Figure 6.
Phosphorylation of Ulk1 Ser 757 by mTORC1 inhibits the Ulk1–AMPK interaction. (a) Ulk1 Ser 757 is required for mTORC1 to regulate the interaction of Ulk1 with AMPK in vivo. CBP/SBP tagged Ulk1 (wild type or S757C) was co-transfected with HA–AMPKα and Rheb into HEK293 cells as indicated. Ulk1 was purified by streptavidin beads and the co-precipitated HA–AMPKα was examined by western blot (Rapa, 50 nM rapamycin treatment for 1 h before cell lysis). (b) Ulk1 Ser 757 is required for rapamycin to enhance the Ulk1–AMPK interaction in vitro. CBP/SBP Ulk1 proteins (wild type or S757C) were prepared from transfected HEK293 cells, which were pre-incubated with 50 nM rapamycin (Rapa, 1h) as indicated. The Ulk1 proteins were purified by streptavidin beads and the resulting Ulk1–bead was incubated with the bacterial purified AMPKα/β/γ complex. AMPKα protein levels in the in vitro pulldown assays were examined by western blot using AMPKα antibody. L.E.; long exposure. (c) Phosphorylation of AMPK sites Ser 317 and Ser 777 in Ulk1 are decreased in Tsc1−/− MEFs. Tsc1+/+ (WT) and Tsc1−/− (KO) MEFs were starved of glucose (4 h), or treated with 50 nM rapamycin (Rapa, 1 h). Ser 317 and Ser 777 phosphorylation of endogenous Ulk1 was examined by western blotting with antibodies against Ulk1 phosphorylated at Ser 317 or Ser 777. (d) Rheb suppresses Ulk1 Ser 317 and Ser 777 phosphorylation in a manner dependent on mTORC1. HA–Ulk1, AMPKα kinase-dead mutant (DN), and Myc–Rheb were co-transfected into HEK293 cells as indicated. The cells were incubated with glucose-free medium (−Glu, 4 h), in which either 20 µM compound C (C.C.) or 50 nM Rapamycin (Rapa) was added. Total cell lysates were probed with antibodies against Ulk1 phosphorylated at Ser 317, Ser 777, Ser 757, and HA, as indicated. (e) Rheb inhibits glucose starvation-induced Ulk1 activation. HA–Ulk1 and Myc–Rheb was transfected into HEK293 cells, which were incubated with glucose-free (−Glu), amino-acid-free (−A.A) medium, or 50 nM rapamycin (Rapa) for 4 h before lysis. HA–Ulk1 was immunoprecipitated and kinase assays were performed. Ulk1 activity was measured by 32P-autoradiogram and the protein level of HA–Ulk1 and GST–Atg13 used in the assay was determined by western blot and by Coomassie staining, respectively. Uncropped images of blots are shown in Supplementary Fig. S5.