ULK1 is a conserved substrate of AMPK. (A) Clustal alignment of four conserved sites in ULK1 and two sites in ULK2 matching the optimal AMPK substrate motif. (B) ULK1 and GST or GST-14-3-3 expression vectors were transfected into human embryonic kidney (HEK)293T cells, and placed in media containing 20µM STO-609 (STO), vehicle (veh), or 5mM phenformin (Phen) for 1h. Cell lysates and GST pulldowns were immunoblotted as indicated. (C) In vitro kinase assays with myc-tagged catalytically inactive (KI: K46I) ULK1 or myc-tagged wild-type raptor which were immunoprecipitated from HEK293T cells and used as substrates for purified active AMPK in the presence of ³²P-γ-ATP. (D) HEK293T cells transfected with myc-tagged wild-type ULK1 or indicated Serine-to-alanine ULK1 mutants were treated with either vehicle or 1mM phenformin for 1h, or were co-transfected with a constitutively active AMPKα1 (aa1-312) mammalian expression vector (). Proteins from lysates were immunoblotted with phospho-specific antibodies as indicated. (E) In vitro kinase assays using myc-ULK1 and purified AMPK as above. Phosphorylation of myc-ULK1 detected by immunoblotting with indicated phospho-specific antibodies. (F) Primary murine embryonic fibroblasts (MEFs) were treated with 2mM AICAR or vehicle for 1h. Lysates immunoblotted as indicated including detection of endogenous ULK1 P-Ser₅₅₅

Genetic deficiency for AMPK or ULK1 in murine liver or primary murine hepatocytes results in autophagy defects. (A) Liver lysates from littermate-matched mice were immunoblotted for the indicated antibodies. p62 to actin ratio calculated from densitometry performed on immunoblots. Data shown as mean +/− SEM. * p<.01 (B) Primary hepatocytes derived from ULK1^+/+ or ULK1^−/− mice or AMPKa1^+/−a2^L/+ or AMPKa1^−/−a2^L/L as described in methods were placed in media containing 2mM metformin (met) or vehicle (veh) for 2h. Lysates were immunoblotted with the indicated antibodies. (C) Transmitting electron microscopy (TEM) was performed on primary murine hepatocytes of the indicated genotypes revealing accumulation of mitochondria in both AMPK- and ULK1-deficient cells. Mitochondria pseudocolored RED, cytoplasm BLUE, nuclei GREEN, and lipid droplets YELLOW. (D) Primary murine hepatocytes of the indicated genotypes stained by immunocytochemistry for the mitochondrial marker TOM20 (red) and nuclei (blue). Scale bar, 10 microns.

AMPK is necessary and sufficient for autophagy induction in C. elegans. (A) Insulin receptor daf-2(e1370) mutant worms expressing GFP::LGG-1 (equivalent to GFP-LC3) were treated with control RNAi or RNAi against bec-1 (Beclin), aak-2 (AMPKα2) or unc-51 (ULK1) and the number of LGG-1/LC3-positive puncta per hypodermal seam cell were quantified. (B) aak-2(ok524) mutants or wild-type N2 (WT) animals expressing GFP::LGG-1 were treated with control or daf-2 RNAi and were scored for LGG-1 positive puncta per seam cell. (C) Transgenic worms expressing constitutively active AAK-2 (ref. ) (amino acids 1-321)::TOMATO (CA-AAK-2::TOMATO_(1-321) fusion or controls were analyzed for LGG-1 positive puncta per seam cell. (D) Animals expressing both CA-AAK-2_(1-321)::TOMATO and GFP::LGG-1 were treated with control or unc-51 RNAi and scored for LGG-1/LC3-positive puncta per seam cell. All panels show relative counts, see for details. Data shown as mean +/− SEM. * P<.0001

AMPK phosphorylation of ULK1 is required for mitophagy and cell survival upon nutrient deprivation (A) U2OS cells stably expressing mouse wild-type (WT) or catalytically inactive (KI) or AMPK non-phosphorylatable (4SA) ULK1 cDNA or the empty retroviral vector (v) along with a shRNA against endogenous human ULK1 and ULK2 were placed in media containing 5mM phenformin (Phen) or vehicle for 1h. Lysates were immunoblotted as indicated. (B) ULK1−/− MEFs stably expressing WT, KI, or 4SA ULK1 cDNA or the empty retroviral vector (v) along with a shRNA against endogenous ULK2 were placed in EBSS starvation media (starv) or control media (ctl) for 6h in the presence or absence of BafilomycinA (BafA) and immunoblotted as indicated. (C) Cells from (B) analyzed by TEM and Inform morphometric software. Mitochondria pseudocolored RED, cytoplasm BLUE, and nuclei GREEN. (D) Flourescence Activated Cell Sorting (FACS) analysis on cells from (B) which were stained with JC-1 under basal conditions, or with the mitochondrial uncoupler CCCP as a control, to measure mitochondrial membrane potential. Compromised mitochondrial membrane potential is shifted to the left, as observed in cells treated with CCCP. (E) Wild-type (WT) MEFs transfected with 20nM siRNA pools to a universal control (ctl), murine Atg5, or murine ULK1 and ULK2 for 72 hours were then placed in starvation medium (starv) or standard media (ctl) for 12h and cell death was scored by AnnexinV- FACS. (F) Cells from (B) were placed in starvation medium (starv) or standard media (ctl) for 12h and cell death was scored by AnnexinV- FACS. (G) Model for AMPK activation of ULK1 in a two-pronged mechanism via direct phosphorylation of ULK1 and inhibition of mTORC1 suppression of ULK1.