1) what isthe main point or purpose of this paper? 2) what research question or hypothesis is being tested? 13) Identify and describe a pivotal experiment from the paper and what was learned in that specific experiment. 4) identify a weakness in the paper and design a control or series of control experiments that could improve that part of the paper; and 5) if this was your research, what experiment (with controls) would you do next?
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, February 1, 2018 Published, Papers in Press, February 21, 2018, DOI 10.1074/jbc.RA117.001379 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 their 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 arrest. 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 for 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- bolic 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 factor/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 cellular 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 V2014-001, American Cancer Society Grant 128436- RSG-15-180-01-LIB, 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 correspondence should be addressed. Tel.: 614-335-2980; Fax: 614-722-5895; E-mail:
[email protected]. 2 The abbreviations used are: AMPK, AMP-activated protein kinase; CDK, cyclin-dependent kinase; mTOR, mammalian target of rapamycin; AICAR, 5-aminoimidazole-4-carboxamide 1-�-D-ribofuranoside; PI, propidium iodide; BrdU, bromodeoxyuridine; 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 Author’s Choice J. Biol. Chem. (2018) 293(14) 5185–5199 5185 © 2018 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. by guest on A pril 14, 2018 http://w w w .jbc.org/ D ow nloaded from http://www.jbc.org/cgi/content/full/RA117.001379/DC1 mailto:
[email protected] http://crossmark.crossref.org/dialog/?doi=10.1074/jbc.RA117.001379&domain=pdf&date_stamp=2018-2-21 http://www.jbc.org/ 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 response to metabolic stress suggests the presence of additional regulatory 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 regulated 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 array 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 inhibitor 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 irreversible 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 because p53 loss of function is common in cancer cells (57–60). Because metabolic stress also causes cell-cycle arrest, 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 arrest imposed by AMPK activation. More- over, pharmacologic abrogation of AMPK-mediated cell-cycle arrest by WEE1 inhibitor induces cell death. These findings reveal a novel AMPK-dependent metabolic checkpoint on cell- cycle G2/M transition, and pharmacological abrogation 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-carboxamide 1-�-D-ribofuranoside (AICAR) or A 769662 (A7) (63). AICAR 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 biosynthetic 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 tricarboxylic 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