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.
