Fig. 7

An AMP-myristoyl switch triggers ULK1 phosphorylation of β1-Ser108. a Adenine nucleotides extracted from HEK293T cells incubated with phenformin (2 mM, 1 h), H₂O₂ (1 mM, 45 min), AZD8055 (1 μM, 1 h), or INK128 (1 μM, 1 h) were quantitated by mass spectrometry. Adenylate energy charge was calculated as described in Online Methods. n = 3. Error bars, mean adenylate energy charge ± s.e.m. Statistical analyses were performed using one-way ANOVA with post hoc Dunnett’s multiple comparison test. ****P > 0.001, *P < 0.05 indicate significant decrease in mean adenylate energy charge compared to basal. b Immunoblots for β1-pSer108 and α-pThr172 in KI-α1β1γ1 or KI-α1β1γ1(D245A) purified from HEK293T cells stimulated with 2 mM phenformin for 1 h. n = 3, representative immunoblots shown. c Immunoblots for ULK1-pSer757, and β1-pSer108 and α-pThr172 in KI-α1β1γ1 purified from HEK293T cells incubated with 1 μM mTOR inhibitors AZD8055 or INK128 for 1 h. n = 3, representative immunoblots shown. C: Bacterial expressed, CaMKK2-treated α1β1γ1 standard. d Immunoblots for β1-pSer108 in KI-α1β1γ1 (myr) or KI-α1β1(G2A)γ1 (non-myr) purified from HEK293T cells incubated with 2 mM phenformin for 1 h (upper) or glucose free medium for 4 h (lower). n = 3, representative immunoblots shown. e Immunoblot for β1-pSer108 in bacterial-expressed, non-myristoylated (non-myr) or myristoylated (myr) KI-α1β1γ1 phosphorylated with ULK1 for 30 min in the presence of 100 μM AMP. n = 3, representative immunoblots shown. C: CaMKK2-treated α1β1γ1 standard. Error bars, mean increase in β1-pSer108 relative to ULK1-untreated ± s.e.m. Statistical analysis was performed using unpaired two-tailed Student’s t-test. ***P < 0.001 indicates significant decrease in β1-pSer108 relative to non-myristoylated AMPK