Regulation of mATG9 trafficking by Src- and ULK1-mediated phosphorylation in basal and starvation-induced autophagy

Abstract

Autophagy requires diverse membrane sources and involves membrane trafficking of mATG9, the only membrane protein in the ATG family. However, the molecular regulation of mATG9 trafficking for autophagy initiation remains unclear. Here we identified two conserved classic adaptor protein sorting signals within the cytosolic N-terminus of mATG9, which mediate trafficking of mATG9 from the plasma membrane and trans-Golgi network (TGN) via interaction with the AP1/2 complex. Src phosphorylates mATG9 at Tyr8 to maintain its endocytic and constitutive trafficking in unstressed conditions. In response to starvation, phosphorylation of mATG9 at Tyr8 by Src and at Ser14 by ULK1 functionally cooperate to promote interactions between mATG9 and the AP1/2 complex, leading to redistribution of mATG9 from the plasma membrane and juxta-nuclear region to the peripheral pool for autophagy initiation. Our findings uncover novel mechanisms of mATG9 trafficking and suggest a coordination of basal and stress-induced autophagy.

Figure 1

The mATG9 N-terminus contains two conserved adaptor protein sorting signals. (A) Alignment of mATG9 N-terminus sequences from different mammalian species reveals that two putative AP sorting signals (red color letters) are highly conserved. (B, C) HEK293T cells were transiently co-transfected with wild-type (WT) mATG9-Myc or the indicated mutants and 3×Flag-AP1/2M1. 24 h after transfection, cells were collected for immunoprecipitation with anti-Flag antibody. M1 is the mu subunit of the AP1 and AP2 complexes. (D) U2OS cells were co-transfected with WT mATG9-Myc or the indicated mutants (red) and TGN46-GFP (green). 24 h after transfection, cells were fixed and immunostained with anti-Myc antibody. Cells were counterstained with DAPI (blue). Scale bar, 10 μm. (E) The distribution of mATG9 or the indicated mutants in cells from D was assessed and quantified in a blind fashion (mean ± SEM; n = 100 cells from three independent experiments, ^**P < 0.01, ^***P < 0.001). (F) Pearson's coefficient was determined for the co-localization between mATG9 or the indicated mutants and TGN46 in cells from D. Data were analyzed by ImageJ (means ± SEM; n = 30 cells from three independent experiments, ^**P < 0.01, ^***P < 0.001). (G) HeLa cells were transfected with mATG9-Myc or the indicated mutants for 24 h, and then incubated at 4 °C with ice-cold Sulfo-NHS-Biotin solution for 30 min, rinsed and lysed. Total mATG9 was immunoprecipitated with anti-Myc antibody and the proportion of biotinylated mATG9 was assessed by immunoblotting with anti-Biotin antibody. (H) HeLa cells were transfected with mATG9-Myc or the SS1/2 mutant for 24 h, and then processed for immunogold electron microscopy with anti-Myc antibody. Boxed areas in the left image are shown enlarged on the right. Gold nanoparticles are indicated by red color arrows. Scale bar, 500 nm. See also Supplementary information, Figure S1.

Figure 2

Src directly phosphorylates the mATG9 N-terminus at Y8. (A) Purified His-ATG9N-HA peptide (aa1-66) was incubated with control lysis buffer or HEK293T cell lysate and incubated at 30 °C for 30 min in kinase buffer. The reaction products were subjected to western blotting using anti-phosphotyrosine and anti-phosphoserine/threonine antibodies to analyze the phosphorylation status of the mATG9 N-terminus. (B) Alignment of mATG9 N-terminal sequences from different mammalian species reveals the conserved consensus motif for Src kinase. The phosphorylated tyrosine (Y) is in red. (C) In vitro Src kinase assay. Immunoprecipitated wild-type (WT) or kinase-dead (KD) Src was incubated with purified His-ATG9N-HA peptide at 30 °C for 30 min in kinase buffer. (D) GST or purified recombinant GST-Src (Sigma) protein was incubated with purified His-ATG9N-HA peptide at 30 °C for 30 min in kinase buffer to assay Src kinase activity in vitro. (E) In vitro Src kinase assay was performed as described in C using His-ATG9N-HA or the indicated point-mutated peptides. (F) HeLa cells were harvested and lysed by NP-40 lysis buffer for immunoprecipitation with anti-mATG9 antibody. (G) HEK293T cell lysate was incubated with immobilized His-ATG9N-HA peptide at 4 °C overnight. (H) HEK293T cells were co-transfected with WT mATG9-Myc or the indicated mutants and Src-GFP or KD Src for 24 h. Total mATG9 was immunoprecipitated with anti-Myc antibody and the phosphotyrosine level was assessed by immunoblotting with anti-phosphotyrosine antibody. See also Supplementary information, Figures S2 and S3A.

Figure 3

Src phosphorylates mATG9 at Y8 to promote constitutive trafficking of mATG9. (A) HeLa cells were transfected with Src-GFP or kinase-dead (KD) Src for 24 h, and collected for immunoprecipitation with anti-mATG9 antibody. (B) HeLa cells were treated with the Src kinase inhibitors PP2 (10 μM) or SU6656 (10 μM) for 12 h and collected for immunoprecipitation with anti-mATG9 antibody. (C, D) HEK293T cells were co-transfected with mATG9-Myc, 3×Flag-AP1/2M1 and GFP vector, Src-GFP or KD Src for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (E, F) HEK293T cells were co-transfected with WT mATG9 or the Y8F mutant, 3×Flag-AP1/2M1 and Src-GFP for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (G) HeLa cells were co-transfected with WT mATG9-Myc or the Y8F mutant and 3×Flag-AP1/2M1 for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (H, I) HeLa cells were co-transfected with mATG9 and 3×Flag-AP1/2M1 for 24 h, and then treated with vehicle or the Src kinase inhibitors PP2 (10 μM) or SU6656 (10 μM) for 12 h. Cells were collected for immunoprecipitation with anti-Flag antibody. (J) U2OS cells were co-transfected with WT mATG9-Myc or the indicated mutants (red) and GFP vector, Src-GFP or KD Src (green) for 24 h, and then fixed and immunostained with anti-Myc antibody. Scale bar, 10 μm. See also Supplementary information, Figure S3B-S3C.

Figure 4

Stimulation of Src kinase activity by EGF promotes retrograde trafficking of mATG9. (A) HeLa cells were serum-starved for 24 h, and then stimulated with 50 ng/ml hEGF for the indicated times. Cells were collected for immunoprecipitation with anti-mATG9 antibody and phospho-mATG9 was assessed by immunoblotting with a specific antibody against Y8. (B) HeLa cells stably expressing mATG9-Myc were transfected with 3×Flag-AP1/2M1, serum-starved for 24 h, and then stimulated with hEGF for the indicated times. Cells were collected for immunoprecipitation with anti-Flag antibody. (C-E) Atg9a KO HeLa cells reconstituted with wild-type mATG9 (C) or the SS1/2 (D) or Y8F (E) mutants were starved of serum for 24 h, and then stimulated with hEGF for the indicated times. Cells were fixed and immunostained with anti-Myc (red) and anti-EGFR (green) antibodies. Nuclei are stained with DAPI (blue). Boxed areas in the left panels are enlarged in the right panels. Scale bar, 10 μm. See also Supplementary information, Figure S3D-S3F.

Figure 5

ULK1 phosphorylates mATG9 at S14 to promote its trafficking under starvation stress. (A) Alignment of the mATG9 N-terminal sequences from different mammalian species reveals the consensus motif for ULK1 kinase. (B) In vitro ULK1 kinase assay. Immunoprecipitated GFP protein, wild-type ULK1 (WT) or kinase-dead ULK1 (KD) was incubated with purified His-ATG9N-HA peptide (aa1-66) at 30 °C for 30 min in kinase buffer. (C) In vitro ULK1 kinase assay was performed as described in B using His-ATG9N-HA or the indicated point-mutated peptides. (D) HeLa cells were treated with or without EBSS for 2 h in the presence or absence of the PI3K inhibitor Wortmannin (100 nM) or the lysosome inhibitor BA1 (20 nM), and then collected for immunoprecipitation with anti-ULK1 antibody. (E) ULK1 protein produced by an in vitro transcription/translation system was incubated with immobilized His-ATG9N-HA peptide at 4 °C overnight. (F) HeLa cells were transfected with GFP-ULK1 or KD ULK1 for 24 h, and collected for immunoprecipitation with anti-mATG9 antibody. (G) HeLa cells were treated with EBSS for the indicated times, and then collected for immunoprecipitation with anti-mATG9 antibody. The proportion of phospho-mATG9 was assessed by immunoblotting with specific antibodies against Y8 or S14. (H) Parental cells or Ulk1 KO HeLa cells were treated with or without EBSS for 2 h. Cells were collected for immunoprecipitation with anti-mATG9 antibody, and phospho-mATG9 was assessed by immunoblotting with a specific antibody against Y8 or S14. (I, J) HEK293T cells were co-transfected with WT mATG9 or the indicated mutants, 3×Flag-AP1/2M1 and GFP, GFP-ULK1 or KD ULK1 for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (K, L) HeLa cells were co-transfected with WT mATG9 or the S14A mutant and 3×Flag-AP1/2M1 for 24 h, and then starved in EBSS for 2 h. Cells were collected for immunoprecipitation with anti-Flag antibody. See also Supplementary information, Figure S4.

Figure 6

Phosphorylation of Y8 and S14 cooperates to regulate mATG9 trafficking and redistribution. (A, B) HEK293T cells were co-transfected with 3×Flag-AP1/2M1 and GFP vector, Src-GFP and/or GFP-ULK1 for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (C) HEK293T cells were co-transfected with WT mATG9-Myc or the FA mutant, 3×Flag-AP1/2M1, Src-GFP and GFP-ULK1 for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (D) HEK293T cells were co-transfected with WT mATG9-Myc or the indicated mutants, 3×Flag-AP1/2M1, GFP vector or GFP-ULK1 for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. (E) HeLa cells were co-transfected with WT mATG9-Myc or the indicated mutants and 3×Flag-AP1/2M1 for 24 h, and then starved in EBSS for 2 h. Cells were collected for immunoprecipitation with anti-Flag antibody. (F) HEK293T cells were co-transfected with 3×Flag-TBC1D5 and GFP vector, Src-GFP and/or GFP-ULK1 for 24 h, and then collected for immunoprecipitation with anti-Flag antibody. See also Supplementary information, Figure S5.

Figure 7

Phosphorylation of both Y8 and S14 is essential for autophagy initiation. (A) U2OS cells were transfected with GFP-LC3 for 24 h, then treated with or without EBSS for 2 h in the presence or absence of the Src inhibitor PP2 (10 μM) and the lysosome inhibitor BA1 (Bafilomycin A1, 20 nM). Scale bar, 10 μm. (B) The number of LC3 dots in cells from A was assessed and quantified in a blind fashion by ImageJ (mean ± SEM; n = 100 cells from three independent experiments, NS, not significant, ^**P < 0.01, ^***P < 0.001). (C) HeLa cells were pretreated with Src inhibitor PP2 (10 μM) or SU6656 (10 μM) for 10 h, and then treated with or without EBSS for 2 h in the presence or absence of Src inhibitor PP2 (10 μM) or SU6656 (10 μM) and the lysosome inhibitor BA1 (20 nM). (D) Atg9a KO HeLa cells stably reconstituted with wild-type mATG9 or the indicated mutants were transfected with GFP-LC3 for 24 h, and then starved in EBSS for 2 h in the presence of the lysosome inhibitor BA1 (20 nM). Cells were fixed and immunostained with anti-Myc antibody. Scale bar, 10 μm. (E) The number of LC3 dots in cells from D was assessed and quantified in a blind fashion by ImageJ (mean ± SEM; n = 100 cells from three independent experiments, ^***P < 0.001). (F) Atg9a KO HeLa cells stablely reconstituted with wild-type mATG9 or the indicated mutants were starved in EBSS for 2 h in the presence of the lysosome inhibitor BA1 (20 nM). (G) Schematic diagram showing the regulation of mATG9 trafficking and autophagy initiation under normal unstressed and starvation conditions. Under normal conditions, mATG9 is phosphorylated by Src at Y8 to sustain its constitutive trafficking and juxta-nuclear localization. In response to starvation stress, mATG9 is phosphorylated at Y8 by Src and at S14 by ULK1 to synergistically promote redistribution of mATG9 from the plasma membrane and TGN to endosomes for autophagy initiation.