FIG 5.

AMPK and ULK1 are differentially required for S761 phosphorylation under basal and stressed conditions. (A) HA-Atg9A was overexpressed in HEK-293 cells and treated with hypoxia for 12 h with the indicated inhibitors (UCN01 [0.3 μM], UCN01 [1 μM], compound C [20 μM], and LY294002 [50 μM]). Immunoprecipitated Atg9A from each treatment was immunoblotted with the phosphorylated 14-3-3 motif (pMotif) antibody. (B) HEK-293 cells expressing HA-Atg9A and transfected with the indicated siRNAs were treated with normoxia or hypoxia for 12 h. HA-Atg9A was immunoprecipitated and immunoblotted for phosphorylated S761. (C) HEK-293 cells expressing HA-Atg9A and transfected with the indicated siRNAs were treated with normoxia. HA-Atg9A was immunoprecipitated and immunoblotted for phosphorylated S761 and the indicated controls. (D) HEK-293 cells expressing HA-Atg9A with or without the AMPK alpha2 K45R mutant (a dominant negative AMPK construct) were treated with hypoxia for 12 h. Immunoprecipitated HA-Atg9A was immunoblotted for endogenous 14-3-3ζ. (E) Cells treated as described above for panel C were subjected to co-IP with HA-agarose resin followed by immunoblotting for phosphorylated S761. (F) HEK-293 cells expressing HA-Atg9A and transfected with siRNAs against the α1 and α2 subunits of AMPK were treated with normoxia or hypoxia for 12 h. HA-Atg9A was immunoprecipitated and immunoblotted for phosphorylated S761. Phosphorylated acetyl-CoA carboxylase (pACC), an AMPK substrate, and AMPK subunits were assessed by Western blotting in lysates to validate AMPK depletion. (G) Resin-bound HA-Atg9A purified from HEK-293 cells was treated with λ phosphatase for 10 min, thoroughly washed, and then incubated with the indicated recombinant enzymes for 30 min. After further washing, resin-bound HA-Atg9A was resolved by SDS-PAGE and immunoblotted with phospho-S761 antibody.