MOLECULAR AND CELLULAR BIOLOGY, June 2009, p. 3134–3150
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 29, No. 11
Functional Dynamics of Polo-Like Kinase 1 at the Centrosome?†
Kazuhiro Kishi,1Marcel A. T. M. van Vugt,1Ken-ichi Okamoto,2
Yasunori Hayashi,2and Michael B. Yaffe1*
David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue,
E18-580, Cambridge, Massachusetts 02139,1and RIKEN-MIT Neuroscience Research Center, The Picower Institute for
Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology,
46-4243A, 43 Vassar Street, Cambridge, Massachusetts 021392
Received 27 October 2008/Returned for modification 20 November 2008/Accepted 12 March 2009
Polo-like kinase 1 (Plk1) functions as a key regulator of mitotic events by phosphorylating substrate proteins
on centrosomes, kinetochores, the mitotic spindle, and the midbody. Through mechanisms that are incom-
pletely understood, Plk1 is released from and relocalizes to different mitotic structures as cells proceed through
mitosis. We used fluorescence recovery after photobleaching to examine the kinetics of this process in more
detail. We observed that Plk1 displayed a range of different recovery rates that differ at each mitotic substruc-
ture and depend on both the Polo-box domain and a functional kinase domain. Upon mitotic entry, centro-
somal Plk1 becomes more dynamic, a process that is directly enhanced by Plk1 kinase activity. In contrast, Plk1
displays little dynamic exchange at the midbody, a process that again is modulated by the kinase activity of
Plk1. Our findings suggest that the intrinsic kinase activity of Plk1 triggers its release from early mitotic
structures and its relocalization to late mitotic structures. To assess the importance of Plk1 dynamic relocal-
ization, Plk1 was persistently tethered to the centrosome. This resulted in a G2delay, followed by a prominent
prometaphase arrest, as a consequence of defective spindle formation and activation of the spindle checkpoint.
The dynamic release of Plk1 from early mitotic structures is thus crucial for mid- to late-stage mitotic events
and demonstrates the importance of a fully dynamic Plk1 at the centrosome for proper cell cycle progression.
This dependence on dynamic Plk1 was further observed during the mitotic reentry of cells after a DNA damage
G2checkpoint, as this process was significantly delayed upon centrosomal tethering of Plk1. These results
indicate that mitotic progression and control of mitotic reentry after DNA damage resides, at least in part, on
the dynamic behavior of Plk1.
Polo-like kinases (Plks) are serine/threonine protein kinases
that play essential roles during the cell cycle. Mammalian Plks
are subdivided into four family members: Plk1, Plk2, Plk3, and
Plk4 (51). Plks have a highly conserved N-terminal kinase
domain and a relatively divergent C-terminal domain, called
the Polo-box domain (PBD), which contains one (Plk4) or two
(Plk1 to Plk3) Polo-boxes. Of the four Plks, the best charac-
terized member is Plk1 (10). Plk1 functions in a diverse num-
ber of processes that are crucial for proper progression
through multiple stages of mitosis, including mitotic entry,
centrosome maturation, bipolar spindle formation, chromo-
some congression and segregation, cytokinesis, and mitotic exit
(3, 10). The kinase domain of Plk1 is regulated in part by
phosphorylation at Thr-210 within the activation loop (26, 32),
likely through the actions of the upstream kinase Aurora-A in
complex with the adaptor protein Bora (45, 60). Mutation of
Thr-210 to Asp (T210D) mimics T-loop phosphorylation and
stimulates kinase activity (26, 32, 56). Expression of an acti-
vated Plk1 T210D mutant can override the G2arrest induced
by DNA damage (62, 72) and allow cells to enter and progress
completely through mitosis, albeit with a slight spindle check-
point-dependent mitotic delay (71).
The kinase domain of Plk1, in its nonphosphorylated less
active state, appears to be negatively regulated through direct
interaction with the PBD (25, 48). We have shown previously
that the PBDs of Plk1 function as a phosphoserine/phospho-
threonine-binding module, recognizing the optimal recogni-
tion sequence motif Ser-[pSer/pThr]-[Pro/X] (15, 16). The
structural basis for this phosphopeptide-specific binding has
been revealed through two high-resolution X-ray structures of
Plk1 PBD:phosphopeptide complexes (7, 16). In addition, we
have shown that binding of phosphopeptides by the PBD in
full-length Plk1 relieves its inhibitory function on kinase activ-
ity (16). These findings imply that priming phosphorylations on
substrates or docking proteins by other mitotic kinases, such as
cyclin-dependent kinases (Cdks), may target Plk1 to these sub-
strates, while simultaneously activating its kinase activity. In
agreement with this, a mass spectrometry study revealed that
many known and potential Plk1 substrates specifically interact
with the PBD of Plk1 in vitro in a phosphorylation- and mito-
sis-specific manner (44), while numerous studies from the
Nigg, Barr, Lee, and Erikson groups, as well as others, have
demonstrated that the PBD plays a crucial function in both
substrate interaction and subcellular targeting of Plk1 in vivo
(5, 26, 32, 33, 35, 48, 50, 54, 61).
Plk1 dynamically localizes to various mitotic structures as
cells progress through different stages of mitosis (3, 10) in a
manner that depends on PBD function (16, 28, 33, 61, 64).
* Corresponding author. Mailing address: Department of Biology
and Biological Engineering, David H. Koch Institute for Integrative
Cancer Research, Massachusetts Institute of Technology, Cambridge,
MA 02139. Phone: (617) 452-2103. Fax: (617) 452-4978. E-mail:
† Supplemental material for this article may be found at http://mcb
?Published ahead of print on 23 March 2009.
During interphase and early prophase Plk1 is found at the
centrosome, where it facilitates ?-tubulin recruitment (11) and
centrosome maturation, separation, and microtubule nucle-
ation during late prophase and prometaphase (2, 3, 8, 31). By
metaphase, a fraction of Plk1 specifically localizes to the ki-
netochores, where it seems to be involved in regulating aspects
of spindle checkpoint function and the metaphase-anaphase
transition (1, 21). During anaphase, Plk1 is concentrated in the
spindle midzone, where it likely facilitates microtubule sliding
(42, 50), while after chromosome segregation in late anaphase,
Plk1 remains located in the central spindle and midbody,
where it participates in ingression of the cleavage furrow dur-
ing cytokinesis (42, 50). Plk1 also resides at the Golgi appara-
tus, presumably linking Golgi fragmentation with mitotic entry
by interacting with the structural Golgi protein GRASP65 (54).
Details of the movement and exchange of Plk1 between
these mitotic structures remains poorly characterized, and
whether Plk1 molecules exhibit similar or different dynamic
behaviors at these distinct mitotic structures is largely un-
known. What controls the kinetics of Plk1 localization during
mitotic progression, and what is the relative importance of the
PBD and kinase domains in this process? To address these
questions, we analyzed the subcellular dynamics of Plk1 ex-
change in wild-type (WT) and mutant Plk1 proteins, at various
mitotic structures, using fluorescence recovery after photo-
bleaching (FRAP). In addition, to investigate whether dynamic
relocalization of Plk1 is critical for proper mitotic progression,
we examined the phenotypes of cells expressing an active but
less dynamic form of Plk1, in the absence or presence of the
endogenous Plk1 protein.
Intriguingly, many of the Plk1-dependent processes involved
in early mitotic events appear to occur on centrosomes. Cen-
trosomes are tiny (1 to 2 ?m3) cytoplasmic organelles consist-
ing of a pair of barrel-shaped microtubule assemblies, the
centrioles, surrounded by pericentriolar material (12). In in-
terphase cells, a single centrosome functions as the microtu-
bule-organizing center, which then divides in S phase and sep-
arates in mitotic prophase to nucleate the bipolar mitotic
spindle in prometaphase. In addition to their role in nucleating
and anchoring the mitotic spindle, centrosomes are increas-
ingly recognized as critical subcellular structures responsible
for coordinately regulating the normal progression from G2
into M. For example, Jackman et al. (24) showed that the early
mitotic activation of the Cdk1-cyclin B complex occurred at
centrosomes, presumably through activation of the phos-
phatase Cdc25B, which has also been shown to localize to the
centrosomes (9, 17, 24, 40). In addition to direct activators of
the G2/M transition, a variety of effectors of the DNA damage-
induced G2/M checkpoint have also been reported to localize
to centrosomes in mammalian cells during the normal cell
cycle, including the kinases Chk1, Chk2, and Plk3 and the
signaling scaffold molecule BRCA1 (43). Consequently, the
centrosome has now emerged as an organelle where signaling
from the DNA damage checkpoint and components of the
Cdk1-cyclin B activation loop are integrated during an unper-
turbed cell cycle to control mitotic entry. Importantly, Plk1 is
involved in Cdk1-cyclin B activation, regulates DNA damage
checkpoint function, and localizes to centrosomes. We there-
fore further investigated whether a centrosome-tethered form
of Plk1 with reduced dynamic exchange would be sufficient to
allow mitotic reentry after a DNA damage-induced G2check-
MATERIALS AND METHODS
Construction of cell lines and plasmids. Human Plk1 cDNAs were subcloned
into pEGFP-C1 (Clontech, Mountain View, CA) (enhanced green fluorescent
protein [EGFP]-Plk1). EGFP-Plk1-AKAP and EGFP-AKAP were generated by
subcloning the PCR-derived AKAP450 fragment corresponding to residues 3644
to 3808 into EGFP-Plk1 and pEGFP-C3, respectively. Spectrin-GFP, the small
interfering RNA vector pS(pSuper), pS-Plk1, and pS-Mad2 have been described
previously (74). EGFP plasmids encoding Plk1 or Plk1-AKAP harboring silent
mutations in the targeting region of pS-Plk1, which renders the protein insensi-
tive to pS-Plk1-mediated degradation, have been described (71).
To isolate the human CENP-A cDNA, total RNAs purified from the human
osteosarcoma U2OS cell line were transcribed to first-strand cDNAs using M-
MLV reverse transcriptase (Promega, Madison, WI). The resulting products
were then used as templates for PCR with a forward primer (5?-CTCTgCggCgT
gTCATgg-3?) and a reverse primer (5?-TCAgCCgAgTCCCTCCTCAAgg-3?)
that were designed to amplify human CENP-A. The PCR fragment was cloned
into pCR2.1-TOPO (Invitrogen, Carsbad, CA) and verified as a fragment con-
taining the CENP-A cDNA by sequencing. The human CENP-A fragment was
subcloned into DsRed2-C1 vector (Clontech). DsRed2-tagged CENP-A
(DsRed2-CENP-A) was amplified by PCR and subcloned into retroviral vector
pLNCX2 (Invitrogen). DsRed2-CENP-A retrovirus was produced by transfec-
tion with pLNCX2-DsRed2-CENP-A, gag-pol, and VSV-g-env DNAs into 293
cells. Filtered tissue culture supernatant containing the virus was mixed with
Polybrene and added to U2OS cells. The infected cells were selected by growth
in the presence of 0.4 mg of G418 (Sigma, St. Louis, MO)/ml for 2 to 3 days, and
U2OS cells stably expressing DsRed2-CENP-A were established. All of the
constructs were confirmed by DNA sequence analysis.
Antibodies and dyes. Thymidine, propidium iodide (PI), doxorubicin, pacli-
taxel, and caffeine were purchased from Sigma. Antibodies were obtained as
follows: rabbit anti-phospho-Ser10-histone H3 (Upstate Biotechnology, Lake
Placid, NY); monoclonal antibody cocktail against Plk1 (Zymed Laboratories,
South San Francisco, CA); rabbit anti-Plk1 (Upstate Biotechnology); mouse
monoclonal anti-GFP (Roche, Manheim, Germany); rabbit anti-GFP (Santa
Cruz Biotechnology, Santa Cruz, CA); mouse monoclonal anti-?-actin, DM1A
mouse monoclonal anti-?-tubulin, and GTU88 mouse monoclonal anti-?-tubulin
(Sigma); human nuclear ANA-centromere autoantibody (CREST; Cortex Bio-
chem, San Leandro, CA); rabbit anti-Mad2 (Covance, Princeton, NJ); mouse
anti-CENP-A (Abcam, Cambridge, MA); peroxidase-conjugated goat anti-rabbit
and rabbit anti-mouse (Dako, Glostrup, Denmark); goat anti-mouse Alexa Fluor
488, anti-rabbit Alexa Fluor 488, anti-mouse Alexa Fluor 594, and anti-rabbit
Alexa Fluor 647 (Molecular Probes, Eugene, OR); and rhodamine-conjugated
anti-human (Jackson Immunoresearch Laboratories, West Grove, PA).
Cells, transfection, synchronization, and recovery from DNA damage. Human
osteosarcoma U2OS cells were purchased from the American Type Culture
Collection. U2OS cells, 293 cells, and 293T cells were grown at 37°C in 5% CO2
in Dulbecco modified Eagle medium supplemented with 100 U of penicillin/ml,
100 ?g of streptomycin/ml, and 10% fetal calf serum. Transfection was per-
formed by using Fugene 6 (Roche), Genejammer (Stratagene, Cedar Creek,
TX), or electroporation (Gene Pulser II; Bio-Rad, Hercules, CA) for U2OS cells
and by using Lipofectamine 2000 (Invitrogen) for 293 cells and 293T cells,
according to the manufacturer’s instructions. For electroporation, cells cultured
in a 150-mm culture dish were transiently transfected with 20 to 40 ?g of plasmid
DNA/cuvette, and then the cells were allowed to adhere to dishes or plates for
24 to 48 h and used for experiments. For immunoblotting in reconstitution
experiments, 10 ?g of pS or pS-Plk1 was cotransfected into U2OS cells with 1 ?g
of pBabe-Puro combined with the indicated amounts of Plk1WT or Plk1-AKAP
where indicated. Then, 2 ?g of puromycin/ml was added about 8 to 16 h after
transfection. After a 24-h selection, cells were harvested and lysed for immuno-
blotting. For flow cytometry analysis in reconstitution experiments, 10 ?g of pS
or pS-Plk1 was cotransfected with 1 ?g of spectrin-GFP or 3 to 15 ?g of the
indicated Plk1 WT or mutant plasmids. Cells were synchronized in thymidine for
24 h and harvested at the indicated times after release. For recovery from DNA
damage, 10 ?g of pS or pS-Plk1 was cotransfected with 3 to 15 ?g of the indicated
Plk1 WT or mutant plasmids. Activation and inactivation of the G2DNA damage
checkpoint was performed as described previously and below (72).
Immunoblotting. Cells were rinsed twice with ice-cold phosphate-buffered
saline (PBS); solubilized in lysis buffer containing 20 mM Tris-HCl (pH 8.0), 20
mM Na4P2O7, 1 mM dithiothreitol, 1% Nonidet P-40, 1 mM MgCl2, 1 mM
CaCl2, 10% glycerol, 0.5 mM Na3VO4, and 20 ?M p-A-phenylmethanesulfonyl
VOL. 29, 2009CENTROSOMAL Plk1 CONTROLS CHECKPOINT RELEASE 3135
fluoride; and centrifuged at 14,000 ? g for 10 min at 4°C. Protein content in the
cell lysates was quantified by using a Bio-Rad protein assay, and aliquots of
protein were boiled for 5 min in sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) sample buffer (50 mM Tris-HCl [pH 6.8], 2% SDS,
2% mercaptoethanol, 10% glycerol). The solubilized proteins were separated by
SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Bio-Rad), and
detected by immunoblotting with the indicated antibodies using Western Lightning
chemiluminescence reagent (Perkin-Elmer Life Sciences, Boston, MA).
Flow cytometry. U2OS cells were transiently transfected with the indicated
plasmids. At 24 to 48 h after transfection, the cells were then incubated with 2.5
mM thymidine in medium for an additional 24 h. After a thymidine block, the
cells were washed three times and incubated with new medium for 20 h. The cells
were harvested by trypsinization, fixed in ice-cold 70% ethanol, and stained with
PI and/or anti-phospho-Ser10-histone H3 antibody in combination with anti-
rabbit Alexa Fluor 647 antibody in 3% bovine serum albumin–PBS–0.05%
Tween 20. Cells were analyzed by using a FACSscan (Becton Dickinson, Fran-
klin Lakes, NJ) or a FACSCalibur (Becton Dickinson). Cell cycle profiles were
generated by using CellQuest (Becton Dickinson) and FlowJo software (Tree
Star, Ashland, OR).
For examining the DNA damage response, U2OS cells were transiently trans-
fected with the indicated plasmids, and at 24 h after transfection the cells were
incubated with 2.5 mM thymidine in medium for an additional 24 h. After the
thymidine block, the cells were washed three times and released into new me-
dium for 6 h to exit from S phase. Cells were then treated with 1 ?M doxorubicin
for 1 h, washed with drug-free medium, and incubated with medium containing
1 ?M paclitaxel for 18 h. The cells were washed again with medium three times
and incubated with new medium in the presence or absence of 5 mM caffeine for
7 h. The cells were harvested and stained as described above.
Immunofluorescence. U2OS cells transfected with the indicated plasmids were
seeded on 18-by-18-mm glass coverslips. At 24 to 48 h after transfection or at the
indicated period after release from thymidine block, the cells were fixed in either
4% paraformaldehyde in PBS or 100% methanol for 15 min at room tempera-
ture, washed with PBS, and permeabilized 0.5% Triton X-100 in buffer (30 mM
Tris buffer [pH 7.8], 75 mM NaCl, 0.3 M sucrose, 3 mM MgCl2) for 15 min at
room temperature. For kinetochore staining, the cells on coverslips were ex-
tracted with PIPES buffer (20 mM PIPES, 0.3% Triton X-100, 1 mM MgCl2, 10
mM EGTA [pH 6.9]) for 5 min and then fixed with 4% paraformaldehyde for 10
min. Coverslips were blocked with 1% bovine serum albumin–1% sucrose in PBS
or 3% bovine serum albumin–1% sucrose in PBS (kinetochore staining) for 1 h
and stained with the indicated antibody at 4°C overnight. After a washing step,
coverslips were stained with Alexa Fluor-conjugated secondary antibody for 1 h
at room temperature. The coverslips were washed five times and then mounted
onto slides with Fluoromount-G (Southern Biotechnologies, Birmingham, AL)
or ProLong Gold antifade reagent (Invitrogen) containing DAPI (4?,6?-di-
amidino-2-phenylindole; Sigma) for DNA staining. Images were collected on an
Axioplan2 microscope (Carl Zeiss, Oberkochen, Germany) and processed using
OpenLab software (Improvision, Waltham, MA). Where indicated, images were
collected on a Deltavision (Applied Precision, Issaquah, WA) and digitally de-
convolved using softWoRx (Applied Precision).
Plk1 kinase assay. Control EGFP or EGFP-Plk1 transfected cells were solu-
bilized in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 1% Triton X-100, 150
mM NaCl, 2 mM EDTA (pH 8.0), 0.5% SDS, 50 mM NaF, 2 mM Na3VO4, 2 mM
dithiothreitol, and protein inhibitors and then centrifuged at 14,000 ? g for 10
min at 4°C. Anti-GFP immunoprecipitates were incubated in kinase buffer con-
taining 50 mM Tris-HCl (pH 7.5), 200 ?M Na3VO4, 5 mM dithiothreitol, 5 mM
NaF, 50 ?M ATP, 2 mM EGTA, and 10 mM MgCl2at 30°C. Then, 20 ?Ci of
[?-32P]ATP and 1 ?g of casein (Sigma) were added, the reaction was incubated
for an additional 30 min at 30°C, and the products were analyzed by SDS-PAGE
Live imaging and FRAP analysis. U2OS cells grown on 12-by-12-mm or
18-by-18-mm coverslips were used for transfection with EGFP-Plk1 constructs.
At 24 to 36 h after transfection, the coverslips were immersed in 37°C prewarmed
PBS containing 0.9 mM CaCl2and 0.33 mM MgCl2, and FRAP images of the
cells then obtained by using an Olympus (Tokyo, Japan) FV 300/IX70 inverted
laser-scanning confocal microscope (objective lens[?60], NA 1.42) with excita-
tion at 488 nm (or 568 nm for DsRed) and 6% laser output intensity. All FRAP
experiments were performed on an ambient temperature stage within 15 min of
removal of the coverslips from the 37°C incubator. Under these conditions, the
temperature of the PBS medium slowly decreased with time but remained at or
above 30°C at the end of the measurement period. For photobleaching of EGFP-
Plk1 constructs, the apertures were adjusted to encompass the smallest possible
area that contained the mitotic substructure of interest, and irradiated using the
488-nm laser line of an argon laser (385-mW actual output). The region of
interest was scanned 20 times with 1-s lapse (mitotic centrosome and kineto-
chore) or 2-s lapse (interphase centrosome and midbody) intervals at the same
wavelength, with 100% laser output intensity after it was scanned 20 times with
the same intervals with 6% intensity to establish a baseline, as described previ-
ously (23). Thereafter, imaging was continued at 6% intensity with the same inter-
vals. In each FRAP experiment a minimum of 30 cells were measured. The mean
fluorescence intensity was analyzed with MetaMorph software (Molecular Devices,
Sunnyvale, CA) and plotted. Recovery was fitted to a single exponential curve (Y ?
TOP*[1 ? exp(?K*X)] using GraphPad Prism software (San Diego, CA).
Statistical analysis. Data are generally expressed as means ? the standard
errors of the mean (SEM) unless otherwise indicated. The data were analyzed by
analysis of variance with the Bonferroni correction for multiple-comparison tests
or t tests (two-tailed) for determination of significance of the differences. A P
value of ?0.05 was considered to be statistically significant.
The subcellular localization pattern of EGFP-tagged Plk1 in
live cells mimics that of the endogenous Plk1 protein. To
analyze the dynamic exchange behavior of Plk1 at various
mitotic structures by FRAP, we constructed an EGFP N-ter-
minally tagged Plk1 (EGFP-Plk1) (Fig. 1A), and examined its
subcellular localization after transient transfection in U2OS
cells as they progressed through mitosis (Fig. 1B). When live
cells were imaged, EGFP-Plk1 was observed at centrosomes in
G2(Fig.1Ba) and persisted at mitotic centrosomes during pro-
metaphase (Fig.1Bb) and metaphase (Fig.1Bc). In addition,
during prometaphase and metaphase, EGFP-Plk1 became as-
sociated with both aligned and unaligned kinetochores, as re-
vealed by the presence of punctate fluorescence in association
with chromatin bodies (Fig. 1Bb and c, small arrows). Upon
anaphase entry, EGFP-Plk1 transiently localized to spindle
microtubules (Fig.1Bd), followed by the appearance of strong
Plk1 fluorescence at the spindle midzone during late anaphase
(Fig.1Be). Finally, during telophase and cytokinesis, EGFP-
Plk1 accumulated at the midbody (Fig.1Bf), although some
residual kinetochore staining was also observed throughout
these late mitotic stages.
When examining U2OS cells in prometaphase, the centroso-
mal localization of EGFP-Plk1 often could not be unambiguously
distinguished from kinetochore localization. This posed a poten-
tial problem for experiments measuring the dynamics of Plk1
exchange at centrosomes versus kinetochores by FRAP during
this mitotic substage. To overcome this difficulty, we therefore
established U2OS cells that stably expressed DsRed2-tagged
CENP-A (DsRed-CENP-A [see Materials and Methods]), a
known marker of kinetochores (29) (Fig. 1C). Expression of
DsRed-CENP-A in these cells was confirmed by immunoblotting
with anti-CENP-A antibodies (Fig. 1C, left panels), while local-
ization of DsRed-CENP-A to kinetochores was verified by live
cell fluorescence microscopy (Fig. 1C, lower right panel). When
EGFP-Plk1 was coexpressed in the cells stably expressing DsRed-
CENP-A, the kinetochore-localized Plk1 signal could be easily
and unambiguously distinguished from the centrosome-localized
Plk1 (Fig. 1C).
These imaging studies, performed in living cells, demon-
strated that EGFP-Plk1 showed patterns of mitotic stage-spe-
cific localization identical to those reported previously in fixed
cells stained for endogenous Plk1 (3, 20, 36) or for a similar
GFP-tagged Plk1 construct (2). In addition, U2OS cells tran-
siently overexpressing low to moderate amounts of EGFP-Plk1
showed normal cell cycle profiles and rates of mitotic progres-
3136KISHI ET AL.MOL. CELL. BIOL.
sion compared to U2OS cells transfected using a spectrin-GFP
control plasmid, although at the highest expression levels Plk1
induced some mitotic cell accumulation (data not shown).
Plk1 present in distinct mitotic structures shows different
rates of exchange with the free Plk1 pool. Next, we probed the
dynamic properties of Plk1 at different mitotic substructures in
living cells during G2- and M-phase using FRAP at 30 to 37°C.
Individual centrosomes, kinetochores, or midbodies in the
EGFP-Plk1-transfected cells showing low to moderate levels of
expression were photobleached, and the recovery of the fluo-
rescence signal in the bleached area was monitored by time-
lapse imaging at 2-s intervals for G2centrosomes and midbod-
ies and at 1-s intervals for mitotic centrosomes and
kinetochores (Fig. 2A and B; see Fig. S9 in the supplemental
material). FRAP analysis revealed that the rate of Plk1 ex-
change was strongly dependent on its subcellular localization
(Table 1). WT EGFP-Plk1 at G2centrosomes, for example,
was highly dynamic, with a mean time for 50% recovery of the
total net fluorescence recovered (t1/2) of 17.9 s and a mean
total net fluorescence recovery intensity of 48.3% (Fig. 2A and
Table 1). At mitotic centrosomes, the rate of exchange of
EGFP-Plk1 was even faster (50% recovery time t1/2? 7.0 ?
1.3 s), although the mean net fluorescence recovery intensity
was somewhat reduced (28.3%) (Fig. 2A and Table 1). Ex-
change rates very similar to those measured for mitotic cen-
trosomes were also observed for mitotic kinetochores (t1/2?
12.4 ? 1.3 s, mean net fluorescence recovery intensity ?
29.1%) (Fig. 2A and Table 1). In marked contrast to the rapid
rate of exchange observed at mitotic centrosomes and kineto-
chores, the exchange rate for WT EGFP-Plk1 localized to the
midbody during the terminal stages of mitosis was significantly
slower (t1/2? 65.7 ? 10.0 s; mean net fluorescence recovery
intensity ? 23.3%) (Fig. 2A and Table 1). Similar half-lives for
recovery were observed over a range of bleaching conditions
(cf. Fig. S1 and S2 in the supplemental material).
Intrinsic protein kinase activity stimulates Plk1 release
from mitotic centrosomes but delays Plk1 release from mid-
bodies. To determine the importance of kinase domain activity
in determining the dynamic behavior of full-length Plk1, con-
structs of EGFP-Plk1 containing a series of mutations and/or
deletions in the kinase domain were transfected into U2OS
cells, and the kinetics of Plk1 exchange at the same G2and
mitotic substructures described above were analyzed by FRAP.
Mutations that were studied included a constitutively active
phosphorylation-mimicking mutation in the T-loop (T210D), a
nonphosphorylatable T-loop mutant (T210A) that should re-
tain basal kinase activity but cannot be further activated by
mitotic phosphorylation (16, 45, 71), a kinase-dead mutant
(D176N), and a truncation mutant solely containing the PBD
(Fig. 1A; see Fig. S3 in the supplemental material) (15, 16, 44).
After transient transfection, all of the constructs were ex-
pressed at similar levels and migrated at the expected sizes
when examined by SDS-PAGE and Western blot analysis (see
Fig. S3 and Table S1 in the supplemental material). The kinase
activities of the WT and mutant EGFP-Plk1 constructs was
directly measured by immunoprecipitating the proteins from
asynchronous cells and assaying their ability to phosphorylate
casein. As expected, roughly similar amounts of kinase activity
were detected in the cells expressing Plk1 WT or T210A, in-
creased by the expression of T210D and absent in cells express-
FIG. 1. Live cell images for FRAP during mitosis in U2OS cells ex-
pressing EGFP-Plk1. (A) Schematic representations of EGFP-tagged
Plk1 constructs that were used for FRAP experiments. From top to
bottom: full-length WT Plk (WT); a phosphorylation-mimicking T-loop
mutation,Thr-210 to Asp (T210D) that renders Plk1 constitutively active;
a kinase-dead Plk1 in which the catalytic base Asp-176, is mutated to Asn
(D176N); a nonphosphorylatable T-loop form of Plk1 displaying only
basal activity in which Thr-210 is mutated to Ala (T210A); an N-termi-
nally truncated construct containing only the C-terminal PBD. (B) U2OS
cells expressing were transfected with EGFP-Plk1 WT. Representative
images of EGFP-Plk1 localization during mitosis are shown. Images rep-
resent G2interphase (a), prometaphase (b), metaphase (c), anaphase (d),
anaphase/telophase (e), and telophase (f), respectively. EGFP-Plk1 local-
ized to the centrosomes in subpanels a, b, and c, as shown by arrowheads.
Arrows indicate EGFP-Plk1 localized at kinetochores in subpanels b and
c, at mitotic spindles in subpanel d, and at the midbody and/or spindle
DsRed2-CENPA. A Western blot analysis of U2OS cell lysates using the
indicated antibodies is shown in the left panel, with ?-actin used as a
loading control. The right panels show fluorescence images of EGFP-Plk1
(upper) and DsRed-CENPA (lower). Arrows indicate kinetochores, and
arrowheads indicate centrosomes. Scale bars in all panels, 5 ?m.
VOL. 29, 2009 CENTROSOMAL Plk1 CONTROLS CHECKPOINT RELEASE3137
FIG. 2. FRAP analysis of Plk1 at centrosomes, kinetochores, and midbodies. (A) Centrosomes during interphase and mitosis, kineto-
chores, and midbodies were targeted for laser photobleaching and monitored by fluorescence time-lapse microscopy. Photobleaching was
performed at 100% laser transmission by scanning the bleached region of interest. Recovery intensity data were collected at 6% laser
transmission at 1- or 2-s intervals. The data points represent the means from 7 to 17 experiments (see Table 1), with ?30 cells measured
in each experiment. Black curves were fitted to data by using GraphPad Prism software. The right panel shows the enlarged plotted
intensities and fitted curves of mitotic centrosomes and kinetochores. (B) The 50% recovery times were calculated from each data curve from
independent experiments. Mean values are indicated with bar graphs (G2phase centrosomes [red], mitotic centrosomes [blue], kinetochores
[yellow], or midbodies [green]). Error bars indicate the SEM. (C) FRAP for EGFP-Plk1 WT (WT) and mutants localized at G2(a) and
mitotic centrosomes (b), kinetochores (c), and midbodies (d) was performed as described in panel A. The fluorescence intensities after
photobleaching are plotted (WT [red squares], T210D [blue triangles], D176N [green triangles], T210A [yellow diamonds], or PBD [pink
diamonds]). Each data point represents the mean of 5 to 19 experiments, with ?25 cells measured in each experiment. (D) The 50% recovery
times for Plk1 localized at G2centrosomes (a), mitotic centrosomes (b), kinetochores (c), and midbodies (d) were calculated from each data
curve from independent experiments. Averages are indicated in the bar graphs with error bars showing the SEM. Statistically significant
differences are indicated by asterisks (?, P ? 0.05; ??, P ? 0.01).
3138KISHI ET AL.MOL. CELL. BIOL.
ing D176N (see Fig. S3 in the supplemental material). FRAP
analysis revealed that the exchange kinetics (t1/2) for the WT,
T210D, and T210A forms of EGFP-Plk1 were roughly similar
at both G2and mitotic centrosomes (Fig. 2C and D and Table
1). Interestingly, for mitotic centrosomes, but not G2centro-
somes, the kinase-dead D176N form of Plk1 showed a signif-
icantly slower recovery time and a reduced percentage of net
fluorescence recovery compared to the T210D form, suggest-
ing that after photobleaching the kinase-dead Plk1 has a re-
duced ability to dissociate from mitotic centrosomes and ex-
change with the free pool of cytoplasmic Plk1 than the
constitutively active form. We interpret this as evidence that
the kinase activity of full-length Plk1 may be important for
stimulating the release of Plk1 from centrosomes as cells
progress through mitosis.
In marked contrast to the behavior of kinase-dead Plk1 at
mitotic centrosomes, the same kinase-dead D176N mutant of
EGFP-Plk1 showed a dramatically enhanced ability to dissoci-
ate from the midbody, exchanging with the free Plk1 pool at a
rate that was nearly eightfold faster than that observed with
WT Plk1 (t1/2? 8.3 ? 4.2 s versus 65.7 ? 10.0 s, respectively)
although, curiously, the mobile fraction of the D176N protein
remains low (17.9%) and comparable to WT Plk1. We inter-
pret this as evidence that intrinsic Plk1 kinase activity is re-
quired to maintain association of this small pool of highly
dynamic Plk1 with the midbody as cells progress through telo-
phase and cytokinesis.
The PBD, located C-terminal to the kinase domain, acts as
a localization domain that recognizes short phosphorylated
sequence motifs on a significant number of Plk1 substrates (15,
16, 44, 70) and has been shown to be critical for Plk1 localiza-
tion to centrosomes, kinetochores, and midbodies (15, 25, 28,
33, 49, 50, 55, 61, 63). An EGFP-PBD construct completely
lacking the kinase domain was found to exhibit a significantly
slower rate of exchange at G2and mitotic centrosomes and
midbodies than that observed with any of the full-length Plk1
constructs, either WT or point mutants (Table 1). In addition,
the mean net fluorescence recovery intensities of the PBD at
these structures were considerably reduced, suggesting that the
photobleached EGFP-PBD remains tightly associated with
these structures. These data suggest that the mere presence of
the Plk1 kinase domain, regardless of its catalytic activity, neg-
TABLE 1. Dynamics of Plk1 at different subcellular locations
Mean 50% recovery
time (s) ? SEM
Mean net fluorescence recovery
intensity (%) ? SEM
No. of independent
17.9 ? 2.2
19.0 ? 3.5
16.6 ? 4.2
14.7 ? 2.0
25.8 ? 1.8
58.2 ? 6.8
48.3 ? 4.1
52.1 ? 4.4
65.8 ? 8.9
42.6 ? 9.1
20.4 ? 5.8
31.7 ? 3.6
7.0 ? 1.3
6.9 ? 0.7
10.0 ? 0.8
9.3 ? 2.2
16.7 ? 1.0
28.3 ? 2.0
30.3 ? 4.6
21.8 ? 1.8
31.8 ? 4.4
17.4 ? 1.8
12.4 ? 1.3
9.4 ? 0.8
15.0 ? 1.5
29.1 ? 2.5
23.2 ? 1.5
17.5 ? 2.0
65.7 ? 10.0
75.4 ? 10.9
8.3 ? 4.2
67.8 ? 19.5
108.1 ? 9.8
23.3 ? 5.5
31.0 ? 4.6
17.9 ? 3.1
31.2 ? 6.0
12.7 ? 1.3
VOL. 29, 2009CENTROSOMAL Plk1 CONTROLS CHECKPOINT RELEASE3139
atively modulates the ability of the PBD to engage its ligands
at these mitotic structures in live cells.
Construction and analysis of a centrosome-localized Plk1
molecule. Given the relatively rapid exchange kinetics of Plk1
at centrosomes and kinetochores (Table 1) and the observation
that kinase activity facilitates the dissociation of Plk1 from
centrosomes as cells move from metaphase to anaphase (Fig.
2B to D), we next investigated whether cells expressing a much
less mobile form of centrosomal Plk1 would be impaired in
their ability to transit through mitosis. To accomplish this, we
created a Plk1 construct that preferentially associates with the
centrosome by fusing the C terminus of full-length Plk1 to
the pericentrin-AKAP450 centrosomal targeting domain of
AKAP450 (19). The resulting construct, EGFP-tagged Plk1-
AKAP (EGFP-Plk1-AKAP), was expressed at moderate levels
after transfection of both U2OS and 293T cells, as confirmed
FIG. 3. Plk1-AKAP expression, kinase activity, and localization. (A) EGFP-Plk1 WT or Plk1-AKAP protein expression in transfected U2OS
cells. Western blotting with the indicated antibodies is shown. (B) Plk1 kinase activities in transfected 293T cells. The amounts of immunopre-
cipitated EGFP-Plk1/EGFP-Plk1-AKAP proteins using Western blot analysis (left) and Plk1 kinase activities against ?-casein (right) are shown.
The extent of casein phosphorylation was quantitated by phosphorimager analysis and are indicated as the percentage observed compared to
EGFP-Plk1, which was set at 100%, and are not normalized for the protein recoveries shown in the left panel. Empty vector was used for control
transfections. (C) EGFP-Plk1/EGFP-Plk1-AKAP localization in transfected U2OS cells. Transfected U2OS cells were fixed and immunostained
with anti-?-tubulin antibody (red). EGFP-Plk1-AKAP is localized almost exclusively at the centrosome (arrows), compared to EGFP-Plk1, which
displays both centrosomal and cytoplasmic localization. Chromosomal DNA is stained with DAPI (blue). In last two rows, transfected U2OS cells
were released at 10 h after thymidine block, fixed, and stained. (D) Quantification of centrosome-localized EGFP-Plk1/EGFP-Plk1-AKAP in live
cells. EGFP fluorescence of a fixed area (5 ?m by 5 ?m) at the centrosome or in the cytosol was analyzed (?25 cells), and the ratio of
centrosome/cytosolic fluorescence was calculated. Representative pictures (upper panels) and the average ratios (lower panel) are shown. Error
bars represent the standard deviations. Statistically significant differences are indicated by asterisks (**, P ? 0.01). All scale bars represent 5 ?m.
3140 KISHI ET AL.MOL. CELL. BIOL.
by immunoblotting with anti-GFP and anti-Plk1 antibodies
(Fig. 3A and B). Although EGFP-Plk1-AKAP appeared at the
expected molecular weight, its expression level was about 5- to
10-fold lower per ?g of transfected DNA than that of EGFP-
Plk1 in both cell types (Fig. 3A and B). EGFP-Plk1 and EGFP-
Plk1-AKAP, however, displayed similar levels of kinase activity
when comparable amounts were assayed (Fig. 3B and see Fig.
S4 in the supplemental material). Next, we examined the sub-
cellular localization of Plk1-AKAP by immunostaining for
EGFP in fixed U2OS cells and by fluorescence microscopy in
live U2OS cells (Fig. 3C and D). Both assays showed that the
Plk1-AKAP strong localized at the centrosomes of interphase
cells, as confirmed by complete overlap with ?-tubulin staining
(Fig. 3C). Importantly, the ratio of centrosomal to cytoplasmic
fluorescence was clearly higher in live U2OS cells expressing
Plk1-AKAP than in those expressing Plk1 WT (Fig. 3D), con-
firming that Plk1-AKAP predominantly localizes to centro-
The mobility of EGFP-Plk1-AKAP was examined using
FRAP (Fig. 4A). At centrosomes, the EGFP-Plk1-AKAP pro-
tein showed a ?3-fold prolongation of the recovery half time
after photobleaching compared to WT EGFP-Plk1 (t1/2?
58.2 ? 6.8 s versus t1/2? 17.9 ? 2.2 s, respectively). Likewise,
the mean net fluorescence intensity recovered at the photo-
bleached sites was reduced for EGFP-Plk1-AKAP (31.7%)
compared to EGFP-Plk1 (48.3%), a finding consistent with
more stable tethering of the Plk1-AKAP construct to centro-
Persistent activity of centrosome-tethered Plk1 causes G2
delay and mitotic arrest. To assess whether altering the sub-
cellular dynamics of Plk1 influences its mitotic function, we
compared the cell cycle profiles of U2OS cells expressing low
to moderate levels of EGFP-Plk1-AKAP, EGFP-Plk1, and
EGFP-PBD (Fig. 4B) at 20 h after release from a thymidine
block. As shown in the upper panel of Fig. 4B, expression of
Plk1 WT-AKAP, but not of Plk1 WT or the AKAP protein
alone, led to an accumulation of 4N DNA-containing cells,
indicating that Plk1-AKAP cells were delayed in G2and/or M
phase. Similar results were also obtained after transfection of
a constitutively active centrosomally tethered form of Plk1,
Plk1T210D-AKAP (data not shown). Further examination re-
vealed that a fraction of the Plk1-AKAP-expressing cells was
blocked in mitosis, since they stained positively for histone H3
phosphorylation (Fig. 4B, lower panel, and 4C) with levels
approximately half of those observed after expression of the
isolated Plk1PBD, a treatment previously established to cause
a preanaphase mitotic arrest (61). The difference in phospho-
histone H3 levels between Plk1-AKAP and Plk1-PBD-express-
ing cells suggests that Plk1-AKAP-expressing cells may have
also undergone a G2delay. This was further supported by
examination of cells at 10 h after thymidine release. As shown
in the bottom two rows of Fig. 3C, at this time point nearly all
of the Plk1-AKAP-expressing cells remained in G2, whereas a
fraction of the Plk1-expressing cells had already entered into
mitosis (phospho-histone H3 positivity of Plk1 WT-expressing
cells, 10.8%; Plk1 WT-AKAP, 1.1% based on fluorescence-acti-
vated cell sorting [FACS] analysis). In addition, Plk1-AKAP-ex-
pressing cells were found to have defects in centrosome separa-
tion, ?-tubulin recruitment, and functional maturation (data not
shown), all of which occur during G2or shortly after the G2/M
In the experiments described above, cells contained endog-
enous Plk1 in addition to the transiently expressed EGFP-
Plk1-AKAP. To exclude the possibility of artifacts contributing
to the observed phenotype, such as dominant inhibitory inter-
actions between endogenous Plk1 and the Plk1-AKAP con-
structs, we studied the effects of anchoring Plk1 to centrosomes
on cell cycle progression in cells depleted of endogenous Plk1.
To accomplish this, we used vector-driven shRNA against Plk1
(pSuper-Plk1, pS-Plk1) (4, 74) and reconstituted these cells
with WT or mutant Plk1. Two silent mutations within the
shRNA-target region of the Plk1 constructs were introduced to
allow expression of the exogenous Plk1, as described previ-
ously (74). We could demonstrate by Western blotting that this
approach was successful, since endogenous Plk1 expression
was eliminated, while simultaneously the expression of EGFP-
Plk1 or EGFP-Plk1-AKAP was observed (see Fig. S5A in the
supplemental material). Combining this reconstitution assay
with flow cytometry analysis, we found that expression of
EGFP-Plk1-AKAP in Plk1-depleted cells resulted in a mitotic
arrest similar to that observed after Plk1-AKAP expression in
Plk1-containing cells, whereas reconstitution with low to mod-
erate levels of WT EGFP-Plk1 did not significantly alter the
cell cycle (see Fig. S5B and C in the supplemental material).
These results are identical to those obtained with U2OS cells
that were not depleted of endogenous Plk1, indicating that
tethering of Plk1 at centrosomes with reduced dynamic ex-
change dominantly interferes with cell cycle progression, re-
gardless of the presence or absence of endogenous Plk1.
Expression of a kinase-dead form of Plk1, D176N, also in-
duced a mitotic arrest (Fig. 4B and C), a finding similar to what
has been reported previously for the K82M hypomorphic allele
of the kinase (61). Intriguingly, when the D176N kinase-dead
Plk1 was tethered to the centrosome by fusion to the pericen-
trin-AKAP450 centrosomal targeting domain of AKAP450,
the population of cells undergoing mitotic arrest was dramat-
ically reduced compared to cells expressing the cytosolic, fully
dynamic form of kinase-dead Plk1 (Plk1D176N), as evidenced
by both a decrease in the population of cells containing 4N
DNA, and the percentage of cells staining positively for phos-
phohistone H3 (Fig. 4B and C). These data indicate that cy-
tosolic Plk1 kinase activity is important for mitotic progression
and that tethering Plk1 to centrosomes causes a dominant cell
cycle arrest phenotype only if kinase activity is present. Taken
together, these data strongly suggest that Plk1 activity at the
centrosome must be extinguished and Plk1 activity in the cy-
toplasm activated in a coordinated manner in order for cells to
progress properly through the various stages of mitosis.
Centrosomally tethered Plk1 induces mitotic spindle defects
and activates the spindle assembly checkpoint to trigger pro-
metaphase arrest. To explore the mitotic arrest phenotype of
Plk1-AKAP cells in detail, the distribution of cells in each
mitotic substage was evaluated by scoring the morphology of
the DAPI-stained DNA masses in GFP-positive cells at 20 h
after release from a thymidine block. More than 95% of the
control EGFP-transfected cells had completed mitosis at this
time and resided in interphase, with only 1.9% of the total cells
population in prometaphase, and smaller percentages in meta-
phase and anaphase/telophase. At this time point cells express-
VOL. 29, 2009CENTROSOMAL Plk1 CONTROLS CHECKPOINT RELEASE3141
ing WT Plk1 showed only a slightly higher percentage of total
cells in mitosis cells (?10%), with most of the cells in anaphase
or telophase. In contrast, expression of the isolated PBD re-
sulted in a significant accumulation of preanaphase mitotic
cells (Fig. 4B and C) (61), with 13.0% of the cells in promet-
aphase and 9.0% in metaphase (Fig. 5A). Similarly, expression
of Plk1-AKAP resulted in the marked accumulation of pro-
metaphase arrested cells compared to the increase in meta-
phase and anaphase/telophase cell populations (Fig. 5A). It
therefore appears that the mitotic arrest induced by the ex-
pression of Plk1-AKAP predominantly consists of promet-
To probe the underlying cause of the prometaphase arrest
induced by Plk1-AKAP expression, mitotic spindle formation
was analyzed by immunostaining the cells using an anti-?-
tubulin antibody at 20 h after release from a thymidine block.
FIG. 4. Expression of Plk1-AKAP induces mitotic arrest. (A) The dynamics of EGFP-Plk1 or EGFP-Plk1-AKAP at the centrosome in
transfected U2OS cells were analyzed by FRAP. Average fluorescence recovery curves (a) and 50% recovery times after photobleaching (b) are
shown. Each data point represents the mean of 14 and 11 experiments, respectively, in which ?30 cells were imaged in each experiment (see Table
1). The black curves in subpanel a show the best single exponential fits to the data. Error bars in subpanel b indicate the SEM with statistically
significant differences are indicated by asterisks (**, P ? 0.01). (B) FACS profiles of DNA content and phospho-histone H3 staining in Plk1 and
Plk1-AKAP cells. U2OS cells were transfected with the indicated EGFP-fused constructs for 24 h and then arrested with a thymidine block for
an additional 24 h. At 24 h after release of the block, progression through the cell cycle was analyzed by FACS. Upper panels show the DNA
profiles (PI) from control (U2OS) cells or from the transfected cells when gated for low to moderate EGFP expression (boxed). Lower panels show
the phospho-histone H3 levels in the GFP-positive cells. The results from a typical experiment are shown. (C) Expression of either the PBD, a WT
form of Plk1 that is tethered to the centrosome, or a freely diffusible kinase-dead Plk1 causes mitotic arrest. The average percentage of
phosphohistone H3-positive cells in the GFP-expressing population from three independent experiments was determined. Error bars represent the
3142 KISHI ET AL.MOL. CELL. BIOL.
As shown in Fig. 5B and C, 93% of the control EGFP-trans-
fected U2OS cells and 87% of the WT Plk1-expressing cells
exhibited bipolar spindle structures, with monopolar or multi-
polar spindles only occasionally observed (Fig. 5C). Expression
of the PBD has been previously reported to induce a defect in
spindle stability (61), presumably by interfering with the re-
cruitment of WT Plk1 to centrosomes, microtubules, and/or
kinetochores. In agreement with this, we observed that 70% of
Plk1-PBD-expressing cells lacked bipolar spindles (Fig. 5C).
Importantly, Plk1 WT-AKAP-expressing cells also displayed
FIG. 5. Expression of Plk1-AKAP induces prometaphase arrest and aberrant spindle morphologies. (A) Transfected U2OS cells prepared as
in Fig. 4 were released at 20 h after a thymidine block and subsequently fixed and stained with DAPI. GFP-positive cells were scored for mitotic
subphases using confocal microscopy. The percentages of prometaphase (red), metaphase (green), and ana/telophase (cyan) cells are shown. The
data are averages of three independent experiments. Error bars represent the standard deviations. (B) Transfected U2OS cells were released at
20 h after a thymidine block and subsequently fixed and stained with anti-?-tubulin antibody and DAPI. Representative images are shown. All scale
bars represent 5 ?m. (C) Cells were stained as in panel B. The percentages of GFP-positive cells displaying normal bipolar or mutant (monopolar
or multipolar) spindle morphologies are shown.
VOL. 29, 2009 CENTROSOMAL Plk1 CONTROLS CHECKPOINT RELEASE3143
both monopolar spindles and multipolar spindles with high
frequency (42 and 26%, respectively), indicating that persistent
centrosomal Plk1 activity results in a set of centrosomal ab-
normalities with defective assembly of bipolar spindles similar
to that observed when Plk1 recruitment is blocked.
Abnormal mitotic spindles can lead to defects in stable mi-
crotubule-kinetochore attachments that persistently activate
the spindle assembly checkpoint. To examine whether the ac-
cumulation of 4N DNA-containing cells after expression of
Plk1-AKAP was the result of activation of the spindle check-
point, we analyzed cells for accumulation of Mad2 at kineto-
chores (Fig. 6). Mad2, a spindle assembly checkpoint protein,
localizes to kinetochores that lack proper microtubule attach-
ment and is released from kinetochores once they form stable
bivalent attachments to microtubules from opposing spindle
poles and equal tension is exerted upon the chromosome arms
(75). U2OS cells expressing WT Plk1 displayed enrichment of
Mad2 on kinetochores during prometaphase but not at meta-
phase, as expected (Fig, 6A, rows 1 and 2). Plk1-AKAP-ex-
pressing cells, as well as PBD-expressing cells, retained Mad2
on most of their kinetochores (Fig. 6A, rows 3 and 4), suggest-
ing that the prometaphase arrest induced by Plk1-AKAP is
spindle checkpoint dependent. To further examine this, we
performed RNA interference-mediated depletion of Mad2
(pS-Mad2) to inactivate the spindle checkpoint (38). As a
control for the efficiency of spindle checkpoint bypass by this
approach, U2OS cells were transfected with the control vector
(pS) or with pS-Mad2, released from a thymidine block into
medium containing the microtubule depolymerizing agent
nocodazole to induce a spindle assembly-dependent mitotic
arrest, and analyzed for phosphohistone H3 staining by flow
cytometry. As shown in the upper panel in Fig. 6B, the Mad2-
depleted cells no longer arrested in mitosis in the presence of
nocodazole, indicating the functional inactivation of the spin-
dle assembly checkpoint. U2OS cells were then transfected
with the various Plk1 constructs in combination with control
vector (pS) or pS-Mad2 and analyzed for spontaneous mitotic
arrest 20 h after release from a thymidine block in the absence
of nocodazole. As shown in the lower panels of Fig. 6B, the
increase in phosphohistone H3 positivity observed after ex-
pression of the isolated Plk1 PBD was significantly suppressed
by pS-Mad2 coexpression, showing that the PBD induces a
spindle checkpoint-dependent mitotic arrest, a finding consis-
tent with the findings of Seong et al. (61). Importantly, deple-
tion of Mad2 also efficiently reversed the accumulation of
phosphohistone H3-positive cells induced by expression of
Plk1-AKAP. Thus, the mitotic arrest, caused by tethering a less
dynamic form of Plk1 to centrosomes is mediated through
activation of the Mad2-dependent spindle assembly check-
Dynamic Plk1 facilitates mitotic reentry after DNA damage.
There is growing evidence that centrosomes are involved in the
cellular response to DNA damage (43). Plk1 is implicated as a
target of DNA damage checkpoint and is also crucial for mi-
totic entry after recovery after DNA damage (62, 72). We
therefore tested whether a less dynamic, centrosomally teth-
ered form of Plk1, which delays progression through the later
stages of mitosis, was still sufficient for release of the DNA
damage checkpoint upon recovery from DNA damage. In
these experiments U2OS cells in which endogenous Plk1 was
silenced by pS-Plk1 were used to examine the effect of exoge-
nous centrosomally tethered Plk1 as the sole source of Plk1.
When control U2OS cells released from a thymidine block are
treated with 1 ?M doxorubicin, a highly synchronous popula-
tion arrests at G2from a DNA damage checkpoint (Fig. 7A,
top row). The addition of 5 mM caffeine, an ATM/ATR inhib-
itor, for 7 h after DNA damage allows recovery from the
checkpoint-induced arrest and a fraction of the cells progress
into mitosis, where they can be trapped by paclitaxel and as-
sayed for phosphohistone H3 by flow cytometry (Fig. 7B, top
row) (72). Cells depleted of endogenous Plk1 by shRNA
showed an identical G2arrest as control U2OS cells after
treatment with doxorubicin (Fig. 7A, rows 2 to 4), regardless of
whether they they also coexpressed Plk1 WT or Plk1 WT-
AKAP. However, the Plk1-depleted cells were unable to exit
from the DNA damage-induced G2arrest (Fig. 7B, row 2), in
agreement with the findings of van Vugt et al. (72). Coexpres-
sion of an RNA interference-resistant form of either Plk1 WT
or Plk1 WT-AKAP in these Plk1-depleted cells allowed them
to override the G2arrest and enter mitosis (Fig. 7B, rows 3 and
4), although the relative percentage of pH3 positivity in cells
that contain the centrosomally tethered Plk1 WT-AKAP was
only ?30% of that seen in cells expressing Plk1 WT protein.
Similar results were also obtained using the constitutively ac-
tive T210D forms of Plk1 and Plk1-AKAP (62; data not
shown). Importantly, doxorubicin treatment had no effect on
the ability of the Plk1-AKAP fusion protein to localize pre-
dominantly to centrosomes (see Fig. S6 in the supplemental
These results indicate that signals emanating from a less
dynamic from of Plk1 tethered at the centrosome reduces the
ability of DNA-damaged cells to progress from G2into M
when upstream signals from the phosphatidylinositol 3-kinase-
like kinases ATM, ATR and DNA-PK are silenced by caffeine.
Thus, it appears that while the critical substrates for Plk1-
directed mitotic reentry likely reside at the centrosome, a fully
dynamic form of Plk1 is required for the process to occur in a
maximally effective manner.
In this study we examined the mobility and exchange of Plk1
at different mitotic substructures using FRAP and explored the
relative roles of the kinase domain and the PBD in controlling
Plk1 dynamics. We found that Plk1 displayed a range of turn-
over rates that were strongly dependent on its subcellular lo-
calization and the mitotic state. At mitotic centrosomes, for
example, Plk1 was highly dynamic with a t1/2of ?7 s, whereas
at the midbody Plk1 was considerably less mobile, with a t1/2of
?66 s. The rapid centrosomal kinetics seen on both G2and
mitotic centrosomes are in good agreement with data for other
centrosomal proteins, including the kinases Aurora-A and
Nek2, which were reported to be in rapid flux with the cyto-
plasmic population (22, 27, 65, 66).
All FRAP experiments were performed between 30 and
37°C, although the microscope stage was not temperature con-
trolled. In a number of our FRAP experiments, the mean net
fluorescence recoveries after photobleaching were low. One
possible explanation for this is that a significant fraction of Plk1
may be stably associated with each of the mitotic substructures
3144 KISHI ET AL.MOL. CELL. BIOL.
FIG. 6. Spindle checkpoint activation in Plk1-AKAP-expressing cells. (A) Mad2 remains associated with kinetochores in Plk1-AKAP expressing
cells, U2OS cells were transfected with the indicated constructs as in Fig. 4, and released at 20 h after thymidine block. Cells were fixed and stained
with anti-Mad2 antibody, CREST, and DAPI. Images were collected and processed using OpenLab (Improvision) or a Deltavision microscope
system (Applied Precision). Plk1 at mitotic centrosomes is indicated by arrowheads. Merged images show Mad2 in green, CREST staining in red,
and DAPI in blue. All scale bars represent 5 ?m. (B) U2OS cells were transfected with the indicated EGFP-tagged Plk1 plasmids with or without
an shRNA-expressing vector targeting Mad2 (pS-Mad2). At 20 h after release from a thymidine block, cells were harvested and fixed for FACS
analysis. In the control experiments examining spindle checkpoint activation by nocodazole, cells were transfected with pS vector control or
pS-Mad2, along with spectrin-GFP, and nocodazole was added for 20 h after release of the thymidine block (top panels). Phospho-histone H3
positivity and DNA content (PI) of GFP-positive cells are shown.
VOL. 29, 2009 CENTROSOMAL Plk1 CONTROLS CHECKPOINT RELEASE3145
we imaged. In agreement with this, the dynamic fraction of
Plk1 that we measured gave essentially identical recoveries
after repeated photobleaching trials (see Fig. S7 in the supple-
mental material). Furthermore, under the conditions that we
could measure, the fluorescence of a similar unbleached mi-
totic structure within the bleached cell did not appear to
change (see Fig. S8 in the supplemental material), arguing
against an apparent reduction in recovery due to nonspecific
bleaching effects. Finally, the extent of fluorescence for that we
observed for Plk1 at centrosomes is similar to that reported for
another centrosomal mitotic kinase, Nek2 (22).
We observed that complete removal of the kinase domain,
leaving only the phospho-binding PBD, resulted in stable as-
sociation of the protein with centrosomes (16, 61, 70), and the
least dynamic form of centrosomal Plk1. These data fit well
with previous in vitro observations of a mutually inhibitory
intramolecular interaction between the PBD and the kinase
domain (15, 25), resulting in enhanced PBD binding when the
kinase domain is absent in our in vivo experiments (16, 25).
Intriguingly, as cells progressed from G2into mitosis, the ex-
change rate of full-length WT Plk1 at the centrosome in-
creased ?2-fold. However, the rate at which kinase-defective
Plk1 mutants at mitotic centrosomes exchanged with the free
cytosolic pool was slower, although the exchange rate of both
WT and kinase-dead Plk1 at G2centrosomes was unchanged.
We interpret these data as evidence that much of the centro-
somal localization of Plk1 during early mitosis results from
strong direct interactions between the PBD of Plk1 and phos-
phorylated centrosomal proteins, likely through the generation
of PBD-binding sites through Cdk1 (49, 50) or Plk1 itself (37,
58), and that these centrosome-localizing interactions are dis-
rupted during subsequent stages of mitosis through Plk1 kinase
activity, facilitating the release and replacement of the photo-
bleached protein. Both cyclin B/Cdk1 and Plk1 kinase activities
are high throughout early to mid mitosis, and Plk1 is thought
to phosphorylate cyclin B and may promote its nuclear trans-
location (67, 76). Thus, during prometaphase or metaphase,
when much of the cyclin B/cdk1 dissociates from centrosomes,
a Plk1 kinase-dependent activity that drives the dissociation of
centrosomal Plk1 may become dominant. Our findings are in
excellent agreement with prior reports of PBD-engaging cen-
trosomal ligands such as hCenexin1 (64). In addition, the ki-
nase-dependent exchange of centrosomal Plk1 that we
observed, probably mediated through phosphorylation of sub-
FIG. 7. Centrosomally tethered less dynamic Plk1 impairs mitotic reentry after a DNA damage G2arrest. (A) Normal activation of the G2DNA
damage checkpoint in the absence or presence of WT or centrosomally tethered Plk1. U2OS cells were cotransfected with the EGFP fusion protein
and shRNA expression vector pair spectrin-GFP/pS, spectrin-GFP/pS-Plk1, EGFP-Plk1 WT/pS-Plk1, or EGFP-Plk1 WT-AKAP/pS-Plk1 at 24 h
from the thymidine block for 6 h, and the samples were then treated with 1 ?M doxorubicin for 1 h, washed, and grown in the presence of paclitaxel
for 18 h. (B) Recovery from G2DNA damage arrest in Plk1-AKAP-expressing cells. After the treatments shown in panel A, caffeine was added
for 7 h. In panels A and B, DNA content (PI) and phospho-histone H3 staining of the GFP-positive cells was examined by FACS as shown.
3146 KISHI ET AL.MOL. CELL. BIOL.
strates at or near the centrosome, may constitute a physiolog-
ical trigger for Plk1 release from the centrosome and retarget-
ing to kinetochores and spindle structures as cells progress
beyond prophase/prometaphase (Fig. 8A).
An alternative possibility is that the rapid exchange behavior
observed for Plk1 at mitotic centrosomes may be a conse-
quence of cyclin B/Cdk1 activation and substrate phosphory-
lation. Plk1 and cyclin B/Cdk1 influence each other heavily
during mitotic entry, as well as during mitotic progression. Plk1
phosphorylates cyclin B and is involved in the robust activation
of cyclin B/Cdk1 at centrosomes (24). Consistent with this,
depletion of Plk1 in both normal cells and cancer cells causes
a delay in progression from G2to M (31, 41, 57). Later in
mitosis, Plk1 is required for correct spindle formation (52, 74),
FIG. 8. Model for Plk1 relocalization during mitosis and effect of centrosomally tethered Plk1 on mitotic progression. (A) Model for Plk1
localization and dynamics during normal mitosis. In prophase and prometaphase, Plk1 kinase activity facilitates its release from the centrosome
and promotes its localization to kinetochores, the spindle midzone and the midbody through self-priming. Some residual Plk1 localization on
kinetochores remains during telophase. (B) Model for the effect of centrosomally tethered Plk1 (Plk1-AKAP) during mitosis. Plk1 tethering to
centrosomes induces spindle checkpoint activation and mitotic arrest, indicating that Plk1 dissociation from centrosome is important for further
mitotic progression. (C) Centrosomally tethered Plk1 (Plk1-AKAP) (bottom, thin arrow) is less effective than fully dynamic WT Plk1 (top, thick
arrow) at stimulating mitotic reentry after DNA damage.
VOL. 29, 2009 CENTROSOMAL Plk1 CONTROLS CHECKPOINT RELEASE3147
as well as in the downregulation of cyclin B/Cdk1 through the
direct activation of the APC/C leading to Cyclin B degradation
(39, 47). Thus, since Plk1 and Cdk1 activities are so interde-
pendent, the protein dynamics we observed for Plk1 may de-
pend on cyclin B/Cdk1 as well. Addition of the Cdk1 inhibitor
roscovitine to cells already in mitosis, however, did not affect
the centrosomal dynamics of Plk1 (data not shown), suggesting
that the role of Cdk1 as a regulator of Plk1 dynamics is limited.
Finally, our observation that the Plk1 kinase domain facilitates
the release, rather than the binding of Plk1 to centrosomes
contrasts with the opposite conclusions reached by Montoya
and coworkers (18), who reported that the isolated Plk1 kinase
domain could localize to centrosomes in asynchronous cells;
however, that study that did not directly examine the effect of
kinase activity on mitotic centrosomal localization of Plk1 in
Surprisingly, although kinase activity seems to promote re-
lease of Plk1 from mitotic centrosomes, kinase activity ap-
peared to dramatically prolong the retention of Plk1 at the
midbody by nearly an order of magnitude. At the midbody, as
at the centrosome, the isolated PBD showed the least dynamic
behavior of all of the constructs examined. Together, these
data suggest that Plk1 kinase activity at the midbody likely
creates its own PBD-binding sites via a self-priming process.
Similar self-priming mechanisms for Plk1 localization have
been elegantly described in a series of studies by Neef et al. and
others, who showed that during anaphase Plk1 generates its
own PBD-binding sites on the microtubule-associated protein
PRC1 and on the mitotic kinesin MKlp2 to localize Plk1 to the
central spindle and drive cytokinesis (3, 49, 50). Plk1 self-
priming for PBD binding has also been recently implicated in
recruiting Plk1 to centromeres/kinetochores via binding to
PBIP1 (28, 34) and, indeed, we observed a statistically signif-
icant increase in Plk1 mobility for the kinase-dead D176N
mutant of Plk1, but not for WT Plk1, compared to the isolated
PBD alone (Fig. 3C). As Neef et al. point out, this process of
self-priming is likely to be especially important in the later
stages of mitosis when Cdk1 activity has declined (49). Fur-
thermore, our observation that Plk1 kinase activity seems to
promote Plk1 release from early mitotic structures and en-
hance relocalization to late mitotic structures helps explain the
prior conundrum of why Plk1 kinase activity, which could self-
prime for PBD binding, does not result in cell cycle arrest by
creating a form of Plk1 that becomes stuck on centrosomes at
early stages in mitosis (49).
The central importance of dynamic release of Plk1 from the
centrosome as cells progress through mitosis was revealed by
the studies shown in Fig. 3 to 6, in which we observed that
centrosome-anchored Plk1 induced both a G2delay and an
M-phase arrest, with significant accumulation of prometaphase
cells (Fig. 8A and B). Why does mild overexpression of cen-
trosome-associated Plk1 prevent mitotic progression, while
overexpression of cytosolic Plk1 does not do the same thing?
One possibility is that there are both stimulatory and inhibitory
pathways for mitotic progression mediated by Plk1. In addition
to stimulating centrosome maturation, centrosome-localized
kinase-active (but not kinase-dead) Plk1 might also trigger an
inhibitory pathway that activates the spindle checkpoint
through phosphorylation of proteins such as Cdc20. Alterna-
tively, inappropriate and excessive phosphorylation of centro-
somal substrates by Plk1-AKAP could result in disruption of
normal Plk1-dependent events at the pericentriolar material,
leading to abnormal numbers of spindle poles. For example,
Plk1 activity directly stimulates the ?-TrCP-dependent degra-
dation of Bora, an adaptor protein that regulates Aurora-A
activation and/or localization (6, 59). Knockdown of hBora
using small interfering RNA, mimicking excessive Plk1-medi-
ated degradation, has been shown to induce a variety of spindle
abnormalities, including multipolar spindles, resulting in acti-
vation of the spindle checkpoint (6). Similarly, excessive and
inappropriate kinase activity could interfere with the normal
regulation of the centrosomal Plk1 substrate Kizuna, the
knockdown of which has been shown to result in dissociation of
the pericentriolar material, multipolar spindle formation, and
prometaphase/metaphase arrest (53). Additional detailed
studies are necessary to further elucidate the exact mechanism
responsible for Plk1-AKAP inhibition of mitotic progression.
The centrosome is increasingly recognized as a critical sub-
cellular structure that regulates mitotic entry during an unper-
tubed cell cycle (13). During normal mitotic entry, cyclin
B/Cdk1 complexes are preferentially activated on centrosomes,
likely through the combined actions of Plk1 and CDC25B (9,
14, 24, 40), while inhibition of mitotic entry at the G2/M tran-
sition seems to involve the specific localization of checkpoint
kinases, including ATM, ATR, Chk1, and/or Chk2, to the
centrosome, where they may inhibit cyclin B/Cdk1 activation
(30, 43, 68, 77). Whether the centrosome also plays a central
role in coordinating the mitotic arrest and reentry functions in
mammalian cells in response to DNA damage is unclear. In
response to DNA damage, Plk1 kinase activity and phosphor-
ylation at Thr210 has been shown to be inhibited in an ATM/
ATR-Chk2/Chk1-dependent manner (62, 69, 73), while reac-
tivation of Plk1 activity and T210 phosphorylation occurs upon
recovery through an hBora:Aurora-A complex (45). Our find-
ing that a centrosomally tethered form of Plk1 with reduced
dynamic behavior impairs the ability of DNA damaged cells to
reenter mitosis from G2arrest (Fig. 8C) is consistent with both
positive and negative regulators of the DNA damage check-
point residing at the centrosome and indicates that fine-tuning
of mitotic reentry from a G2checkpoint requires communica-
tion between the centrosome and surrounding factors.
We thank D. Lim, J. Alexander, M. Stewart, E. Vasile, and M.
Hayashi for reagents, assistance with techniques, and data analysis; S.
Munro (MRC Laboratory of Molecular Biology, Cambridge, United
Kingdom) for providing the AKAP 450 cDNA; and Rene Medema for
K.K. was supported by the Manpei Suzuki Diabetes Foundation.
M.V. was supported by an EMBO fellowship. This study was supported
by NIH grants GM60594 and CA112967 to M.B.Y.
1. Ahonen, L. J., M. J. Kallio, J. R. Daum, M. Bolton, I. A. Manke, M. B. Yaffe,
P. T. Stukenberg, and G. J. Gorbsky. 2005. Polo-like kinase 1 creates the
tension-sensing 3F3/2 phosphoepitope and modulates the association of
spindle-checkpoint proteins at kinetochores. Curr. Biol. 15:1078–1089.
2. Arnaud, L., J. Pines, and E. A. Nigg. 1998. GFP tagging reveals human
Polo-like kinase 1 at the kinetochore/centromere region of mitotic chromo-
somes. Chromosoma 107:424–429.
3. Barr, F. A., H. H. Sillje, and E. A. Nigg. 2004. Polo-like kinases and the
orchestration of cell division. Nat. Rev. Mol. Cell. Biol. 5:429–440.
4. Brummelkamp, T. R., R. Bernards, and R. Agami. 2002. A system for stable
expression of short interfering RNAs in mammalian cells. Science 296:550–
3148 KISHI ET AL.MOL. CELL. BIOL.
5. Casenghi, M., P. Meraldi, U. Weinhart, P. I. Duncan, R. Korner, and E. A.
Nigg. 2003. Polo-like kinase 1 regulates Nlp, a centrosome protein involved
in microtubule nucleation. Dev. Cell 5:113–125.
6. Chan, E. H., A. Santamaria, H. H. Sillje, and E. A. Nigg. 2008. Plk1 regulates
mitotic Aurora A function through ?TrCP-dependent degradation of hBora.
7. Cheng, K. Y., E. D. Lowe, J. Sinclair, E. A. Nigg, and L. N. Johnson. 2003.
The crystal structure of the human polo-like kinase-1 polo box domain and
its phospho-peptide complex. EMBO J. 22:5757–5768.
8. De Luca, M., P. Lavia, and G. Guarguaglini. 2006. A functional interplay
between Aurora-A, Plk1 and TPX2 at spindle poles: Plk1 controls centro-
somal localization of Aurora-A and TPX2 spindle association. Cell Cycle
9. De Souza, C. P., K. A. Ellem, and B. G. Gabrielli. 2000. Centrosomal and
cytoplasmic Cdc2/cyclin B1 activation precedes nuclear mitotic events. Exp.
Cell Res. 257:11–21.
10. Donaldson, M. M., A. A. Tavares, I. M. Hagan, E. A. Nigg, and D. M. Glover.
2001. The mitotic roles of Polo-like kinase. J. Cell Sci. 114:2357–2358.
11. Donaldson, M. M., A. A. Tavares, H. Ohkura, P. Deak, and D. M. Glover.
2001. Metaphase arrest with centromere separation in polo mutants of Dro-
sophila. J. Cell Biol. 153:663–676.
12. Doxsey, S. 2001. Re-evaluating centrosome function. Nat. Rev. Mol. Cell.
13. Doxsey, S., W. Zimmerman, and K. Mikule. 2005. Centrosome control of the
cell cycle. Trends Cell Biol. 15:303–311.
14. Dutertre, S., M. Cazales, M. Quaranta, C. Froment, V. Trabut, C. Dozier, G.
Mirey, J. P. Bouche, N. Theis-Febvre, E. Schmitt, B. Monsarrat, C. Prigent,
and B. Ducommun. 2004. Phosphorylation of CDC25B by Aurora-A at the
centrosome contributes to the G2-M transition. J. Cell Sci. 117:2523–2531.
15. Elia, A. E., L. C. Cantley, and M. B. Yaffe. 2003. Proteomic screen finds
pSer/pThr-binding domain localizing Plk1 to mitotic substrates. Science 299:
16. Elia, A. E., P. Rellos, L. F. Haire, J. W. Chao, F. J. Ivins, K. Hoepker, D.
Mohammad, L. C. Cantley, S. J. Smerdon, and M. B. Yaffe. 2003. The
molecular basis for phosphodependent substrate targeting and regulation of
Plks by the Polo-box domain. Cell 115:83–95.
17. Gabrielli, B. G., C. P. De Souza, I. D. Tonks, J. M. Clark, N. K. Hayward,
and K. A. Ellem. 1996. Cytoplasmic accumulation of cdc25B phosphatase in
mitosis triggers centrosomal microtubule nucleation in HeLa cells. J. Cell
Sci. 109(Pt. 5):1081–1093.
18. Garcia-Alvarez, B., G. de Carcer, S. Ibanez, E. Bragado-Nilsson, and G.
Montoya. 2007. Molecular and structural basis of polo-like kinase 1 substrate
recognition: implications in centrosomal localization. Proc. Natl. Acad. Sci.
19. Gillingham, A. K., and S. Munro. 2000. The PACT domain, a conserved
centrosomal targeting motif in the coiled-coil proteins AKAP450 and peri-
centrin. EMBO Rep. 1:524–529.
20. Golsteyn, R. M., K. E. Mundt, A. M. Fry, and E. A. Nigg. 1995. Cell cycle
regulation of the activity and subcellular localization of Plk1, a human
protein kinase implicated in mitotic spindle function. J. Cell Biol. 129:1617–
21. Goto, H., T. Kiyono, Y. Tomono, A. Kawajiri, T. Urano, K. Furukawa, E. A.
Nigg, and M. Inagaki. 2006. Complex formation of Plk1 and INCENP
required for metaphase-anaphase transition. Nat. Cell Biol. 8:180–187.
22. Hames, R. S., R. E. Crookes, K. R. Straatman, A. Merdes, M. J. Hayes, A. J.
Faragher, and A. M. Fry. 2005. Dynamic recruitment of Nek2 kinase to the
centrosome involves microtubules, PCM-1, and localized proteasomal deg-
radation. Mol. Biol. Cell 16:1711–1724.
23. Hayashi, M. K., H. M. Ames, and Y. Hayashi. 2006. Tetrameric hub structure
of postsynaptic scaffolding protein homer. J. Neurosci. 26:8492–8501.
24. Jackman, M., C. Lindon, E. A. Nigg, and J. Pines. 2003. Active cyclin
B1-Cdk1 first appears on centrosomes in prophase. Nat. Cell Biol. 5:143–
25. Jang, Y. J., C. Y. Lin, S. Ma, and R. L. Erikson. 2002. Functional studies on
the role of the C-terminal domain of mammalian polo-like kinase. Proc.
Natl. Acad. Sci. USA 99:1984–1989.
26. Jang, Y. J., S. Ma, Y. Terada, and R. L. Erikson. 2002. Phosphorylation of
threonine 210 and the role of serine 137 in the regulation of mammalian
polo-like kinase. J. Biol. Chem. 277:44115–44120.
27. Kallio, M. J., V. A. Beardmore, J. Weinstein, and G. J. Gorbsky. 2002. Rapid
microtubule-independent dynamics of Cdc20 at kinetochores and centro-
somes in mammalian cells. J. Cell Biol. 158:841–847.
28. Kang, Y. H., J. E. Park, L. R. Yu, N. K. Soung, S. M. Yun, J. K. Bang, Y. S.
Seong, H. Yu, S. Garfield, T. D. Veenstra, and K. S. Lee. 2006. Self-regulated
Plk1 recruitment to kinetochores by the Plk1-PBIP1 interaction is critical for
proper chromosome segregation. Mol. Cell 24:409–422.
29. Kingwell, B., and J. B. Rattner. 1987. Mammalian kinetochore/centromere
composition: a 50-kDa antigen is present in the mammalian kinetochore/
centromere. Chromosoma 95:403–407.
30. Kramer, A., N. Mailand, C. Lukas, R. G. Syljuasen, C. J. Wilkinson, E. A.
Nigg, J. Bartek, and J. Lukas. 2004. Centrosome-associated Chk1 prevents
premature activation of cyclin-B-Cdk1 kinase. Nat. Cell Biol. 6:884–891.
31. Lane, H. A., and E. A. Nigg. 1996. Antibody microinjection reveals an
essential role for human polo-like kinase 1 (Plk1) in the functional matura-
tion of mitotic centrosomes. J. Cell Biol. 135:1701–1713.
32. Lee, K. S., and R. L. Erikson. 1997. Plk is a functional homolog of Saccha-
romyces cerevisiae Cdc5, and elevated Plk activity induces multiple septation
structures. Mol. Cell. Biol. 17:3408–3417.
33. Lee, K. S., T. Z. Grenfell, F. R. Yarm, and R. L. Erikson. 1998. Mutation of
the polo-box disrupts localization and mitotic functions of the mammalian
polo kinase Plk. Proc. Natl. Acad. Sci. USA 95:9301–9306.
34. Lee, K. S., J. E. Park, Y. H. Kang, W. Zimmerman, N. K. Soung, Y. S. Seong,
S. J. Kwak, and R. L. Erikson. 2008. Mechanisms of mammalian polo-like
kinase 1 (Plk1) localization: self- versus non-self-priming. Cell Cycle 7:141–
35. Lee, K. S., S. Song, and R. L. Erikson. 1999. The polo-box-dependent
induction of ectopic septal structures by a mammalian polo kinase, plk, in
Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 96:14360–14365.
36. Lee, K. S., Y. L. Yuan, R. Kuriyama, and R. L. Erikson. 1995. Plk is an
M-phase-specific protein kinase and interacts with a kinesin-like protein,
CHO1/MKLP-1. Mol. Cell. Biol. 15:7143–7151.
37. Lenart, P., M. Petronczki, M. Steegmaier, B. Di Fiore, J. J. Lipp, M. Hoff-
mann, W. J. Rettig, N. Kraut, and J. M. Peters. 2007. The small-molecule
inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase
1. Curr. Biol. 17:304–315.
38. Lens, S. M., R. M. Wolthuis, R. Klompmaker, J. Kauw, R. Agami, T. Brum-
melkamp, G. Kops, and R. H. Medema. 2003. Survivin is required for a
sustained spindle checkpoint arrest in response to lack of tension. EMBO J.
39. Lindon, C., and J. Pines. 2004. Ordered proteolysis in anaphase inactivates
Plk1 to contribute to proper mitotic exit in human cells. J. Cell Biol. 164:
40. Lindqvist, A., H. Kallstrom, A. Lundgren, E. Barsoum, and C. K. Rosenthal.
2005. Cdc25B cooperates with Cdc25A to induce mitosis but has a unique
role in activating cyclin B1-Cdk1 at the centrosome. J. Cell Biol. 171:35–45.
41. Liu, X., M. Lei, and R. L. Erikson. 2006. Normal cells, but not cancer cells,
survive severe Plk1 depletion. Mol. Cell. Biol. 26:2093–2108.
42. Liu, X., T. Zhou, R. Kuriyama, and R. L. Erikson. 2004. Molecular interac-
tions of Polo-like-kinase 1 with the mitotic kinesin-like protein CHO1/
MKLP-1. J. Cell Sci. 117:3233–3246.
43. Loffler, H., J. Lukas, J. Bartek, and A. Kramer. 2006. Structure meets
function: centrosomes, genome maintenance, and the DNA damage re-
sponse. Exp. Cell Res. 312:2633–2640.
44. Lowery, D. M., K. R. Clauser, M. Hjerrild, D. Lim, J. Alexander, K. Kishi,
S. E. Ong, S. Gammeltoft, S. A. Carr, and M. B. Yaffe. 2007. Proteomic
screen defines the Polo-box domain interactome and identifies Rock2 as a
Plk1 substrate. EMBO J. 26:2262–2273.
45. Macurek, L., A. Lindqvist, D. Lim, M. A. Lampson, R. Klompmaker, R.
Freire, C. Clouin, S. S. Taylor, M. B. Yaffe, and R. H. Medema. 2008.
Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery.
46. Meraldi, P., and E. A. Nigg. 2002. The centrosome cycle. FEBS Lett. 521:
47. Moshe, Y., J. Boulaire, M. Pagano, and A. Hershko. 2004. Role of Polo-like
kinase in the degradation of early mitotic inhibitor 1, a regulator of the
anaphase promoting complex/cyclosome. Proc. Natl. Acad. Sci. USA 101:
48. Mundt, K. E., R. M. Golsteyn, H. A. Lane, and E. A. Nigg. 1997. On the
regulation and function of human Polo-like kinase 1 (PLK1): effects of
overexpression on cell cycle progression. Biochem. Biophys. Res. Commun.
49. Neef, R., U. Gruneberg, R. Kopajtich, X. Li, E. A. Nigg, H. Sillje, and F. A.
Barr. 2007. Choice of Plk1 docking partners during mitosis and cytokinesis
is controlled by the activation state of Cdk1. Nat. Cell Biol. 9:436–444.
50. Neef, R., C. Preisinger, J. Sutcliffe, R. Kopajtich, E. A. Nigg, T. U. Mayer,
and F. A. Barr. 2003. Phosphorylation of mitotic kinesin-like protein 2 by
Polo-like kinase 1 is required for cytokinesis. J. Cell Biol. 162:863–875.
51. Nigg, E. A. 1998. Polo-like kinases: positive regulators of cell division from
start to finish. Curr. Opin. Cell Biol. 10:776–783.
52. Ohkura, H., I. M. Hagan, and D. M. Glover. 1995. The conserved Schizo-
saccharomyces pombe kinase plo1, required to form a bipolar spindle, the
actin ring, and septum, can drive septum formation in G1 and G2 cells.
Genes Dev. 9:1059–1073.
53. Oshimori, N., M. Ohsugi, and T. Yamamoto. 2006. The Plk1 target Kizuna
stabilizes mitotic centrosomes to ensure spindle bipolarity. Nat. Cell Biol.
54. Preisinger, C., R. Korner, M. Wind, W. D. Lehmann, R. Kopajtich, and F. A.
Barr. 2005. Plk1 docking to GRASP65 phosphorylated by Cdk1 suggests a
mechanism for Golgi checkpoint signalling. EMBO J. 24:753–765.
55. Qi, W., Z. Tang, and H. Yu. 2006. Phosphorylation- and polo-box-dependent
binding of Plk1 to Bub1 is required for the kinetochore localization of Plk1.
Mol. Biol. Cell 17:3705–3716.
56. Qian, Y. W., E. Erikson, and J. L. Maller. 1999. Mitotic effects of a consti-
VOL. 29, 2009CENTROSOMAL Plk1 CONTROLS CHECKPOINT RELEASE3149
tutively active mutant of the Xenopus Polo-like kinase Plx1. Mol. Cell. Biol. Download full-text
57. Qian, Y. W., E. Erikson, F. E. Taieb, and J. L. Maller. 2001. The Polo-like
kinase Plx1 is required for activation of the phosphatase Cdc25C and cyclin
B-Cdc2 in Xenopus oocytes. Mol. Biol. Cell 12:1791–1799.
58. Santamaria, A., R. Neef, U. Eberspacher, K. Eis, M. Husemann, D. Mum-
berg, S. Prechtl, V. Schulze, G. Siemeister, L. Wortmann, F. A. Barr, and
E. A. Nigg. 2007. Use of the novel Plk1 inhibitor ZK-thiazolidinone to
elucidate functions of Plk1 in early and late stages of mitosis. Mol. Biol. Cell
59. Seki, A., J. A. Coppinger, H. Du, C. Y. Jang, J. R. Yates III, and G. Fang.
2008. Plk1- and ?-TrCP-dependent degradation of Bora controls mitotic
progression. J. Cell Biol. 181:65–78.
60. Seki, A., J. A. Coppinger, C. Y. Jang, J. R. Yates, and G. Fang. 2008. Bora
and the kinase Aurora a cooperatively activate the kinase Plk1 and control
mitotic entry. Science 320:1655–1658.
61. Seong, Y. S., K. Kamijo, J. S. Lee, E. Fernandez, R. Kuriyama, T. Miki, and
K. S. Lee. 2002. A spindle checkpoint arrest and a cytokinesis failure by the
dominant-negative polo-box domain of Plk1 in U-2 OS cells. J. Biol. Chem.
62. Smits, V. A., R. Klompmaker, L. Arnaud, G. Rijksen, E. A. Nigg, and R. H.
Medema. 2000. Polo-like kinase-1 is a target of the DNA damage check-
point. Nat. Cell Biol. 2:672–676.
63. Song, S., T. Z. Grenfell, S. Garfield, R. L. Erikson, and K. S. Lee. 2000.
Essential function of the polo box of Cdc5 in subcellular localization and
induction of cytokinetic structures. Mol. Cell. Biol. 20:286–298.
64. Soung, N. K., Y. H. Kang, K. Kim, K. Kamijo, H. Yoon, Y. S. Seong, Y. L.
Kuo, T. Miki, S. R. Kim, R. Kuriyama, C. Z. Giam, C. H. Ahn, and K. S. Lee.
2006. Requirement of hCenexin for proper mitotic functions of polo-like
kinase 1 at the centrosomes. Mol. Cell. Biol. 26:8316–8335.
65. Stenoien, D. L., S. Sen, M. A. Mancini, and B. R. Brinkley. 2003. Dynamic
association of a tumor amplified kinase, Aurora-A, with the centrosome and
mitotic spindle. Cell Motil. Cytoskel. 55:134–146.
66. Thompson, H. M., H. Cao, J. Chen, U. Euteneuer, and M. A. McNiven. 2004.
Dynamin 2 binds gamma-tubulin and participates in centrosome cohesion.
Nat. Cell Biol. 6:335–342.
67. Toyoshima-Morimoto, F., E. Taniguchi, N. Shinya, A. Iwamatsu, and E.
Nishida. 2001. Polo-like kinase 1 phosphorylates cyclin B1 and targets it to
the nucleus during prophase. Nature 410:215–220.
68. Tsvetkov, L. 2004. Polo-like kinases and Chk2 at the interface of DNA
damage checkpoint pathways and mitotic regulation. IUBMB Life 56:449–
69. Tsvetkov, L., and D. F. Stern. 2005. Phosphorylation of Plk1 at S137 and
T210 is inhibited in response to DNA damage. Cell Cycle 4:166–171.
70. van de Weerdt, B. C., D. R. Littler, R. Klompmaker, A. Huseinovic, A. Fish,
A. Perrakis, and R. H. Medema. 2008. Polo-box domains confer target
specificity to the Polo-like kinase family. Biochim. Biophys. Acta 1783:1015–
71. van de Weerdt, B. C., M. A. van Vugt, C. Lindon, J. J. Kauw, M. J. Ro-
zendaal, R. Klompmaker, R. M. Wolthuis, and R. H. Medema. 2005. Un-
coupling anaphase-promoting complex/cyclosome activity from spindle as-
sembly checkpoint control by deregulating polo-like kinase 1. Mol. Cell. Biol.
72. van Vugt, M. A., A. Bras, and R. H. Medema. 2004. Polo-like kinase-1
controls recovery from a G2 DNA damage-induced arrest in mammalian
cells. Mol. Cell 15:799–811.
73. van Vugt, M. A., V. A. Smits, R. Klompmaker, and R. H. Medema. 2001.
Inhibition of Polo-like kinase-1 by DNA damage occurs in an ATM- or
ATR-dependent fashion. J. Biol. Chem. 276:41656–41660.
74. van Vugt, M. A., B. C. van de Weerdt, G. Vader, H. Janssen, J. Calafat, R.
Klompmaker, R. M. Wolthuis, and R. H. Medema. 2004. Polo-like kinase-1
is required for bipolar spindle formation but is dispensable for anaphase
promoting complex/Cdc20 activation and initiation of cytokinesis. J. Biol.
75. Waters, J. C., R. H. Chen, A. W. Murray, and E. D. Salmon. 1998. Local-
ization of Mad2 to kinetochores depends on microtubule attachment, not
tension. J. Cell Biol. 141:1181–1191.
76. Yuan, J., F. Eckerdt, J. Bereiter-Hahn, E. Kurunci-Csacsko, M. Kaufmann,
and K. Strebhardt. 2002. Cooperative phosphorylation including the activity
of polo-like kinase 1 regulates the subcellular localization of cyclin B1.
77. Zhang, S., P. Hemmerich, and F. Grosse. 2007. Centrosomal localization of
DNA damage checkpoint proteins. J. Cell Biochem. 101:451–465.
3150 KISHI ET AL.MOL. CELL. BIOL.