phosphatase 1 nuclear targeting subunit (pnuts) regulates aurora … · phosphatase 1 nuclear...

Cell Cycle and Senescence

Phosphatase 1 Nuclear Targeting Subunit(PNUTS) Regulates Aurora Kinases and MitoticProgressionFeifei Wang1,2,3, Ling Wang2, Laura A. Fisher2, Chunling Li3,Weidong Wang3,and Aimin Peng2

Abstract

Mitotic progression is regulated largely by reversible phos-phorylation events that are mediated by mitotic kinasesand phosphatases. Protein phosphatase 1 (PP1) has beenshown to play a crucial role in regulation of mitotic entry,progression, and exit. We previously observed, in Xenopusegg extracts, that phosphatase 1 nuclear targeting subunit(PPP1R10/PNUTS) acts as a mitotic regulator by negativelymodulating PP1. This study investigates the role of PNUTSin mitotic progression in mammalian cells, and demon-strates that PNUTS expression is elevated in mitosis anddepletion partially blocks mitotic entry. Cells that entermitosis after PNUTS knockdown exhibit frequent chromo-some mis-segregation. Aurora A/B kinase complexes andseveral kinetochore components are identified as PNUTS-

associated proteins. PNUTS depletion suppresses the acti-vation of Aurora A/B kinases, and disrupts the spatiotem-poral regulation of the chromosomal passenger complex(CPC). PNUTS dynamically localizes to kinetochores, andis required for the activation of the spindle assembly check-point. Finally, PNUTS depletion sensitizes the tumor cellresponse to Aurora inhibition, suggesting that PNUTS is apotential drug target in combination anticancer therapy.

Implications: Delineation of how PNUTS governs themitotic activation and function of Aurora kinases willimprove the understanding of the complex phospho-regu-lation in mitotic progression, and suggest new options toenhance the therapeutic efficacy of Aurora inhibitors.

IntroductionMitotic kinases, particularly cyclin-dependent kinase 1

(CDK1), polo-like kinase 1 (Plk1), and Aurora A/B, are wellestablished as central regulators of mitosis. Activation of thesekinases triggers the phosphorylation of various substrates tomodulate all aspects of mitotic cell reorganization and progres-sion (1–4). Thus, regulated activation and deactivation of thesekinases are defined asmolecular events that dictateM-phase entryand exit. Among mitotic kinases, Aurora A and Aurora B kinasesphosphorylate a number of substrates to regulate the dynamics ofcentrosomes, microtubules, and chromatin during mitotic pro-gression. Inactivation of Aurora A and B causes spindle defects,chromosomemissegregation, cytokinesis failure, and aneuploidy(4–7). Owing to the crucial role of mitotic kinases in cell prolif-eration, and the profound toxicity of their inhibition, mitotickinases are appreciated as potential drug targets for cancer

therapy. Over the recent decade, numerous small-molecule inhi-bitors of Aurora kinases have been identified, and studies usingthese inhibitors demonstrated promising antitumor activities.Many of these inhibitors have entered clinical trials for breastcancer, prostate cancer, leukemia, and other malignancies (6, 8).

Protein phosphatases antagonize the action of kinases, but thefunction and regulation of mitotic phosphatases are considerablyless studied (1, 2, 4, 9–12). The two major groups of serine andthreonine phosphatases, protein phosphatase 1 (PP1) and 2A(PP2A), were both known to play important roles in mitoticregulation. For example, it has been shown that PP1 depho-sphorylates numerousmitotic factors and thereby regulatesmitot-ic entry, metaphase–anaphase transition, chromatin condensa-tion, and mitotic exit (13–15). A major gap in knowledge,however, is how PP1 is regulated to achieve these dynamic andspecific actions during mitotic progression.

Among the mitotic proteins dephosphorylated by PP1 wereAurora A andBkinases and their substrates, and the general role ofPP1 as an antagonizer of Aurora kinases has been shown inmultiple experimental systems (16–19). In yeast, the codeletionof PP1/Glc7withAurora/Ipl1 rescuedmitotic progression and cellproliferation from Aurora/Ipl1 deletion (16). Similarly, PP1suppression in mammalian cells prevented mitotic defectsinduced by the inhibition of Aurora B (17–19). The opposingrelationship between PP1 andAurora is perhaps best illustrated inthe regulation of spindle assembly checkpoint signaling at kine-tochores. The spindle checkpoint is a surveillancemechanism thatensures the proper, bipolar kinetochore–microtubule attach-ments (20, 21). It has been shown that reversible phosphorylationof kinetochore components plays a pivotal role in governingspindle attachment and the activation/deactivation of the spindle

1Institute of Physical Science and Information Technology, Anhui University,Hefei, China. 2Department of Oral Biology, College of Dentistry, University ofNebraska Medical Center, Lincoln, Nebraska. 3Institute of Hypertension,Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Aimin Peng, Department of Oral Biology, College ofDentistry, University of Nebraska Medical Center, 40th & Holdrege, Lincoln, NE68516. Phone: 402-472-5903; Fax: 402-472-2551;E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-17-0670

�2018 American Association for Cancer Research.

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checkpoint (22). On one hand, centromeric Aurora B is respon-sible for the activation of the spindle checkpoint via both therecruitment of spindle checkpoint kinases, such as Mps1 andBub1, and the subsequent phosphorylation of BubR1, Mad1,Ndc80/Hec1, Knl1, and other kinetochore proteins (21, 22). Onthe other hand, several kinetochore components, especially Knl1,yield PP1-binding activities, and were reported to recruit PP1 tokinetochores (19). After the proper spindle–kinetochore attach-ment, PP1 dephosphorylates Aurora B, Knl1, BubR1, and otherkinetochore proteins to silence the spindle checkpoint and triggermetaphase–anaphase transition (22).

Phosphatase 1 nuclear subunit (PNUTS), also known asPPP1R10, was originally described as a nuclear regulator of PP1that retains a portion of PP1 in the nucleus (23). PNUTS has beenimplicated in transcription and RNA processing (24, 25), DNAdamage response andmaintenance of telomere stability (26–30),andmodulation of RB and PTEN (31–33). Inmost cases, with theexception of RNAPol II, PNUTS acts to inhibit PP1 toward specificsubstrates. The earliest evidence that linked PNUTS to mitosiscame from the observation that PNUTS enhanced the in vitrochromosome decondensation in a PP1-dependent manner (34).Interestingly, our recent study in Xenopus egg extracts suggested arole of PNUTS as an essential regulator of mitotic progression.Overexpression of PNUTS in Xenopus egg extracts inhibitedmitot-ic exit. PNUTS depletion disruptedmitotic maintenance, whereascodepletion of PP1 rescued the defect (35). The level of PNUTSoscillated in cycling extracts and peaked in mitosis. In this study,we sought to examine the role of PNUTS in human cells, and toreveal further mechanistic insights into how PNUTS governsmitotic signaling. Our results confirmed a role of PNUTS inmammalian mitotic progression, characterized PNUTS-mediatedregulation of Aurora kinases and spindle checkpoint signaling,and suggested PNUTS as a potential target to enhance the cyto-toxicity of Aurora inhibitors.

Materials and MethodsAntibodies and other reagents

Cdc27 antibody was purchased from BD Transduction Labo-ratories; Aurora A, Aurora B, Bub1, Cenp-E, PP1, and PNUTSantibodies were purchased from Bethyl Laboratories; histoneH3, phospho-H3 Ser-10, phospho-Aurora A/B/C, Aurora B, andb-actin antibodies were purchased from Cell Signaling Technol-ogy; PP1antibodywaspurchased fromSantaCruzBiotechnology.PNUTS siRNA was obtained from Integrated DNA Technologies,with the targeting sequence of GGCGGCUACAAACUUCUU.PNUTS shRNAwas generatedusingapSUPERvector (Oligoengine),with the targeting sequence of CAGTGGTGGTTTCTGACAA.

Cell culture and treatmentHuman cervix carcinoma (HeLa) cells, authenticated by

ATCC, were maintained in DMEM (Hyclone) with 10% FBS(Hyclone). Breast cancer ZR75-1 and BT20 cells were authen-ticated at the University of Colorado Tissue Culture Core, andcultured in DMEM/F12 with 10% FBS. Cell-cycle synchroniza-tion at G1–S was performed using two rounds of thymidinetreatment (at a final concentration of 2 mmol/L). For mitoticarrest, HeLa cells were treated with nocodazole (100 ng/mL) for12 hours. Transfection was performed using Lipofectamine(Thermo Fisher Scientific), following a protocol recommendedby the manufacturer.

Immunoblotting and immunoprecipitationSDS-PAGE and immunoblotting were performed as described

previously (36). For immunoprecipitation, anti-rabbit magneticbeads (New England Biolabs) were conjugated to the primaryantibody, and then incubated in cell lysates for 2 hours. The beadswere collected using a magnet, washed, eluted with Laemmlisample buffer, and then analyzed by immunoblotting.

Immunofluorescence and imagingImmunofluorescence was performed as described previously

(37). Briefly, cells were fixed in a fixation buffer (3% formalde-hydewith 0.1% Triton X-100), washed, and blocked in a blockingbuffer (10% goat serum in PBS). The primary antibodies werediluted in the blocking buffer, and incubated with the cells for2 hours. The cells were thenwashed, and incubatedwith the AlexaFluor 594 and 488 secondary antibodies (Invitrogen) for 1 hour.Imaging was performed using a Zeiss Axiovert 200M invertedfluorescence microscope at the UNMC Advanced MicroscopyCore Facility. Live-cell imagingwas performed using theMarianasLive Cell system based around a Zeiss Axiovert 200Mmicroscopestand, and the SlideBook6 software (Intelligent Imaging Innova-tions, Inc.). Images were collected with 10� objective lens mag-nification. Once the live-cell microscopy was completed, thecaptured images were loaded into SlideBook Reader Software(Intelligent Imaging Innovations).

Protein expression, pull-down, andmass spectrometry analysisMBP-tagged PNUTS was constructed and expressed as in our

previous study (35). The recombinant protein was expressed inBL21 bacterial cells and purified on amylose beads. For the pull-down assay, amylose beads conjugated with MBP–PNUTS wereincubated in Xenopus egg extracts that were prepared as in ourprevious study (35). The beads were reisolated, washed, eluted,and then resolved by SDS-PAGE for immunoblotting or massspectrometry (Taplinmass spectrometry facility, HarvardMedicalSchool, Boston, MA).

ResultsPNUTS regulates the mitotic progression of mammalian cells

To investigate the expression level of PNUTS during the cellcycle, HeLa cells were released into cell-cycle progression fromthymidine arrest. Interestingly, PNUTS expression is stronglyelevated inmitosis (Fig. 1A), consistent with our previous findingin Xenopus egg extracts (35). Compared with cyclin B1, whichgradually accumulates prior to mitotic entry, PNUTS is moreabruptly upregulated in M-phase (Fig. 1A). We then sought toexamine the role of PNUTS in mammalian cell-cycle progression.siRNA-mediated PNUTS knockdown significantly reduced thepercentage of mitotic cells (Fig. 1B and C), suggesting a role ofPNUTS in mitotic entry. Consistently, a synchronized mitoticentrywas not evident in cells treatedwith PNUTS siRNA, and thenreleased from thymidine arrest (Fig. 1D). In addition to siRNA, theexpression of PNUTS was also suppressed by shRNA, whichtargeted a distinct sequence of PNUTS (Fig. 1E). Real-timemicros-copy revealed blocked mitotic entry in cells expressing PNUTSshRNA (Fig. 1F). Moreover, although a smaller portion of cellswith PNUTS knockdown still enteredmitosis, they exhibited highlevels of chromosomemisalignment inmetaphase and chromatinbridges in anaphase (Fig. 2A and B). Collectively, these lines of

Aurora Kinases and Mitosis Are Regulated by PNUTS

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evidence demonstrated an important role of PNUTS in mitoticregulation.

PNUTS associates with Aurora kinases and kinetochorecomponents

We speculated that PNUTSmodulates the action of PP1 towarda subset of mitotic substrates. Thus, to reveal mechanistic insightsinto the specific function of PNUTS,weusedmass spectrometry toidentify proteins that copurified with PNUTS. Interestingly, theanalysis identified Aurora A/Tpx2, Aurora B/Incenp complexes,and a number of kinetochore proteins, including Cenp-E andBub1 (Fig. 3A; Supplementary Table S1). Immunoprecipitation in

human cell lysates confirmed PNUTS association with Aurora A,Aurora B, Cenp-E, Bub1, and PP1 at the endogenous level (Fig.3B). Consistently, the reciprocal immunoprecipitation of AuroraB recovered both PNUTS and PP1 (Fig. 3C).

PNUTS modulates PP1-dependent dephosphorylation ofAurora A/B

Identification of Aurora A and B complexes as associatedproteins of PNUTS prompted us to examine the potential roleof PNUTS in regulation of Aurora kinases. It has been shown thatPP1 antagonizes the functions of both Aurora A and Aurora B (14,15, 22, 38). Interestingly, PNUTS blocked the PP1-dependent

Figure 1.

PNUTS is upregulated in mitosis and plays an essential role in mammalian mitotic progression. A, PNUTS upregulation in mitotic cells. HeLa cells weresynchronizedby thymidine arrest, and then released for 0 to8 hours. Cellswere harvested andanalyzedby immunoblotting.B,HeLa cellswere treatedwith control orPNUTS siRNA, and then analyzed by immunoblotting. C, PNUTS knockdown reduced mitotic entry. HeLa cells with control or PNUTS siRNA were stained with DAPIand analyzed microscopically. At least 1,000 cells in multiple fields were examined. Mitotic cells exhibiting condensed chromosomes and nuclear envelopebreakdown were morphologically identified. The percentage of mitotic cells in control or PNUTS siRNA–treated cells was quantified. The mean value and SD werecalculated from three independent experiments. Statistical significance was analyzed using an unpaired two-tailed Student t test. P < 0.05 was consideredstatistically significant (�). D, HeLa cells were transfected with control or PNUTS siRNA. Thymidine release was performed as in A. Cells were harvestedat the indicated time points, and analyzed by immunoblotting. The phosphorylation (band-shift) of Cdc27 indicates mitosis. E, HeLa cells were treated with PNUTSshRNA as described in Materials and Methods. The shRNA-expressing vector also contains a GFP-expressing cassette. As expected, the GFP-positive cell exhibitsa lower level of PNUTS expression. F, PNUTS knockdown led to deficient mitotic entry. HeLa cells were treated with PNUTS shRNA as in E. These cells weresynchronized by thymidine arrest and then released, as in A. Live cell imaging was performed as described in Materials and Methods to monitor mitoticprogression. Representative images are shown. G, As in F, HeLa cells were released from thymidine arrest and analyzed by live cell imaging. Mitotic progressionwas monitored morphologically, as illustrated in F. The percentage of cells with mitotic entry within 10 hours is shown. At least 20 cells in multiple fields wereexamined. The mean value and SD were calculated from three independent experiments. Statistical significance was analyzed using an unpaired two-tailedStudent t test. P < 0.01 was considered statistically significant (��).

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dephosphorylation of Aurora B kinase in a reconstitutive phos-phatase assay (Fig. 4A and B). A similar impact of PNUTS on PP1-mediated dephosphorylation of Aurora A kinase was alsoobserved (Fig. 4C). Moreover, the collection of mitotic cells withPNUTS knockdown exhibited lower levels of Aurora A and Bactivation, as judged by autophosphorylation (Fig. 4D). Thedeficient Aurora phosphorylation was rescued with reexpressionof WT, but not W401A, PNUTS (Supplementary Fig. S1). Becausethe W401A mutation disrupts the PNUTS–PP1 association (24,30, 35), our findings indicate that PNUTS modulates Aurorakinases in a PP1-dependent manner. Furthermore, immunoflu-orescent analysis of individual mitotic cells showed those thatexhibited metaphase and anaphase defects due to PNUTS knock-down lacked efficient phosphorylation of Aurora A/B (Fig. 5A).Histone H3 Ser-10 is hyperphosphorylated in mitosis, mediatedprimarily by Aurora B (39). We observed that PNUTS depletionsignificantly lowered the level of H3 Ser-10 phosphorylation inmetaphase and anaphase (Fig. 5B).

PNUTS depletion disrupts the mitotic localization of thechromosomal passenger complex

Upon metaphase-to-anaphase transition, the Aurora B–containing chromosomal passenger complex (CPC) relocatesfrom anaphase chromosomes to the cell equator where it med-iates mitotic exit (3, 4, 40). Interestingly, we observed alteredlocalization of Aurora B in anaphase with PNUTS knockdown: inanaphase cells with PNUTS knockdown, Aurora Bwas trapped onchromosomes rather than being released to spindles at the cellequator (Fig. 5C). The dysregulation of Aurora B localizationcorrelated withmitotic chromosomemissegregation after PNUTSsilencing (Fig. 5D). Moreover, a similar defect of INCENP local-ization was observed in PNUTS-depleted cells (Fig. 5E).

The spatiotemporal regulationof PNUTS inmitotic progressionThe role of PNUTS in mitosis prompted us to ask how PNUTS

itself is spatiotemporally modulated. As expected, PNUTS isexclusively localized in the cell nucleus in interphase. From late

Figure 2.

PNUTS knockdown disrupts mitoticchromosome segregation. HeLa cellswere treated with control or PNUTSsiRNA for 24 hours, and analyzedmicroscopically for chromosomemisalignment in metaphase (A) andanaphase bridge (B). The percentagesof mitotic cells (N > 50) exhibiting thesedefects, and representative images(with DAPI stain) are shown. Misalignedchromosomes and anaphasebridges aremarked by arrow heads. Themean valueand SD were calculated from threeindependent experiments. Statisticalsignificance was analyzed using anunpaired two-tailed Student t test.P < 0.01 was considered statisticallysignificant (��).

Figure 3.

PNUTS associates with multiple mitotic regulators. A, As described in Materials and Methods, PNUTS pull-down was performed in Xenopus egg extracts thatare particularly amenable for the biochemical isolation of large protein complexes, and analyzed by mass spectrometry to identify binding partners ofPNUTS. The selected mitotic proteins and the number of identified peptides are shown. B, PNUTS immunoprecipitation (IP) was performed in HeLa cell lysatesand analyzed by immunoblotting to confirm its association with Aurora A/B, Bub1, Cenp-E, and PP1. A control immunoprecipitation was performed usingblank beads, and a 20% input lysate was included. C, Aurora B immunoprecipitation was performed in HeLa cell lysates and analyzed by immunoblotting to confirmits association with PNUTS and PP1. A control immunoprecipitation was performed using blank beads, and a 20% input lysate was included.






prometaphase to early telophase, the majority of PNUTS isremoved from chromatin, with a small portion resides on spin-dles and poles (Supplementary Fig. S2). The reaccumulation ofPNUTS on chromosomes begins in telophase (SupplementaryFig. S2), suggesting a potential involvement of PNUTS in thedecondensation of mitotic chromosome. Moreover, consistentwith the protein association of PNUTS with kinetochore proteinsCenp-E and Bub1 (Fig. 3), a portion of PNUTS colocalizes withkinetochore proteins in interphase cells, as marked by Cenp-Eand ACA. However, PNUTS is delocalized from kinetochores inmetaphase (Fig. 6A and B), indicating the dynamic dissociation ofPNUTS from kinetochores.

PNUTS depletion attenuates the activation of the spindlecheckpoint

The role of PNUTS in regulation of Aurora kinases, and thedynamic kinetochore-association of PNUTS suggest a role ofPNUTS in kinetochore signaling and the activation of the spindleassembly checkpoint. Interestingly, PNUTS depletion acceleratedthe mitotic progression from nuclear envelope breakdown(NEBD) to the onset of anaphase (Fig. 6C). Moreover, PNUTSsiRNA impaired metaphase arrest after nocodazole treatment(Fig. 6D), and diminished the phosphorylation and kinetochorelocalization of BubR1 (Fig. 6E and F). Together, these lines ofevidence indicate a role of PNUTS in the activation of the spindlecheckpoint.

PNUTS is a potential drug target to overcome the cancerresistance to Aurora inhibition

An important lesson learned from existing genetic and cellularstudies is that, although Aurora kinases play important functionsin mitosis, depletion of Aurora kinases can be well tolerated viadownregulation of the counteracting PP1 (16–19). As we discov-ered PNUTS as a PP1-modulator toward Aurora kinases and theirsubstrates, we speculated that PNUTS targeting would synergizewithAurora inhibition in conferring cytotoxicity. Interestingly, weobserved that, compared with the treatment of an Aurora inhib-itor, ZM447439, alone, the combination of PNUTS siRNA andZM447439 more profoundly decreased the viability of breastcancer ZR75-1 cells (Fig. 7A). This breast cancer cell line wasselected because it was previously shown to be resistant to Aurorakinase inhibition (41). Consistently, PNUTS siRNA andZM447439 synergistically induce cell death (Fig. 7B). The syner-gistic effect between PNUTS siRNA and ZM447439 was alsoconfirmed in BT20, another breast cancer cell line, and HeLa(Supplementary Fig. S3A and B).

DiscussionPNUTS modulates PP1 during mitotic progression

An evolutionarily conserved role of PP1 in mitotic regulationhas been well established in yeast, Drosophila, Xenopus, andmammalian cells (14, 15, 42, 43). Mammalian PP1 exhibits

Figure 4.

PNUTS suppresses PP1-dependent inactivation of Aurora kinases. A, PNUTS prevents PP1-dependent dephosphorylation of active Aurora B. An in vitrophosphatase assay was performed using purified PP1, Aurora B, and PNUTS, as indicated. Samples were taken at the indicated time points and immunoblotted,as indicated. B, The PP1 phosphatase assay was performed as in A. The level of Aurora B phosphorylation was quantified using NIH ImageJ, and normalizedto that of PP1. The mean value and SD were calculated from three independent experiments. Statistical significance was analyzed using an unpairedtwo-tailed Student t test. P < 0.01 was considered significant (��). C, An in vitro phosphatase assay was performed using purified PP1, Aurora A, and PNUTS,as indicated. Samples were taken at the indicated time points and immunoblotted, as indicated. D, PNUTS knockdown reduced mitotic phosphorylation ofAurora A/B. HeLa cells with or without PNUTS siRNA were synchronized by thymidine block and released for 9 hours for mitotic entry. Mitotic cells werecollected by shake-off and analyzed by immunoblotting.

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Figure 5.

PNUTS knockdown disrupts the mitoticactivation and localization of Aurora kinases.A, HeLa cells with control or PNUTS siRNAwere analyzed by immunofluorescence forthe phosphorylation of Aurora kinases.Misaligned chromosomes and anaphasebridges are marked by arrow heads. B, HeLacells with control or PNUTS siRNA wereanalyzed by immunofluorescence for thephosphorylation of H3 Ser-10. Misalignedchromosomes and anaphase bridges aremarked by arrow heads. C, HeLa cells withcontrol or PNUTS siRNA were analyzed byimmunofluorescence for Aurora B.Misaligned chromosomes and anaphasebridges are marked by arrow heads. D, As inC, 20 anaphase cells with either control orPNUTS knockdown were analyzed byimmunofluorescence for Aurora Blocalization at either anaphase cell equator oranaphase chromosomes. Within the controland PNUTS siRNA–treated groups, cells werefurther classified into subgroups thatexhibited either normal anaphasemorphology or anaphase bridges. E, HeLacells with control or PNUTS siRNA wereanalyzed by immunofluorescence for Incenp.Misaligned chromosomes and anaphasebridges are marked by arrow heads.






dynamic and isoform-specific localization at kinetochores, cen-trosomes, chromosomes, and midbodies. Functionally, PP1modulates the cell-cycle transitions of mitotic entry and exit, andcontrols various aspects of mitotic progression, including nuclearenvelope assembly, kinetochore signaling and spindle check-point, chromosome architecture, and cytokinesis (14, 15, 42,43). The complex functions of PP1 in mitosis rely on manytargeting subunits that direct PP1 to specific mitotic substrates.For example, Repo-Man, KNL1, SKA, AKAP149, and Ki67 bindPP1, and recruit PP1 to anaphase chromosome, kinetochore,nuclear lamins, and other structures to governmitotic progression(19, 43–47).

Interestingly, mitotic regulation of PP1 involves a number ofmechanisms that negativelymodulate PP1 activities. For example,PP1 is mitotically phosphorylated by CDK1, resulting in suppres-sion of PP1 activity (48). Moreover, a specific inhibitor of PP1,inhibitor-1, binds PP1 during mitosis to reinforce PP1 inhibition(49). Another PP1 inhibitor, inhibitor-2, modulates the action ofPP1 toward multiple mitotic kinases, including Nek2A, Aurora, Aand Aurora B (18, 50, 51). Our previous study in Xenopus eggextracts suggested a role of PNUTS in the mitotic modulation ofPP1 (35). In this study, we first confirmed the role of PNUTS inmammalian mitosis: PNUTS expression oscillates during the cellcycle and peaks in mitosis; PNUTS knockdown reduced mitotic

Figure 6.

PNUTS colocalizes with kinetochoresin a cell cycle–dependent manner, andis required for the activation of thespindle checkpoint. A, HeLa wereanalyzed by immunofluorescence forPNUTS and Cenp-E. Representativeimages of cells in interphase andmitosis (metaphase) are shown. B,HeLa were analyzed byimmunofluorescence for PNUTS andACA (anticentromere antibody).Representative images of cells ininterphase and mitosis (metaphase)are shown. C, HeLa cells with orwithout PNUTS knockdown wereanalyzed by live-cell imaging formitotic progression. The time fromNEBD to anaphase onset wasdocumented. The mean values and SDare shown (N ¼ 20). Statisticalsignificance was analyzed using anunpaired two-tailed Student t test.P < 0.05 was considered statisticallysignificant (�). D, HeLa cells weretreated with nocodazole for 12 hoursand analyzedwith lightmicroscope formitotic index, as in Fig. 1C. The meanvalue and SD were calculated fromthree independent experiments.Statistical significance was analyzedusing an unpaired two-tailed Studentt test. P < 0.001 was considered highlysignificant (��). E, PNUTS knockdowndisrupts BubR1 phosphorylationinduced by nocodazole. HeLa cellswere treated with nocodazole andPNUTS siRNA as indicated, andanalyzed by immunoblotting. F,PNUTS knockdown reduces thekinetochore localization of BubR1.HeLa cells with or without PNUTSsiRNA were treated with nocodazoleand analyzed by immunofluorescencefor BubR1. Representative images ofmetaphase cells are shown.

Wang et al.





entry, and induced severe chromosome missegregation. We thenidentified Aurora A, Aurora B-containing complexes, and severalkinetochore proteins as specific binding patterns of PNUTS.PNUTS depletion suppressed the activation of Aurora A andAurora B kinases, inhibited the relocalization of the Aurora B–containing chromosomal passenger complex from anaphasechromosomes to the cell equator, and bypassed the activationof the spindle checkpoint. Thus, our findings revealed a newmechanism involved in the inhibitory regulation of mitotic PP1.In particular, this mechanism is responsible for the efficientactivation and function of Aurora kinases in mitotic progression.Although the experimentswere primarily carriedout inHeLa cells,owing to the high efficiency of siRNA transfection in these cells, aportion of the studies were confirmed in additional breast cancercell lines. Moreover, the role of PNUTS inmitotic progression wasfirst revealed in Xenopus egg extracts (35), indicating the well-conserved nature of this regulatory mechanism.

It remains a very intriguing question why the cell employsmultiple distinct mechanisms to antagonize the activity of PP1in mitosis. We speculate that the complex pattern of PP1modulation is necessary to achieve the proper regulation ofPP1 during mitotic progression. On one hand, the bulk of PP1activity needs to be inhibited to allow the phosphorylation ofmitotic substrates, which would otherwise be dephosphory-lated by PP1. To this end, the antimitotic function of PP1 hasbeen well established as an essential mechanism that promotesmitotic exit (42, 49). As an example, recent studies discovered arole of PP1 in the dephosphorylation of MASTL (also known asGreatwall kinase) during mitotic exit (36, 52–54). On the otherhand, portions of PP1 bind specific mitotic structures andsubstrates, and play an active role in promoting several keysteps of mitotic progression. For example, it has been shownthat mitotic PP1 governs the proper microtubule–kinetochoreattachment, silences the spindle checkpoint, and maintains theproper phosphorylation gradient and chromosome architecture(15, 42, 43). Interestingly, general inhibition of mitotic PP1 viaoverexpression of nuclear inhibitor of PP1 (NIPP1) in HeLacells caused prometaphase arrest, spindle-formation and chro-mosome-congression defects, and hyperactivation of the spin-dle checkpoint (55). In principle, the involvement of multipleinhibitory mechanisms allows for the dynamic, localization-dependent, and substrate-specific modulation of PP1, although

additional molecular details are needed to further delineate thismodel.

PNUTSmediates kinetochore signaling and spindle checkpointactivation

Our study revealed PNUTS association with kinetochore pro-teins, Cenp-E and Bub1. A portion of PNUTS colocalizes withCenp-E and kinetochores in interphase, whereas PNUTS is exclud-ed from kinetochores and chromosomes in metaphase. Thedynamic kinetochore association and dissociation of PNUTS ispotentially interesting. It is well recognized that reversible phos-phorylation of kinetochore components plays a pivotal role ingoverning spindle attachment and the activation/deactivation ofthe spindle checkpoint. Several kinetochore components, espe-cially Knl1, yield PP1-binding activities, and were reported torecruit PP1 to kinetochores (19). After the proper spindle–kinetochore attachment, PP1 dephosphorylates Aurora B, Knl1,BubR1, and other kinetochore proteins to silence the spindlecheckpoint and trigger metaphase–anaphase transition (22). Thisswitch-like, abrupt activation of PP1 may involve mechanismsthat prevent the premature action of PP1. Thus, it is plausible thatPNUTS plays a role in suppressing the premature activation ofPP1, and that the kinetochore dissociation of PNUTS allows thetimely activation of PP1 on kinetochores, which then triggersmetaphase–anaphase transition. Further investigations are need-ed to better delineate the cell cycle–dependent kinetochore local-ization of PNUTS, as well as the detailed function of PNUTS inkinetochore signaling.

PNUTS is a potential drug target to overcome the resistance toAurora kinase inhibition

Small-molecule inhibitors of Aurora kinases have been sug-gested as promising therapeutics for breast, ovarian, lung, andcolon cancers, as well as hematologic malignancies (6, 8). Unfor-tunately, the clinical benefits of these therapeutic agents are oftenlimited by drug resistance. Therefore, revealing mechanisms thatallow cells to tolerate Aurora kinase inhibition is a crucial steptoward development of strategies that overcome drug resistance.Interestingly, existing genetic and cellular studies showed that,although Aurora kinases play important functions in mitosis,depletion of Aurora kinases can be almost completely toleratedvia downregulation of the counteracting PP1 (16–19). As we

Figure 7.

PNUTS targeting renders ZR75-1 breast cancer cells sensitive to Aurora inhibition. (A) ZR75-1 cells were treated with PNUTS siRNA and ZM447439 as indicated.The relative cell number (actual cell number/the starting cell number in day 1) is shown. The mean value and standard deviation were calculated from 3independent experiments. Statistical significance was analyzed using one-way ANOVA and Turkey post hoc test. P < 0.001 was considered statistically significant(��). (B) ZR75-1 cells were treated with PNUTS siRNA and ZM447439, as indicated, for two days, and measured by the trypan blue exclusion assay for cell death.The mean value and standard deviation were calculated from 3 independent experiments. Statistical significance was analyzed using an unpaired 2-tailedStudent t-test. P < 0.001 was considered highly significant (��).






discoveredPNUTS as aPP1-modulator towardAurora kinases andsubstrates, we speculated that that mitotic modulation of PP1 byPNUTS may contribute to the cellular resistance to Aurora inhi-bition. We confirmed that PNUTS depletion sensitized resistantbreast cancer cells to Aurora inhibition. Thus, it is plausible thatPNUTSupregulation constitutes amechanismof cancer resistanceto Aurora inhibition. As such, PNUTS expression may serve as aprognostic indicator for Aurora resistance, and targeting PNUTSexpression or its PP1 interaction, in conjunction with Aurorainhibition, may expand the therapeutic window, particularly incancer cells that respondpoorly to the current clinical inhibitors ofAurora kinases.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: F. Wang, A. PengAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F. Wang, L. Wang, L.A. Fisher

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F. Wang, L. Wang, A. PengWriting, review, and/or revision of themanuscript: F.Wang,W.Wang, A. PengAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. LiStudy supervision: C. Li, W. Wang, A. Peng

AcknowledgmentsMicroscopic analysis was performed at the UNMC Advanced Microscopy

Core Facility, supported by the Nebraska Research Initiative, the Fred andPamela Buffett Cancer Center support grant (P30CA036727), and an Institu-tional Development Award (IDeA) from the National Institute of GeneralMedical Sciences of the NIH (P30GM106397). This work was supported byNIH grant R01CA172574 to A. Peng.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 15, 2017; revised May 31, 2018; accepted August 27,2018; published first September 6, 2018.

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2019;17:10-19. Published OnlineFirst September 6, 2018.Mol Cancer Res Feifei Wang, Ling Wang, Laura A. Fisher, et al. Aurora Kinases and Mitotic ProgressionPhosphatase 1 Nuclear Targeting Subunit (PNUTS) Regulates

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