Plk1-dependentPhosphorylationRegulatesFunctionsof ... · topoII , and resulted in accumulation of catenated DNA. Finally, we showed that wild type topoII , but not the unphos-phorylatable

Plk1-dependent Phosphorylation Regulates Functions ofDNA Topoisomerase II� in Cell Cycle Progression*

Received for publication, November 1, 2007, and in revised form, December 28, 2007 Published, JBC Papers in Press, January 2, 2008, DOI 10.1074/jbc.M709007200

Hongchang Li, Yun Wang, and Xiaoqi Liu1

From the Department of Biochemistry and the Cancer Center, Purdue University, West Lafayette, Indiana 47907

Plk1 (Polo-like kinase 1) has been documented as a criticalregulator of manymitotic events. However, increasing evidencesupports the notion that Plk1 might also have functions outsideof mitosis. Using biochemical fractionation and RNA interfer-ence approaches, we found that Plk1 was required for both G1/Sand G2/M phases and that DNA topoisomerase II� (topoII�)was a potential target for Plk1 in both interphase and mitosis.Plk1 phosphorylates Ser1337 andSer1524 of topoII�. Overexpres-sion of an unphosphorylatable topoII� mutant led to S phasearrest, suggesting that Plk1-associated phosphorylation firstoccurs in S phase. Moreover, overexpression of the unphospho-rylatable topoII�mutant activated theATM/R-dependentDNAdamage checkpoint, probably due to reduced catalytic activity oftopoII�, and resulted in accumulation of catenated DNA.Finally, we showed that wild type topoII�, but not the unphos-phorylatable mutant, was able to rescue topoII� depletion-in-duced defects in sister chromatid segregation, indicating thatPlk1-associated phosphorylation is essential for the functions oftopoII� in mitosis.

The cell cycle entails a series of macromolecular events thatlead to cell division and the production of two daughter cells,each maintaining genetic information identical to that of theparental cell. DNA replication occurs during the S phase of thecell cycle and the duplicated chromosomes are distributed intotwo daughter cells during mitosis or the M phase. Precise tem-poral control of the cell cycle ensures a high fidelity of chromo-some duplication/segregation, which occurs only when allcovalent DNA links between replicated sister chromatids havebeen removed. The enzyme that decatenates covalently inter-linked DNA molecules to disentangle intertwined chromo-somes is DNA topoisomerase II (1).There are two known isoforms of DNA topoisomerase II in

mammalian cells, topoII�2 and topoII� (1). It has been shownthat the two isoforms are differentially regulated during cell

cycle progression. Although the level of topoII� remains fairlyconstant across different phases of the cell cycle, topoII� levelsrise significantly in S phase, peak in G2/M phase, and then fallrapidly following mitosis (2). In addition, the phosphorylationof topoII� and topoII� is also regulated throughout the cellcycle. Mammalian topoII� and topoII� are hyperphosphoryla-ted at mitosis, and several M phase-specific phosphorylationsites have been identified (2). In Chinese hamster ovary cells,distinct topoII phosphorylation sites have been observed inmitosis and interphase (3).The role of phosphorylation in regulating topoII� has been

the subject of several publications, but no consistent patternhas emerged (2). InDrosophila, topoII� was phosphorylated bya number of protein kinases, including casein kinase II, proteinkinase C, and Cdc2 kinase. In all cases, phosphorylation stimu-lated enzyme activity (4). In budding yeast, it was reported thatdephosphorylation of topoII� resulted in a loss of catalyticactivity, suggesting that at least some phosphorylation wasrequired for its activity (5). However, studies with mammaliantopoII� yielded conflicting results. In one study, dephosphoryl-ation of topoII� by �-phosphatase treatment had essentially noeffect on its decatenation activity (6). In a separate study, bymutating serine to alanine, Chikamori and colleagues showedthat phosphorylation at Ser1106 in topoII� positively regulatedits enzymatic activity, and a kinase likely to be responsible forthe phosphorylation was casein kinase II (7).In addition to cyclin-dependent kinases, Plk1 (Polo-like

kinase 1) has also emerged as a key regulator involved in manycell cycle-related events, such as centrosomematuration, bipo-lar spindle formation, and cytokinesis (8, 9). Sufficient evidencealso indicates that Plk1 plays a critical role in chromosome seg-regation at the onset of anaphase. In budding yeast, Cdc5-asso-ciated phosphorylation of cohesin subunit Scc1 stronglyenhances its cleavage by separase, which leads to sister chroma-tid separation (10). Using depletion experiments, Plx1, the Plk1homolog in Xenopus, was shown to be required for cohesindisplacement from chromosome arms in a phosphorylation-dependent manner (11). In human cells, cohesin depletion-in-ducedmitotic delay can be rescued by inhibition of topoII, sug-gesting that the accumulation of catenations by topoIIinhibition in preseparated sister chromatids may overcome thereduced tension arising from cohesin depletion (12). Similarly,Plk1-associated phosphorylation of PICH, a centromere pro-tein, also causes it to dissociate from chromatid arms to cen-tromeres and lead to the formation of DNA threads connect-ing sister kinetochores. Remarkably, these PICH-positivethreads are exacerbated by the inhibition of topoII or cohe-sin, suggesting that they represent stretched centromeric

* The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Recipient of NCI, National Institutes of Health, Howard Temin Award K01CA114401. To whom correspondence should be addressed: Dept. of Bio-chemistry, Purdue University, 175 S. University St., West Lafayette, IN47907. Tel.: 765-496-3764; Fax: 765-494-7897; E-mail: [email protected].

2 The abbreviations used are: topoII�, DNA topoisomerase II�; topoII�, DNAtopoisomerase II�; IF, immunofluorescence; IP, immunoprecipitation;RNAi, RNA interference; PBS, phosphate-buffered saline; GFP, green fluo-rescent protein; WT, wild type; DAPI, 4�,6�-diamidino-2-phenylinodole;kDNA, kinetoplast DNA; FACS, fluorescence-activated cell sorting; BrdUrd,bromodeoxyuridine; aa, amino acid(s); ATM, ataxia telangiectasia-mutat-ed; ATR, ATM and Rad3-related.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 10, pp. 6209 –6221, March 7, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

MARCH 7, 2008 • VOLUME 283 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6209

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chromatin (13). The data accumulated so far are consistentwith the model that topoII may act in the last step of sisterkinetochore separation (13). In this paper, we show thattopoII� is also a Plk1 substrate both in vitro and in vivo, andthe essential topoII� functions during cell cycle progressionmight be regulated by Plk1-associated phosphorylation.

EXPERIMENTAL PROCEDURES

Reagents—The phosphohistone H3 antibody (06-570) andCdc2 antibody (06-923) were fromUpstate Biotechnology, Inc.The rabbit antibodies against TopoII� and TopoII� wereobtained from TopoGene. The mouse Plk1 antibody wasordered from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).The Ki-67 antibody was purchased from BD TransductionLaboratories.Vector Construction—To specifically deplete endogenous

topoII� inmammalian cells, plasmid pBS/U6-topoII�was con-structed as previously described (14). The targeting sequence ofhuman topoII� (accession number NM_001067) was GGT-GAAGTTTAAGGCCCAAG, corresponding to 1242–1261 ofthe coding region relative to the first nucleotide of the startcodon. Plasmid pBS/U6-topoII�-1st half (sense strand) wasused as a control vector. This control vector produces RNA thatcannot form a hairpin structure to generate interfering RNA(RNAi). Plasmid pBS/U6-Plk1 was previously described (14).Cell Culture and Synchronization—HeLa, U2OS, and

hTERT-RPE1 cells were maintained in Dulbecco’s modifiedEagle’s medium supplemented with 10% (v/v) fetal bovineserum, 100 units ml�1 penicillin, and 100 units ml�1 strepto-mycin at 37 °C in 8%CO2. To synchronizeHeLa cells, cells weretreated with 2.5 mM thymidine for 16 h, released for 8 h, andthen treated with thymidine a second time for 16 h. After twowashes with phosphate-buffered saline (PBS), cells were cul-tured for different times as indicated in each experiment andharvested. Based on our experience, cells enter S phase after 4 hof release and accumulate in G2 phase after 8 h of release intonormalmedium.Most cells arrest atmitosis after 12 h of releasein the presence of 100 ng ml�1 of nocodazole, and 13.5 h ofrelease in the absence of nocodazole results in at least 50% ofcells at telophase/cytokinesis. Alternatively, cells were treatedwith 0.3 mM mimosine for 20 h, 4 mM hydroxyurea for 24 h, or200 ng ml�1 nocodazole for 12 h to arrest at G1, S, or M phase,respectively.DNA Transfections—For phenotype analysis of gene deple-

tion in randomly growing cells, HeLa cells were co-transfectedwith pBS/U6-topoII� or pBS/U6-Plk1 and pBabe-puro at aratio of 8:1 using GenePorter reagents. After 2 days of selectionfor transfection-positive cells with 2 �gml�1 puromycin, float-ing cells were washed away with PBS, and the attached cellswere incubated until harvesting for phenotypic analysis. Todeplete topoII� in well synchronized cells, cells were treatedwith thymidine for 16 h, co-transfected with pBS/U6-topoII�and pBabe-puro, incubated for 8 h, and blockedwith the seconddose of thymidine in the presence of puromycin for 20 h. Aftersynchronization/selection, the floating cells were removed, andthe remaining cells were released into fresh medium for differ-ent times. To rescue the topoII� depletion-induced pheno-types, cells growing on coverslips were co-transfected with

pBS/U6-topoII� andRNAi-resistantGFP-topoII� (WTor Plk1unphosphorylatable mutant) at a ratio of 5:1 and subjected tothe thymidine block. Upon release into fresh medium for dif-ferent times, cells were stained with 4�,6�-diamidino-2-pheny-linodole (DAPI), and GFP-positive cells were analyzed.Isolation of Nuclear and Chromosome-binding Fractions—

Cytosolic, nuclear, and chromosome-binding fractions fromcell lysates were prepared by using a Qproteome nuclear pro-tein kit (Qiagen). Briefly, harvested cells were resuspended inlysis buffer supplemented with detergent solution NP. Aftercentrifugation for 5 min, the supernatant was collected as thecytoplasmic fraction. The nuclei pellet was resuspended innuclear protein lysis buffer NL and centrifuged for 5 min. Afterremoving the supernatant, the nuclear pellet was resuspendedin extraction buffer NX1. After a 10-min spin, the supernatantwas collected as the soluble nuclear fraction. The insoluble pel-let was resuspended in extraction buffer NX2 supplementedwith benzonase and incubated for 1 h with gentle agitation.After another 10-min spin, the supernatant was collected as thechromosome-binding fraction.Kinase Assay—Cdc2 was immunoprecipitated from cell

lysates with a Cdc2 antibody and resuspended in TBMD buffer(50 mM Tris, pH 7.5, 10 mM MgCl2, 5 mM dithiothreitol, 2 mMEGTA, 0.5 mM sodium vanadate, 20 mM p-nitrophenyl phos-phate) supplemented with 25 �M ATP and 50 �Ci of[�-32P]ATP. The reaction mixtures were incubated at 30 °C for30min in the presence of histoneH1 as a substrate and resolvedby SDS-PAGE. The gels were stained with Coomassie BrilliantBlue, dried, and subjected to autoradiography.Mitotic Chromosome Spread—Chromosome spread analysis

was performed as described (12). HeLa cells were transfectedwith GFP-topoII� (WT or Plk1 unphosphorylatable mutant)and blocked at M phase with nocodazole treatment for 12 h.Mitotic cells were mechanically shaken off of plates, washedwith PBS, and swollen in a hypotonic solution (75 mM KCl),followed by spreading using a 1000-rpm spin for 5 min. Spreadcells were fixed by paraformaldehyde and subsequently stainedwith DAPI.Topoisomerase II�Activity Assay—TopoII� enzymatic activ-

ity was assayed by measuring the decatenation of kinetoplastDNA (kDNA) as described (15). A standard assay was carriedout in a total volume of 20 �l, including 50 mM Tris-HCl, pH7.9, 88 mM KCl, 10 mM MgCl2, 0.5 mM EDTA, 10 mM ATP, 10mMdithiothreitol, 100�gml�1 bovine serum albumin, and 125ng of kDNA. The reaction mixture containing equal amountsof topoII� (WT, S1337A/S1524A, or S1337E/S1524E) wasincubated at 37 °C for different times, and the reaction wasstopped by the addition of 5 �l of stop solution (5% SDS, 25%Ficoll, and 0.05% bromphenol blue). The samples wereresolved by electrophoresis at 115 V using a 1% agarose gel ina Tris acetate-EDTA buffer. Following electrophoresis, thegel was stained with ethidium bromide and photographedunder UV illumination.

RESULTS

Plk1 Functions in G1/S Phase—It has been well documentedthat Plk1 is involved in manymitotic processes, such as mitoticentry, bipolar spindle formation in metaphase, and cytokinesis

Plk1 Targets TopoII�

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(8, 9). However, several recent reports indicate that Plk1 mighthave additional functions outside of mitosis. For example, Plk1is required for recovery from the DNA replication checkpointresponse, which occurs in S phase (16). Based on immunofluo-rescence (IF) staining, it has long been believed that Plk1 startsto express in the cytoplasm at late S or early G2 phase (17). Tofurther examine the possible non-M phase functions of Plk1,

HeLa cells were synchronizedwith adouble thymidine block protocol(16 h of thymidine treatment and8 h of release, followed by a second16-h incubationwith thymidine). Atdifferent times after release fromthe block, cells were harvested andfractionated into cytoplasmic,nuclear, and chromosome-bindingfractions. Different subcellular frac-tions were then analyzed by West-ern blot. Based on FACS analysis,cells were arrested at late G1 afterthe block, went though S phase at4–6 h postrelease, reached G2phase at 8 h postrelease, and enteredmitosis at 10 h postrelease (data notshown). As shown in Fig. 1A, Plk1was clearly detected at early S phasein HeLa cells (4 h after release). Sig-nificantly, Plk1 wasmainly localizedin the nucleus during S phase andG2 phase and was also localized tochromosomes during G2/M phase(Fig. 1A). This apparent inconsis-tency might be due to the fact thatonly cytoplasmic fractions wereanalyzed in the previous study. Todetermine whether the nuclearlocalization of Plk1 was a generalphenomenon, U2OS, another tu-mor cell line, and hTERT-RPE1, anontransformed cell line, were usedto further analyze Plk1 localizationand expression. Cells were treatedwith mimosine, hydroxyurea, ornocodazole to block at G1, S, or Mphase, respectively. Cytoplasmicand nuclear fractions were preparedfor anti-Plk1 Western blot analysis.Nuclear localization of Plk1 duringinterphase was clearly detected inHeLa cells andU2OS cells but not inhTERT-RPE1 cells (Fig. 1B), indi-cating that the nuclear localizationof Plk1might be tumor cell-specific.Moreover, two tumor cell linesshowed much higher overall levelsof Plk1 than that of RPE1 cells, inagreement with the notion that Plk1overexpression might be correlated

with transformation (18). Nuclear localization of Plk1 in HeLacells was also observed by IF staining, using a modified perme-abilization/fixation protocol (Fig. 1C).Next, RNAi was used to test whether Plk1 was required for

cell cycle progression in the early stages, such as G1 and Sphases. Plk1 was depleted by using a vector-based RNAiapproach in randomly growing cells as previously described

FIGURE 1. Plk1 functions in G1/S phase. A, HeLa cells were synchronized using the double thymidine block. Atdifferent times after release from the block (Rel. Time), cells were harvested, fractionated into cytoplasmic,nuclear, and chromosome fractions and analyzed by Western blot. B, HeLa, U2OS, and hTERT-RPE1 cells weretreated with mimosine (Mimo), hydroxyurea (HU), or nocodazole (Noc) to block at G1, S, or M phase, respec-tively. Cytoplasmic and nuclear fractions were isolated and analyzed by Western blot. C, cells growing oncoverslips were stained with a Plk1 antibody and analyzed by IF microscopy. DNA is visualized with DAPIstaining. Scale bar, 5 �m. D–F, HeLa cells were co-transfected with pBS/U6-Plk1 and pBabe-puro at a ratio of 8:1.At 1 day post-transfection, puromycin was added for an additional 36 h to select for transfection-positive cells.After floating cells were removed, the remaining attached cells were treated with 200 ng ml�1 nocodazole fordifferent times as indicated and harvested. For each plate, half of the cells were prepared for FACS (D and E), andthe other half of the cells were harvested for Western blot (F) analysis. G, cells were depleted of Plk1 using theprotocol described in D, treated with nocodazole for different times, and stained with a phosphohistone H3antibody. The ratio between phosphohistone H3-positive cells and cells with 4 N DNA content was calculatedto follow the G2/M transition.




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(14). Upon nocodazole treatment, control cells quickly accu-mulated at the G2/M phase, as shown by the increase in cellpopulation with 4 N DNA content by FACS. In contrast, Plk1-depleted cells were much more resistant to nocodazole treat-ment (Fig. 1, D and E). To confirm this observation, we alsoexamined the degradation rate of cyclin E, a G1/S marker pro-tein. In control cells, cyclin E was almost completely degradedafter 6 h of treatment with nocodazole. However, a significantamount of cyclin Ewas still detected in Plk1-depleted cells evenafter 12 h of nocodazole treatment (Fig. 1F), indicating that Plk1might be required for G1/S phase. Finally, Plk1-depleted cellswere also treated with nocodazole for shorter times and stainedwith a phosphohistone H3 antibody, a mitotic marker. Theratio between phosphohistone H3-positive cells and the cellpopulation with 4 N DNA content was used to follow the G2/Mtransition. For control cells, the percentage of phosphohistoneH3-positive cells out of cells with 4 N DNA content clearly

increased upon nocodazole treat-ment. However, such an increasewas not detected in the Plk1-de-pleted cells, supporting the notionthat Plk1 is required for mitoticentry (Fig. 1G).To further appreciate the poten-

tial involvement of Plk1 in the earlystages of cell cycle, we next tried todeplete Plk1 in a synchronized cul-ture. Accordingly, HeLa cells weredepleted of Plk1 and treated withthymidine for 24 h to block at theG1/S boundary. Cells were thenreleased into fresh medium for dif-ferent times in the presence orabsence of nocodazole and har-vested. The double thymidine blockprotocol is not ideal for these exper-iments, since Plk1 depletion alsocauses dramatic G2/M block. Asindicated, Plk1 depletion was veryefficient using such a protocol (Fig.2A). To be consistent with theresults obtained with an asynchro-nous culture as described above,Plk1-depleted cells showed an obvi-ous G1 peak during the entirereleasing period, even in the pres-ence of nocodazole, whereas controlcells quickly entered mitosis (Fig. 2,B and C).It has been documented that Plk1

depletion leads to cell cycle arrest,followed by apoptosis in HeLa cells(14). Thus, in addition to the normal2 N, 4 N peaks, cells with sub-G1DNA content (1 N peak) weredetected at later stages of nocoda-zole treatment after Plk1 depletion(Fig. 1D). In a synchronized culture

after Plk1 depletion, apoptotic cells with 1 NDNA content werealso detected after 8 h of release from the thymidine block, evenin the absence of nocodazole (Fig. 2B), indicating that the apo-ptotic cell deathwe observed in Fig. 1D is not due to nocodazoletreatment.TopoII� Interacts with Plk1 in Vivo—To search for a possible

Plk1 target during interphase, we turned our attention to DNAtopoII�, which is well known to be overexpressed in tumor cellsand has functions in both S and M phases (1). In addition,topoII� was also found to be one of several potential Plk1substrates in a yeast two-hybrid screen to search for Plk1-interacting proteins. To test whether topoII� is a bindingpartner of Plk1, cells were treated with mimosine, hydroxyu-rea, or nocodazole to block at G1, S, or M phase, respectively.Soluble nuclear and chromosome-binding fractions werecombined and subjected to anti-Plk1 immunoprecipitation(IP), followed by anti-topoII� Western blot analysis. As

FIGURE 2. Plk1 depletion in synchronized cells. HeLa cells were co-transfected with pBS/U6-Plk1 and pBabe-puro at a ratio of 8:1. At 1 day post-transfection, cells were treated with thymidine for 24 h in the presence ofpuromycin. After floating cells were removed, the remaining attached cells were released in the absence (B) orpresence (C) of 200 ng ml�1 nocodazole (Noc) for different times as indicated and harvested for Western blot (A)or FACS analysis (B and C).




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shown in Fig. 3A, topoII� was co-immunoprecipitated withPlk1 in both hydroxyurea and nocodazole-treated cells, but notin mimosine-treated cells, indicating that the binding betweentopoII� and Plk1 occurs during S and G2/M phase in vivo. BothtopoII� and Plk1 were clearly detected in the nuclei of ran-domly growing cells byWestern blot analysis (Fig. 3B). TopoII�and Erk2 were used as loading controls to indicate efficientsubcellular fractionation. The nuclear co-localization oftopoII� and Plk1 was further confirmed by IF analysis (Fig. 3C).Based on these data, we hypothesized that topoII� might be asubstrate of Plk1 in both interphase and mitosis.TopoII� Is Required for Cell Proliferation—To investigate the

functions of topoII� during normal cell cycle progression, wefirst used vector-based RNAi to specifically deplete topoII� in

HeLa cells. As indicated by Western blot analysis, topoII� wasefficiently depletedwith this approach (Fig. 4A).Wenext deter-mined whether topoII� depletion influences the proliferationof HeLa cells. Although transfectionwith the control vector didnot affect the growth rate of cells, transfection with the plasmidpBS/U6-topoII� strongly inhibited cell proliferation (Fig. 4B).We also examined the viability of topoII�-depleted cells.Transfection with the control vector showed little effect on cellviability, whereas �10% of topoII�-depleted cells were stillattached to the culture dishes at 6 days post-transfection (Fig.4C). To characterize the inhibition of cell growth by topoII�depletion, cell cycle progression was analyzed by FACS. Asshown in Fig. 4D, transfection with the control vector did notaffect the cell cycle profile, whereas topoII�depletion induced aslight increase of cell population in G2/M phase and obviouscell cycle arrest at S phase. Alternatively, these results may bedue to the possibility that topoII�-depleted cells undergo anaberrantmitosis, resulting in daughter cellswith highly unequalDNA content. Starting from 5 days post-transfection, topoII�-depleted cells showed a significant sub-G1 population (Fig. 4D),suggesting that these cells were undergoing apoptosis. To fur-ther analyze this phenotype in topoII�- depleted cells, an anti-caspase 3Western blot was performed (Fig. 4E). Caspase 3, theexecutioner caspase in apoptosis, was clearly activated intopoII�-depleted cells, as shown by the cleavage of full-lengthprotein. Finally, a BrdUrd labeling approach was used to con-firm the S phase arrest induced by topoII� depletion. As shownin Fig. 4F, at 3 days post-transfection, topoII�-depleted cells

FIGURE 3. TopoII� interacts with Plk1 in vivo. A, HeLa cells were treated withmimosine, hydroxyurea or nocodazole to block at G1, S, or M phase, respec-tively. Soluble nuclear extracts were subjected to anti-Plk1 IP, followed byanti-topoII� Western blot analysis. B, lysates from randomly growing cellswere fractionated for Western blot analysis using antibodies as indicated.C, cells growing on coverslips were co-stained with antibodies against Plk1and topoII� and analyzed by IF. Scale bar, 5 �m.

FIGURE 4. TopoII� is required for HeLa cell proliferation and survival.A, cells were co-transfected with pBS/U6-topoII� and pBabe-puro at a ratio of8:1. After 1 day of incubation, puromycin was added for an additional 2 daysto select for transfection-positive cells. After the removal of dead cells bywashing, the remaining attached cells were harvested for Western blot anal-ysis. B and C, cells were depleted of topoII� as in A, and cell proliferation (B)and viability (C) were measured. D and E, FACS profiles (D) and anti-caspase 3Western blot analysis (E) of topoII�-depleted cells. F, BrdUrd incorporation oftopoII�-depleted cells at 3 days post-transfection.




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showed a slightly higher percentage of BrdUrd-positive cellscompared with that of control cells, indicating that topoII� isnot required for DNA synthesis per se but might be involved inother interphase functions.Depletion of TopoII� Leads to Multiple Cell Cycle Defects—

Considering that topoII� is involved in chromosome conden-sation and segregation (1), we next examined the possiblemitotic defects induced by topoII� depletion. For that purpose,topoII� was depleted in synchronized cells using the protocolshown in Fig. 5A. TopoII�-depleted cells showed obviousdefects in chromosome behavior during mitosis, especially in

sister chromatid separation. Asshown in Fig. 5B, topoII�-depletedcells were eventually able to gothrough mitosis but with obviousconnected DNA bridges betweenseparated sister chromatids throughall late mitotic stages, includinganaphase, telophase, and cytokinesis(Fig. 5B). To confirm the formationof DNA bridges, topoII�-depletedcells were treated with either DNaseor RNase (Fig. 5C). We found thatthese bridges were sensitive toDNase but not RNase treatment,indicating that they contain DNA.To further analyze topoII� deple-tion-induced phenotypes, mitoticprogression was followed by stain-ing with a phosphohistone H3 anti-body. Although no dramatic differ-ence between control cells andtopoII�-depleted cells was detected,topoII�-depleted cells showed aslight delay in mitotic exit (Fig. 5D).Interestingly, phosphohistone H3staining was positive in the DNAbridges connecting the separatingsister chromatids, even long aftercell division (Fig. 5E).We also assessed the percentage

of cells expressing the proliferationmarker Ki67, which is normallyexpressed in cells inG1, S,G2, andMphases but not in G0 (19). Almost100% of control cells were detectedas Ki67-positive, whereas onlyabout 33% of topoII�-depleted cellswere Ki67-positive, indicating that asignificant portion of topoII�-de-pleted cells had exited the cell cycle(Fig. 5F).Abnormal nuclear morphology

was also observed in topoII�-de-pleted cells. Based on DAPI stain-ing, cells can be further categorizedinto three groups: cells with a nor-mal nucleus, cells with a deformed

nucleus, andmultinucleated cells. For control cells, �95% con-tained normal nuclei, �5% of cells were multinucleated, andvery few cells with deformed nuclei were detected. In strikingcontrast, almost 30% of topoII�-depleted cells had deformednuclei, and 20% of topoII�-depleted cells were multinucleated(Fig. 5G). Taken together, these results indicate that topoII� isrequired for chromosome segregation in mitosis.As a different approach, two topoII inhibitors were also uti-

lized to study the effects on cell cycle progression. Accordingly,HeLa cells were synchronized using the double thymidineblock, released for different times in the presence of VP16 or

FIGURE 5. Cell cycle defects induced by topoII� depletion. A, the protocol used to deplete topoII� in a wellsynchronized culture. B, representative images of topoII�-depleted cells during late mitosis/cytokinesis. DNAwas stained with DAPI. C, topoII�-depleted cells were permeabilized and incubated for 15 min with buffercontaining RNase or DNase (5 �g ml�1) prior to fixation and stained with DAPI. Histograms showed thepercentage of the cells with DNA bridges. D and E, cells were depleted of topoII� as in A, released for differenttimes, and stained with a phosphohistone H3 antibody. F, cells were depleted of topoII� as in Fig. 4A andstained with a Ki67 antibody. G, cells were depleted of topoII� as in Fig. 4A and stained with DAPI. Based onnuclear morphology, topoII�-depleted cells were categorized into three groups: normal, deformed, and mul-tiple nuclei. Both representative images from each category (top) and quantification results (bottom) areshown. Scale bars for B, C, E, and G, 5 �m. Scale bar for F, 20 �m.




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ICRF193, and harvested for FACS (Fig. 6). Since a much morestringent synchronization protocol was used here (double thymi-dine block in Fig. 6 versus a single thymidine block in Fig. 2), FACSprofiles of control samples at 0 h points are slightly different, witha better synchronization result after the double thymidine block.Cells treated with VP16 were blocked in S phase over the entirereleasing period, probably due to the activation of the DNA dam-agecheckpoint.By inhibiting the religationactivityof topoII,VP16treatment leads to DNA double strand breaks (20). In contrast,ICRF193 is a topoII inhibitor thatdoesnot causeDNAdamagebutarrests the enzyme at a point in its catalytic cycle after strand pas-sageandreligationbutbefore releaseof thepassedDNA(21).Cellstreated with ICRF193 were able to progress into mitosis andblocked there. Therefore, topoII activity is not essential for DNAreplication but is absolutely required for mitosis. That BrdUrdincorporation is not affected in topoII�-depleted cells also sup-ports such a notion (Fig. 4F). However, we do observe an increaseof S phase population in topoII�-depleted cells, indicating thattopoII� may have additional interphase functions independent ofits enzymatic activity (Fig. 4D).

Plk1 Phosphorylates TopoII� in Vitro—To investigatewhether Plk1 directly regulates topoII�, we first examinedwhether Plk1 phosphorylates topoII� in vitro. PurifiedPlk1-WT or Plk1-KM (kinase-defective mutant) was incubatedwith purified topoII� in the presence of [�-32P]ATP. The reac-tion mixture was resolved by SDS-PAGE, followed by autora-diography. As shown in Fig. 7A, wild type Plk1 phosphorylatedtopoII� efficiently, whereas the corresponding kinase inactive

FIGURE 6. TopoII inhibition in synchronized cells. HeLa cells were synchro-nized using the double thymidine block (16-h treatment with thymidine, 8-hrelease, and a second thymidine block for 16 h), released into medium con-taining VP16 (25 �g/ml) or ICRF193 (0.5 �g/ml) for different times, and har-vested for FACS analysis.

FIGURE 7. Identification of Ser1337 and Ser1524 of topoII� as two Plk1phosphorylation sites. A, purified GST-Plk1-WT or GST-Plk1-Km (kinase-de-fective mutant) was incubated with purified topoII� in the presence of[�-32P]ATP. The reaction mixtures were resolved by SDS-PAGE, followed byautoradiography. B, Plk1 was incubated with purified GST-topoII� C-terminalfragments as in A. (aa 1079 –1350 and 1345–1532). C, Plk1 was incubated withfour GST-topoII� fragments (aa 1079 –1258, 1259 –1350, 1345–1438, or1439 –1532) as in A. D, Plk1 was incubated with topoII�-(1259 –1350) withvarious serine to alanine mutations as in A. E, Plk1 was incubated with topoII�-(1439 –1532) with various serine to alanine mutations as in A. F, two identifiedPlk1 phosphorylation sites: Ser1337 and Ser1524.




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form did not. Considering that most known phosphorylationsites in topoII� are localized to the C-terminal domain (2), wenext purified two overlapping C-terminal fragments of topoII�(aa 1079–1350 and 1345–1532) for kinase assay in vitro. Bothfragments were good substrates for Plk1 (Fig. 7B). To furthernarrow down the sites, four GST fusion topoII� fragments (aa1079–1258, 1259–1350, 1345–1438, and 1439–1532) werepurified and subjected to the kinase assay. We found that thefragments containing aa 1259–1350 and 1439–1532 yieldedstrong phosphorylation signals (Fig. 7C). Using phosphoaminoacid analysis, themajor phosphate-accepting residue of topoII�

in vivo was shown to be serine (22).Therefore, we generated a series ofsingle or multiple serine to alaninemutationswithin aa 1259–1350 and1439–1532 of topoII�. Kinaseassays showed that a single muta-tion of Ser1337 or Ser1524 to alaninewas sufficient to completely abolishthe phosphorylation signal withinaa 1259–1350 or 1439–1532 oftopoII�, respectively (Fig. 7, D andE). It has been reported thatthe sequence (D/E)X(S/T)�X(D/E)(where X represents any amino acidand � is a hydrophobic amino acid)is an optimal phosphorylationsequence targeted by Plk1 (23). Thetwo phosphorylation sites identifiedin topoII� fit verywell with this Plk1target consensus sequence (Fig. 7F).Altogether, these data indicate thatSer1337 and Ser1524 of topoII� aretwo major Plk1 phosphorylationsites in vitro.TopoII�-2A-expressing Cells Showed

G1/S Phase Arrest—To probe thepossible function of Plk1-associatedphosphorylation of topoII�, wecompared the phenotypes resultingfrom overexpression of topoII�with different phosphorylationstates. A series of constructs wereused in this study and are shown inFig. 8A. FACS profiles indicatedthat overexpression of topoII� ledto obvious apoptosis, whereasexpression of topoII�-2A (S1337A/S1524A) did not show any sign ofcell death (Fig. 8B). Further analysisof this phenotype showed that apo-ptosis was due to expression of theC-terminal, but not the N-terminal,domain of topoII�, and the phos-phorylation state of both sites wasimportant in the process (Fig. 8C).We also found that hydroxyureatreatment rescued topoII� expres-

sion-induced apoptosis, indicating that the cell deathwas prob-ably due to defects in G2/M phase (Fig. 8C). Induction of apo-ptosis by overexpression of topoII� was reported previously(24). In that study, topoII� expression-induced apoptosis wasblocked by coexpression of a dominant-negative form of thecyclin-dependent kinase Cdk2 but not by Cdk1. Overexpres-sion of dominant negative forms of Cdk2 and Cdk1 leads to cellcycle arrest in G1 and G2/M phases, respectively. Thus, it wasproposed that topoII� expression-induced cell death is due to aprematuremitotic entry (24). Based on these studies, onewouldpredict that treatment of topoII�-expressing cells with drugs to

FIGURE 8. TopoII�-2A-expressing cells arrest at G1/S phase. A, various topoII� constructs used in the over-expression study. B, At 2 days post-transfection with GFP-topoII� or GFP-topoII�-2A, HeLa cells were harvestedand analyzed by FACS. C, cells were transfected with different topoII� constructs, harvested, and analyzed byFACS to determine the degree of apoptosis. D, cells were co-transfected with topoII� (WT or 2A mutant) andpBabe-puro at a ratio of 7:1. At 1 day post-transfection, puromycin was added for 30 h to select for transfection-positive cells. After the floating cells were removed, attached cells were treated with 200 ng ml�1 nocodazolefor 12 h, harvested, and subjected to FACS analysis. A 4 N DNA line in the right diagrams was used to visualizethe S phase arrest of topoII�-2A-expressing cells, especially after nocodazole treatment. E, after expression oftopoII� as in D, cells were treated with nocodazole for 6 h, harvested, and analyzed by Western blot. F, afterexpression of topoII� as in D, cells were treated with nocodazole for 12 h, harvested, and analyzed by ananti-Cdc2 IP/kinase assay using histone H1 as a substrate.




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block cells at interphase should rescue the cell death. Ourexperimental results with hydroxyurea are consistent with thisprediction.To further explore themechanism, FACSprofiles of topoII�-

and topoII�-2A-expressing cells were carefully analyzed (Fig.8D). Compared with topoII�-expressing cells, topoII�-2A-ex-pressing cells showed a lower percentage of cells with 4 N DNAcontent both in the presence or absence of nocodazole, indicat-ing that the expression of topoII�-2A probably leads to G1/Sphase arrest. In addition, slightly higher cyclin E levels (Fig. 8E)and obvious lowerCdc2 kinase activities (Fig. 8F) were detectedin topoII�-2A-expressing cells, further supporting the notionthat expression of topoII�-2A might lead to G1/S arrest.Cell Cycle Arrest in TopoII�-2A-expressing Cells Might Be

Due to Activation of the DNA Damage Checkpoint—To furtherdistinguish whether topoII�-2A-expressing cells arrest at G1 orS phase, cells transfected with GFP-topoII�-2Awere incubatedin medium containing BrdUrd reagent. After 2 h of incubation,cells were stained with an anti-BrdUrd antibody and analyzedby microscopy. Compared with that of control cells, thetopoII�-2A-expressing cells showed a slightly higher percent-age of BrdUrd-positive staining, indicating that these cellsmight have a prolonged Sphase (Fig. 9A). In addition, inhibitionof mitotic entry was detected in topoII�-2A-expressing cells,which is probably a secondary effect of S phase arrest (Fig. 9B).

Based on DAPI staining, abnormalities in nuclear morphol-ogy were also observed in topoII�-2A-expressing cells. Almost30% of topoII�-2A-expressing cells contained micronuclei,whereas only 5% of topoII�-expressing cells had this phenotype(Fig. 9C). Next, we tried to test the possibility that the S phasearrest in topoII�-2A-expressing cellsmight be due to activationof the DNA damage checkpoint. Accordingly, we performedWestern blot analysis with an antibody against phosphohistoneH2AX, a marker for DNA double strand breaks (25). Positivephosphohistone H2AX signals were detected in cell lysatesfrom topoII�-expressing cells, topoII�-2A-expressing cells,and topoII�-depleted cells (Fig. 9D). Since both expression ofwild type topoII� and depletion of topoII� led to apoptosis, butexpression of topoII�-2Adid not showany sign of cell death,wepropose that the positive phosphohistone H2AX signals intopoII�-expressing cells and topoII�-depleted cells werecaused by the activation of caspases, which subsequently cleaveDNA, whereas the positive phosphohistone H2AX signalobserved in the topoII�-2A-expressing cells is probably due todirect DNA damage. To further confirm that topoII�-2Aexpression-induced S phase arrest is due to activation of theDNA damage checkpoint, caffeine, an ATM/R inhibitor, wasused to treat the topoII�-2A-expressing cells. As expected, wefound that the addition of caffeine led to obvious cell death intopoII�-2A-expressing cells as well as that in topoII�-express-ing cells (Fig. 9E). Our initial phenotypic analysis showed thattopoII� expression-induced apoptosis occurs during G2/Mphase (Fig. 8C). The results we show here indicate that theaddition of caffeine promoted the topoII�-2A-expressing cellsto enter into G2/M phase, further supporting the notion thatthe S phase arrest in topoII�-2A-expressing cells might beinduced by the ATM/R-mediated DNA damage checkpoint.

Plk1 Is a Positive Regulator of TopoII� Activity—Consid-ering that the essential functions of topoII� in cell cycleprogression rely on its enzymatic activity, it is intriguing totest whether Plk1-dependent phosphorylation regulates thedecatenation activity of topoII�. Toward that end, HEK293cells were transfected with GFP-topoII� with different phos-phorylation states (wild type, S1337A/S1524A (unphospho-rylatable mutant), and S1337E/S1524E (phospho-mimeticmutant)) and blocked with thymidine at G1 phase. Nuclearand chromosome-binding fractions from these cells wereincubated with purified Plk1 under kinase reaction condi-tions and subjected to anti-GFP IP, followed by a topoIIactivity assay. The enzymatic activity of topoII� was ana-lyzed by an ATP-dependent decatenation assay using kDNAas a substrate (15). Compared with that of wild type topoII�,the enzymatic activity of the topoII�-2A and -2E mutantswas significantly decreased and increased, respectively (Fig.10, A and B), suggesting the hypothesis that Plk1 might be apositive regulator for the enzymatic activity of topoII�.

FIGURE 9. Cell cycle arrest in topoII�-2A-expressing cells might be due toactivation of the DNA damage checkpoint. A, at 2 days post-transfectionwith GFP-topoII�-2A, cells were labeled with BrdUrd for 2 h. Only GFP-positivecells were counted for quantification. B, cells were transfected with GFP-to-poII�-2A and synchronized by using the double thymidine block. At differenttimes after release from the block, cells were stained with a phosphohistoneH3 antibody. C, cells were transfected with GFP-topoII�-2A and stained withDAPI, and nuclear morphology was analyzed. Scale bar, 5 �m. D, cells wereeither depleted of topoII� as described above, or topoII� (WT or 2A mutant)was overexpressed and analyzed by phospho-H2AX (P-H2AX) Western blot.E, at 2 days post-transfection with GFP-topoII�-2A, cells were treated with 2mM caffeine for an additional 10 h and harvested for FACS analysis.




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Next, we tried to test whether the activity of topoII� is cellcycle-dependent. At 16 h post-transfection with GFP-topoII�,HEK293 cells were treated with thymidine to avoid topoII�

expression-induced cell death, released for different times, andharvested. Nuclear and chromosome-binding fractions fromcells enriched at different phases were prepared and subjected

FIGURE 10. Plk1 is a positive regulator of topoII� activity. A, HEK293 cells were transfected with GFP-topoII� (WT or 2A or 2E mutant), incubated withthymidine for 1 day, and harvested. Soluble nuclear and chromosome fractions were prepared, and the amounts of GFP-topoII� were quantified by Westernblot analysis. The nuclear and chromosome fractions containing equal amounts of GFP-topoII� were incubated with purified Plk1 under kinase reactionconditions and subjected to anti-GFP IP, followed by incubation with kDNA for different times as indicated. B, histograms quantifying the results of A to showthe percentage of catenated kDNA remaining after different incubation times. C, HEK293 cells were transfected with GFP-topoII� and incubated with thymi-dine for 1 day. G1, S, and M phase cells were harvested after 0, 4, and 10 h release from the thymidine block, respectively. After the amounts of GFP-topoII� werequantified by Western blot analysis, the nuclear and chromosome fractions containing equal amounts of GFP-topoII� were subjected to anti-GFP IP, followedby incubation with kDNA for different times, as indicated. D, HEK293 cells transfected with GFP-topoII� were released to different cell cycle stages as describedin C. Nuclear extracts were incubated with or without purified Plk1, and immunoprecipitated with GFP antibody, followed by topoII activity analysis. Tocompare the effects of Plk1 on topoII� activity, the amounts of GFP-topoII� used for IP from S and M phase cells were 30 and 10% of that from G1 phase cells,respectively. E, HEK293 cells transfected with GFP-topoII� or GFP-topoII�-2A were released to different cell cycle stages as described in C. Equal amounts ofGFP-topoII� and topoII�-2A from different phases of the cell cycle were subjected to anti-GFP IP, followed by topoII activity analysis.




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to anti-GFP IP, followedby a decatenation assay. TopoII� activ-ity was detected in G1 phase, significantly increased at S phase,and reached a peak at M phase, suggesting that topoII� activityis regulated in a cell cycle-dependent manner (Fig. 10C). Wefurther examined the effects of Plk1-dependent phosphoryla-tion on the activity of topoII� prepared from cells at differentphases. Accordingly, nuclear extracts from different phases ofthe cell cycle were incubated with or without purified Plk1under kinase reaction conditions and subjected to anti-GFP IP,followed by topoII activity analysis. The most dramatic differ-ence after Plk1 incubation was detected in samples preparedfromG1 cells, whereas no detectable difference was observed insamples prepared fromSphase orMphase cells (Fig. 10D, com-pare �Plk1 and �Plk1 samples). To capture a potential minoreffect of Plk1, the amounts of GFP-topoII� used from S and Mphase cells were reduced, corresponding to about 30 and 10%ofthat fromG1 phase cells, respectively. These data indicated thatthe Plk1-associated phosphorylation of topoII� occurs as earlyas S phase, and the phosphorylation positively regulates itsenzymatic activity. Finally, the activity of topoII�-WT and -2Aat different cell cycle stages was also examined (Fig. 10E).Dramatic differences were observed at all stages of the cellcycle, further confirming that topoII� activity is positivelyregulated by Plk1-associated phosphorylation during the cellcycle in vivo.Plk1-dependent Phosphorylation in TopoII� Is Required for

Sister Chromatid Segregation—Dynamic relocation of topoII�on chromosomes during M phase has been previouslydescribed. It was believed that topoII� was evenly distributedover the whole chromosome at prophase but concentrated tothe kinetochore at metaphase (26). Recently, a number of stud-ies showed that Plk1was involved in the dynamic localization oftwo chromosome structural proteins, cohesin and PICH, bothof which participate in the process of sister chromatid separa-tion (11, 13). Considering that the localization of topoII� dur-ing mitosis was very similar to that of cohesin and PICH, wetestedwhether Plk1-associated phosphorylation of topoII� alsoaffects its dynamic distribution on chromosomes. Toward thatend, HeLa cells were transfected with GFP-topoII� (WT or 2A)and mitotic chromosomes were spread. Both topoII�-WT andtopoII�-2A were observed to evenly spread on the chromo-somes, and no significant localization differenceswere detectedbetween them (Fig. 11A). Thus, Plk1-dependent phosphoryla-tion in topoII� might not be required for its relocalization dur-ing mitosis.Next, we tested whether the Plk1-associated phosphoryla-

tion of topoII� was essential for its function in sister chromatidsegregation. For that purpose, HeLa cells were co-transfectedwith pBS/U6-topoII� and RNAi-resistant GFP-topoII� (WT-rand 2A-r) at a ratio of 5:1 to express topoII� with differentphosphorylation states in the absence of endogenous protein(Fig. 11B).OnlyGFP-positive cells in cytokinesiswere analyzed.After topoII� depletion, �74% of GFP positive cells in cytoki-nesis had DNA bridges. Expression of RNAi-resistanttopoII�-WTwas able to reduce GFP-positive cells in cytokine-sis with DNA bridges to 24%, whereas �71% of GFP-topoII�-2A-r-positive cells in cytokinesis still hadDNAbridges, indicat-ing that WT topoII�, but not the 2A mutant, can rescue the

topoII� depletion-induced DNA bridge formation between theseparating sister chromatids (Fig. 11C). Taken together, weconcluded that Plk1-dependent phosphorylation in topoII�was required for normal sister chromatid segregation in latemitosis. Finally, to understand the topoII�-depletion-inducedphenotypes as described in Fig. 5G, we monitored the FACSprofiles of synchronized HeLa cells after long term release inthe presence of ICRF193.After 24 h of release, cellswith S phasearrest, 8 N DNA content, and sub-G1 population were accumu-lated (Fig. 11D), indicating that the abnormal nuclei morphol-ogy we observed in topoII�-2A expression cellsmight be due tomitotic defects.

DISCUSSION

The essential role of Plk1 during mitosis has been well estab-lished. However, increasing evidence suggests that Plk1 mighthave additional functions outside of M phase. Plk1 has beenshown to control recovery from G2 phase DNA damage-in-duced arrest in mammalian cells (27). Although the detailedmechanism is still unclear, the p38 mitogen-activated proteinkinase pathway-mediated phosphorylation of Plk1might play acritical role in this process (28). Moreover, Xenopus Plx1 isessential for the adaptation of a DNA replication checkpointresponse, which occurs in S phase (16). Plx1-dependent phos-phorylation of a DNA damage adaptor protein, claspin, causesit to dissociate from chromatin and be degraded, which leads totermination of a DNA replication checkpoint response (16, 29,30). Here, we provide evidence to show that Plk1 is alsorequired for G1/S phase progression even in the absence ofstress.So, what could be a potential substrate for Plk1 in interphase?

DNA topoisomerase II� is a likely candidate for the followingreasons. First, both proteins are localized to the nucleus ininterphase and detected in chromosome-binding fractions dur-ing mitosis. Second, topoII� was co-immunoprecipitated withPlk1 in both hydroxyurea- and nocodazole-treated cells, thehighest binding affinity being observed in S phase. Third, deple-tion of topoII� using vector-based RNAi led to defects in both Sphase andmitosis. Fourth, Plk1 directly phosphorylates Ser1337and Ser1524 of topoII� in vitro. Fifth, overexpression oftopoII�-2A (S1337A/S1524A) led to S phase arrest, reminis-cent of the interphase defects induced by Plk1 depletion. Sixth,introduction of alanine mutations in two Plk1 phosphorylationsites inhibited the decatenation activity of topoII�.By using direct transfection of double-stranded RNA target-

ing topoII�, it was previously shown that topoII� is involved insister chromatid segregation, as indicated by the presence ofmassive chromatin bridges in topoII�-depleted cells (31, 32).This phenotype is in agreement with what we have describedhere using the vector-based RNAi approach. In addition, wealso found that topoII� depletion led to S phase arrest and inhi-bition of cell proliferation. Direct transfection with double-stranded RNA targeting topoII� did not show any sign of cellgrowth inhibition, probably due to the relatively low depletionefficiency of that approach (32). In the vector-based RNAiapproach, pBabe-puro, containing a puromycin resistancegene, was co-transfected with the vector generating short hair-pin RNA. Subsequent selection of transfection-positive cells




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with puromycin led to much more efficient topoII� depletion,thus providing an opportunity to detect additional phenotypesthat are not observed using the direct transfection of double-stranded RNA. The S phase defect observed in topoII�-de-pleted cells is also supported by the phenotype associated

with overexpression of the Plk1unphosphorylatablemutant. Proba-bly due to a dominant negativeeffect, the catalytically inactivetopoII� mutant blocks cells at Sphase.As described above, the role of

phosphorylation in regulatingtopoII� has been controversial (2).In this study, we showed that thephosphorylation of topoII� by Plk1substantially increased the catalyticactivity of topoII�. We are confi-dent to draw such a conclusion forthe following reasons. First, phos-phorylation by casein kinase II, pro-tein kinase C, and Cdc2 stimulatetopoII activity in flies and buddingyeast (4, 5). Second, phosphoryla-tion at Ser1106 by casein kinase IIpromotes topoII� activity in mam-malian cells (7). Third, overexpres-sion of the Plk1 unphosphorylatabletopoII� mutant led to cell cyclearrest at S phase, a phenotype that isstrikingly different to that associ-ated with overexpression of wildtype topoII�, strongly suggestingthat Plk1-associated phosphory-lation regulates its catalytic activity.To our knowledge, we are the firstgroup to report the phenotype foroverexpression of a topoII�unphosphorylatable mutant andanalyze phosphorylation-dependentfunctions in vivo. Fourth, althoughthe C-terminal domain of eukary-otic topoII� does not contain thecatalytically functional regions,such as the ATP binding sites or theactive tyrosine for DNA breakageand religation, some previous stud-ies indicated that this region stillmight be related to the regulation ofits enzymatic activity. For example,binding to a PT1342 antibody,which recognizes phospho-Thr1342in topoII�, completely abolishedtopoII� activity. It was proposedthat the catalytically active sites andThr1342 were close in secondarystructure and might interact,although they were separated in the

primary structure (33). Two Plk1 phosphorylation sites weidentified are located in the C-terminal region of topoII�, andthe phosphorylation states of these two sites affected its activityboth in vitro and in vivo (Fig. 10). One possible explanation isthat phosphorylation in the C-terminal domain of topoII�

FIGURE 11. Plk1-dependent phosphorylation at Ser1337 and Ser1524 in topoII� might be required forsister chromatid segregation. A, chromosome spreading of GFP-topoII� and GFP-topoII�-2A-expressing HeLacells. B and C, after co-transfection with pBS/U6-topoII� and RNAi-resistant GFP-topoII� (WT or 2A) at a ratio of 5:1 for16 h, cells were blocked with thymidine for 24 h. At 15 h after release from the thymidine block, cells were harvestedand analyzed by Western blot (B) or stained with DAPI (C). The arrows indicate formation of DNA threads due totopoII� depletion. Scale bar, 5 �m. D, HeLa cells were synchronized with the double thymidine block, released intomedium containing ICRF193 for the times indicated, and harvested for FACS. E, a model illustrating the possiblefunction of Plk1-dependent phosphorylation in topoII� during the cell cycle.




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might change its secondary structure, through which the enzy-matic activity is regulated. Another possibility is that phospho-rylation in the C-terminal domain makes this region negativelycharged and therefore subsequently affects its interactions withDNA or other proteins.In summary, Plk1 first interacts with and phosphorylates

topoII� at Ser1337 and Ser1524 in S phase, and the maximumlevel of phosphorylation occurs inmitosis. Although Plk1-asso-ciated phosphorylation of topoII� does not affect the dynamiclocalization of topoII� in chromosomes, it is required for theessential role of topoII� in sister chromatid segregation. Over-expression of a Plk1 unphosphorylatable topoII� mutant leadsto ATM/R-dependent activation of the DNA damage check-point, leading to S phase arrest, probably due to the DNA dam-age formation in the previous M phase (Fig. 11E).

Acknowledgments—We are grateful to Dr. Raymond Erikson, inwhose laboratory the preliminary experiments were performed, forgenerously providing many cell lines and plasmids. We thank Drs.Gerald Goldenberg, Peter McPherson, Yin-Yuan Mo, and WilliamBeck for various topoII� expression constructs. We also thank Drs.Jiabin Tang and Zhaoqiu Wu for helpful discussions.

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Hongchang Li, Yun Wang and Xiaoqi Liuin Cell Cycle Progression

αPlk1-dependent Phosphorylation Regulates Functions of DNA Topoisomerase II

doi: 10.1074/jbc.M709007200 originally published online January 2, 20082008, 283:6209-6221.J. Biol. Chem.

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Plk1-dependentPhosphorylationRegulatesFunctionsof ... · topoII , and resulted in accumulation of catenated DNA. Finally, we showed that wild type topoII , but not the unphos-phorylatable