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.