Nutrient starvation elicits an acute autophagic response mediated by Ulk1 dephosphorylation and its subsequent dissociation from AMPK.
ABSTRACT Macroautophagy (herein referred to as autophagy) is an evolutionarily conserved self-digestive process cells adapt to starvation and other stress responses. Upon starvation, autophagy is induced, providing cells with needed nutrient supplies. We report here that Unc-51-like kinase 1 (Ulk1), a key initiator for mammalian autophagy, undergoes dramatic dephosphorylation upon starvation, particularly at serine 638 and serine 758. Phosphorylations of Ulk1 are mediated by mammalian target-of-rapamycin (mTOR) kinase and adenosine monophosphate activated protein kinase (AMPK). AMPK interacts with Ulk1 in a nutrient-dependent manner. Proper phosphorylations on Ulk1 are crucial for Ulk1/AMPK association, as a single serine-to-alanine mutation (S758A) at Ulk1 impairs this interaction. Compared to the wild-type ULK1, this Ulk1-S758A mutant initiates starvation-induced autophagy faster at an early time point, but does not alter the maximum capacity of autophagy when starvation prolongs. This study therefore revealed previously unnoticed acute autophagy response to environmental changes.
- SourceAvailable from: Roberto Bravo[Show abstract] [Hide abstract]
ABSTRACT: Autophagy is a highly regulated catabolic process that involves lysosomal degradation of proteins and organelles, mostly mitochondria, for the maintenance of cellular homeostasis and reduction of metabolic stress. Problems in the execution of this process are linked to different pathological conditions, such as neurodegeneration, aging, and cancer. Many of the proteins that regulate autophagy are either oncogenes or tumor suppressor proteins. Specifically, tumor suppressor genes that negatively regulate mTOR, such as PTEN, AMPK, LKB1, and TSC1/2 stimulate autophagy while, conversely, oncogenes that activate mTOR, such as class I PI3K, Ras, Rheb, and AKT, inhibit autophagy, suggesting that autophagy is a tumor suppressor mechanism. Consistent with this hypothesis, the inhibition of autophagy promotes oxidative stress, genomic instability, and tumorigenesis. Nevertheless, autophagy also functions as a cytoprotective mechanism under stress conditions, including hypoxia and nutrient starvation, that promotes tumor growth and resistance to chemotherapy in established tumors. Here, in this brief review, we will focus the discussion on this ambiguous role of autophagy in the development and progression of cancer.BioMed Research International 01/2014; 2014:603980. · 2.71 Impact Factor
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ABSTRACT: Reactive oxygen species (ROS) have an important role in regulating various cellular processes. Our previous study confirmed that selenite, an anti-tumour agent, triggered apoptosis through the production of ROS in multiple types of cancer cells. In this study, we discovered that ROS also inhibited protective autophagy by decreasing the expression of ULK1, an initiator of autophagy, in selenite-treated NB4 cells. Further experiments demonstrated that p-p53 (S392), a phosphorylation event promoted by p70S6K, bound to the promoter of ULK1 and modulated its expression. Experiments in a mouse tumour model with NB4 cells provided in vivo confirmation of the alterations in the p70S6K/p53/ULK1 axis. Collectively, our results show that ROS inhibited autophagy by downregulating the p70S6K/p53/ULK1 axis in selenite-treated NB4 cells.Cell Death & Disease 11/2014; 5:e1542. · 5.18 Impact Factor
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ABSTRACT: Accumulating evidence reveals that metabolic and cell survival pathways are closely related, sharing common signaling molecules. Hexokinase catalyzes the phosphorylation of glucose, the rate-limiting first step of glycolysis. Hexokinase II (HK-II) is a predominant isoform in insulin-sensitive tissues such as heart, skeletal muscle, and adipose tissues. It is also upregulated in many types of tumors associated with enhanced aerobic glycolysis in tumor cells, the Warburg effect. In addition to the fundamental role in glycolysis, HK-II is increasingly recognized as a component of a survival signaling nexus. This review summarizes recent advances in understanding the protective role of HK-II, controlling cellular growth, preventing mitochondrial death pathway and enhancing autophagy, with a particular focus on the interaction between HK-II and Akt/mTOR pathway to integrate metabolic status with the control of cell survival.Cell Death and Differentiation advance online publication, 17 October 2014; doi:10.1038/cdd.2014.173.Cell Death and Differentiation 10/2014; 22(2). · 8.39 Impact Factor
Nutrient starvation elicits an acute autophagic
response mediated by Ulk1 dephosphorylation
and its subsequent dissociation from AMPK
Libin Shanga, She Chenb, Fenghe Dua, Shen Lib, Liping Zhaoa,b, and Xiaodong Wanga,b,1
aHoward Hughes Medical Institute and Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines
Boulevard, Dallas, TX 75390; and
bNational Institute of Biological Sciences, Zhongguancun Life Science Park, Beijing 102206, China
Contributed by Xiaodong Wang, January 28, 2011 (sent for review December 5, 2010)
Macroautophagy (herein referred to as autophagy) is an evolutio-
narily conserved self-digestive process cells adapt to starvation
and other stress responses. Upon starvation, autophagy is induced,
providing cells with needed nutrient supplies. We report here that
Unc-51-like kinase 1 (Ulk1), a key initiator for mammalian autop-
hagy, undergoes dramatic dephosphorylation upon starvation,
particularly at serine 638 and serine 758. Phosphorylations of Ulk1
are mediated by mammalian target-of-rapamycin (mTOR) kinase
and adenosine monophosphate activated protein kinase (AMPK).
AMPK interacts with Ulk1 in a nutrient-dependent manner. Proper
phosphorylations on Ulk1 are crucial for Ulk1/AMPK association,
as a single serine-to-alanine mutation (S758A) at Ulk1 impairs this
interaction. Compared to the wild-type ULK1, this Ulk1-S758A mu-
tant initiates starvation-induced autophagy faster at an early time
point, but does not alter the maximum capacity of autophagy
unnoticed acute autophagy response to environmental changes.
calcium ∣ LC3 ∣ ATG13 ∣ SILAC ∣ PI3K
changes. Among these, macroautophagy (herein referred to as
autophagy) is an evolutionarily conserved self-digestive process
cells adapt to nutrient starvation (1, 2). Autophagy plays crucial
roles in development, innate immune defense, protein quality
control, tumor suppression, and cell death (3, 4). During autop-
hagy, portions of cytoplasmic materials are engulfed into specia-
lized double-membrane structures to form autophagosomes,
which then fuse with lysosomes to degrade their cargos and re-
generate nutrients (5, 6). This process is highly inducible and
tightly regulated. Under normal growth conditions when nutri-
ents are abundant, autophagy is kept at a basal level mainly
for house-keeping purposes such as degradation of long-lived
proteins and turn-over of damaged cellular organelles; under
stress conditions like nutrient starvation, autophagy is further
induced toprovide cells withadditional internal nutrient supplies.
This induction is largely due to inhibition of TOR (target-of-
rapamycin) complex 1 (TORC1), a kinase complex whose activity
is regulated through integrating upstream PI3K (phosphatidyli-
nositol 3-kinase)/AMPK (adenosine monophosphate activated
protein kinase) activities and other nutrient-sensing signalings
(1, 7, 8).
Initiation of autophagy has been extensively studied in budding
yeast Saccharomyces cerevisiae. Atg1 kinase actively participates
in cytoplasm-to-vacuole targeting pathway under nutrient-rich
condition and switches to induce autophagy upon starvation by
forming an autophagy-initiating complex with Atg13, Atg17,
Atg2, and Atg31 (2). This event is composed of a signaling cas-
cade including inhibition of yeast TORC1, dephosphorylation of
Atg13, increased association between Atg13 and Atg1, and final-
ly, increased Atg1 kinase activity to trigger downstream events. In
higher eukaryotes including mammals, though similar to their
yeast counterpart, detailed machinery and regulation involved
ukaryotic cells have evolved various signaling cascades
and cellular processes in response to rapid environmental
in autophagy initiation have not been investigated until recently.
Unc-51-like kinase 1 (Ulk1), as well as less-studied Ulk2, are two
mammalian functional homologs of yeast Atg1 kinase. Same as
yeast Atg1, Ulk1 kinase interacts with several autophagy-related
partners, including mammalian Atg13 (mAtg13), Atg101, and
FIP200/RB1CC1 (focal adhesion kinase family interacting
protein of 200 kD, or retinoblastoma 1-inducible coiled-coil 1)
Despite these similarities between yeast and mammalian
autophagy-initiating complexes, significant differences, however,
remain between these two systems. FIP200/RB1CC1 and Atg101
are not present in budding yeast S. cerevisiae. As for FIP200/
RB1CC1, it may functionally overlap with both yeast Atg11
and yeast Atg17 (16). Most importantly, mAtg13 constantly
associates with Ulk1 regardless of nutrient availability, which is
different from the starvation-induced association between Atg1
and Atg13 in S. cerevisiae (10–12, 14). Because this dynamic in-
teraction between Atg1 and Atg13 is a key regulatory step for
yeast autophagy initiation and is lacked in Ulk1 complex, the
mammalian autophagy needs to be initiated in a way that, at least
partially, differs from that in yeast.
Functional outputs of autophagy are generally considered as
accumulative and relatively slow processes. Variety of assays
monitors autophagic readouts in wide time windows, normally
hours after treatments that perturb cellular signalings. Yet, up-
stream regulations of autophagy are mainly through kinase cas-
cades that are inherently prompt. It is therefore conceptually
plausible that autophagy can be detected at earlier time points,
especially when cultured cells are treated with harsh conditions
such as total medium/serum withdrawal.
In this report, we address these issues by focusing on the phos-
phorylation status of Ulk1. Our data reveal Ulk1 is globally
dephosphorylated upon starvation. 13 phosphorylation sites were
mapped. AMPK, a key energy-surveillance kinase complex that is
considered to act upstream of mTOR, directly associates with
Ulk1 complex in a nutrient-dependent manner. Ulk1/AMPK
association is determined by Ulk1 phosphorylation and conse-
quently, by nutrient availability. This interaction may serve as a
sequestering reservoir to confine Ulk1 during normal growth con-
dition, and prepares cells to promptly initiate autophagy upon
Author contributions: L.S., S.C., F.D., and X.W. designed research; L.S., S.C., and F.D.
performed research; L.S., S.L., and L.Z. contributed new reagents/analytic tools; L.S.,
S.C., and X.W. analyzed data; and L.S. and X.W. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
whomcorrespondence should beaddressed.E-mail:xiaodong.wang@
This article contains supporting information online at www.pnas.org/lookup/suppl/
4788–4793 ∣ PNAS ∣ March 22, 2011 ∣ vol. 108 ∣ no. 12www.pnas.org/cgi/doi/10.1073/pnas.1100844108
Ulk1 Is Rapidly Dephosphorylated upon Starvation. Recent studies
show mammalian TORC1 (mTORC1), the upstream regulator
of autophagy, may directly interact with Ulk1 complex under
nutrient-rich (fed) conditions and possibly prevent autophagy
initiation through inhibitory phosphorylations (10, 12, 14). More
specific information including critical phosphorylation residues
has not been provided and functional consequences not charac-
terized. We used SILAC (stable isotope labeling with amino
acids in cell culture) to quantitatively monitor phosphorylation
changes of Ulk1 complex in response to nutrient availability.
Unexpectedly, mAtg13 showed little change before and after star-
vation. Total phosphorylation levels of mAtg13 were low when
cells were fed and stayed largely unaltered after starvation
(Table S1). In contrast, Ulk1 was extensively phosphorylated at
many residues under nutrient-rich condition and dramatically
dephosphorylated upon medium withdrawal (Table 1). These
results suggest that instead of mAtg13, Ulk1 could be the major
regulatory component during mammalian autophagy initiation.
In particular, SILAC experiment observed more than 10-fold
decreases of phosphorylation at serine 638 and serine 758 of
Ulk1, the most significant changes among all phosphorylation
sites identified. Phospho-specific antibodies against these two
sites were generated and the phosphorylation statuses of these
two sites in response to nutrient availability were confirmed. Both
serine 638 and serine 758 of Ulk1 were dephosphorylated after
starvation as verified by Western blotting (Fig. S1A). Mutating
these two residues to alanine (S638/758A) mimics dephosphory-
lation status, as phospho-specific antibodies could no longer
pick up signal on mutant proteins. Mutating these two residues
to aspartic acid (S638/758D) could not properly mimic phosphor-
ylation status of these two sites; the S638/758D mutant behaved
similarly to the S638/758A mutant as tested by the phospho-
specific antibodies (Fig. S1B).
Phosphorylations of Ulk1 at Serine 638 and Serine 758 Are Differen-
tially Regulated. Time course experiments were performed to
monitor dynamics of phosphorylation changes at serine 638 and
serine 758 in response to nutrient availability. Serine 638 was
found to respond faster than serine 758. As shown in Fig. 1A,
5 min after medium withdrawal, serine 638 was largely depho-
sphorylated. For serine 758, this process took 30 min. Similarly,
when cells were replenished with medium, serine 638 was rapidly
rephosphorylated in 5 min, while it took 30 min for serine 758 to
be phosphorylated again.
Phosphorylations at serine 638 and serine 758 exhibited differ-
ent kinetics, suggesting these two sites are regulated differently.
We next investigated what are the specific nutritional triggers for
phosphorylations at these two sites. As shown in Fig. 1B, DMEM
(Dulbecco’s modified eagle medium) withdrawal remarkably
decreases phosphorylation at serine 638, while serum withdrawal
has a minor effect on this residue. Nevertheless, ULK1 protein
was downshifted. In contrast, phosphorylation at serine 758 was
severely affected by either serum or DMEM withdrawal.
Each component from DMEM was further screened for its
role in impact ULK1 phosphorylation (for DMEM formula,
see Table S2). Unexpectedly, among all components in DMEM,
calcium showed the biggest effect on serine 638 phosphoryla-
tion. Simply adding calcium to total starvation medium largely
restored phosphorylation of Ulk1 at this site (Fig. 1C, compare
lane 3 with lane 2). Single calcium drop-out from DMEM led to
dephosphorylation at serine 638, but not to the same extent as in
total starvation medium. These results suggest calcium is critical
for proper phosphorylation of Ulk1 at serine 638, yet there
are other nutritional factors in DMEM which also contribute.
In contrast, phosphorylation of Ulk1 at serine 758 is not influ-
enced by calcium availability.
mTOR Mediates Phosphorylation at both Serine 638 and Serine 758,
that both serine 638 and serine 758 are regulated by multiple
nutrients made us suspect the involvement of mTOR kinase,
which can integrate various nutritional signals and has been
reported to interact with Ulk1. Indeed, rapamycin treatment
induced rapid dephosphorylation at serine 638 and serine 758.
Both sites started to be dephosphorylated 10 min after treat-
ment (Fig. 2A). Furthermore, knockdown of mTOR also led
to dephosphorylation at these two sites (Fig. 2B). We conclude
that mTOR signaling is required for phosphorylations at both
serine 638 and serine 758 of Ulk1.
Calcium signal only affects phosphorylation at serine 638,
suggesting that this residue is also under regulation of other
Table 1. Quantification of the Ulk1 phosphorylated peptides
reveals that phosphorylation of Ulk1 is globally decreased after
Phospho-sites*Phosphorylation ratio (fed/starved) SD
Thirteen phosphorylated residues were identified. Quantitative changes
of phosphorylation level were recorded by SILAC. Serine 638 and serine 758
showed more than 10-fold decrease in phosphorylation level upon
starvation. See also Fig. S3.
530 120 5 30
- serum- DMEM
total starvation + Ca2+
differently. (A) Dynamics of Ulk1 phosphorylation. U2OS cells were starved in
total starvation medium (HBSS solution containing 1% rich medium), and
then replenished with rich medium (DMEM plus 10% dialyzed FBS) for dura-
tions as indicated. Cell extracts were analyzed by Western blotting to visua-
lize phosphorylations of Ulk1 at serine 638 and serine 758. (B) U2OS cells were
treated for 2 h as indicated. For serum withdrawal, cells were cultured in
DMEM without FBS; for DMEM withdrawal, cells were cultured using 10%
dialyzed FBS in HBSS. Phosphorylations of Ulk1 at Ser638 and Ser758 were
analyzed using phospho-specific antibodies against these two sites. (C) Cal-
cium is crucial for phosphorylation of Ulk1 at serine 638, but not serine 758.
U2OS cells are treated for 2 h as indicated. Lane 3: calcium was added
into total starvation medium to reach the same calcium concentration as
in regular DMEM (0.2 g∕L); Lane 4: regular DMEM medium (containing
10% dialyzed FBS) with single drop-out of calcium. See also Fig. S1.
Phosphorylations of Ulk1 at serine 638 and serine 758 were regulated
Shang et al.PNAS
March 22, 2011
kinase(s) besides mTOR. We noticed that knockdown of either
AMPKα1/α2 or AMPKβ1/β2 subunits led to dephosphorylation
at serine 638 but not at serine 758 (Fig. 2C). Moreover, in any
nutrient condition tested, there is a fine correlation between
serine 638 phosphorylation and the kinase activity of AMPK
as indicated by the phosphorylation at threonine 172 of AMPKα,
and at serine 79 of acetyl-CoA carboxylase (ACC), a well studied
AMPK substrate (Fig. 2D). Taken together, these results indicate
that besides mTOR, AMPK signaling is also required for the
phosphorylation of Ulk1 at serine 638.
Nutrient-Dependent Phosphorylation of Ulk1 Is Required for Ulk1/
AMPK Interaction. We next investigated whether phosphoryla-
glutinin (HA) tandem immunoprecipitation (IP) assays were
performed in HeLa cells that stably express Flag-HA-Ulk1. The
identified mAtg13, FIP200, and Atg101 to be constantly associated
only under nutrient-rich condition and dissociated from Ulk1
after starvation. Another AMPK subunit AMPKβ did not stain
with silver but was indentified there as well by Western blotting
(see below). We also performed Flag-HA tandem IP in HeLa cells
Atg101 were successfully pulled down; however, mAtg13 did not
interact with AMPK subunit under either fed or starved condition
(Fig. 3A). Similar association patterns were confirmed in HEK
293Tcells: Ulk1 interacted strongly with all three AMPK subunits
(AMPKα, AMPKβ, and AMPKγ), while mAtg13 did not associate
with any subunit of AMPK complex (Fig. S2, compare A with B).
We then used SILAC to further confirm these observed
changes in ULK1 complexes. Amount of Ulk1-associated
AMPKα and AMPKγ1 decreased the most when medium was
withdrawn (Table S3). Association between Ulk1 and mAtg13
only slightly increased. Associations of Ulk1 with FIP200 and
Atg101 stayed largely unchanged.
Ulk1 did not interact with constitutively active form of AMP-
Kα1 (aa1 to aa312) (Fig. S2C). Neither did Ulk1 interact with
full-length AMPKα is required for its association with Ulk1
(Fig. S2 D and E). Kinase activity of AMPK seems not required
for its interaction with Ulk1, as Ulk1 was able to interact with
kinase-dead form of AMPKα in the same nutrient-dependent
manner (Fig. S2F). As to Ulk1, kinase domain of Ulk1 (aa1—
a278) is not required for its interaction with AMPK (Fig. S2G).
Association between AMPK and the kinase-domain-deleted
Ulk1 (aa279—C-terminal end) was still regulated by nutrient
availability (Fig. S2G, compare lane 6 with lane 5).
Interestingly, AMPK started to dissociate from Ulk1 5 min
after starvation, and completely reassociated with Ulk1 within
30 min after medium replenishment (Fig. 3B). This result is in
accordance with the time course experiment for Ulk1 phosphor-
ylation. A series of Ulk1 mutants were then generated to test
whether Ulk1/AMPK dissociation upon medium withdrawal is
due to dephosphorylation of Ulk1 at these sites. As shown in
Fig. S3, mutations at most of residues did not alter Ulk1/AMPK
interaction. However, phosphorylation of Ulk1 at serine 758, one
of the most regulated sites as identified by SILAC, is critical for
AMPK association. Single mutation at this site (Ulk1-S758A) im-
paired Ulk1/AMPK interaction, but the residue interaction was
still regulated by starvation (Fig. 3C). In contrast, single mutation
at serine 638 (Ulk1-S638A), the other most regulated site, did not
alter Ulk1/AMPK interaction, but it helped further dissociate
Ulk1/AMPK when combined with the S758A mutation (Fig. 3D).
Ulk1/AMPK Dissociation Primes Cells for Faster Response to Starva-
tion-Induced Autophagy. Both SILAC and Western blotting data
show AMPK associates with Ulk1 but not mAtg13. Recent stu-
dies also reported that various AMPK subunits are able to inter-
act with mammalian autophagy-initiating factors such as Ulk1/2,
FIP200, and Atg101, but not mAtg13 (17). These observations
raise a possibility that Ulk1 may exist in two mutually exclusive
complexes: both will have core components such as Ulk1/2,
FIP200 and Atg101, and either mAtg13 or AMPK bind to these
core factors. Both may contribute to autophagy induction and it is
therefore difficult to characterize the functional outputs from
one particular complex in the presence of the other. Indeed, for
autophagy level as tested by long-lived protein degradation
(LLPD) assay, no significant difference was observed between
wild-type Ulk1 and the S758A mutant defective in AMPK asso-
ciation (Fig. S4 D–F).
We tried to resolve this issue by knocking down mAtg13 and
therefore eliminating the activity generated from the mAtg13
complex. Interestingly, absence of mAtg13 led to dramatic Ulk1
destabilization (Fig. S4 A and B; also see in ref. 12). We then used
this cellular background (absence of both Ulk1 and mAtg13) to
study Ulk1/AMPK interaction and performed rescue experiments
with either wild-type Ulk1 or Ulk1-S758A mutant. Autophagy is
dampened if protein level of mAtg13 and/or Ulk1 is decreased.
Nonetheless, LLPD assay showed that in the absence of mAtg13,
autophagy could still be greatly induced when cells were starved.
In the first 10 min during starvation, U2OS cells expressing Ulk1-
S758A mutant induced much more protein degradation than
those expressing wild-type Ulk1 (Fig. 4A). Thirty minutes after
starvation, the mutant still exhibited more than 20% excess of
activity compared to the wild type (Fig. S4G). This difference
in LLPD between wild-type Ulk1 and S758A mutant disappeared
after prolonged starvation for 120 min (Fig. S4H). These results
differently by mTOR and AMPK. (A) Rapamycin treatment induced depho-
sphorylation of Ulk1 at both serine 638 and serine 758. U2OS cells were
cultured in rich medium and rapamycin was added (100 nM). Cells were then
collected at given time points after rapamycin treatment. (B) Knockdown of
mTOR induced dephosphorylation of Ukl1 at both serine 638 and serine 758.
U2OS cells were transfected with either control siRNA (Luc) or mTOR siRNA
oligos. 72 h after transfection, cells were collected and extracts analyzed by
Western blotting. (C) Knockdown of AMPK induced dephosphorylation of
Ulk1 at serine 638, but not serine 758. U2OS stable cell lines that inducibly
knock down AMPKα (α1 and α2) or AMPKβ (β1 and β2) upon addition of
doxycycline (Dox) were generated. 72 h after addition of Dox, phosphoryla-
tions of Ulk1 at Ser638 and Ser758 were visualized by Western blotting.
(D) AMPK activity correlates with phosphorylation of Ulk1 at serine 638 but
ted for 2 h as indicated and cell extracts were analyzed by Western blotting.
Phosphorylations of Ulk1 at serine 638 and serine 758 were mediated
www.pnas.org/cgi/doi/10.1073/pnas.1100844108 Shang et al.
correlate with the dynamics between Ulk1 and AMPK interac-
tion, as it takes about 30 min of starvation for wild-type Ulk1
to dissociate with AMPK. After that time point, wild-type Ulk1
should behave the same as the mutant in terms of AMPK-
association pattern and consequent functional outputs.
The Class III PI3K, hVps34, phosphorylates the inositol ring
of phosphatidylinositol (PI) at the D3 position to generate PI3P,
a step essential for autophagosome formation. The hVps34
proteins from cells expressing Ulk1-S758A mutant has higher
in vitro activity compared to those from cells expressing wild-type
Ulk1 protein under fed condition (Fig. 4B). This result indicates
that the Ulk1 mutant defective in AMPK association may better
prime cells for autophagy induction with higher Class III PI3K
activity. Upon starvation, the PI3K activity of hVps34 further
increased, yet differences between the wild type and the mutant
became less obvious as starvation prolonged (Table S4).
Microtubule associated protein light chain 3 (LC3) is the
mammalian homologue of yeast Atg8 protein. During autophagy,
cytoplasmic LC3 (LC3I) is translocated to autophagosomes,
where LC3II is generated by proteolysis and lipidation at its C
terminus. This conversion of LC3I to LC3II represents activation
of autophagy. Thirty minutes after starvation, cells expressing
Ulk1-S758A mutant induced more LC3II conversion as indicated
by increased ratio of LC3II to LC3I, while the wild-type control
stays largely unchanged (Fig. 4C). Prolonged starvation (120 min)
abolishes this difference of LC3II/LC3I ratio between the wild
type and the mutant.
Taken together, these data show Ulk1-S758A mutant defective
in Ulk1/AMPK interaction initiates starvation-induced autop-
hagy faster at an early time point, but does not change the
Proper Phosphorylation at Serine 638 Facilitates Phosphorylation at
Serine 758 and Proper Ulk1/AMPK-Association. Alteration in serine
638, the other most regulated residue besides serine 758, does
not affect apparent Ulk1/AMPK-association. Consequently, it
is difficult to observe the functional impacts of serine 638 phos-
phorylation toward autophagy using LLPD or other assays.
Instead, because serine 638 always responds to nutrient signals
faster compared to serine 758 as shown in Fig. 1A, we asked
whether phosphorylation at serine 638 facilitates such change at
serine 758. The experiment was carried out in U2OS cells in
which endogenous Ulk1 was stably knocked down. The cells were
then rescued with either wild-type Ulk1 or Ulk1-S638A mutant.
No obvious difference was observed between the wild type and
the mutant in terms of dephosphorylation rate at serine 758
upon starvation (Fig. 4D). However, when cells were replenished
with rich medium, rephosphorylation at serine 758 was much
stronger in the wild-type background compared to that in the
S638A mutation background. This result indicates proper phos-
phorylation at serine 638 promotes faster recovery of phosphor-
ylation at serine 758 (Fig. 4D, compare lane 9 and 10 with lane 4
and 5). In accordance with these dynamics of phosphorylation,
reassociation between Ulk1 and AMPK was also decreased in
S638A mutation background (Fig. 4E, compare lane 6 with
lane 5). Therefore, serine 638 may indirectly contribute to better
regulation of autophagy by helping proper phosphorylation of
serine 758 and Ulk1/AMPK interaction.
We discovered Ulk1 undergoes dramatic dephosphorylation
upon starvation, particularly at serine 638 and serine 758. Phos-
phorylation of Ulk1 is regulated by mTOR and AMPK, and is
crucial for Ulk1/AMPK association. Ulk1 dissociates with AMPK
when cells are deprived of nutrients. A single serine-to-alanine
mutation (S758A) on Ulk1 impairs Ulk1/AMPK interaction.
Upon starvation, this mutant Ulk1 induces autophagy much
faster compared to the wild type.
Our report presented here reveals several previously unknown
regulatory steps occurred on Ulk1 during starvation-induced
availability. (A) Only Ulk1, but not mAtg13, associates with
AMPK when cells are fed. In HeLa cells, Flag-HA-Ulk1, and
Flag-HA-Atg13 stable lines were generated. Cells were trea-
ted for 2 h with either rich medium or total starvation med-
ium. Flag-HA tandem IP assays were performed using cell
extracts prepared from above treatments. Proteins in the fi-
nal eluates were separated with 4–12% SDS-PAGE gel and
were visualized by silver staining. Arrows indicates major
coimmunoprecipitated proteins identified by MS. (B) Dy-
namics of Ulk1/AMPK interaction in response to nutrient
availability. Cells were starved and repleted with rich med-
ium for durations as indicated. Flag IP assays were then per-
formed. Ulk1/AMPK association patterns were analyzed by
Western blotting. (C) Phosphorylation of Ulk1 at serine 758
was required for Ulk1/AMPK association. HEK 293T cells
were transfected with either wild-type Flag-Ulk1 or Flag-
Ulk1 mutants as indicated, and treated for 2 h in rich or total
starvation medium. Cell extracts were immunoprecipitated
with anti-Flag beads, and the eluates were analyzed by Wes-
tern blotting. (D) Phosphorylation of Ulk1 at serine 638 was
not required for Ulk1/AMPK association. Experiments were
performed the same as in (C) with either wild-type Ulk1 or
indicated mutants. See also Fig. S2.
Ulk1 interacts with AMPK in response to nutrient
Shang et al.PNAS
March 22, 2011
autophagy. As shown schematically in Fig. 5, Ulk1 is hyper-phos-
phorylated at many serine/threonine residues including S638
and S758. Upon starvation, serine 638 is firstly dephosphorylated
then serine 758 follows. Dephosphorylation at serine 758 leads
Ulk1 to dissociate from AMPK and become more active in au-
tophagy induction. When cells are replenished with nutrients,
mTOR is reactivated and phosphorylates Ulk1 at multiple sites
such as S638 and S758. Proper phosphorylation of Ulk1 then
leads to Ulk1/AMPK association. Though kinase activity of
AMPK is considered relatively low under nutrient-rich condition,
when in close proximity, AMPK may help maintain phosphoryla-
tion of Ulk1 at serine 638 and strengthen its association with
AMPK has basal level activity when cells are fed and will be
further activated upon aminoacid starvationor glucose starvation
(Fig. 2D, compare lane 6 and 7 with lane 1). Interestingly, calcium
seems to be crucial for AMPK to properly exhibit its kinase activ-
ity. Basal AMPK activity will be further decreased if calcium is
removed from rich medium (Fig. 2D, compare lane 5 with lane
1).Thisresult explainswhytotalmedium withdrawal,theharshest
starvation condition, leads to lower AMPK activity, because
calcium was also removed under this circumstance (Fig. 2D, com-
pare lane 2 with lane 1). The kinase activity of AMPK may also
contribute to autophagy induction besides classical AMPK-TSC1/
2-mTOR signaling, as suggested recently by J W Lee et al. that
AMPK can lift the inhibitory effect of mTOR on Ulk1 by phos-
phorylating raptor, the key adaptor in mTORC1 (18). Taken to-
gether, weenvision AMPKmay have dual roles toward autophagy
regulation: it promotes autophagy when cells are starved as pre-
viously well documented, while suppressing autophagy when cells
are fed by forming a complex with and confining portions of Ulk1.
Functional readouts of Ulk1/AMPK interaction cannot be well
detected in the presence of high “background noise” due to the
existence of too much Ulk1/mAtg13 complex. We resolved this
issue by knocking down mAtg13. Paradoxically, it seems the pre-
sence of mAtg13 facilitates efficient phosphorylations of Ulk1
(Fig. S4C). Therefore knockout (or knockdown) of mAtg13
may impair Ulk1/AMPK interaction due to less phosphorylation
of Ulk1 at serine 758. This result leads to a situation where
presence of mAtg13 brings high background noise, while absence
of mAtg13 brings less Ulk1/AMPK interaction. In either way, it
is difficult to pinpoint the actual contribution of Ulk1/AMPK
interaction toward autophagy induction. Moreover, Ulk1/AMPK
interaction is promptly regulated. The rapid kinetics makes
most of qualitative assays unsuitable for monitoring autophagic
effects caused by Ulk1/AMPK interaction. If nutrient deprivation
prolongs, the differences between wild-type Ulk1 and AMPK-
are fed, Ulk1 is hyper-phosphorylated at serine 638 and serine 758. Upon star-
vation, serine 638 is firstly dephosphorylated; then followed by dephosphor-
ylation at serine 758. Dephosphorylation of Ulk1 leads to dissociation of Ulk1/
AMPK. When cells are replenished with rich medium, mTOR is activated; it
phosphorylates serine 638 and serine 758. The phosphorylation of Ulk1 at
serine 758 then leads to reassociation between Ulk1 and AMPK. When in
close proximity, AMPK functions to maintain phosphorylation Ulk1 at serine
Model of Ulk1 phosphorylation in response to nutrients. When cells
638 and serine 758. (A) LLPD assays showed Ulk1-S758A initiates
starvation-induced autophagy faster compared to wild-type Ulk1
(*P ¼ 0.003 in one-tailed Student’s t-test with equal variances,
n ¼ 3). Error bar represents SD. U2OS stable cell line that inducibly
knock downmAtg13 upon addition ofDoxwas generated. Absence
of mAtg13 also led to absence of Ulk1. 72 h after addition of Dox,
cells were transfected with either wild-type Ulk1 orUlk1-S758A con-
struct, and cultured for another 48 h in rich medium containing no
3H-labeled but excessive cold leucine. Cells were than cultured in
either rich or total starvation medium for 10 min. Percentages of
3H-labeled leucine released from cells into medium were calculated.
(B) In either fed condition or10min after starvation,cells expressing
Ulk1-S758A exhibited higher hVps34 activity compared to the wild
type. Using same cells and same transfections as in (A), kinase
activity of hVps34 was assayed by quantifying conversion from PI
to PI3P in given time as described in Materials and Methods. Error
and 30 or 120 min after starvation; lower: Quantitative analysis of
the ratio of LC3II to LC3I (n ¼ 3). (D) In Ulk1-S638A background, ser-
ine 758 could not be properly rephosphorylated 60 min after med-
ium replenishment, while in wild-type Ulk1 background this process
took less than 15 min. U2OS stable cell line that inducibly knock
downUlk1uponaddition ofDox was generated.72h after addition
of Dox, cells were transfected with either wild-type Ulk1 or Ulk1-
S638A construct, and cultured for another 48 h in rich medium
to allow expression. Cells were then treated as indicated. (E) In
S638A background, Ulk1/AMPK reassociation is slowed down. Using
same cell line and transfections as in (D), cells were starved for 1 h
and recovered for 20 min. Extracts were immunoprecipitated
with anti-Flag beads, and the eluates were analyzed by Western
blotting. See also Fig. S4.
Functional outputs of Ulk1 phosphorylations at serine
www.pnas.org/cgi/doi/10.1073/pnas.1100844108Shang et al.
association-defective Ulk1 mutant (Ulk1-S758A) gradually disap-
pears. Time windows for studying function of Ulk1/AMPK inter-
action is limited, only at the very early time point of a given
environmental change. In our hands, it is less than 30 min after
starvation. Fortunately, despite all these difficulties, the func-
tional differences between wild-type and S758A mutant Ulk1
can be distinguished by using more quantitative approaches, such
as LLPD assay and ELISA for hVps34 activity.
Though autophagy is in general conserved from yeast to
human (particularly in later stages including membrane expan-
sion, autophagosome formation, fusion with lysosome, and recy-
cling), regulations of autophagy initiation in mammals are much
more complicated and differ from yeast in many aspects. In this
study, we try to elaborate this complexity by discovering a more
rapid form of autophagy induction. For prolonged starvation,
there is little difference between mutant Ulk1-S758A and the
wild type, indicating interaction between Ulk1/AMPK mainly
responds to acute nutritional changes. This acute mechanism
not only functions as prompt response to nutrient deprivation,
but may also be a crucial exit strategy to effectively down-regulate
autophagy when cells leave harsh environments and need to
resume proper growth/proliferation. In summary, with this layer
of regulation, mammalian autophagy is capable of responding to
environmental changes more rapidly than previously considered.
Materials and Methods
Mass Spectrometry Analysis. The protein gel bands were digested in gel with
sequencing grade trypsin (10 ng∕μL trypsin, 50 mM ammonium bicarbonate,
pH8.0) overnight at 37 °C. Peptides were extracted with 5% acetic acid/50%
acetonitrile and 0.1% acetic acid/75% acetonitrile sequentially and then con-
centrated to ∼20 μL. The extracted peptides were separated by a homemade
analytical capillary column (50 μm 10 cm) packed with C18 reverse phase
material (YMC 5 μm spherical particles). An Agilent 1100 series binary pump
was used to generate HPLC gradient as follows: 0%–5% B in 5 min, 5%–40%
B in 25 min, and 40%–100% B in 15 min (A ¼ 0.1 M acetic acid in water,
B ¼ 0.1 M acetic acid/80% methanol). The eluted peptides were sprayed into
a QSTAR XL mass spectrometer (MDS SCIEX) equipped with a nano-ESI ion
source. The mass spectrometer was operated in Information Dependent
Acquisition mode. Ion spray voltage was 2.1 KV. The MS scan was from
m∕z 400 to 2,000. From each MS scan, top three most abundant peaks were
selected for MS/MS (tandem mass spectrometry) fragmentation. Each scan
was accumulated for 1 sec. The dynamic exclusion time was set as 20 sec.
Protein Database Search and Peptide Quantification. The mass spectra were
searched against IPI-human database on an in-house Mascot server (Version
2.2, Matrix Science Ltd.). Carbamidomethyl on cysteine was set as fixed
modification. Variable modifications included oxidation on methionine,
phosphorylation on serine, threonine, and tyrosine. Quantification mode
was SILAC double labeled [13C6, 15N2]-lysine and [13C6, 15N4]-arginine. A
maximum of three miscleavages was allowed for search. Mass tolerance
was 0.2 Da for precursor ion, 0.25 Da for fragment ions. Mascot search results
were imported to the open source software MSQuant (http://msquant.
sourceforge.net) to calculate ratios of the heavy/light peptide pairs. Quanti-
fication results from each peptide were manually checked to ensure their
Long-Lived Protein Degradation Assay. Detailed procedure has been described
previously(19). In brief, U2OS cells were plated in 6-well dish with a density of
40,000 cells per well and let grow for 3 d. The growth medium was leucine-
drop-out DMEM (USBiological) supplied by addition of 65 μM cold leucine
and 1 μCi∕mL3H-labeled leucine (Perkin Elmer). In this assay, dialyzed FBS
was used to eliminate leucine from other source. The cells then were washed
with regular DMEM medium and let grow for another 48 h in regular DMEM
medium containing 2 mM cold leucine. On the next day, the cells were
washed and treated with either regular DMEM or total starvation medium
for time durations as indicated (10 min, 30 min, or 120 min). After treatment,
for the growth medium, we collected 1 mL from each well and added with
112 μL of 100% trichloroacetic acid (TCA) to reach 10% TCA concentration;
we centrifuged the samples at 12;000× g for 2 min; and took 400 μL of super-
natant to measure3H readout with scintillation counter. For the cells, we first
washed with DPBS, then 1 mL of 10% TCA was added to each well and we
incubated the cells at room temperature for 5 min; after fixation, the cells
were washed with 10% TCA and dissolved in 0.2 M NaOH; and we took
400 μL of the lysates to measure3H readout. We calculated the total3H read-
out in the medium and total3H readout within the cells. Then we calculated
the ratio of released3H inthe medium to thetotal3H readout; this represents
the degradation percentage in a given time duration.
Phosphatidylinositol 3-Phosphate (PI3P) Quantification (ELISA for hVps34
Activity). For details, refer to the protocol for class III PI3-Kinase kit (96-well
ELISA assay for detection of PI3P) by Echelon Inc. In brief, endogenous hVps34
proteins were immunoprecipitated and incubated with PI substrate for 2 h to
catalyze the synthesis of PI3P. The reaction (containing synthesized PI3P
product) was then added to 96-well coated with PI3P. Detector proteins that
recognize PI3P were also added to the wells. In the next 2 h, the PI3P immo-
bilized on the 96-well plate competed with free PI3P in the reaction for
detector binding. The amount of detector proteins bound to the plate is
determined through colorimetric detection at 450 nm absorbance. The read-
ing was inversely proportional to the amount of PI3P produced by hVps34.
ACKNOWLEDGMENTS. We thank Dr. K-L Guan for kindly providing cDNA
and various constructs of AMPK subunits. Drs. Lai Wang, Wenhua Gao, and
Sudan He for helpful discussions. The work is also supported by the Welch
Foundation Grant I-1412 and the National High Technology Projects 863 from
Chinese Ministry of Science and Technology.
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Shang et al.PNAS
March 22, 2011