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• W3152114230 abstract "•AMPKα2 is selectively activated during mitosis by CDK1 and PLK1•A conserved motif in AMPKα2 determines its interaction with and activation by PLK1•Mitotic AMPK activation contributes to maintain genomic stability in normal mitosis AMP-activated protein kinase (AMPK) senses energy status and impacts energy-consuming events by initiating metabolism regulatory signals in cells. Accumulating evidences suggest a role of AMPK in mitosis regulation, but the mechanism of mitotic AMPK activation and function remains elusive. Here we report that AMPKα2, but not AMPKα1, is sequentially phosphorylated and activated by CDK1 and PLK1, which enables AMPKα2 to accurately guide chromosome segregation in mitosis. Phosphorylation at Thr485 by activated CDK1-Cyclin B1 brings the ST-stretch of AMPKα2 to the Polo box domain of PLK1 for subsequent Thr172 phosphorylation by PLK1. Inserting of the AMPKα2 ST-stretch into AMPKα1, which lacks the ST-stretch, can correct mitotic chromosome segregation defects in AMPKα2-depleted cells. These findings uncovered a specific signaling cascade integrating sequential phosphorylation by CDK1 and PLK1 of AMPKα2 with mitosis to maintain genomic stability, thus defining an isoform-specific AMPKα2 function, which will facilitate future research on energy sensing in mitosis. AMP-activated protein kinase (AMPK) senses energy status and impacts energy-consuming events by initiating metabolism regulatory signals in cells. Accumulating evidences suggest a role of AMPK in mitosis regulation, but the mechanism of mitotic AMPK activation and function remains elusive. Here we report that AMPKα2, but not AMPKα1, is sequentially phosphorylated and activated by CDK1 and PLK1, which enables AMPKα2 to accurately guide chromosome segregation in mitosis. Phosphorylation at Thr485 by activated CDK1-Cyclin B1 brings the ST-stretch of AMPKα2 to the Polo box domain of PLK1 for subsequent Thr172 phosphorylation by PLK1. Inserting of the AMPKα2 ST-stretch into AMPKα1, which lacks the ST-stretch, can correct mitotic chromosome segregation defects in AMPKα2-depleted cells. These findings uncovered a specific signaling cascade integrating sequential phosphorylation by CDK1 and PLK1 of AMPKα2 with mitosis to maintain genomic stability, thus defining an isoform-specific AMPKα2 function, which will facilitate future research on energy sensing in mitosis. Mitosis is an energy-consuming process essential for equal chromosome segregation. Accurate chromosome separation safeguards faithful inheritance of genetic information from a mother cell to two daughter cells (Godek et al., 2015Godek K.M. Kabeche L. Compton D.A. Regulation of kinetochore-microtubule attachments through homeostatic control during mitosis.Nat. Rev. Mol. Cell Biol. 2015; 16: 57-64Crossref PubMed Scopus (95) Google Scholar). Mounting evidence over the last three decades has established exquisite and dynamic regulation kinase cascades such as CDK1, PLK1, Aurora A, and Aurora B in accurate cell division control (Joukov and De Nicolo, 2018Joukov V. De Nicolo A. Aurora-PLK1 cascades as key signaling modules in the regulation of mitosis.Sci. Signal. 2018; 11: eaar4195Crossref PubMed Scopus (66) Google Scholar). AMPK orchestrates in a myriad of fundamental cellular processes, including autophagy, cell polarity, and mitosis (Dasgupta and Chhipa, 2016Dasgupta B. Chhipa R.R. Evolving lessons on the complex role of AMPK in normal physiology and cancer.Trends Pharmacol. Sci. 2016; 37: 192-206Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). AMPK is a heterotrimeric protein complex composed of one catalytic α subunit (α1 or α2) and two regulatory subunits, β and γ (β1 or β2, and γ1, γ2 or γ3). Combination of these multiple subunit isoforms in mammals constitutes up to 12 distinct AMPK holo-enzymes (α1β1γ1, α1β1γ2, α1β1γ3, etc.) (Lin and Hardie, 2018Lin S.C. Hardie D.G. AMPK: sensing glucose as well as cellular energy status.Cell Metab. 2018; 27: 299-313Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). It is postulated that different regulatory isoform combinations with a given catalytic subunit constitute the basis for context-dependent function of AMPK (Dasgupta and Chhipa, 2016Dasgupta B. Chhipa R.R. Evolving lessons on the complex role of AMPK in normal physiology and cancer.Trends Pharmacol. Sci. 2016; 37: 192-206Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Under nutrient or energy starvation, activation of AMPK requires the presence of its canonical upstream kinase serine/threonine kinase 11 (LKB1) or calmodulin-dependent protein kinase kinase-β (CAMKKβ). When the cellular AMP/ATP ratio increases, excess AMP binds to the γ subunit of AMPK inducing allosteric changes of the γ subunit, which promotes Thr172 phosphorylation of AMPKα by LKB1, leading to the subsequent increase in AMPK kinase activity (Woods et al., 2003Woods A. Johnstone S.R. Dickerson K. Leiper F.C. Fryer L.G. Neumann D. Schlattner U. Wallimann T. Carlson M. Carling D. LKB1 is the upstream kinase in the AMP-activated protein kinase cascade.Curr. Biol. 2003; 13: 2004-2008Abstract Full Text Full Text PDF PubMed Scopus (1270) Google Scholar; Shaw et al., 2004Shaw R.J. Kosmatka M. Bardeesy N. Hurley R.L. Witters L.A. DePinho R.A. Cantley L.C. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress.Proc. Natl. Acad. Sci. U S A. 2004; 101: 3329-3335Crossref PubMed Scopus (1350) Google Scholar). AMPK can also be activated via direct phosphorylation at Thr172 in response to calcium flux, through CAMKKβ (Hawley et al., 2005Hawley S.A. Pan D.A. Mustard K.J. Ross L. Bain J. Edelman A.M. Frenguelli B.G. Hardie D.G. Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase.Cell Metab. 2005; 2: 9-19Abstract Full Text Full Text PDF PubMed Scopus (1204) Google Scholar). The active form AMPK localizes to the mitotic apparatus from centrosomes to spindle midzone during mitotic progression (Vazquez-Martin et al., 2009Vazquez-Martin A. Oliveras-Ferraros C. Menendez J.A. The active form of the metabolic sensor: AMP-activated protein kinase (AMPK) directly binds the mitotic apparatus and travels from centrosomes to the spindle midzone during mitosis and cytokinesis.Cell Cycle. 2009; 8: 2385-2398Crossref PubMed Scopus (81) Google Scholar, Vazquez-Martin et al., 2011Vazquez-Martin A. Oliveras-Ferraros C. Cufi S. Menendez J.A. Polo-like kinase 1 regulates activation of AMP-activated protein kinase (AMPK) at the mitotic apparatus.Cell Cycle. 2011; 10: 1295-1302Crossref PubMed Scopus (41) Google Scholar, Vazquez-Martin et al., 2012Vazquez-Martin A. Cufi S. Oliveras-Ferraros C. Menendez J.A. Polo-like kinase 1 directs the AMPK-mediated activation of myosin regulatory light chain at the cytokinetic cleavage furrow independently of energy balance.Cell Cycle. 2012; 11: 2422-2426Crossref PubMed Scopus (15) Google Scholar; Tripodi et al., 2018Tripodi F. Fraschini R. Zocchi M. Reghellin V. Coccetti P. Snf1/AMPK is involved in the mitotic spindle alignment in Saccharomyces cerevisiae.Sci. Rep. 2018; 8: 5853Crossref PubMed Scopus (10) Google Scholar). Employing a chemical genetic screening, a study identified 28 novel substrates of AMPKα2, some of which have known roles in mitosis and cytokinesis (Banko et al., 2011Banko M.R. Allen J.J. Schaffer B.E. Wilker E.W. Tsou P. White J.L. Villen J. Wang B. Kim S.R. Sakamoto K. et al.Chemical genetic screen for AMPKalpha2 substrates uncovers a network of proteins involved in mitosis.Mol. Cell. 2011; 44: 878-892Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). AMPK regulates the mitotic spindle orientation by phosphorylating the myosin regulatory light chain (MRLC) (Thaiparambil et al., 2012Thaiparambil J.T. Eggers C.M. Marcus A.I. AMPK regulates mitotic spindle orientation through phosphorylation of myosin regulatory light chain.Mol. Cell Biol. 2012; 32: 3203-3217Crossref PubMed Scopus (56) Google Scholar) and promotes mitotic entry by phosphorylating Golgi-Brefeldin-A-resistant GBF1, a guanine nucleotide exchange factor that is critical for Golgi disassembly during the entrance of mitosis (Mao et al., 2013Mao L. Li N. Guo Y. Xu X. Gao L. Xu Y. Zhou L. Liu W. AMPK phosphorylates GBF1 for mitotic Golgi disassembly.J. Cell Sci. 2013; 126: 1498-1505Crossref PubMed Scopus (32) Google Scholar; Miyamoto et al., 2008Miyamoto T. Oshiro N. Yoshino K. Nakashima A. Eguchi S. Takahashi M. Ono Y. Kikkawa U. Yonezawa K. AMP-activated protein kinase phosphorylates Golgi-specific brefeldin A resistance factor 1 at Thr1337 to induce disassembly of Golgi apparatus.J. Biol. Chem. 2008; 283: 4430-4438Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Activation of AMPK in mitosis requires its upstream kinase LKB1 or CAMKKβ (Thaiparambil et al., 2012Thaiparambil J.T. Eggers C.M. Marcus A.I. AMPK regulates mitotic spindle orientation through phosphorylation of myosin regulatory light chain.Mol. Cell Biol. 2012; 32: 3203-3217Crossref PubMed Scopus (56) Google Scholar; Zhao et al., 2019Zhao H. Li T. Wang K. Zhao F. Chen J. Xu G. Zhao J. Li T. Chen L. Li L. et al.AMPK-mediated activation of MCU stimulates mitochondrial Ca(2+) entry to promote mitotic progression.Nat. Cell Biol. 2019; 21: 476-486Crossref PubMed Scopus (47) Google Scholar; Lee et al., 2015Lee I.J. Lee C.W. Lee J.H. CaMKKbeta-AMPKalpha2 signaling contributes to mitotic Golgi fragmentation and the G2/M transition in mammalian cells.Cell Cycle. 2015; 14: 598-611Crossref PubMed Scopus (15) Google Scholar). PLK1, an evolutionarily conserved serine/threonine kinase, is essential for mitotic processes (Macurek et al., 2008Macurek L. Lindqvist A. Lim D. Lampson M.A. Klompmaker R. Freire R. Clouin C. Taylor S.S. Yaffe M.B. Medema R.H. Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery.Nature. 2008; 455: 119-123Crossref PubMed Scopus (492) Google Scholar; Seki et al., 2008Seki A. Coppinger J.A. Jang C.Y. Yates J.R. Fang G. Bora and the kinase Aurora a cooperatively activate the kinase Plk1 and control mitotic entry.Science. 2008; 320: 1655-1658Crossref PubMed Scopus (433) Google Scholar). Chemical inhibition of AMPK activation by PLK1 inhibitor GW843682X, together with the spatiotemporal co-localization of PLK1 and activated AMPK, suggests important role of PLK1-AMPK interaction during mitosis (Vazquez-Martin et al., 2011Vazquez-Martin A. Oliveras-Ferraros C. Cufi S. Menendez J.A. Polo-like kinase 1 regulates activation of AMP-activated protein kinase (AMPK) at the mitotic apparatus.Cell Cycle. 2011; 10: 1295-1302Crossref PubMed Scopus (41) Google Scholar). As a classical mitotic kinase, PLK1 or its upstream kinase Aurora A have not yet been demonstrated to be energy sensing, challenging the view that mitotic AMPK activation is solely attributable to Thr172 phosphorylation by LKB1 elicited by ATP reduction. In fact, low energy status produces a “stop” signal that prevents cells from entering into energy-consuming mitosis (Dasgupta and Chhipa, 2016Dasgupta B. Chhipa R.R. Evolving lessons on the complex role of AMPK in normal physiology and cancer.Trends Pharmacol. Sci. 2016; 37: 192-206Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Based on these emerging evidences, we postulate that mitotic AMPK is uncoupled from its energy sensing function in normal mitosis and may be activated by PLK1 through an alternative signaling cascade. Here, we show that AMPK is indeed activated by PLK1 during mitosis. Neither LKB1 nor CAMKKβ are needed for normal mitotic AMPK activation. Furthermore, we found that PLK1-mediated AMPK activation requires another crucial mitotic kinase, CDK1, to prime the cascade via phosphorylation of the C-terminus of AMPKα2 at Thr485, which promotes the interaction between AMPK and PLK1. Interestingly, activation of AMPK in mitosis is limited to the α2 subunit. Although the α1 subunit can also be phosphorylated by CDK1 at the equivalent site corresponding to Thr485 of α2, the phosphorylated motif located on α1 is poorly recognized by PLK1. Finally, we demonstrate that activation of AMPKα2 in mitosis is essential for accurate mitotic progression and genomic integrity. Our findings reveal a distinctive sequential CDK1/PLK1-dependent and isoform-specific AMPK activation and function in mitosis. To investigate the potential mechanism for AMPK activation in mitosis, HeLa or U2OS cells were synchronized in prometaphase with nocodazole, and cell lysates were collected and analyzed by immunoblotting. We observed dramatically increased phosphorylation of endogenous AMPK at Thr172 in the activation loop with a parallel rise in phosphorylation of its substrate, acetyl-CoA-carboxylase (ACC) at Ser79 in mitotic cells (Lin and Hardie, 2018Lin S.C. Hardie D.G. AMPK: sensing glucose as well as cellular energy status.Cell Metab. 2018; 27: 299-313Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar), indicating that AMPK is active in mitosis (Figure 1A). We next determined the subcellular distribution of active AMPK by immunofluorescence with the activation loop-specific (pT172) phospho-AMPK antibody. Centrosomal localization of active AMPK was readily apparent in mitotic, but not interphase cells (Figure 1B). We also confirmed the localization of AMPK by staining HeLa cells that stably express GFP-AMPK with ACA or γ-tubulin antibody (Figures S1A and S1B). We found that AMPK shows strong centrosomal (γ-tubulin) localization, which was consistent with pT172-AMPK antibody staining. To further characterize the temporal dynamics of AMPK during cell cycle, synchronized HeLa cells were collected at various time points upon release, followed by western blot analysis. Levels of AMPK itself remained stable throughout the cell cycle, whereas the levels of both pT172 in AMPK and its substrate site, pS79 in ACC, exhibited cell-cycle-dependent dynamic changes with a peak at mitosis (Figure 1C). The consistent result was obtained by immunofluorescence staining with pT172-AMPK antibody in different mitotic stages, indicating a potential role of AMPK in mitosis (Figures S1C and S1D). This cyclic pattern of AMPK activity was reminiscent of those of Cyclin B1, PLK1, and Aurora A (Golsteyn et al., 1995Golsteyn R.M. Mundt K.E. Fry A.M. Nigg E.A. Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function.J. Cell Biol. 1995; 129: 1617-1628Crossref PubMed Scopus (384) Google Scholar; Hutterer et al., 2006Hutterer A. Berdnik D. Wirtz-Peitz F. Zigman M. Schleiffer A. Knoblich J.A. Mitotic activation of the kinase Aurora-A requires its binding partner Bora.Dev. Cell. 2006; 11: 147-157Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar; Jackman et al., 2003Jackman M. Lindon C. Nigg E.A. Pines J. Active cyclin B1-Cdk1 first appears on centrosomes in prophase.Nat. Cell Biol. 2003; 5: 143-148Crossref PubMed Scopus (480) Google Scholar), supporting the proposed role of AMPK in mitosis. Furthermore, suppression of AMPK activity with AMPK antagonist compound C in mitotic cells resulted in severe mitotic defects (Lee et al., 2020Lee H. Zandkarimi F. Zhang Y. Meena J.K. Kim J. Zhuang L. Tyagi S. Ma L. Westbrook T.F. Steinberg G.R. et al.Energy-stress-mediated AMPK activation inhibits ferroptosis.Nat. Cell Biol. 2020; 22: 225-234Crossref PubMed Scopus (132) Google Scholar), whereas the untreated cells progressed normally (Figures S1E–S1G). This suggests that AMPK activity is essential for normal mitotic progression. Recently, selective activation of AMPK catalytic subunits has been discovered to control specific physiological activities including fatty acid oxidation, epigenetic silencing, and endothelial proliferation (Yang et al., 2018Yang Q. Xu J. Ma Q. Liu Z. Sudhahar V. Cao Y. Wang L. Zeng X. Zhou Y. Zhang M. et al.PRKAA1/AMPKalpha1-driven glycolysis in endothelial cells exposed to disturbed flow protects against atherosclerosis.Nat. Commun. 2018; 9: 4667Crossref PubMed Scopus (32) Google Scholar; Lopez-Mejia et al., 2017Lopez-Mejia I.C. Lagarrigue S. Giralt A. Martinez-Carreres L. Zanou N. Denechaud P.D. Castillo-Armengol J. Chavey C. Orpinell M. Delacuisine B. et al.CDK4 Phosphorylates AMPKalpha2 to inhibit its activity and repress fatty acid oxidation.Mol. Cell. 2017; 68: 336-349Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar; Wan et al., 2018Wan L. Xu K. Wei Y. Zhang J. Han T. Fry C. Zhang Z. Wang Y.V. Huang L. Yuan M. et al.Phosphorylation of EZH2 by AMPK suppresses PRC2 methyltransferase activity and oncogenic function.Mol. Cell. 2018; 69: 279-291Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). We therefore sought to ascertain whether AMPKα1 and AMPKα2 are selectively activated during mitosis. To this end, we employed a lentivirus-based gene silencing approach to specifically deplete AMPKα1 or AMPKα2 subunit. Interestingly, knockdown AMPKα2, but not AMPKα1, dramatically reduced AMPK phosphorylation at pT172, suggesting that Thr172 phosphorylation mainly occurs in AMPKα2 (Figure 1D). To confirm this unexpected finding, AMPKα2 and AMPKα1 were immunoprecipitated from asynchronous and synchronized HeLa cells, respectively and were analyzed by western blotting for phosphorylation status. As shown in Figures 1E and S1H, AMPKα2, but not AMPKα1, phosphorylation at Thr172 was dramatically increased in the mitotic immunoprecipitates. To explore the role of AMPKα2 in mitosis, we performed a knockdown/rescue experiment and observed the resulting phenotype under time-lapse microscopy (Figure 1F). Clearly, with the depletion of AMPKα2, cells exhibited a high frequency of chromosomal segregation defects, including chromosomal misalignment and lagging chromosomes. Expression of wild-type AMPKα2, but not the kinase-deficient mutant containing a T172A substitution, reversed the effect of AMPKα2 knockdown (Figures 1G, S1I, and S1J). Thus, we conclude that activated AMPKα2 is essential for accurate chromosomal segregation. We next tested whether LKB1 or CAMKKβ were required for AMPKα2 activation in mitosis using shRNA-mediated knockdown. Surprisingly, neither CAMKKβ nor LKB1 appear to be involved in AMPKα2 activation in mitosis, as determined by the phosphorylation levels of Thr172 of AMPKα2 and of Ser79 of ACC (Figures 2A and S2A). We further examined if any other mitotic kinases regulate AMPKα2 activity using chemical inhibition. As shown in Figure 2B and Figure S2B, BI2536, a selective inhibitor of PLK1 (Steegmaier et al., 2007Steegmaier M. Hoffmann M. Baum A. Lenart P. Petronczki M. Krssak M. Gurtler U. Garin-Chesa P. Lieb S. Quant J. et al.BI2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo.Curr. Biol. 2007; 17: 316-322Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar), almost completely suppressed AMPKα2 activity judged by pT172-AMPK and pS79-ACC without affecting the levels of AMPKα2 protein. In addition, the Cdk inhibitor roscovitine attenuated AMPKα2 activity, albeit mild compared with that of BI2536 (Figures 2B and S2B). We further verified the impact of PLK1 on AMPKα2 activation by using lentivirus-based PLK1 gene silencing in cells. Consistent with the results observed in Figures 2C and S2C, the centrosomal signal of pT172-AMPKα2, rather than AMPKα2 itself (Figure S2D), was abolished in PLK1 shRNA-treated cells (Figures 2D, S2E, and S2F). Collectively, these correlative studies suggest that PLK1 is responsible for AMPKα2 activation in mitosis. Our data demonstrated that PLK1 regulates AMPKα2 activation at the centrosomes during mitosis. To examine if nutrient starvation would regulate AMPK activity in mitosis, we subjected nocodazole-synchronized U2OS cells to nutrient starvation followed by western blot analysis. As shown in Figures 2E and S2G, chemical inhibition of PLK1 dramatically reduced AMPK activity in normal mitotic cells judged by pT172 abundance but not in nutrient starved cells in mitosis, suggesting that AMPK activity in mitotic cells could be contextually regulated by PLK1-dependent and PLK1-independent pathways. To further characterize if centrosomal AMPK activity is a function of PLK1 during mitotic starvation, synchronized HeLa cells were then fixed and examined for phosphorylation of AMPK in cells at metaphase. As shown in the studies above, suppression of PLK1 prevented AMPK activation at the centrosomes of control cells (Figures 2F, 2G, and S2H). However, in the starvation group, suppression of PLK1 did not alter AMPK activity at the centrosomes. The AMPK activation by nutrient starvation in mitosis was eliminated only when both CAMKKβ and PLK1 were knocked down (Figures 2F, 2G, and S2H). Thus, these data indicate that PLK1 is responsible for AMPKα2 activation during regular mitotic processes, whereas LKB1 or CAMKKβ participates in stress response in nutrient-deficient mitosis. To further examine the role of PLK1 in regulating AMPKα2, we first characterized the subcellular distribution of PLK1 relative to activated AMPKα2 during cell division. Several studies demonstrated this co-distribution of PLK1 with activated AMPK at each stage of mitosis (Vazquez-Martin et al., 2011Vazquez-Martin A. Oliveras-Ferraros C. Cufi S. Menendez J.A. Polo-like kinase 1 regulates activation of AMP-activated protein kinase (AMPK) at the mitotic apparatus.Cell Cycle. 2011; 10: 1295-1302Crossref PubMed Scopus (41) Google Scholar). Our immunofluorescence study experiments demonstrated that PLK1 and activated AMPKα2 are co-localized to the centrosomes in mitotic cells (Figure 3A), thus assigning a distinct role for AMPKα2 in mitosis. When PLK1 was knocked down, the level of activated AMPKα2 was dramatically reduced (Figures 3B–3D), indicating the activated AMPKα2 is a function of PLK1 in mitosis. And pT172 signal was abolished in cells depleted of AMPKα2, suggesting the specificity of pT172-AMPK antibody (Figure S3A). Consistent with this notion, chemical inhibition of PLK1 activity by BI2536 also abolished AMPK activation during mitosis (Figures 3E–3G, S3B, and S3C). Our western blotting analyses show that BI2536 inhibits PLK1 activity without alteration of PLK1 protein level (Figure 3E). These results suggest that PLK1 kinase activity, rather than the protein itself, is necessary for AMPKα2 activation. Indeed, exogenous expression of wild-type PLK1 in HeLa cells deficient in endogenous PLK1 restored AMPK activity in mitosis, whereas expression of a kinase-dead mutant of PLK1 failed to restore AMPK activity judged by western blotting analyses of pT172-AMPK and pS79-ACC (Figures 3H and S3D). Thus, we conclude that PLK1 kinase activity is essential for mitotic AMPKα2 activation. To understand the mechanisms underlying PLK1-elicited AMPKα2 activation in mitosis, we examined the interaction between PLK1 and AMPKα2. As shown in Figures 4A and S4A, endogenous PLK1 was co-precipitated with AMPKα2, but not AMPKα1, which is consistent with the finding above that AMPKα1 is not activated during mitosis. Early study claimed that PLK1 is co-localized with activated AMPK at each stage of mitosis (Vazquez-Martin et al., 2011Vazquez-Martin A. Oliveras-Ferraros C. Cufi S. Menendez J.A. Polo-like kinase 1 regulates activation of AMP-activated protein kinase (AMPK) at the mitotic apparatus.Cell Cycle. 2011; 10: 1295-1302Crossref PubMed Scopus (41) Google Scholar), and our experiments demonstrate the co-localization of PLK1 and AMPKα2 at centrosomes during mitosis by stable cell line and PLK1 antibody (Figure 4B). Interestingly, we found that PLK1 disappears from centrosomes earlier than AMPKα2 in anaphase (Figure 4B). In addition, as shown in Figure 3A, pT172-AMPK signal was abolished in anaphase, confirming the role of PLK1 in regulating AMPK activity during mitosis. To further characterize PLK1-AMPKα2 interaction and pinpoint the binding interface between the AMPKα2 and PLK1 proteins, FLAG-PLK1 and various GFP-AMPKα2 deletion mutants were constructed and were co-transfected with FLAG-PLK1 into HEK293T cells. Our experiments demonstrated that the C-terminus of AMPKα2 was required for its interaction with PLK1 (Figures 4C, S4B, and S4C). Because PLK1 possesses several functionally distinct domains, we next examined the region of PLK1 which mediates its binding to AMPKα2. To this end, different recombinant GST-PLK1 proteins bound to glutathione agarose beads were used as affinity matrix to bind GFP-AMPKα2-C from HEK293T cell lysates. As shown in Figure 4D, the C-terminus of PLK1 retained GFP-AMPKα2-C, indicating that the polo-box binding domain (PBD) mediates the interaction of PLK1 and GFP-AMPKα2. To determine their physical interaction, bacterially expressed recombinant GST-PBD or GST-PBD-2A mutant (H538A/K540A; negative control) (Garcia-Alvarez et al., 2007Garcia-Alvarez B. de Carcer G. Ibanez S. Bragado-Nilsson E. Montoya G. Molecular and structural basis of polo-like kinase 1 substrate recognition: implications in centrosomal localization.Proc. Natl. Acad. Sci. U S A. 2007; 104: 3107-3112Crossref PubMed Scopus (88) Google Scholar) were incubated with mitotic HeLa cell lysates. GST pull-down analyses showed that AMPKα2 interacts with PBD but not PBD-2A mutant (Figure 4E). Furthermore, we immunoisolated FLAG-AMPKα2 protein from stably expressed HeLa cells and performed in vitro kinase assays. Mitotic AMPKα2 exhibits a basal phosphorylation at Thr172 without addition of PLK1. However, the phosphorylation at Thr172 was markedly augmented in the presence of addition of PLK1. In parallel experiment, we failed to detect apparent phosphorylation signals of AMPKα2 from asynchronous cell lysates, AMPKα1 from mitotic cell lysates, or AMPKα1 from asynchronous cell lysates, with or without PLK1 kinase (Figure 4F). Thus, we conclude that PLK1 interacts with the C-terminal region of AMPKα2 via its PBD and selectively phosphorylates AMPKα2 isolated from mitotic but not interphase cells. The PBD of PLK1 is a signaling module that recognizes a conserved consensus motif S-(pT/pS)-(P/X) on its effectors, and CDK1 priming phosphorylation is essential for PBD domain binding (Elia et al., 2003aElia Andrew E.H. Cantley L.C. Yaffe Michael B. Proteomic screen finds pSer/pThr–binding domain localizing Plk1 to mitotic Substrates.Science. 2003; 299: 1228-1231Crossref PubMed Scopus (560) Google Scholar, Elia et al., 2003bElia Andrew E.H. Rellos P. Haire Lesley F. Chao Jerry W. Ivins Frank J. Hoepker Katja Mohammad Duaa Cantley Lewis C. Smerdon Stephen J. Yaffe Michael B. The molecular basis for phosphodependent substrate targeting and regulation of plks by the polo-box domain.Cell. 2003; 115: 83-95Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar). Given the suppression of pT172 level by the CDK inhibitor roscovitine (Figures 2B and S2B), we hypothesized that CDK1 may phosphorylate AMPKα2 at a priming site (or sites), which promotes the interaction between PBD and AMPKα2 for optimal activation of AMPKα2 by PLK1. Sequence alignment analyses revealed that AMPKα2 in multicellular organisms contained potential phosphorylation sites (Thr85, Ser345, and Thr485) for CDK1, and these sites are consistent with the PLK1 docking motif (Figure 5A). Recent work suggested that CDK1 can phosphorylate multiple subunits of AMPK (Stauffer et al., 2019Stauffer S. Zeng Y. Santos M. Zhou J. Chen Y. Dong J. Cyclin-dependent kinase 1-mediated AMPK phosphorylation regulates chromosome alignment and mitotic progression.J. Cell Sci. 2019; 132: jcs236000Crossref PubMed Scopus (8) Google Scholar). Thus, we proposed that AMPKα2 was phosphorylated by CDK1 before PLK1-mediated phosphorylation at Thr172. To test this hypothesis, we first examined if AMPKα2 interacts with the CDK1-Cyclin B1 complex. As shown in Figure S5A, immunoprecipitation of FLAG-AMPKα2 from HeLa cell lysates co-precipitated CDK1-Cyclin B1. Further, utilizing recombinant MBP-AMPKα2 purified from Escherichia coli as a substrate and pCDK-sub, an antibody that specifically recognizes the phosphorylated substrates of CDK kinase (Whalley et al., 2015Whalley H.J. Porter A.P. Diamantopoulou Z. White G.R. Castaneda-Saucedo E. Malliri A. Cdk1 phosphorylates the Rac activator Tiam1 to activate centrosomal Pak and promote mitotic spindle formation.Nat. Commun. 2015; 6: 7437Crossref PubMed Scopus (31) Google Scholar), in vitro kinase assay indicated that CDK1 was able to phosphorylate AMPKα2 and the phosphorylation level was significantly reduced in the presence of CDK1 inhibitor RO-3306 (Figure S5B). To pinpoint the phosphorylated sites in AMPKα2, we subjected purified AMPKα2 phosphorylated by CDK1-Cyclin B1 to mass spectrometric analysis and identified three sites: Ser345, Ser377, and Thr485 (Figures S5C and S5E). To confirm that these three identified sites are substrates of CDK1, we created AMPKα2 mutants by replacing the three residues with alanine and performed in vitro kinase assays. Direct phosphorylation of AMPKα2 by CDK1 was detected with 32P-labeled ATP and pCDK-sub. However, single residue replacement moderately reduced AMPKα2 phosphorylation, and triple residue replacement eliminated AMPKα2 phosphorylation, confirming that they are the major CDK1 phosphorylation sites on AMPKα2 (Figures S5F and S5G). To further test whether these sites are all responsible for AMPK activation, we generated a series of AMPKα2 mutants in which the three phosphorylated sites were individually mutated to alanine. To our surprise, FLAG-AMPKα2 T485A mutant isolated from mitotic cells contained minimal level of phospho-Thr172, whereas the other two FLAG-AMPKα2 site-specific mutants (S345A or S377A) exhibited no alteration of the pT172 level (Figures 5B and S5H), suggesting that Thr485 phosphorylation was required as prim" @default.
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• W3152114230 title "AMPKα2 activation by an energy-independent signal ensures chromosomal stability during mitosis" @default.
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