Figure 1.
Glucose starvation activates Ulk1 protein kinase through AMPK-dependent phosphorylation. (a) HEK293 cells were starved of glucose (4 h) as indicated, endogenous Ulk1 was immunoprecipitated and an autophosphorylation assay was performed. Proteins were resolved by SDS–PAGE and visualized with autoradiography (top) or western blotting (WB; bottom). (b) Cells were incubated in glucose-free medium (4 h) as indicated and lysed. Lysates were incubated with lambda phosphatase (λ PPase) as indicated. Endogenous Ulk1 mobility was examined by western blotting. (c) HA–Ulk1 was transfected into HEK293 cells together with wild-type (WT) AMPKα1 or a kinase-dead (DN) mutant. Cells were starved of glucose (4 h; Glu) or amino acids (−A.A) and treated with compound C (20 µM, C.C) as indicated. Ulk1 mobility as well as phosphorylation levels of ACC and S6K were determined by western blotting. (d) HA–Ulk1 proteins were immunopurified from transfected HEK293 cells, which had undergone glucose starvation (4 h) as indicated. The HA–Ulk1 proteins were treated with λ PPase, and in vitro kinase assays were performed in the presence of GST–ATG13. Proteins were resolved by SDS–PAGE; phosphorylated proteins were visualized with autoradiography, HA–Ulk1 by western blotting and GST–Atg13 by Coomassie staining. (e) HA–Ulk1 was immunopurified from transfected HEK293 cells under glucose-rich media and treated with AMPK in the presence of cold ATP for 15 min, followed by kinase assays as described in d. (f) AMPK wild-type (WT) and α1/α2 double knockout (DKO) MEFs were incubated with or without glucose (4 h). Endogenous Ulk1 was immunoprecipitated and autophosphorylation was measured (mean ± s.d., n = 3). Autophosphorylation activity was normalized to Ulk1 protein level; relative activity is calculated by normalization to Ulk1 activity from AMPK wild-type MEFs in glucose-rich conditions. (g) HA–Ulk1 was transfected into HEK293 cells together with vector (Vec) or an AMPKα1 kinase-dead mutant (DN). The cells were starved of glucose (−Glu) or amino acids (−A.A), or treated with 50 nM rapamycin (Rapa) for 3 h before lysis. Left: autophosphorylation activity was assessed and normalized as in f (mean ± s.d., n = 3). Right: fold induction in Ulk1 autophosphorylation, compared with Ulk1 autophosphorylation from cells under nutrient-rich conditions. Uncropped images of blots are shown in Supplementary Fig. S5.