ULK1-mediated phosphorylation of ATG16L1 promotes xenophagy, but destabilizes the ATG16L1 Crohn's mutant

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.

Keywords: ATG16L1; Crohn's disease; ULK1; autophagy; caspase.

Figure 1. ATG16L1 is phosphorylated by ULK1

1. 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).

2. 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.

3. 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.

4. 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.

5. 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.

Figure EV1. ATG16L1 is a target of ULK1 kinase

1. Mass spectrometry data for ULK1‐mediated ATG16L1 phosphorylation.

2. 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.

3. 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.

Figure 2. ULK1/2 is required for phosphorylation of ATG16L1 and xenophagy induction

1. 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.

2. 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.

3. 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.

4. 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.

Figure EV2. ULK1 is required for phosphorylation of ATG16L1 and xenophagy induction

1. Full scan for WB data for phospho‐ATG16L1(S278) is shown in Fig 2A.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

Figure 3. ULK1 promotes cleavage of T300A ATG16L1 through phosphorylation on S278

1. 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).

2. 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).

3. 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.

4. 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.

5. 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.

Figure EV3. ULK1 promotes cleavage of caATG16L1 through phosphorylation on S278

1. 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.

2. 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.

3. ATG16L1 knock‐out cells were validated by direct sequencing.

4. 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.

5. Inputs for CFU assays in Fig 3E. ATG16L1 knock‐out HEK293A transfected with tagged ATG16L1 as indicated were lysed and examined by WB.

Figure 4. ULK1‐mediated phosphorylation is required for ATG16L1 localization to Salmonella site and bacterial clearance

1. Wild‐type MEF cells were infected with Salmonella for 25 min. Phospho‐ATG16L1, total ATG16L1 and LPS were stained and analysed by immunofluorescence. Representative immunofluorescent images are shown (scale bars, 10 and 1 μm).

2. Wild‐type and ULK1/2 dKO cells were infected with Salmonella for 25 min. Immunofluorescence was performed using antibodies against LPS and ATG16L1. Representative immunofluorescent images are shown on the left panel (scale bars, 10 and 2 μm). Quantification of ATG16L1‐positive bacteria from seven fields of view from a representative experiment is shown in the right panel.

3. ATG16L1 knock‐out HCT116 cells transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 h. Bacteria were stained using anti‐LPS antibodies to analyse localization in addition to ATG16L1. Representative immunofluorescent images of ATG16L1 and LPS are shown (scale bars, 5 and 1 μm). Quantification of ATG16L1 localizing to bacteria from seven fields of view from a representative experiment is shown in the lower panel.

4. ATG16L1 knock‐out HCT116 cells transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 h. Bacteria were stained using anti‐LPS antibodies to analyse localization in addition to the autophagy marker LC3B. Representative immunofluorescent images of LC3B and LPS are shown (scale bars, 5 and 1 μm). Quantification of bacteria undergoing autophagic clearance from seven fields of view from a representative experiment is shown in the lower panel.

5. A diagram demonstrating our working model for the role of ULK1‐mediated phosphorylation at S278 in wild‐type and T300A ATG16L1 background.

Data information: Unless otherwise indicated, experiments were performed twice. Data are represented as mean, and P‐values were determined by Student's t‐test.

Figure EV4. ULK1‐mediated phosphorylation is required for ATG16L1 localization to Salmonella site

1. Larger field of view for images shown in Fig 4A. Bacteria staining observable in white. MEF cells were infected with Salmonella for 25 min. Phospho‐ATG16L1 (red) and total ATG16L1 (green) were visualized (scale bars, 20 and 10 μm) by immunofluorescence. Dashed boxes represent the cells selected for enlarged display in Fig 4A.

2. Quantification of ATG16L1 localization to the bacteria of Fig 4A biological replicate. Wild‐type MEF cells were infected with Salmonella for 25 min. Phospho‐ATG16L1, total ATG16L1 and LPS were stained and analysed by immunofluorescence. Data are represented as mean, and P‐values were determined by Student's t‐test.

3. Larger field of view for images shown in Fig 4B. Extracellular bacteria staining observable in white. MEF cells were infected with Salmonella for 25 min. Endogenous ATG16L1 (red) puncta were visualized (scale bars, 30 and 10 μm) by immunofluorescence. Dashed boxes represent the cells selected for enlarged display in Fig 4B.

4. Quantification of ATG16L1 puncta of Fig 4B biological replicate. Wild‐type and ULK1/2 dKO cells were infected with Salmonella for 25 min. Immunofluorescence was performed using antibodies against LPS and ATG16L1. Data are represented as mean, and P‐values were determined by Student's t‐test.

5. Larger field of view for images shown in Fig 4C and extra data from the same experiment were also included. ATG16L1 knock‐out HCT116 cells transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 h. ATG16L1 (red) puncta were analysed by immunofluorescence (scale bars, 10, 5 and 1 μm). The experiments were repeated twice. Data are represented as mean ± standard deviation from seven unique fields of view, and P‐values were determined by Student's t‐test.

6. Quantification of ATG16L1‐positive bacteria of Fig 4C biological replicate. ATG16L1 knock‐out HCT116 cells transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 h. Bacteria were stained using anti‐LPS antibodies to analyse localization in addition to ATG16L1. Data are represented as mean, and P‐values were determined by Student's t‐test.

Figure EV5. ULK1‐mediated phosphorylation is required for xenophagy and bacterial clearance

1. Larger field of view for images shown in Fig 4D, and extra data from the same experiment were also included. ATG16L1 knock‐out HCT116 cells transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 h. LC3B (red) puncta were analysed by immunofluorescence (scale bars, 10, 5 and 1 μm). The experiments were repeated twice. Data are represented as mean ± standard deviation from seven unique fields of view, and P‐values were determined by Student's t‐test.

2. Quantification of LC3B‐positive bacteria of Fig 4D biological replicate. ATG16L1 knock‐out HCT116 cells transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 h. Bacteria were stained using anti‐LPS antibodies to analyse localization in addition to the autophagy marker LC3B. Data are represented as mean and P‐values were determined by Student's t‐test.

3. 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 middle panel. Expression of ATG16L1 was examined by WB (bottom panel). The experiments were repeated three times. Data are represented as mean ± standard deviation, and P‐values were determined by Student's t‐test.

4. ATG16L1 KO HCT116 cells with or without the indicated reconstituted OLLAS ATG16L1 were incubated with HBSS media in the presence of bafilomycin A1 for 1 h. LC3B flux was analysed by WB. The experiments were repeated three times. Data are represented as mean ± standard deviation, and P‐values were determined by Student's t‐test.