PHA-793887 BMS-777607 Peptide synthesis

autophagy initiating kinase, plays a central role in general autophagy,
in particular in autophagosome formation [11]. However, recent
studies indicate that it has a selective function in mitophagy uponstimulation by Peptide synthesis different environmental cues. ULK1 has been identi-
?ed as the substrate of AMPKa1which connects cellular energy sens-
ing to mitophagy in glucose or nutrient starvation-induced
autophagy [16,17,22]. Several potential ULK1 phosphorylation sites
have been identi?ed in these studies, but the dynamic changes in
the phosphorylation status of ULK1 during hypoxia are not clear. In
our study, hypoxia signi?cantly induced AMPK a1 activation as indi-
cated by phosphorylation at Thr-172 (Fig.

1A), which is consistent
with AMPK activation in response to other stimuli including glucose
withdrawal or energy depletion [25,26,32,35].Phosphorylationof
ULK1 at Ser-555 and Ser-317 also increased dramatically under
hypoxia, while phosphorylation at Ser-757 decreased (Fig. 1A).
Interestingly, the phosphorylation pattern of ULK1 under
hypoxic conditions selleckchem is similar to that in glucose or nutrient
starvation-induced autophagy, suggesting that a common mecha-
nism of ULK1 activation is shared between general autophagy and
mitophagy [25,26,30,32,35].
The changes in phosphorylation status are possibly due to the
actions of the different binding partners of ULK1 during hypoxia,
since the ULK1/AMPK a1 interaction is increased in hypoxic cells
while the ULK1/mTOR interaction is decreased (Fig. 1C). InFig. 4.

AMPK a1 is required for ULK1-dependent mitophagy induced by hypoxia. (A) MEF cells were exposed to hypoxic conditions for 24 h, and the rescuing effect of HA-
AMPK, HA-AMPK KD, HA-ULK1 WT, HA-ULK1 (S555A) or HA-ULK1 (S555D) was examined after endogenous AMPK was knocked down with si-AMPK. Cell lysates were
immunoblotted for the indicated proteins. (B) Quanti?cation of the ratio of p62, VDAC1, TIM23 and TOM20 to Actin in (A). Data were fromthree independent experiments. (C)
MEFs were cultured under hypoxia for 6 h and treated with Dorsomorphin 2HCl (1 lM) to inhibit AMPK, si-AMPK to knock down endogenous AMPK, or Metformin (2 mM) to
activate AMPK. The cells selleck screening library were ?xed in 4% paraformaldehyde and stained with anti-TIM23 (mouse) and anti-LC3 (rabbit) primary antibodies then Alexa Fluor 488-labeled
donkey anti-mouse IgG and Alexa Fluor 555-labeled donkey anti-rabbit IgG secondary antibodies before analysis by immuno?uorescence microscopy.

Bar, 10 lm. (D)
Quanti?cation of the translocated ULK1 puncta (mean = S.D.; n = 20 cells from three independent experiments;
P < 0.01;
P < 0.001). (E) Cells were treated as in (C), and
then analyzed by electron microscopy. Red asterisks mark mitochondrion-containing mitophagosomes. Red arrows indicate autolysosomes.mitophagy, the phosphorylation of ULK1 at Ser-555 and Ser-317 is
increased, which may be due to the strengthened binding of AMPK
a1 with ULK1 (Fig.