Phosphorylation of CDC25C by AMP-activated protein kinase
mediates a metabolic checkpoint during cell-cycle
G2/M-phase transition
Received for publication, December 11, 2017, and in revised form, Fe
uary 1, 2018 Published, Papers in Press, Fe
uary 21, 2018, DOI XXXXXXXXXX/jbc.RA XXXXXXXXXX
Yuqing Shen‡§, John William Sherman‡, Xuyong Chen‡, and Ruoning Wang‡1
From the ‡Center for Childhood Cancer and Blood Diseases, Hematology/Oncology and BMT, Research Institute at Nationwide
Children’s Hospital, Ohio State University, Columbus, Ohio 43205 and the §Department of Microbiology and Immunology, Key
Laboratory of Developmental Genes and Human Disease, Ministry of Education, Medical School, Southeast University,
Nanjing 210009, China
Edited by Phyllis I. Hanson
From unicellular to multicellular organisms, cell-cycle pro-
gression is tightly coupled to biosynthetic and bioenergetic
demands. Accumulating evidence has demonstrated the G1/S-
phase transition as a key checkpoint where cells respond to thei
metabolic status and commit to replicating the genome. How-
ever, the mechanism underlying the coordination of metabo-
lism and the G2/M-phase transition in mammalian cells remains
unclear. Here, we show that the activation of AMP-activated
protein kinase (AMPK), a highly conserved cellular energy sen-
sor, significantly delays mitosis entry. The cell-cycle G2/M-
phase transition is controlled by mitotic cyclin-dependent
kinase complex (CDC2-cyclin B), which is inactivated by WEE1
family protein kinases and activated by the opposing phospha-
tase CDC25C. AMPK directly phosphorylates CDC25C on ser-
ine 216, a well-conserved inhibitory phosphorylation event,
which has been shown to mediate DNA damage–induced
G2-phase a
est. The acute induction of CDC25C or suppression
of WEE1 partially restores mitosis entry in the context of AMPK
activation. These findings suggest that AMPK-dependent phos-
phorylation of CDC25C orchestrates a metabolic checkpoint fo
the cell-cycle G2/M-phase transition.
Somatic cell-cycle progression involves a doubling and then
equal distribution of cellular components and macromolecules
into the two daughter cells. As such, interphase (G1, S, and G2
phases) represents a long period of cellular growth (accumula-
tion of mass due to anabolic processes), whereas mitosis is the
period of division, which is short and accompanied by meta-
olic suppression (1). Consequently, a fundamental problem in
mammalian cells is coordination of the metabolic status with
cell-cycle progression (2–6). The progression through the G1
phase in the mammalian cell cycle is regulated by growth
facto
mitogen–mediated signals and metabolic status. The
latter remotely resembles a mechanism in yeast known as
START and represents a nutrient-sensing metabolic check-
point (7–11). The signaling network behind the G1-phase met-
abolic checkpoint coordinates the cell-cycle machinery and
metabolic activities, thus ensuring the availability of energy and
nucleotide precursors for genome replication and a timely tran-
sition from G1 to S phase (12–15). Also, it has been suggested
that a sufficient storage of energy and biosynthetic materials
may enable the execution of mitosis in a robust and all-or-none
fashion (16 –19). It is conceivable that a cell size–sensing mech-
anism may play a role in coordinating metabolic status (growth)
and the G2/M-phase transition. This mechanism would allow
cells to keep biosynthetic activity in check, ensuring suffi-
cient biomass accumulation to produce daughter cells with
the proper size (20 –24). These studies implicate the exist-
ence of metabolic checkpoints during the G1/S- and G2/M-
phase transition.
The AMP-activated protein kinase (AMPK)2 complex is a
central signaling node that keeps the cellular metabolic status
in check by sensing changes in cellular AMP and other cellula
metabolites, indicative of energy and nutrient status. Upon its
activation, AMPK acts to maintain ATP homeostasis by rewir-
ing metabolic programs to produce more energy and mean-
while suppressing many energy-consuming cellular processes,
including cell-cycle progression (25, 26). It has been known that
AMPK activation inhibits cell proliferation by increasing p21
and p27, two inhibitors of cyclin-dependent kinase (CDK) com-
plex. Under conditions of insufficient nutrients, such as low
glucose in cell culture medium, AMPK phosphorylates tran-
scription factor p53, and this phosphorylation event mediates
the suppression of G1-phase progression under glucose restric-
tion (27–30). The mammalian target of rapamycin (mTOR), an
This work was supported by National Institute of Health Grant 1R01AI114581,
V-Foundation Grant V XXXXXXXXXX, American Cancer Society Grant 128436-
RSG XXXXXXXXXXLIB, and a research grant from the CancerFree KIDs Foun-
dation (to R .W). The authors declare that they have no conflicts of interest
with the contents of this article. The content is solely the responsibility of
the authors and does not necessarily represent the official views of the
National Institutes of Health.
Author’s Choice—Final version free via Creative Commons CC-BY license.
This article contains Tables S1–S3 and Figs. S1–S5.
1 To whom co
espondence should be addressed. Tel.: XXXXXXXXXX; Fax:
XXXXXXXXXX; E-mail: XXXXXXXXXX.
2 The a
eviations used are: AMPK, AMP-activated protein kinase; CDK,
cyclin-dependent kinase; mTOR, mammalian target of rapamycin; AICAR,
5-aminoimidazole-4-ca
oxamide 1-�-D-ribofuranoside; PI, propidium
iodide; BrdU,
omodeoxyuridine; PPP, pentose phosphate pathway; ERK,
extracellular signal-regulated kinase; ATP�S, adenosine 5�-O-(thiotriphos-
phate); AS, analog-specific; thioP, thiophosphate; 2DG, 2-deoxyglucose;
LKB1, liver kinase B1; DMEM, Dulbecco’s modified Eagle’s medium; GST,
glutathione S-transferase; MBP, maltose-binding protein.
croARTICLE
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evolutionarily conserved protein kinase, integrates environ-
mental cues to coordinately regulate many fundamental cellu-
lar processes, including cell-cycle progression through the G1
phase. AMPK has been reported to directly phosphorylate key
components of mTORC1 and consequently suppress mTORC1
signaling and the G1/S-phase transition (31–36). These find-
ings clearly implicate AMPK as a key player in coupling the
cellular metabolic status to the regulation of the G1/S-phase
transition. However, the robustness of AMPK-dependent reg-
ulation on a myriad of fundamental cellular processes in
esponse to metabolic stress suggests the presence of additional
egulatory steps coupling AMPK and cell-cycle progression,
and the molecular mechanisms behind these unrevealed regu-
latory steps remain to be explored.
The G2/M-phase transition is driven by a series of tightly
egulated and coordinated signaling events that eventually lead
to the activation of CDC2-cyclin B (37–40). Among these
events, the rate-limiting step in directing mitosis entry is the
activation of dual-specificity protein phosphatase CDC25C.
The activation of CDC25C generally involves two steps, initia-
tion and amplification (41, 42). The latter requires an a
ay of
protein kinases that can extensively phosphorylate CDC25C
and change its conformation (43–50). Likewise, the initiation
step of CDC25C activation requires multiple coordinated
events, including dephosphorylation of serine 216, a conserved
inhibitory phosphorylation, dissociation from the inhibito
14-3-3, and change in the subcellular location (51–55). The
amplification step of CDC25C activation is part of a positive-
feedback loop that enables a rapid, robust, and i
eversible
mitosis entry, whereas the initiation step represents a surveil-
lance mechanism that ensures the order and integrity of the
cell-cycle machinery (56). Supporting this idea, the DNA
damage–induced G2-phase checkpoint is largely mediated
through inhibition of CDC25C, thus suppressing CDC2-cyclin
B. Importantly, this is a p53-independent mechanism that is
critical for the DNA damage response in most cancer cells
ecause p53 loss of function is common in cancer cells (57–60).
Because metabolic stress also causes cell-cycle a
est, it is con-
ceivable that CDC25C may also represent a critical target of the
metabolic checkpoint on cell-cycle progression.
In this study, we report a crucial role of AMPK in regulating
the G2/M-phase transition. Unlike AMPK-dependent regula-
tion on the G1/S transition, AMPK activation delays mitosis
entry independently from its regulation on p21, p27, and
mTORC1. Instead, AMPK directly phosphorylates CDC25C on
serine 216, an inhibitory phosphorylation event that has been
previously shown to retain CDC25C in the cytosol and keep it
inactive (51, 53, 54, 61, 62). Either acute overexpression of
CDC25C-S216A mutant or inhibition of WEE1 can reverse
cell-cycle G2-phase a
est imposed by AMPK activation. More-
over, pharmacologic a
ogation of AMPK-mediated cell-cycle
a
est by WEE1 inhibitor induces cell death. These findings
eveal a novel AMPK-dependent metabolic checkpoint on cell-
cycle G2/M transition, and pharmacological a
ogation of this
checkpoint may represent a new therapeutic approach to treat
cancers.
Results and discussion
Activation of AMPK at G2 phase delays mitosis entry
Previous studies have demonstrated an AMPK-dependent
cell-cycle checkpoint at the G1/S-phase boundary, which may
ensure the coordination of DNA synthesis in S phase with the
availability of nutrients for nucleotide biosynthesis in G1 phase
(27, 33). However, it is still unclear whether the G2/M-phase
transition is regulated by AMPK and represents a checkpoint
for the coordination of cell metabolism and cell-cycle progres-
sion. For this, we treated HeLa cells overnight with two mech-
anistically distinct pharmacologic activators of AMPK, 5-
aminoimidazole-4-ca
oxamide 1-�-D-ribofuranoside (AICAR)
or A XXXXXXXXXXA XXXXXXXXXXAICAR is considered as an AMP-mi-
metic compound that directly binds to a nucleotide-binding
pocket in the AMPK� subunit and promotes AMPK kinase
activities; A7 binds to a cleft between the AMPK� and � sub-
units and causes allosteric activation of the AMPK kinase com-
plex (63–66). We found that both AICAR and A7 increased the
percentage of cells in the G1 and G2 phases, as indicated by PI
staining in combination with BrdU incorporation (Fig. 1A). By
contrast, the percentage of cells in mitosis indicated by phos-
phorylation of histone H3 (pH3) is reduced following AICAR
and A7 treatment (Fig. 1A). Notably, the disappearance of BrdU
incorporation in AICAR group is probably due to the substrate
competition between BrdU and AICAR, both of which are nucle-
otide analogs. Next, we repeated the experiment in the presence of
nocodazole, a reversible inhibitor of microtubule polymerization,
which blocks mitosis exit and therefore highlights the changes of
mitotic entry following treatments. Both AICAR and A7 reduced
the percentage of cells in mitosis compared with the control group
(Fig. 1B).
We next applied radiochemical-based approaches to deter-
mine the activity of major catabolic pathways that could fuel the
iosynthetic programs in cells released into G1 phase or G2
phase. We also included cells starved by serum removal as a
control to indicate the baseline metabolic activity. Compared
with cells at G1 phase or serum-starved cells, cells at G2 phase
significantly up-regulated glycolysis, indicated by the detritia-
tion of [5-3H]glucose; glucose consumption via the pentose
phosphate pathway (PPP), indicated by 14CO2 release from
[1-14C]glucose; and glutamine consumption through oxidative
catabolism (glutaminolysis), indicated by 14CO2 release from
[U-14C]glutamine (Fig. 2A). In contrast, both mitochondria-de-
pendent pyruvate oxidation through the trica
oxylic acid
(TCA) cycle, indicated by 14CO2 release from [2-14C]pyruvate,
and fatty acid �-oxidation, indicated by the detritiation of [9,10-
3H]palmitic acid, were comparable among all three groups (Fig.
2B). These data suggest that cells at G2 phase actively engage
glucose and glutamine catabolic programs to meet their bioen-
ergetic and biosynthetic demands.
Next, we sought to determine whether the acute activation of
AMPK at G2 phase would cause a delay