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.
