Journal: Molecular and Cellular Biology

Article Title: Metabolic-Stress-Induced Rearrangement of the 14-3-3ζ Interactome Promotes Autophagy via a ULK1- and AMPK-Regulated 14-3-3ζ Interaction with Phosphorylated Atg9

doi: 10.1128/MCB.00740-14

Figure Lengend Snippet: 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.

Article Snippet: The ULK1 enzyme system (catalog number U3521) was purchased from Promega.

Techniques: Immunoprecipitation, Expressing, Transfection, Mutagenesis, Dominant Negative Mutation, Construct, Co-Immunoprecipitation Assay, Western Blot, Purification, Incubation, Recombinant, SDS Page

Journal: Molecular and Cellular Biology

Article Title: Metabolic-Stress-Induced Rearrangement of the 14-3-3ζ Interactome Promotes Autophagy via a ULK1- and AMPK-Regulated 14-3-3ζ Interaction with Phosphorylated Atg9

doi: 10.1128/MCB.00740-14

Figure Lengend Snippet: Model of ULK1- and AMPK-mediated regulation of Atg9A via phosphorylation at S761. Under nutrient-replete conditions, a low level of phosphorylation is dependent on ULK1 and AMPK, suggesting a relationship between these kinases under basal conditions (42). We favor the hypothesis that AMPK is directly responsible for Atg9A phosphorylation, with ULK1 playing an ancillary role (see Discussion). We posit that this low level of phosphorylation maintains a basal level of autophagy. Under nutrient deprivation, an increased AMP-to-ADP ratio results in the full activation of AMPK and increased phosphorylation at S761 independent of ULK1, leading to increased autophagy, localization of Atg9A to autophagosomes, and autophagosome biogenesis.

Article Snippet: The ULK1 enzyme system (catalog number U3521) was purchased from Promega.

Techniques: Activation Assay