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• W2949137984 abstract "Report24 May 2019free access Transparent process ULK1-mediated phosphorylation of ATG16L1 promotes xenophagy, but destabilizes the ATG16L1 Crohn's mutant Reham M Alsaadi Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada Search for more papers by this author Truc T Losier orcid.org/0000-0001-9785-2389 Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada Search for more papers by this author Wensheng Tian Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada Search for more papers by this author Anne Jackson Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Zhihao Guo Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada Search for more papers by this author David C Rubinsztein Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK UK Dementia Research Institute, Cambridge, UK Search for more papers by this author Ryan C Russell Corresponding Author ryan.russel[email protected] orcid.org/0000-0003-3364-8869 Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada Center for Infection, Immunity and Inflammation, University of Ottawa, Ottawa, ON, Canada Search for more papers by this author Reham M Alsaadi Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada Search for more papers by this author Truc T Losier orcid.org/0000-0001-9785-2389 Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada Search for more papers by this author Wensheng Tian Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada Search for more papers by this author Anne Jackson Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Zhihao Guo Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada Search for more papers by this author David C Rubinsztein Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK UK Dementia Research Institute, Cambridge, UK Search for more papers by this author Ryan C Russell Corresponding Author [email protected] orcid.org/0000-0003-3364-8869 Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada Center for Infection, Immunity and Inflammation, University of Ottawa, Ottawa, ON, Canada Search for more papers by this author Author Information Reham M Alsaadi1,‡, Truc T Losier1,‡, Wensheng Tian1, Anne Jackson2, Zhihao Guo1, David C Rubinsztein2,3 and Ryan C Russell *,1,4 1Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada 2Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK 3UK Dementia Research Institute, Cambridge, UK 4Center for Infection, Immunity and Inflammation, University of Ottawa, Ottawa, ON, Canada ‡These authors contributed equally to this work *Corresponding author. Tel: +1 613 568 5800; E-mail: [email protected] EMBO Rep (2019)20:e46885https://doi.org/10.15252/embr.201846885 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Autophagy is a highly regulated catabolic pathway that is potently induced by stressors including starvation and infection. An essential component of the autophagy pathway is an ATG16L1-containing E3-like enzyme, which is responsible for lipidating LC3B and driving autophagosome formation. ATG16L1 polymorphisms have been linked to the development of Crohn's disease (CD), and phosphorylation of CD-associated ATG16L1 T300A (caATG16L1) has been hypothesized to contribute to cleavage and autophagy dysfunction. Here we show that ULK1 kinase directly phosphorylates ATG16L1 in response to infection and starvation. Phosphorylated ATG16L1 localizes to the site of internalized bacteria and stable cell lines harbouring a phospho-dead mutant of ATG16L1 have impaired xenophagy, indicating a role for ATG16L1 phosphorylation in the promotion of anti-bacterial autophagy. In contrast to wild-type ATG16L1, ULK1-mediated phosphorylation of caATG16L1 drives its destabilization in response to stress. In summary, our results show that ATG16L1 is a novel target of ULK1 kinase and that ULK1 signalling to ATG16L1 is a double-edged sword, enhancing the function of the wild-type ATG16L1, but promoting degradation of caATG16L1. Synopsis ULK1 phosphorylates ATG16L1 on S278 to promote autophagic activity of wild-type ATG16L1. In contrast, this regulation enhances caspase-mediated degradation of Crohn-associated (T300A) ATG16L1, thereby reducing xenophagy and bacterial clearance. ULK1 phosphorylates ATG16L1 on S278. Starvation and bacterial infection induce ULK1-mediated ATG16L1 regulation. ULK1 signalling to ATG16L1 enhances the function of wild-type ATG16L1 while degrading Crohn-associated (T300A) ATG16L1. Introduction Macroautophagy (hereafter referred to as autophagy) is a cellular degradative process capable of degrading a vast array of substrates including cytoplasm, organelles, aggregated macromolecules and pathogens 1. Autophagic cargo is first sequestered by the formation a double-membraned vesicle called an autophagosome, which matures into a degradative vesicle after fusion with lysosomes. Autophagosome formation is driven by a set of autophagy-related (ATG) genes, which include a protein kinase (Unc 51-like kinase 1; ULK1), a lipid kinase (vacuolar protein sorting 34; VPS34) and a trimeric E3-like enzyme (ATG5-ATG12/ATG16L1) 1. These enzymes are all required for autophagy initiation and are tightly regulated by upstream stress-sensitive signalling. One of the best characterized upstream regulators of the autophagy pathway is mTORC1, which potently inhibits autophagy induction through direct phosphorylation of the ULK1 and VPS34 kinase complexes 2-5. mTORC1 activity is repressed, thereby allowing autophagy induction, in response to a myriad of stressors including nutrient or cytokine starvation, reactive oxygen species or infection 6-8. Mammals have two homologues of the yeast ATG1, ULK1 and ULK2, which are largely functionally redundant for autophagy induction 9. Under basal conditions, mTORC1-mediated phosphorylation represses ULK1 activity; however, starvation releases this inhibitory phosphorylation and upregulates ULK1 2. Activated ULK1 then phosphorylates several components of the pro-autophagic ATG14-containing VPS34 complexes 10-12. Autophagic VPS34 complexes are recruited to the phagophore where they phosphorylate phosphatidylinositol (PtdIns) to produce phosphatidylinositol(3)phosphate (PtdIns(3)P) 13. PtdIns(3)P functions as a platform bridging downstream components like the ATG16L1 complex to promote autophagosome formation. Additionally, mTORC1 has been shown to directly mediate the activity of VPS34 complexes, thereby allowing a tight regulation of autophagy initiation in response to stresses 3. Downstream of VPS34, ATG16L1 forms a trimeric complex with ATG5 and ATG12. ATG16L1 is the subunit responsible for recruiting the E3-like enzyme to the phagophore 1, 14. ATG12 acts to recruit microtubule-associated protein 1 light chain 3 (LC3) to the expanding autophagosomal membrane, and ATG5 catalyzes the conjugation of the ubiquitin-like LC3 to phosphatidylethanolamine in membranes of nascent autophagosomes, thereby driving their development. Activation of anti-bacterial autophagy (hereafter referred to as xenophagy) involves these 3-key enzymes in the autophagy pathway, but also requires xenophagy-specific proteins involved in pathogen-sensing that signal to the autophagy machinery during infection 8. For instance, galectin-8 detects damaged Salmonella-containing vacuoles (SCV) and subsequently activates xenophagy through recruitment of the autophagy receptor NDP52 15. Immunity-related GTPase M (IRGM) has been shown to act as a scaffold bringing together ULK1, Beclin-1-containing VPS34 complexes and ATG16L1 to promote xenophagy initiation 16. In addition to IRGM, ATG16L1-containing enzyme is also regulated by activation of intracellular (NOD2) sensors of bacterial peptidoglycan, where NOD2 binds ATG16L1 recruiting the LC3-lipidating enzyme to the site of bacterial infection 17. Interestingly, several of the proteins involved in xenophagy induction (ATG16L1 and IRGM) and pathogen detection (NOD2 and TLR4) have been linked to Crohn's disease (CD), but are not found in the related chronic inflammatory bowel disease ulcerative colitis (UC) 18. Genome-wide association studies have linked a non-synonymous single nucleotide polymorphism (SNP) in ATG16L1 that substitutes threonine 300 for alanine with an increased susceptibility for CD 19. Molecular characterization of the CD-associated ATG16L1 (caATG16L1) has shown that stresses such as starvation or pathogen infection enhance the susceptibility of caATG16L1 to caspase-mediated cleavage 20-23. Enhanced cleavage of caATG16L1 has been shown to lead to an increase in inflammatory cytokine secretion and a decrease in xenophagy, which are thought to contribute to CD 21, 24-26. Interestingly, a recent study has found that IκB kinase subunit IKKα is capable of phosphorylating ATG16L1 on serine 278 (S278), which regulates the sensitivity of caATG16L1 to caspase cleavage 24. The caspase cleavage site on ATG16L1 lies in between the S278 phosphorylation site and the T300A Crohn's SNP. This raises the interesting possibility that phosphorylation of ATG16L1 in response to infection leads to inappropriate cleavage if the site is in close proximity to the T300A mutation. ATG16L1 contains several conserved serine/threonine residues proximal to T300, which may also be phosphorylated and may potentially regulate ATG16L1 function. However, it remains to be seen what effect phosphorylation has on wild-type ATG16L1 and if other stressors or kinases regulate ATG16L1 phosphorylation. Results and Discussion ATG16L1 is phosphorylated by ULK1/2 Starvation has been described to trigger caspase-mediated cleavage of ATG16L1 containing a common amino acid substitution (T300A) 21. However, IKKα has not been implicated in starvation-induced autophagy. Interestingly, ATG16L1 has been shown to bind FIP200, an essential co-factor of the ULK1 kinase complex. The interaction of ATG16L1 with FIP200 has been shown to be involved in regulating ATG16L1 localization in autophagy induction 27, 28. Therefore, we hypothesized that ULK1/2, the only protein kinases in the autophagy pathway, may phosphorylate ATG16L1 under starvation. To test this hypothesis, we performed an in vitro kinase assay using either purified ULK1 or ULK2 with recombinant ATG16L1 as substrate. We found that both ULK1 and ULK2 were capable of phosphorylating ATG16L1 in vitro (Fig 1A). In order to narrow down the site of phosphorylation, we repeated the kinase assay using truncations of ATG16L1. We found that the truncation mutant lacking amino acids 254–294 was a very poor substrate for ULK1, indicating that the primary site(s) of ULK1-mediated phosphorylation are located in this region (Fig 1B). Amino acids 254–294 are serine/threonine rich, containing 10 conserved residues (Fig 1C). Therefore, to identify the residue(s) that are phosphorylated by ULK1 in this region we repeated the kinase assay on full-length ATG16L1 and performed mass spectrometry analysis. Our results revealed a single high confidence phosphorylation site on serine 278 (Fig EV1A and marked in green in Fig 1C) and another of slightly lower confidence on serine 287 (Fig EV1A and marked in grey in Fig 1C), both of which map to the region of ATG16L1 we previously identified as required for ULK1-mediated phosphorylation (Fig 1B). Peptide coverage in the mass spectrometry was 80% across the whole protein, and only two S/T residues were missed in the putative 254–294 region. To confirm the major site(s) of phosphorylation on ATG16L1, we mutated S278 and S287 singly in the full-length protein and performed another in vitro ULK1 kinase assay. Interestingly, we observed a significant loss of ULK1-mediated phosphorylation in the S278A mutant and little reduction in the S287A mutant (Fig 1D). This indicates that the major site of phosphorylation on ATG16L1 is S278, which is the same residue previously identified as a site for IKKα–mediated phosphorylation 24. Next, we created phospho-specific antibodies against S278 or S287 of ATG16L1 and tested its specificity by co-transfection of wild-type or mutant ULK1 and ATG16L1. Excitingly, we observed that ULK1 phosphorylates ATG16L1 on S278 in cells and that our antibody was specific to the phosphorylated form of the protein with little to no signal against ATG16L1 (S278A) or wild-type ATG16L1 co-transfected with kinase-dead ULK1 (Fig 1E). Despite good specificity for our S287 antibody (Fig EV1B and C), we observed that the lower probability site obtained by mass spectrometry, S287, was not phosphorylated in an ULK1-dependent manner (Fig 1E). Collectively, these results show that ATG16L1 is a direct target of ULK1 and that the primary site of phosphorylation is S278. Figure 1. ATG16L1 is phosphorylated by ULK1 in vitro kinase assays were performed using purified recombinant kinases (ULK1 and ULK2) and substrate (ATG16L1) in the presence of radiolabelled ATP. ULK and ATG16L1 inputs were examined by western blot (WB), and substrate phosphorylation was analysed by autoradiography (AR). Full-length or truncated versions of ATG16L1 were subjected to an in vitro ULK1 kinase assay. ULK1 and ATG16L1 inputs were examined by western blot and target phosphorylation by autoradiography. ATG16L1 was phosphorylated in an in vitro ULK1 kinase reaction and analysed by mass spectrometry. Phosphorylation of S278 and S287 in humans (S278 marked in green, S287 marked in grey) was identified with high and low confidence, respectively. Conservation of amino acids 254–294 is shown using the Shapely colour scheme. Mass spectrometry was performed on a single experiment. Full-length or mutated HA-ATG16L1 was purified from mammalian cells and subjected to an in vitro ULK1 kinase assay. Inputs were analysed by WB and target phosphorylation by AR. HEK293A cells were transfected with wild-type or phospho-dead ATG16L1 in the presence of wild-type or kinase-dead ULK1. Phosphorylation of ATG16L1 (S278 or S287) and inputs were examined by WB. Data information: Unless otherwise indicated, experiments were performed three times. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. ATG16L1 is a target of ULK1 kinase Mass spectrometry data for ULK1-mediated ATG16L1 phosphorylation. ATG16L1 knock-out HEK293A cells were transfected with either flag-tagged wild-type or S287A ATG16L1. Phosphorylation of ATG16L1 at S287 was determined by WB. Wild-type ATG16L1 substrate and ULK1 were incubated with or without lambda phosphatase. Phospho-specificity of ATG16L1(S287) antibody was determined by immunoblot for total- and phospho-ATG16L1. Download figure Download PowerPoint ULK1 is required for phosphorylation of ATG16L1 and xenophagy induction We next sought to determine whether ULK1 regulated ATG16L1 phosphorylation endogenously and whether this signalling was responsive to starvation. ULK1/2 wild-type or ULK1/2 double-knockout (dKO) cells were starved for amino acids, either with amino acid-free DMEM or HBSS, followed by analysis of pATG16L1 levels by western blot of whole-cell extracts. Starvation potently inhibits mTORC1 signalling, as demonstrated by loss of S6K phosphorylation, which is a prerequisite for ULK1 activation. Importantly, we observed that starvation resulted in a clear increase in endogenous ATG16L1 phosphorylation only in cells containing ULK1 (Figs 2A and EV2A, lanes 1–6). We found that ablation of ULK1-mediated phosphorylation of ATG16L1 had no effect on the stability of the ATG16L1/5-12 complex (Fig EV2B). Notably, our phospho-antibody only recognizes the slower migrating ATG16L1β isoform and is observed as a single band. As IKKα was previously described to phosphorylate ATG16L1 on S278 under infection, we also tested the requirement for IKKα in starvation-induced ATG16L1 phosphorylation. However, we observed that IKKα deficiency had no detectable effect on starvation-induced ATG16L1 phosphorylation (Fig 2A, lanes 7–9). This is perhaps expected as IKKα has no known role in starvation-induced autophagy. This result indicates that the ATG16L1 subunit of the LC3-lipidating enzyme is a direct and physiological target of ULK1 under starvation. We next asked if ULK1/2 or IKKα contributed to ATG16L1 phosphorylation upon infection or TNFα treatment. ULK1/2 wild-type, ULK1/2 dKO or IKKα KO cells were infected with Salmonella enterica serovar Typhimurium (hereafter referred to as Salmonella) or treated with TNFα, and ATG16L1 phosphorylation was examined by western blot. Surprisingly, we observed that Salmonella and TNFα-induced ATG16L1 phosphorylation was abolished in ULK1/2 dKO cells, but was still observed in IKKα knockout cells (Figs 2B and EV2C). Of note, phospho-ATG16L1 signal is consistently lower under infection as only a small minority of cells are subjected to the stress of internalized bacteria (Fig EV2D). These results clearly indicate that ULK1/2 is required for phosphorylation of ATG16L1 under starvation, inflammatory cytokine signalling and infection. Figure 2. ULK1/2 is required for phosphorylation of ATG16L1 and xenophagy induction Wild-type, ULK1/2 double-knockout (dKO) or IKKα KO mouse embryonic fibroblasts (MEFs) cells were incubated with either complete medium, amino acid-deficient DMEM or HBSS for 1 h. Samples were immunoblotted using the indicated antibodies. Wild-type, ULK1/2 dKO or IKKα KO MEFs cells were infected with log phase Salmonella for 2 h; bacteria-containing media was then removed, and cells were incubated with gentamycin (50 μg/ml)-containing DMEM for 2 h. Samples were immunoblotted using the indicated antibodies. Wild-type, ULK1/2 dKO or IKKα KO MEFs cells were infected with Salmonella for 1 h. Autophagic capture of Salmonella was analysed by immunostaining for LPS and LC3B. Representative images are shown (scale bars, 10 and 3 μm). Quantification was generated from eight fields of view from a representative experiment. The experiments were repeated twice. Wild-type, ULK1/2 dKO and IKKα KO MEFs cells were infected with Salmonella for 1 h. Xenophagy rates were examined through colony-forming unit (CFU) assays. Quantification of infection rates by immunofluorescence is demonstrated in the right panel. Data information: Unless otherwise indicated, experiments were performed three times. Data are represented as mean ± standard deviation, and P-values were determined by Student's t-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. ULK1 is required for phosphorylation of ATG16L1 and xenophagy induction Full scan for WB data for phospho-ATG16L1(S278) is shown in Fig 2A. ATG16L1 knock-out HEK293A cells transfected with the indicated GST HA ATG16L1 plasmids were immunoprecipitated for HA. WB was used to examine the binding of ATG5/ATG12 to ATG16L1. Wild-type, ULK1/2 dKO or IKKα KO MEFs cells were treated with either amino acid-free media or the indicated amounts of TNFα for 3 h. Samples were immunoblotted using the indicated antibodies. Levels of ATG16L1 phosphorylation were quantified from three biological replicates. Data are represented as mean ± standard deviation, and P-values were determined by Student's t-test. Wild-type, ULK1/2 dKO and IKKα KO MEFs cells were infected with Salmonella for 1 h. Quantification of infected cells was examined through immunofluorescence of two biological repeats. Data are represented as mean ± standard deviation from seven unique fields of view, and P-values were determined by Student's t-test. Larger field of view for images shown in Fig 2C. Extracellular bacteria staining observable in white. MEF cells were infected with Salmonella for 1 h in the presence of bafilomycin A1. Endogenous LC3B (red) puncta were visualized (scale bars, 20 and 10 μm) by immunofluorescence. Dashed boxes represent the cells selected for enlarged display in Fig 2C. Quantification of LC3B-positive bacteria of Fig 2C biological replicate. Wild-type, ULK1/2 dKO or IKKα KO MEFs cells were infected with Salmonella for 1 h. Autophagic capture of Salmonella was analysed by immunostaining for LPS and LC3B. Data are represented as mean, and P-values were determined by Student's t-test. Download figure Download PowerPoint We next sought to determine the requirement for ULK1/2 and IKKα in promoting xenophagy. Xenophagic clearance of Salmonella is very well established and its intracellular growth is restricted by the pathway, making it an ideal model pathogen for this analysis. Wild-type or knockout cells were infected with Salmonella, and the number of LC3B-positive Salmonella was quantified. LC3B is conjugated to the autophagosomal membrane and colocalizes with bacteria targeted for clearance by xenophagy and can be used at early time points to monitor xenophagy induction. We found that ULK1/2-deficient cells exhibited a potent decrease in LC3B-positive bacteria, while IKKα loss did not significantly affect xenophagy (Figs 2C and EV2E and F). In order to confirm the roles for ULK1/2 and IKKα in xenophagy induction and suppression of invasive bacteria, we performed colony-forming unit (CFU) assays in our wild-type or knockout lines. CFU assays measure bacterial viability after internalization and are inversely correlated with xenophagy rates 29. Analysis of Salmonella viability 4 h postinfection revealed that ULK1/2 dKO cells harboured a much higher number of viable internalized bacteria, indicative of an autophagy defect, when compared to wild-type and IKKα knockout cells (Fig 2D). Surprisingly, our results indicate that ULK1/2, but not IKKα, is required for ATG16L1 phosphorylation and xenophagy induction. ULK1 promotes cleavage of caATG16L1 through phosphorylation on S278 Multiple groups have shown that the T300A substitution in caATG16L1 renders it sensitive to caspase cleavage under stress conditions including nutrient starvation and infection 21, 24, 30. Moreover, it was shown that mutation of serine 278 of ATG16L1 to alanine is involved in stress-induced caspase cleavage in the caATG16L1 background 24. Our data indicate that ULK1 is responsible for the phosphorylation of wild-type ATG16L1 on S278 under nutrient starvation and infection. Therefore, we next sought to determine whether ULK1 signalling was involved in the stress-induced destabilization of caATG16L1. HEK293A cells were transfected with either wild-type ATG16L1 or caATG16L1 co-transfected with increasing amounts of ULK1 kinase. Importantly, overexpression of ULK1 is known to result in autoactivation and induction of downstream signalling in the absence of stress, thereby allowing us to determine the isolated effect of ULK1 signalling on ATG16L1 stability independent of other stress-responsive pathways. Interestingly, we observed that ULK1 is capable of stimulating ATG16L1 cleavage and the level of cleavage is elevated in the caATG16L1 background (Fig 3A). In order to determine whether ATG16L1 cleavage was a result of ULK1-mediated phosphorylation on S278, we transfected HEK293A cells with wild-type, T300A or S278/T300A mutants of ATG16L1 in the presence or absence of ULK1. Excitingly, we observed that single mutation of the ULK1 phosphorylation site was sufficient to reduce ULK1-driven cleavage (Fig 3B). As expected mutation of S287, the low confidence ULK1 phosphorylation site identified by mass spectrometry, had no impact on cleavage in the T300A background (Fig EV3A). These results indicate that caATG16L1 is preferentially cleaved through ULK1-mediated phosphorylation of S278. Conversely, we found that T300A did not have any effect on ATG16L1 phosphorylation (Fig EV3B). Lastly, we repeated this experiment in the presence or absence of Z-VAD-FMK, a pan-caspase inhibitor, to confirm the faster migrating form of ATG16L1 was indeed a product of caspase-mediated cleavage. Treatment with a pan-caspase inhibitor resulted in a potent reduction in the levels of the faster migrating ATG16L1 band, confirming that the ULK1-driven cleavage product was a caspase cleavage product (Fig 3C). Increasing evidence in vitro and in vivo has shown that caspase-mediated destabilization of caATG16L1 is a critical event associated with the pathobiology of this SNP 21, 24. Moreover, in unstressed conditions caATG16L1 is known to have the same stability as wild type 21. To study the effect of ULK1-mediated caspase cleavage of ATG16L1 in cells, we knocked out ATG16L1 using CRISPR/Cas9 (Fig EV3C) and transfected ATG16L1(T300A) in HEK293A cells and infected cells in the presence or absence of ULK inhibitor. Interestingly, we observed Salmonella treatment destabilized the T300A mutant, which could be reversed with ULK inhibitor (Fig 3D). However, ATG16L1(WT) stability was not drastically affected by either Salmonella or ULK inhibition (Fig 3D). We also found ATG16L1(T300A) was stabilized by ULK inhibitors under TNFα treatment (Fig EV3D). We next sought to determine the function of S278 phosphorylation of ATG16L1 in both the wild-type and T300A background. ATG16L1 knockout cells were transfected with ATG16L1 (WT, S278A, T300A or S278A/T300A) at similar levels and treated with Salmonella (Fig EV3E). Quantification of Salmonella at 4 h postinfection showed that mutation of S278 phosphorylation in the wild-type background resulted in an increase in Salmonella, indicating ULK1 phosphorylation may act to promote xenophagy in wild-type ATG16L1 (Fig 3E, columns 1 and 2). Conversely, in the T300A background S278A mutation improved Salmonella clearance, indicating ULK1 phosphorylation is detrimental in this background (Fig 3E, columns 3 and 4). Figure 3. ULK1 promotes cleavage of T300A ATG16L1 through phosphorylation on S278 HEK293A cells were transfected with either flag-tagged WT ATG16L1 or T300A ATG16L1. ULK1 was co-transfected in increasing amounts where indicated. Cleavage of ATG16L1 was analysed by WB of whole-cell lysates. Levels of ATG16L1 cleavage were quantified from three biological repeats (right panel). HEK293A cells were transfected with either tagged wild-type, T300A or S278/T300A ATG16L1 in the presence or absence of ULK1. Cleavage of ATG16L1 was analysed by WB. Levels of ATG16L1 cleavage were measured from three biological repeats (right panel). HEK293A cells were transfected with the indicated plasmids in the presence or absence of a pan-caspase inhibitor Z-VAD-FMK (15 μM) for 4 h. Cleavage of ATG16L1 was analysed by WB of three biological repeats. Wild-type or T300A-expressing HEK293A were treated with Salmonella in the presence or absence of ULK1/2 inhibitor (16 μM) for the indicated time points. Expression of ATG16L1 was analysed by WB. The experiments were performed twice. ATG16L1 knock-out HEK293A cells transfected with the indicated HA GST ATG16L1 plasmids were infected with Salmonella for 1 h. Xenophagy rates were examined through CFU assays. Quantification of infection rates by immunofluorescence is demonstrated in the right panel. Data information: Unless otherwise indicated, experiments were performed three times. Data are represented as mean ± standard deviation, and P-values were determined by Student's t-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. ULK1 promotes cleavage of caATG16L1 through phosphorylation on S278 ATG16L1 knock-out HEK293A cells were transfected with the indicated GST HA ATG16L1 plasmids in the presence or absence of Z-VAD-FMK (15 μM) for 4 h. Cleavage of ATG16L1 was analysed by WB of two biological replicates. Data are represented as mean, and P-values were determined by Student's t-test. ATG16L1 knock-out HEK293A cells were transfected with the indicated GST HA ATG16L1 plasmids in the presence or absence of Z-VAD-FMK (15 μM) for 4 h. Phosphorylation of ATG16L1 was analysed by WB. ATG16L1 knock-out cells were validated by direct sequencing. ATG16L1 knock-out HCT116 cells transfected with the tagged T300A ATG16L1 plasmids were treated with TNFα (20 ng/ml) in the presence or absence of ULK1/2 inhibitor for 4 h. ATG16L1 levels were examined by WB. Inputs for CFU assays in Fig 3E. ATG16L1 knock-out HEK293A transfected with tagged ATG16L1 as indicated were lysed and examined by WB. Download figure Download PowerPoint Collectively, our data shed light on the relationship between stress and caATG16L1 cleavage showing that: (i) ULK1-mediated phosphorylation of ATG16L1 is increased under infection and starvation, which are known to promote the cleavage of caATG16L1, (ii) caATG16L1 is preferentially cleaved upon ULK1 activation, and (iii) mutating the ULK1 phosphorylation site reduces ULK1-driven cleavage and improves xenophagy in the caATG16L1 background. ULK1-mediated phosphorylation is required for ATG16L1 localization to Salmonella site and bacterial clearance ULK1 kinase has a well-established role in stimulating autophagy, making it unlikely that the primary function of ULK1-induced ATG16L1 p" @default.
• W2949137984 created "2019-06-27" @default.
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• W2949137984 date "2019-05-24" @default.
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• W2949137984 title "ULK1‐mediated phosphorylation of ATG16L1 promotes xenophagy, but destabilizes the ATG16L1 Crohn's mutant" @default.
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• W2949137984 doi "https://doi.org/10.15252/embr.201846885" @default.
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