications exert their reported sites, as neither Ser863 nor Ser877 matches function, and how the identified factors cooperate in the ULK1 phosphorylation motif. Strikingly, Ser863 is autophagy regulation. How does phosphorylation areportedtargetofmTor[123]. The upregulation of by AMPK increase ULK1 activity? What molecular phosphorylationatanmTORtargetsiteinRaptorand changes are induced in ULK1 upon phosphoryla- an autophosphorylation site in mTOR by ULK1 tion by TORC1, rendering it inactive? Comprehen- overexpression indicate that ULK1 does not repress sive and rigorous biochemical analyses together the catalytic activity of TORC1 per se [122].TOR- with structural approaches will help to answer such C1-activating cues have been shown to induce questions. conformational changes in TORC1, weakening the Progress in identifying downstream effectors of binding of Raptor to the complex [123,124]. Converse- Atg1/ULK1 has been slow in the beginning, but has ly, both ULK1 and ULK2 have been reported to stabilise picked up the pace over the last few years. Today, a Raptor in TORC1, implying that they might regulate range of Atg1/ULK1 targets in several organisms are mTOR kinase domain accessibility rather than activity known (Fig. 3C), including several in not autopha- [81]. Whether this is achieved by phosphorylation of gy-related pathways [125,126], which we did not Raptor Ser855 and Ser859 remains to be determined. cover in this review. In line with its role as an ULK1 has been found to phosphorylate AMPK and autophagy signaling hub, Atg1/ULK1 acts at many to negatively regulate AMPK activity, constituting a levels of autophagy, phosphorylating cytoskeletal negative feedback loop which attenuates autopha- organizers and regulators of motor proteins, cargo gy-inducing signaling [99]. ULK1 has been reported receptors, other kinase complexes and core autoph- to phosphorylate AMPK in vitro at three sites in the agy proteins in different organisms (Fig. 4; see the AMPKα1, four sites in AMPKβ2 and two sites in appropriate sections for details). Knowledge of the AMPKγ1(Fig. 3C). However, no direct evidence for a Atg1 and ULK1 consensus motifs will facilitate the functional role of these sites has been provided, and discovery of additional Atg1/ULK1 substrates and for several of them, direct targeting by ULK1 is hard regulatory pathways downstream of these kinases. to conceive. These include threonine residues and Similar to studies focusing on the regulation upstream serine residues in an unfavourable sequence context, of Atg1/ULK1, future studies on downstream effectors most of which are not conserved in either D. should not only identify Atg1/ULK1 target sites but melanogaster or S. cerevisiae. Ser260 and Ser269 in also characterize their mechanistic role in the pro- rat AMPKγ1 might be the most promising targets, as gression of autophagy. Asking “how?” more often than they are conserved and correspond well to the ULK1 “what?” when studying potential targets of this central consensus motif in human AMPKγ1, and are present autophagy kinase will allow deciphering the mecha- also in yeast Snf4, albeit flanked by sequences nistic events underlying autophagy signaling. matching the Atg1 phosphorylation motif only moder- In addition to further clarifying the mechanistic roles ately (Fig. 3c and e). of phosphorylation in Atg1/ULK1 regulation and Regulation of autophagy by Atg1/ULK1 signaling 1737

PKA AMPK TORC1 AMPK PKA

YEAST MAMMALS ULK1 Atg1

Atg13 Beclin-1 Atg9 Atg2 Atg6 Atg101 AMBRA1 FUNDC1 FIP200 Vps34

PI3K complex 1 ULK1 complex PI3KC3 complex 1

activation inhibition function unknown potentially indirect

Fig. 4. Regulation of autophagy by signaling through the Atg1/ULK1 complex. The activity of Atg1 and ULK1 is directly or indirectly regulated by TORC1, AMPK and PKA. In yeast, Atg1 phosphorylates Atg9 to promote PAS organization and isolation membrane formation. Atg1 also targets Atg2 and the PI3K complex member Atg6, but the role of these phosphorylation events has not yet been elucidated. In mammals, ULK1 phosphorylates its complex partners Atg13, Atg101 and FIP200. Phosphorylation of Atg13 and the cargo receptor protein FUNDC1 is required for mitophagy. ULK1 activates the PI3KC3 complex 1 by phosphorylating Beclin-1 and AMBRA1; the role of Vps34 phosphorylation has remained unclear. ULK1 also phosphorylates the TORC1 component Raptor and several AMPK subunits and thus regulates the activity of the upstream signaling complexes. Note that crosstalk between TORC1, AMPK and PKA exists, but is not shown in this model.

function, the identification of counteracting protein is supported by a “Vienna Research Groups for phosphatases should be an important aim of future Young Investigators” grant from the Vienna Science autophagy research. Knowledge on protein phospha- and Technology Fund (WWTF VRG10-001) and two tases involved in autophagy pathways is very limited. grants from the Austrian Science Fund (FWF P This is in stark contrast to their likely important role, 25522-B20 and P 28113-B28). especially considering the rapid dephosphorylation events observed upon autophagy induction in yeast. Received 30 January 2016; Received in revised form 25 March 2016; Accepted 28 March 2016 Available online 6 April 2016

Acknowledgments Keywords: Autophagy; The authors thank Reuben J. Shaw and Benjamin Autophagy signaling; E. Turk for providing a positional score matrix of the Atg1/ULK1 regulation; ULK1 consensus motif. Work in the Kraft laboratory Atg1/ULK1 phosphorylation sites; 1738 Regulation of autophagy by Atg1/ULK1 signaling

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