Uncoupling Anaphase-Promoting Complex/Cyclosome Activity from Spindle Assembly Checkpoint Control by Deregulating Polo-Like Kinase 1

Division of Molecular Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands.
Molecular and Cellular Biology (Impact Factor: 4.78). 04/2005; 25(5):2031-44. DOI: 10.1128/MCB.25.5.2031-2044.2005
Source: PubMed
ABSTRACT
Polo-like kinase 1 (Plk1) plays a role in numerous events in mitosis, but how the multiple functions of Plk1 are separated is poorly understood. We studied regulation of Plk1 through two putative phosphorylation residues, Ser-137 and Thr-210. Using phospho-specific antibodies, we found that Thr-210 phosphorylation precedes Ser-137 phosphorylation in vivo, the latter occurring specifically in late mitosis. We show that expression of two activating mutants of these residues, S137D and T210D, results in distinct mitotic phenotypes. Whereas expression of both phospho-mimicking mutants as well as of the double mutant leads to accelerated mitotic entry, further progression through mitosis is dramatically different: the T210D mutant causes a spindle assembly checkpoint-dependent delay, whereas the expression of the S137D mutant or the double mutant results in untimely activation of the anaphase-promoting complex/cyclosome (APC/C) and frequent mitotic catastrophe. Using nonphosphorylatable Plk1-S137A and Plk1-T210A mutants, we show that both sites contribute to proper mitotic progression. Based on these observations, we propose that Plk1 function is altered at different stages of mitosis through consecutive posttranslational events, e.g., at Ser-137 and Thr-210. Furthermore, our data show that uncontrolled Plk1 activation can uncouple APC/C activity from spindle assembly checkpoint control.

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Available from: René H Medema
MOLECULAR AND CELLULAR BIOLOGY, Mar. 2005, p. 2031–2044 Vol. 25, No. 5
0270-7306/05/$08.000 doi:10.1128/MCB.25.5.2031–2044.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Uncoupling Anaphase-Promoting Complex/Cyclosome Activity from
Spindle Assembly Checkpoint Control by Deregulating Polo-Like
Kinase 1†
Barbara C. M. van de Weerdt,
1
Marcel A. T. M. van Vugt,
1
Catherine Lindon,
2
Jos J. W. Kauw,
1
Marieke J. Rozendaal,
1
Rob Klompmaker,
1
Rob M. F. Wolthuis,
1
and Rene´ H. Medema
1
*
Division of Molecular Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands,
1
and Wellcome Trust/Cancer
Research UK Institute, Cambridge, United Kingdom
2
Received 11 May 2004/Returned for modification 14 June 2004/Accepted 24 November 2004
Polo-like kinase 1 (Plk1) plays a role in numerous events in mitosis, but how the multiple functions of Plk1
are separated is poorly understood. We studied regulation of Plk1 through two putative phosphorylation
residues, Ser-137 and Thr-210. Using phospho-specific antibodies, we found that Thr-210 phosphorylation
precedes Ser-137 phosphorylation in vivo, the latter occurring specifically in late mitosis. We show that
expression of two activating mutants of these residues, S137D and T210D, results in distinct mitotic pheno-
types. Whereas expression of both phospho-mimicking mutants as well as of the double mutant leads to
accelerated mitotic entry, further progression through mitosis is dramatically different: the T210D mutant
causes a spindle assembly checkpoint-dependent delay, whereas the expression of the S137D mutant or the
double mutant results in untimely activation of the anaphase-promoting complex/cyclosome (APC/C) and
frequent mitotic catastrophe. Using nonphosphorylatable Plk1-S137A and Plk1-T210A mutants, we show that
both sites contribute to proper mitotic progression. Based on these observations, we propose that Plk1 function
is altered at different stages of mitosis through consecutive posttranslational events, e.g., at Ser-137 and
Thr-210. Furthermore, our data show that uncontrolled Plk1 activation can uncouple APC/C activity from
spindle assembly checkpoint control.
Since the identification of Polo in Drosophila over 15 years
ago (54), the functions proposed for Polo-like kinases have
been numerous. Most of the proposed actions of Plk are im-
portant for entry into, progression through, and exit from mi-
tosis (16, 41). For instance, Polo-like kinase 1 (Plk1) has been
suggested to promote mitotic entry by activating cyclin B1/
Cdk1 in multiple ways: by phosphorylating cyclin B1 itself, by
phosphorylating the Cdk1-activating phosphatase Cdc25C, and
by phosphorylating the Cdk1-inhibiting kinases Myt1/Wee1 (1,
23, 30, 39, 45, 55, 58). Furthermore, Drosophila Polo and hu-
man Plk1 have been implicated in centrosome maturation and
separation, with defects giving rise to monopolar spindles (31,
42, 45, 54). Proposed Plk targets regulating centrosome func-
tion are Hsp90, Asp, and Nlp (5, 9, 11). Additionally, budding
yeast (Saccharomyces cerevisiae) Plk Cdc5 and Xenopus Plx1
phosphorylate a cohesin complex subunit, Scc1, thereby en-
hancing cleavage and regulating sister chromatid separation (2,
36, 53). Studies of budding yeast, Xenopus, murine, and human
cells indicated a role for Plk1 in activating the anaphase-pro-
moting complex/cyclosome (APC/C) (7, 10, 17, 29, 48). For
one, Plk1 in combination with cyclin B1/Cdk1 can phosphory-
late and activate the APC/C in vitro (17, 29). In addition,
budding yeast Cdc5 has been proposed to contribute to the
activation of the APC/C coactivator Cdh1, possibly by phos-
phorylating Cdc14 phosphatase (7, 47, 57). Cdc5 was shown to
activate Cdc14 by releasing it from the nucleolus, which ulti-
mately leads to mitotic exit and the onset of cytokinesis (21,
52). In fission and budding yeast, Plk1 homologues Plo1 and
Cdc5 were implicated in the actual formation of the cytokinetic
ring and septum (32, 42). Recently, MKlp2 and NudC were
identified as possible direct Plk1 targets involved in cytokinesis
in human cells (40, 59).
Looking at all of the above-listed functions of Plks, we won-
dered how these different actions are regulated. One level of
regulation may be Plk’s subcellular distribution. Plk1 is located
mostly in the cytoplasm during interphase and translocates to
the nucleus in early mitosis. Plk1 was shown to associate with
centrosomes from G
2
up to metaphase, to translocate to ki
-
netochores at metaphase, and to locate at the midbody from
anaphase to telophase (18, 33). The Polo box domain located
in the C terminus is required for the localization of Plk1 to the
centrosome and midbody (24, 51) and for the interaction with
cell cycle regulators, e.g., Cdc25C in human cells and APC/C
subunits in fission yeast (12, 46). The Polo box binds prefer-
entially to phosphorylated serine/threonine motifs (12, 13),
suggesting that the timely recruitment of Plk1 to a given sub-
strate requires a priming phosphorylation event, e.g., by cyclin
B1/Cdk1, that yields a high-affinity binding site for the Polo
box. As such, sequential functions of Plk1 may require timed
activation of different priming kinases.
Besides changes in subcellular localization, different phos-
phorylation events might also regulate the kinase Plk1 (19, 33,
38). Several kinases have been reported as upstream regulators
of Polo-like kinases, e.g., Cdk1, mitogen-activated protein ki-
nase, and protein kinase A (PKA). At different embryonic
* Corresponding author. Present address: Department of Medical
Oncology, University Medical Center, Str. 2.103 universiteitsweg 100,
3584 CG Utrecht, The Netherlands. Phone: 31-030-2539689. Fax: 31-
30-2538479. E-mail: roh.medema@med.uu.nl.
† Supplemental material for this article may be found at http://mcb
.asm.org/.
2031
Page 1
phases in starfish, distinct upstream kinases for Plk1 were iden-
tified, i.e., cyclin B/Cdk1 at meiosis I, mitogen-activated pro-
tein kinase as well as Cdk1 at meiosis II, and cyclin A/Cdk1 at
embryonic M phase (43). In Xenopus, both xPlkk1 and PKA
were shown to phosphorylate and activate Plx1 (28, 45). As
with xPlkk1, related Ste20-like kinases were shown to target
Plk1. For instance, human Ste20-like kinase phosphorylated
mouse Plk1 in vitro (14) and mouse lymphocyte-oriented ki-
nase was shown to phosphorylate Plk1 in vitro (25).
Possible target residues in Plk1 for upstream kinases are the
highly conserved Ser-137 and Thr-210, latter being located in
the so-called activation or T loop. When one or both residues
are mutated into Asp to mimic phosphorylation, kinase activity
is increased severalfold (32, 44). So far, no upstream kinase
responsible for phosphorylating Ser-137 has been identified,
and conflicting data exist about the kinase upstream of Thr-
210, with xPlkk1 and PKA as candidates (25, 28, 44).
In this study, we examine the role of Ser-137 and Thr-210 in
regulating Plk1 function in human cells. We show that phos-
phorylation of these residues in vivo occurs with different tim-
ing. Expression of activated Plk1 mutated at either residue
affects mitotic progression differentially and demonstrate a
role for Plk1 in APC/C activation. Our observations suggest
that consecutive posttranslational modifications regulate sep-
arate Plk1 actions and that the order of events needs to be
tightly controlled to prevent uncoupling of APC/C activation
from spindle assembly checkpoint control.
MATERIALS AND METHODS
Construction of cell lines and plasmids. UTA6 cells are a clonal population of
human osteosarcoma U2OS cells stably transfected with the tetracycline-repress-
ible transactivator tTA (provided by C. Englert, Karlsruhe, Germany) (15).
UTA6 cells were grown in Dulbecco’s modified Eagle’s medium supplemented
with 6 to 10% fetal calf serum, 100 U of penicillin/ml, 100 g of streptomycin/ml,
and 1 g of tetracycline/ml. When washing and trypsinizing cells, 1 g of tetra-
cycline/ml was routinely added to the phosphate-buffered saline (PBS) and
trypsin. pCMV-plasmids encoding wild-type (wt) Plk1, S137D, T210D, and
S137D/T210D with an N-terminal Myc tag were a gift from E. Nigg (Martinsried,
Germany). These (mutant) Myc-Plk1 constructs were subcloned into the tetra-
cycline-repressible expression vector pUHD10-3 (provided by M. Gossen and H.
Bujard, Berlin, Germany). To generate tetracycline-inducible (mutant) Plk1 cell
lines, UTA6 cells were transfected with 10 g of pUHD10-3 (mutant) Plk1 and
1 g of pBabepuro (8) by the standard calcium phosphate transfection protocol.
After 1 day, fresh medium containing 1 g of puromycin/ml and 1 g of tetra-
cycline/ml was added. Two weeks later, individual colonies were picked and
analyzed for Myc-Plk1 expression after induction by washing cells three times
with PBS.
The murine nondegradable cyclin B1 plasmid pEF-B1DM-green fluorescent
protein (GFP) was a gift of M. Brandeis (Jerusalem, Israel). Histone H2B-GFP
(27), spectrin-GFP (26), the small interfering RNA (siRNA) vector pS (3),
pS-Mad2 (34), and pS-Plk1 (56) have all been described previously, and pS-
BubR1 was provided by G. Kops (La Jolla, Calif.). In short, the 19-mer targeting
regions of pS-Plk1, pS-Mad2, and pS-BubR1 siRNA vectors were CGGCAGC
GTGCAGATCAAC, GGAAGAGTCGGGACCACAG and AGATCCTGGCT
AACTGTTC, respectively. A pRcCMV plasmid encoding Myc-wt Plk1 harbor-
ing silent mutations in the targeting region of pS-Plk1 (Plk1-sil) has been
described elsewhere (56), rendering the protein insensitive to pS-Plk1-mediated
degradation. These silent mutations were introduced in pRcCMV plasmids en-
coding Myc-Plk1-S137D and T210D (described above) by PCR-based mutagen-
esis (Stratagene) with the following primers: forward, 5-AGCAACCGGCAGT
GTTCAGATCAACTTC-3 (silent mutations are indicated in boldface type);
and reverse, 5-GAAGTTGATCTGAACACTGCCGGTTGCT-3. Plk1-S137A
and T210A mutations were introduced into pRcCMV-Myc-Plk1-sil (56) with the
following primers: S137A forward, 5-TGCCGCCGGAGGGCCCTCCTGGAG
C-3; S137A reverse, 5-GCTCCAGGAGGGCCCTCCGGCGGCA-3; T210A
forward, 5-GGAGAGGAAGAAGGCCCTGTGTGGGAC-3; and T210A re-
verse, 5-GTCCCACACAGGGCCTTCTTCCTCTCC-3.
Growth curves. To assess proliferation in the (mutant) Plk1-inducible cell
lines, 80,000 cells were plated in duplicate at day 0. Cells were either plated in the
presence of tetracycline (noninduced) or were washed twice with PBS and plated
in the absence of tetracycline (induced). Cells were harvested 1, 2, and 4 days
after plating, after which the numbers of live, single cells with 12- to 30-m
diameters were determined by using a Casy 1 cell counter (RJM Sales, Scotch
Plains, N.J.).
Immunofluorescence microscopy. Cells grown on coverslips were fixed in 20
mM piperazine-N,N-bis(2-ethanesulfonic acid) (PIPES; pH 6.8), 0.2% Triton
X-100, 1 mM MgCl
2
, 10 mM EGTA, and 4% formaldehyde for 10 min at room
temperature. After blocking in 3% bovine serum albumin-PBS, cells were stained
with mouse anti-Myc clone 4A6 (Upstate Biotechnology, Lake Placid, N.Y.),
followed by chicken anti-mouse Alexa 488 and TO-PRO-3 (both from Molecular
Probes, Eugene, Oreg.) for DNA staining.
Antibodies. Rabbit anti-phospho-histone H3, rabbit anti-Plk1, and mouse anti-
Myc clone 9E10 (used for immunoblotting and immunoprecipitation [IP]) and
clone 4A6 (used for immunohistochemistry) were from Upstate Biotechnology;
rabbit anti-Cdk4, rabbit anti-cyclin A, and mouse anti-cyclin B1 were from Santa
Cruz Biotechnology (Santa Cruz, Calif.); phospho-Plk (Ser-137) antibody was
from Cell Signaling Technology, Inc. (Beverly, Mass.). At our request, phospho-
Plk1–Thr-210 antibody no. 98 was made by PhosphoSolutions (Aurora, Colo.).
Donkey anti-rabbit antibody–Cy5 conjugate was from Jackson Immunoresearch
Laboratories (Westgrove, Pa.). Peroxidase-conjugated goat anti-rabbit antiserum
was from DAKO (Glostrup, Denmark). Propidium iodide (PI) was from Sigma
(St. Louis, Mo.). Chicken anti-mouse antibody–Alexa 488 and TO-PRO-3 were
from Molecular Probes.
Synchronization and spindle assembly checkpoint activation. Plk1 (mutant)
cell lines were synchronized at the G
1
/S transition by a double thymidine block.
To this end, cells were treated with thymidine (2.5 mM; Sigma) for 24 h. Cells
were released from the thymidine block by washing twice with PBS and then
placed in the incubator for 15 min in fresh, warm medium before the medium
was replaced again. Twelve hours after release, thymidine was added for another
24 h. To keep cells in a noninduced state, cells were always washed and cultured
in the presence of 1 g of tetracycline/ml. Next, cells were released from the
second thymidine block as described above, either in the presence of tetracycline
(noninduced) or in the absence of tetracycline (induced). When indicated, 250 ng
of nocodazole/ml was added immediately after release or a 5 M concentration
of proteasome inhibitor MG132 (N-CBZ-Leu-Leu-Leu-Ala) (both from Sigma)
was added for the last 3 h before harvesting. By using a cytocentrifuge (Shandon
Elliot Cytospin; Frankfurt am Main, Germany) at 4,000 g for 4 min, PI-stained
cell suspension was spun on a microscope slide. The chromosome condensation
state was assessed for 300 cells per condition.
For analysis with phospho-specific antibodies, cells were released from thymi-
dine for 15 h in the presence of nocodazole. Mitotic cells were collected by
shake-off and harvested or released from nocodazole by washing three times with
PBS. Thirty minutes later, MG132 was added for 2.5 h to arrest cells in late
mitosis. Only mitotic cells were harvested through shake-off. Plk1-depleted cells
arrested in mitosis were harvested by shake-off 22 h after release from the
thymidine block. For phosphatase treatment, cell lysates after nocodazole release
(see above) were incubated at 30°C for 30 min in the presence of buffer and
MnCl
2
with or without -phosphatase (New England BioLabs, Inc., Beverly,
Mass.). To test phospho-specificity of the Thr-210 antibody, we compared its
recognition of peptides containing phospho- and dephospho-Thr-210 by densi-
tometric analysis of dot blots with different amounts of both peptides.
Transfections. Transient transfections in UTA6 cells were performed by using
the standard calcium phosphate transfection protocol. Four micrograms of non-
degradable cyclin B1 plasmid was transfected into Plk1-S137D cells. Directly
after washing away the calcium phosphate precipitate, cells were arrested in
thymidine for 24 h and used for time-lapse analysis after release.
For immunoblotting in reconstitution experiments, 10 g of pS or pS-Plk1 was
cotransfected into UTA6 cells with 1 g of pBabepuro combined with the
indicated amounts of wt Plk1-sil when indicated. Puromycin was added about 6 h
after the calcium phosphate precipitate was washed away. After 24-h selection
for transfected cells, cells were washed and puromycin-free medium was added.
Another 24 h later, cells were harvested and lysed for immunoblotting.
For flow cytometry analysis in reconstitution experiments, 10 gofpSor
pS-Plk1 was cotransfected with 0.5 g of spectrin-GFP combined with 0.5 gof
wt Plk1-sil, 0.5 g of S137A, 10 g of S137D, 0.5 g of T210A, or 1 g of T210D
plasmid. Different amounts of reconstitution plasmids were used to equalize
expression levels. Cells were synchronized in thymidine for 24 h and harvested
2032 VAN DE WEERDT ET AL. MOL.CELL.BIOL.
Page 2
24 h after release. After 10 to 12 h of release, thymidine was re-added to collect
cells at the G
1
/S transition.
For time-lapse analysis in reconstitution experiments, cells were cotransfected
with 10 g of pS-Plk1, 0.5 g of H2B-GFP, and 0.5 g of wt Plk1-sil or Plk1-
S137A and monitored after synchronization with a single thymidine block.
Kinase assays, flow cytometry and immunoblotting. Immunoblotting and in
vitro kinase assays on cyclin B1 and Myc immunoprecipitates were performed as
described previously (50) by using histone H1 (Roche, Basel, Switzerland) or
dephosphorylated -casein (Sigma) as a substrate. Tina 2.0 software (Raytest,
Straubenhardt, Germany) was used to quantify kinase activity. Phospho-histone
H3 and PI staining were performed as described previously (34).
Time-lapse analysis. Plk1 (mutant) cell lines were plated on 35-mm-diameter
glass-bottom culture dishes (WillCo-dish; WillCo Wells, Amsterdam, The Neth-
erlands). When indicated, Plk1 (mutant) expression was induced the next day by
washing three times with PBS. Cells were monitored with a Zeiss Axiovert 200 M
microscope equipped with a 0.55 numerical aperture (NA) condenser and a 20,
0.75 NA Plan-Apochromat objective in medium containing 33 M HEPES (pH
7.4) and, when indicated, 250 ng of nocodazole (Sigma)/ml. Cells were heated to
a temperature of 37°C with a microincubator ring (22). Images were taken by
using Axio Vision 3.1 software (Zeiss, Oberkochen, Germany).
Plk1-depleted cells transfected with either wt Plk1 or Plk1-S137A in combi-
nation with H2B-GFP were monitored by time-lapse microscopy after release
from thymidine block. Dishes were transferred to a heated culture chamber
(37°C, 5% CO
2
) of a Zeiss Axiovert 200 M microscope equipped with a 0.55 NA
condenser and a 40, 1.3 NA Plan-Neo differential interference contrast (DIC)
objective. Twelve-bit DIC and green fluorescence images were captured by using
a Photometrics CoolSNAP HQ charged coupled device camera set at gain 1.0
(Scientific, Tucson, Ariz.) and a GFP filter cube (Chroma Technology Corp.,
Rockingham, Vt.) to select specific fluorescence. Images were processed by using
MetaMorph software (Universal Imaging, Downingtown, Pa.).
RESULTS
Plk1 Ser-137 and Thr-210 are phosphorylated at different
intervals in the cell cycle. To study the regulation of Plk1 by
phosphorylation, we examined the timing of in vivo Plk1 phos-
phorylation on the putative phosphorylation residues Ser-137
and Thr-210. Using phospho-specific antibodies, we analyzed
phosphorylation at different points in the cell cycle in U2OS
cell lysates. Expression of total Plk1 was most abundant at 12
and 15 h after thymidine release, at time points when the
percentage of mitotic cells was at a maximum as measured by
phospho-histone H3 positivity (Fig. 1A). Phosphorylation of
Ser-137 was found mostly at 15 h after thymidine release,
whereas Thr-210 phosphorylation was detected at both 12 and
15 h after thymidine release. This finding suggests that both
residues are phosphorylated in vivo and that Thr-210 phos-
phorylation may precede phosphorylation of Ser-137. Adding
the proteasome inhibitor MG132 for the last 3 h before har-
vesting resulted in increased percentages of mitotic cells and
increased levels of Ser-137- and Thr-210-phosphorylated Plk1.
We also examined cells arrested in mitosis by the microtubule-
destabilizing agent nocodazole and cells released from nocoda-
zole but blocked in late mitosis by the addition of MG132, thus
comparing prometaphase cells with cells in late mitosis. In
these two situations, Plk1 expression levels were equal (Fig.
1A, lower right panel). Ser-137 phosphorylation was not de-
tected in nocodazole-blocked cells, but was found only in cells
released from nocodazole. This finding indicates that Ser-137
phosphorylation occurs only in late mitosis. In contrast, phos-
phorylation of Thr-210 was found both in nocodazole and
nocodazole release. These data indicate that Thr-210 phos-
phorylation precedes phosphorylation of Ser-137, which takes
place only in late mitosis.
The Ser-137 phospho-specific antibody is a commercially
available antibody and its phospho-specificity was demon-
FIG. 1. Different timing of Plk1 Ser-137 and Thr-210 phosphorylation. (A) U2OS cells were released from a thymidine block for the indicated
times, and MG132 was added during the last 3 h before harvesting where indicated (left panel). In fixed cells, PI staining was combined with
phospho-histone H3 staining to detect mitotic cells by flow cytometry (bottom left panel). Cells were collected from nocodazole arrest (noco), after
release from nocodazole for 3 h with MG132 present for the last 2.5 h to keep cells in mitosis (noco release), or after Plk1 depletion (pS-Plk1)
(right panel). Only mitotic cells were harvested by mitotic shake-off. Whole-cell lysates (WCL) were used for IP and immunoblotting (IB) with the
indicated antibodies. (B) Densitometric analysis of dot blots of the indicated amounts of peptides containing phospho-Thr-210 (Thr-210-p) or
dephospho-Thr-210 (Thr-210). (C) Cell lysates of cells released from nocodazole (see legend to panel A) were treated with -phosphatase before
IP and immunoblotting as indicated.
V
OL. 25, 2005 SEPARATING THE ROLES OF Plk1 IN MITOSIS 2033
Page 3
strated by the supplier by using an enzyme-linked immunosor-
bent assay (Cell Signaling Technology). At our request, a
Thr-210 phospho-specific antibody was generated (Phospho-
Solutions). This antibody was tested for phospho-specificity by
densitometric analysis of dot blots with peptides containing
phospho- or dephospho-Thr210 (Fig. 1B). We also tested the
specificities of both phospho-specific antibodies by phospha-
tase treatment. Cells released from nocodazole but blocked in
mitosis by MG132 were lysed and incubated with or without
-phosphatase. Phosphorylation of both Ser-137 and Thr-210
was decreased after phosphatase treatment, demonstrating
that the phospho-specific antibodies are indeed phosphoryla-
tion-specific (Fig. 1C).
Expression of mutant Plk1 induces proliferation defects.
We wanted to study phosphorylation of Ser-137 and Thr-210 in
more detail. Mutation of Ser-137 and Thr-210 to a phospho-
mimicking residue has been described to result in elevated
Plk1 kinase activity (25, 32, 44). When these mutants were
expressed in U2OS cells and immunoprecipitated, we found
that kinase activity of Plk1 in the respective mutants was in-
deed increased (Fig. 2A). We next generated inducible U2OS-
derived cell lines that express (mutant) Plk1 under the control
of a tetracycline-repressible promoter. Multiple clones ex-
pressing wt Plk1, Plk1-S137D, Plk1-T210D, or the double mu-
tant Plk1-S137D/T210D, all N-terminally Myc-tagged, were
generated. To compare expression and kinase activity in all cell
lines, expression of Plk1 was induced by washing away the
tetracycline-containing medium. After overnight induction,
cells were harvested, lysed, and used for both immunoblotting
and Myc-IP kinase assays using -casein as a substrate. For our
studies, we selected four cell lines with comparable Plk1 ex-
pression levels (Fig. 2B, middle panel). Kinase activity of the
Plk1 mutants was elevated compared to wt Plk1 (Fig. 2B, upper
panel). Noninduced control cells showed no or hardly any
detectable expression and kinase activity (Fig. 2B). Induction
of Plk1 expression in cells expressing Plk1-S137D was analyzed
and was found to be very rapid (Fig. 2C, upper panel), with
kinase activity reaching maximum levels around 8 h after in-
duction (Fig. 2C, lower panel).
To examine whether expression of (mutant) Plk1 affects
proliferation, we obtained growth curves of all cell lines (Fig.
2D). Cells induced to express wt Plk1 proliferated more rapidly
than noninduced cells. Remarkably, the expression of Plk1-
S137D, Plk1-T210D, or Plk1-S137D/T210D resulted in se-
verely impaired cell growth, indicating that the expression of
constitutive active mutants of Plk1 is incompatible with normal
cell proliferation.
Mutant Plk1 induces premature mitotic entry. Plk1 has
been implicated in promoting mitotic entry via activation of
cyclin B1/Cdk1 complexes through activation of Cdc25C and
inhibition of Myt1/Wee1. To determine if the timing of mitotic
entry is affected in the cell lines expressing active mutants of
Plk1, cells were released from a double thymidine block with
Plk1 expression induced at the moment of release. At different
time points after release, the cells were harvested and stained
for phospho-histone H3 positivity to allow examination of mi-
totic cells by flow cytometry. Mitotic entry in noninduced con-
trol cells was observed around 12 to 14 h after release (Fig.
3A). Cells expressing wt Plk1 started to enter mitosis at 10 to
12 h after release. Plk1-T210D-expressing cells showed a more
accelerated mitotic entry starting at 8 to 10 h after release,
while cell expression of Plk1-S137D or S137D/T210D forced
cells to enter mitosis even earlier, at 6 to 8 h after release (Fig.
3A). These data indicate that expression of active Plk1 can
induce premature mitotic entry in human cells.
Furthermore, a very slow S phase progression was observed
with the Plk1-S137D and Plk1-S137D/T210D cell lines when
expression was induced 12 or 24 h before release from the
second thymidine block instead of at the time of release (data
not shown). This result indicates that inducing expression of
constitutive active Plk1-S137D or Plk1-S137D/T210D at the
G
1
/S transition, at a time when Plk1 is normally not expressed,
inhibits DNA replication. Indeed, a function for Ser-137 in S
phase was proposed earlier based on a reduction in the per-
centage of cells in S phase in HeLa cells overexpressing Plk1-
S137D (25).
Different mitotic defects induced by S137D and T210D mu-
tants. To study cell cycle progression of the Plk1 cell lines in
more detail, we monitored cells by time-lapse microscopy. Af-
ter synchronization in G
1
/S by using a double thymidine block,
cells expressing wt Plk1 entered mitosis slightly earlier than
noninduced cells, with mitotic entry starting at 9 to 11 h and 12
to 14 h, respectively (Fig. 3B). The expression of wt Plk1 did
not result in mitotic abnormalities (data not shown). Cells
stably expressing Plk1-S137D or Plk1-S137D/T210D entered
mitosis prematurely with kinetics similar to those we observed
by flow cytometry (Fig. 3A and B). This finding confirms that
the phospho-histone H3 positivity we used as a mitotic marker
in flow cytometry indeed correlates with mitotic entry. Many of
these cells underwent severe blebbing and subsequent cell
death while in mitosis or shortly after exit from mitosis, a
finding which suggests that these cells undergo mitotic catas-
trophe (Fig. 3B; Fig. S1 in the supplemental material). Indeed,
approximately 50% of the cells expressing Plk1-S137D died
during the course of the experiment. We observed 41% cell
death with the Plk1-S137D/T210D-expressing cells. Of the mi-
totic cells expressing Plk1-S137D or Plk1-S137D/T210D that
did not die during mitosis, 71 or 69%, respectively, failed at
cytokinesis, ultimately giving rise to only one (multinucleated)
daughter cell (Fig. 3C). Some mitotic cells expressing Plk1-
S137D (7%, n 42) started normal formation of a cleavage
furrow, which subsequently regressed, while other mitotic cells
(64%, n 42) rounded only partially and flattened without
undergoing a proper mitotic division. These partially rounded
cells appeared not to degrade the nuclear envelope and in most
cases died shortly after flattening (data not shown), which
suggests that they underwent a very-early-onset mitotic catas-
trophe. Thus, expression of Plk1-S137D appears to result in
mitotic catastrophe or aberrant cytokinesis in cells that do
complete the early stages of mitosis.
In contrast to what we observed in cells expressing Plk1-
S137D or the double mutant, cell lines expressing wt Plk1 or
T210D showed only few dying cells (Fig. 3B) or cytokinesis
defects (Fig. 3C). Interestingly, in Plk1-T210D cells, we found
mitotic progression to be delayed. In these cells, the time from
nuclear envelope breakdown until anaphase onset ranged from
50 min to approximately 10 h, with an average of 150 min (n
91) compared to an average of 50 min in cells expressing wt
Plk1 (n 78). This finding suggests that Thr-210 in Plk1 may
have to be dephosphorylated for proper mitotic progression.
2034 VAN DE WEERDT ET AL. MOL.CELL.BIOL.
Page 4
Plk1-T210D cells underwent successful cytokinesis, although it
was often accompanied by excessive blebbing of the cell mem-
brane coincident with cleavage furrow ingression (data not
shown). Thus, cell lines expressing different active mutants of
Plk1 give rise to distinct mitotic defects. The effect seen with
the S137D/T210D mutant is very similar to that with the S137D
mutant, while the T210D mutation appears to cause a very
different mitotic defect, indicating that the S137D mutation
has a dominant effect over the T210D mutation.
As described above, cells expressing Plk1-S137D or Plk1-
S137D/T210D showed considerable cell death during mitosis
or shortly after mitotic exit. Also, some cells seemed to die in
late G
2
or prophase, but before nuclear envelope breakdown.
This finding suggests that the observed cell death occurs as a
consequence of mitotic defects. Alternatively, the high expres-
sion of mutant Plk1 around this time may induce cell death
independent of the cell cycle state of the cells. Thus, to test
whether mere high expression of Plk1-S137D could lead to cell
FIG. 2. Expression of active Plk1 mutants results in proliferation defects. (A) wt Plk1, S137D, T210D, or S137D/T210D mutants were
transfected in U2OS cells and subjected to Myc-IP kinase assays using -casein as a substrate (left panel). Casein phosphorylation was quantified
with wt Plk1 phosphorylation set as the reference. The amount of precipitated Myc-Plk1 was determined by anti-Myc immunoblotting (IB; right
panel). (B) Expression of wt Plk1, S137D, T210D, or S137D/T210D mutants was induced in cell lines for 16 h by washing away tetracycline ( tet)
or in samples left untreated (noninduced; tet). Cell lysates were used for Myc-Plk1 IP kinase reactions using -casein as a substrate (upper panel)
or for immunoblotting and probed for Myc-Plk1 (middle panel) or Cdk4 as a loading control (lower panel). (C) Plk1-S137D expression was induced
in asynchronous cells by washing away tetracycline. Cells were harvested and lysed at indicated time points. Lysates were used for immunoblotting
and probed for Plk1 (upper panel) and used for Myc-Plk1 IP kinase assays with -casein as a substrate (lower panel). Casein phosphorylation was
quantified by phosphorylation with 0 h induction set as the reference. (D) Cell lines were induced ( tet) or noninduced ( tet) and plated in
duplicate on day 0. Cell numbers were counted 1, 2, and 4 days after plating.
V
OL. 25, 2005 SEPARATING THE ROLES OF Plk1 IN MITOSIS 2035
Page 5
FIG. 3. Expression of active Plk1 mutants induces premature mitotic entry and different mitotic defects. (A) Expression of wt Plk1, S137D,
T210D, or S137D/T210D was induced (or not induced) at the time of release from a double thymidine block. Cells were harvested at indicated
time points after release. PI staining was combined with phospho-histone H3 staining to detect mitotic cells by flow cytometry. (B) Cells were plated
on coverslips, released from the double thymidine (T/T) block, and replated in medium without (induced; tet) or with (noninduced; tet)
tetracycline. After release, cells were analyzed for 18 h by using time-lapse microscopy. Mitotic and dead cells were counted and plotted as
cumulative numbers. (C) Normal mitotic division and cytokinesis defects observed in time-lapse experiments were counted and plotted as
percentages of the total mitotic population. (D) wt Plk1 and S137D cell lines were blocked in thymidine for 24 h, and expression was induced for
12 or 24 h in the presence of nocodazole, continuously arresting cells in thymidine. DNA profiles of fixed cells were analyzed together with
phospho-histone H3 positivity by using flow cytometry. M, mitotic cells.
2036
VAN DE WEERDT ET AL. MOL.CELL.BIOL.
Page 6
death, we induced expression in S phase-arrested cells. But
contrary to the experimental setting described above, we now
monitored the faith of these cells when they were kept arrested
at the G
1
/S transition in the continued presence of thymidine.
wt Plk1 or Plk1-S137D cell lines were arrested with thymidine
for 24 h, after which expression was induced for 12 or 24 h in
the continued presence of thymidine to keep cells in G
1
/S
during the experiment (Fig. 3D). After 12 h induction, neither
cell line showed less than 2N DNA content, indicative of cell
death. After 24 h of induction, some cells were found to con-
tain less than 2N DNA content, but percentages were similar in
wt Plk1 and Plk1-S137D-expressing cells. Also, Plk1-S137D-
expressing cells did not enter mitosis (Fig. 3D). Thus, mere
high expression of Plk1-S137D could not account for the ob-
served levels of cell death or mitotic entry, indicating that
progression through the cell cycle is needed for these effects.
Expression of Plk1-S137D induces spindle assembly check-
point failure. We wanted to examine other mitotic aspects in
the mutant cell lines. The spindle assembly checkpoint moni-
tors attachment and tension between chromosomes and the
mitotic spindle to ensure that anaphase takes place only when
all chromosomes are attached and proper tension is generated.
We checked for proper spindle assembly checkpoint arrest by
arresting cells in mitosis with nocodazole, which prevents spin-
dle assembly checkpoint inactivation by depolymerizing micro-
tubules. Again, we observed an early mitotic entry of all cell
lines expressing mutant Plk1 compared to those expressing wt
Plk1 (Fig. 4A). Surprisingly, however, cells expressing Plk1-
S137D or Plk1-S137D/T210D could not be properly blocked in
mitosis by nocodazole, in contrast to wt Plk1- and Plk1-T210D-
expressing cells. A small but significant increase in the percent-
ages of mitotic cells was consistently observed with the Plk1-
S137D- and Plk1-S137D/T210D-expressing cultures at 9 to
12 h after release, but the percentages of mitotic cells markedly
decreased at later time points. Concomitantly, cyclin B1/Cdk1
activity increased over time in the presence of nocodazole in
cells expressing wt Plk1 or Plk1-T210D, but not in Plk1-S137D-
or Plk1-S137D/T210D-expressing cells (Fig. 4B).
We determined cyclin B1 expression levels in cells released
from a single thymidine block for 20 h in the presence of
nocodazole. We observed decreased expression of cyclin B1
when Plk1-S137D or Plk1-S137D/T210D mutants were ex-
pressed (Fig. 4C, upper panel). This result suggests that these
cells have failed to establish spindle assembly control of the
APC/C, leading to premature cyclin B1 degradation.
To confirm mitotic progression past an active spindle assem-
bly checkpoint in Plk-S137D- or Plk1-S137D/T210D-express-
ing cells, we filmed these cells in the presence of nocodazole
after synchronization by a double thymidine block. Even
though nocodazole was present, cells could exit mitosis (Fig.
4D and E). Cells showed defective cytokinesis, similar to the
cytokinesis defects observed without nocodazole. These results
confirm that the expression of Plk1-S137D or Plk1-S137D/
T210D results in a defective spindle assembly checkpoint-me-
diated arrest.
Plk1-S137D expression leads to untimely activation of the
APC/C. Spindle assembly checkpoint inactivation allows acti-
vation of the APC/C, which targets its substrates for degrada-
tion by the 26S proteasome. We investigated APC/C activation
by examining proteasomal degradation of its target proteins.
Therefore, we determined the expression level of several
APC/C substrates with or without the addition of a proteasome
inhibitor (MG132). Because adding MG132 for longer than 3 h
was toxic to U2OS cells, we added the proteasome inhibitor
only the last 3 h before harvesting.
After induction of Plk1-S137D expression and release for
12 h, both expression of cyclin A and B1 and cyclin B1/Cdk1
kinase activity were dramatically reduced (Fig. 5A). The de-
creased expression and kinase activity was restored by adding
MG132 (even though it was added only for 3 h), indicating that
cyclin A and B1 had been actively degraded by the proteasome
(Fig. 5A). We examined histone H3 phosphorylation to check
whether Plk1-S137D-expressing cells were indeed blocked in
mitosis after the addition of MG132. We observed a signifi-
cantly higher phospho-histone H3 positivity after the addition
of MG132, indicative of a mitotic arrest (Fig. 5A). Again, the
addition of nocodazole at the time of release only marginally
restored the expression of APC/C target proteins and histone
H3 phosphorylation (Fig. 5A), similar to paclitaxel (Taxol)
(data not shown), another spindle poison that blocks progres-
sion through mitosis. We quantified the number of mitotic cells
and observed that 32% of cells were mitotic after3hofMG132
addition. In sharp contrast, only 9% of cells were mitotic after
nocodazole treatment for 12 h (Fig. 5B). In comparison,
around 40% of the cells expressing wt Plk1 were mitotic at 18 h
after release into nocodazole (Fig. 4A). In Plk1-S137D-expressing
cells, we also observed micronucleated cells (6%), indicative of a
defective spindle checkpoint, defective cytokinesis, or cell death
(Fig. 5B and C). These data confirm a spindle assembly check-
point defect and suggest untimely APC/C activation in cells ex-
pressing Plk1-S137D or Plk1-S137D/T210D.
Thus far, our data suggest that cells expressing Plk-S137D
mutants enter mitosis prematurely, but fail to maintain the
mitotic state in the presence of spindle poisons. Concomi-
tantly, we observe untimely degradation of APC/C substrates,
such as cyclin B1, that are normally degraded only upon mi-
totic exit. Therefore, we wanted to test if we could prevent
mitotic exit by the expression of a nondegradable cyclin B1
mutant shown to arrest cells in late mitosis (60). To this end,
we transfected a plasmid encoding nondegradable cyclin B1
and released cells from a thymidine block while inducing Plk1-
S137D expression at release. Time-lapse microscopy demon-
strated that Plk1-S137D-expressing cells could be arrested in
mitosis for an extensive period of time by the expression of
nondegradable cyclin B1 (Fig. 5D). This result was confirmed
by flow cytometry analysis (Fig. 5E). So, Plk1-S137D-express-
ing cells can indeed be arrested in mitosis, as long as the
degradation of cyclin B1 is prevented.
Plk1-T210D causes a spindle assembly checkpoint-depen-
dent delay. We next investigated whether the mitotic delay in
cells expressing Plk1-T210D was possibly due to a spindle
assembly checkpoint-dependent arrest. To this end, we si-
lenced the expression of the essential checkpoint components
Mad2 or BubR1 to see if we could rescue the mitotic delay.
Knocking down expression of either Mad2 or BubR1 was re-
ported to be sufficient to decrease the duration of mitosis by
approximately half and to lead to mitotic exit before full con-
gression (34). We transfected cells with a control vector (pS) or
a vector encoding small interfering RNA (siRNA) for Mad2 or
BubR1 (pS-Mad2 or pS–BubR1) (34) and released cells from
VOL. 25, 2005 SEPARATING THE ROLES OF Plk1 IN MITOSIS 2037
Page 7
a single thymidine block, leading to down-regulation of expres-
sion (Fig. 6A). Mitotic cells were visualized with anti-phospho-
histone H3 antibody and measured by flow cytometry. About
25% of the control-transfected Plk1-T210D cells were phos-
pho-histone H3 positive 14 h after release (Fig. 6B). In con-
trast, only 3 to 5% of Plk1-T210D cells were in mitosis after
inhibiting spindle assembly checkpoint function by knocking
down Mad2 or BubR1 expression (Fig. 6B). These cells did not
arrest in G
2
or undergo apoptosis, but progressed to G
1
as
shown by DNA profiles (Fig. 6C). From this finding, we con-
clude that the spindle assembly checkpoint is functional in
Plk1-T210D cells, in contrast to Plk1-S137D mutants that show
a checkpoint override. Thus, mimicking phosphorylation at
Ser-137 and Thr-210 in Plk1 has distinct and partly opposing
effects on the spindle assembly checkpoint and consequent
mitotic progression.
Ser-137 and Thr-210 phosphorylation both contribute to
proper mitotic progression. To study the function of Ser-137 in
more detail, we compared a nonphosphorylatable S137A mu-
tation to the phospho-mimetic S137D mutation in a back-
ground devoid of endogenous Plk1. To this end, we depleted
endogenous Plk1 in U2OS cells by vector-driven siRNA (pS-
Plk1) (56) and reconstituted these cells with the respective
Plk1 mutants. Two silent mutations in the siRNA-target region
of the Plk1 constructs were introduced to allow expression of
the exogenous Plk1, without possible interference of endoge-
FIG. 4. Plk1-S137D and S137D/T210D mutants undergo a defective spindle assembly checkpoint arrest. (A) Expression of wt Plk1, S137D,
T210D, or S137D/T210D was induced upon release from a double thymidine (T/T) block in the presence of nocodazole or was left noninduced.
Cells were analyzed for phospho-histone H3 positivity combined with DNA profiles by flow cytometry. (B) See legend for panel A. In parallel, cells
were lysed and cyclin B1-IP kinase assays were performed with histone H1 as a substrate. (C) Plk1 expression in cell lines was induced for 20 h
by washing away tetracycline ( tet) or left noninduced ( tet) in the presence of nocodazole. Immunoblots of cell lysates were probed for cyclin
B1 and Cdk4. (D) Plk1-S137D cells synchronized with thymidine were monitored by time-lapse microscopy. At release, expression was induced
and nocodazole was added. Elapsed time (h:min) is indicated in the upper left corner of each panel. noco, nocodazole. (E) Conditions were as for
panel D, but Plk1-S137D/T210D-expressing cells were used.
2038
VAN DE WEERDT ET AL. MOL.CELL.BIOL.
Page 8
nous Plk1. We could indeed demonstrate that this approach
results in the replacement of endogenous Plk1 with the exog-
enous Plk1 (Fig. 7A). Routinely, we obtain a knockdown effi-
ciency of more than 95% (56). To avoid possible overexpres-
sion artifacts, we used 0.5 g of nontargetable wt Plk1, since
this approach resulted in wt Plk1 expression comparable to or
slightly lower than endogenous Plk1 expression (Fig. 7A).
Next, cells were reconstituted with the different Ser-137 and
Thr-210 mutants and harvested 48 h after transfection. Com-
pared to wt Plk1, mutating Ser-137 or Thr-210 into Asp in-
creased kinase activity approximately fivefold, whereas kinase
activity was lowered after mutation into Ala (Fig. 7B). These
kinase activities are comparable to those of the same Plk1
mutants as those described in other experiments with Xenopus
and U2OS cells (25, 32, 44).
Next, we examined whether the Plk1-Ser-137 mutants could
reconstitute Plk1-depleted cells, which arrest in G
2
/M (Fig.
7C) (56). Using 0.5 g of wt Plk1, we almost fully restored
normal cell cycle progression in Plk1-depleted cells as analyzed
by flow cytometry (Fig. 7C). Interestingly, we found that the
expression of Plk1-S137A could not induce a rescue of cell
cycle progression to the level we observed with wt Plk1. The
cultures reconstituted with Plk1-S137A had a higher content of
4N cells (Fig. 7C), indicating that they are delayed at some
point in G
2
/M, suggesting that Ser-137 phosphorylation does
contribute to normal Plk1 function. In contrast, reconstitution
with Plk1-S137D seemed to result in a better rescue than with
wt Plk1, as judged by an increased G
1
content and decreased
mitotic population (Fig. 7C). However, it should be noted that
about one-third of the Plk1-S137D-expressing cells did not
progress through the cell cycle properly after release from a
thymidine block and remained arrested at the G
1
/S transition
(data not shown). This finding is consistent with the result
described above, that expression of the active Plk1-S137D dur-
ing a thymidine block can inhibit DNA replication.
By time-lapse analysis, we found that Plk1-S137D-reconsti-
tuted cells that did enter mitosis underwent either a normal
division (79%, n 19) or one with defective cytokinesis (11%,
n 19), and approximately 20% of the cells died. Percentages
of cells showing these phenotypes are lower than in the Plk1-
FIG. 5. The APC/C is untimely activated by Plk1-S137D expression. (A) Plk1-S137D cells were released for 12 h from a double thymidine (T/T)
block in medium with (noninduced) or without (induced) tetracycline. When indicated, nocodazole was added at release. Proteasome inhibitor
MG132 was added only 3 h before harvesting. Cell lysates were used for the indicated immunoblots (IB) and Myc-IP or cyclin B1-IP kinase assays
with -casein or histone H1 as a substrate, respectively. (B) The experimental setup was as for panel A. More than 300 cells were counted by
cytospinning in an examination of DNA condensation to identify mitotic and micronucleated cells. (C) See legend to panel B. Examples of a mitotic
cell (arrow) and micronucleated cells (arrowheads) are shown. (D) The Plk1-S137D cell line was transfected with a nondegradable cyclin B1
plasmid combined with H2B-GFP. After synchronization, Plk1-S137D expression was induced and cells were monitored by time-lapse microscopy.
Numbers in the upper left corners are the elapsed time (h:min). (E) The Plk1-S137D cell line was transfected with spectrin-GFP alone ()orin
combination with nondegradable cyclin B1 (nd-B1). At release from thymidine block, Plk1-S137D expression was induced ( tet) or not induced
( tet) and cells were harvested for flow cytometric analysis of mitotic cells at the indicated time points.
V
OL. 25, 2005 SEPARATING THE ROLES OF Plk1 IN MITOSIS 2039
Page 9
S137D-inducible cell line (Fig. 3B and C), possibly due to the
observed G
1
/S arrest or to a difference in expression levels of
the mutant protein.
In addition, we tested Plk1 Thr-210 mutants in the reconsti-
tution assay. Plk1-T210A could not substitute for wt Plk1 in
Plk1-depleted cells, and those cells arrested in G
2
/M (Fig. 7C).
Plk1-T210D had somewhat delayed cell cycle progression com-
pared to wt Plk1, possibly due to slower mitotic progression, as
described above. These data indicate that Thr-210 is indeed a
crucial in vivo phosphorylation site necessary for proper Plk1
function and mitotic progression. Concomitantly, we examined
Myc-Plk1 localization in cells reconstituted with Plk1-S137A,
Plk1-S137D, Plk1-T210A, or Plk1-T210D by immunohisto-
chemistry. Like wt Plk1, all mutants were able to localize to
centrosomes and kinetochores (see Fig. S2 in the supplemental
material). For the mutants for which we could find cells at late
stages of mitosis, we also observed localization at the midbody.
The different mutant Plk1 effects can thus not be attributed to
obvious differences in subcellular localization.
The above data suggested that Plk1-S137A could reconsti-
tute Plk1-depleted cells but to a lesser extent than wt Plk1.
Since the above in vivo data with a phospho-specific antibody
suggested that Ser-137 is phosphorylated in late mitosis, we
hypothesized that a late mitotic function of Plk1 might be
defective in Plk1-S137A-expressing cells and therefore that
they might show problems during late mitotic stages. We ex-
amined Plk1-S137A-reconstituted cells by cotransfecting GFP-
tagged histone H2B and analyzed mitotic progression in these
cells by time-lapse microscopy. We measured the duration of
mitosis starting from prophase until either anaphase or cell
death. In wt Plk1-reconstituted cells, the average duration of
mitosis was 1.0 h (n 10). In Plk1-S137A-reconstituted cells,
the average duration of mitosis was at least 6.4 h (Fig. 7D).
This estimate is low because 5 of 13 Plk1-S137A-reconstituted
cells were still in mitosis at the end of the experiment. Cells
reconstituted with wt Plk1 underwent a normal mitosis (Fig.
7E), although it was sometimes accompanied by severe bleb-
bing during anaphase and telophase (data not shown). Plk1-
S137A-reconstituted cells reached mostly metaphase align-
ment and consequently arrested (8 of 13 cells), although
sometimes one or more chromosomes did not congress (4 of
13). Rotation of the metaphase plate was frequently observed
(data not shown). During metaphase arrest, single chromo-
somes detached shortly from the metaphase plate and re-
aligned, or multiple chromosomes detached and then moved
towards one or both spindle poles (see Fig. 7F and Video S3 in
the supplemental material). Eventually, Plk1-S137A-reconsti-
tuted cells divided (5 of 13), died while in mitosis (3 of 13), or
stayed in arrest for the duration of imaging (5 of 13). Thus, the
expression of Plk1-S137A gives rise to errors in the late stages
of mitosis, suggesting that Ser-137 phosphorylation may be
needed at this time. These data confirm a late mitotic function
as suggested by our in vivo data with a phospho-specific anti-
body (Fig. 1A).
DISCUSSION
Here, we have studied the role of the conserved Ser-137 and
Thr-210 residues in Plk1. Our data indicate that phosphoryla-
tion of Ser-137 and Thr-210 on Plk1 occurs with distinct timing.
Cells expressing phospho-mimicking mutations on these resi-
dues displayed different mitotic phenotypes, suggesting that
timed activation of distinct Plk1 functions may be regulated by
consecutive posttranslational modifications. Furthermore, our
results suggest that tight control of Plk1 activity is important to
FIG. 6. Plk1-T210D induces a spindle assembly checkpoint-dependent delay. Plk1-T210D-expressing cells were transfected with control siRNA
vector pS, pS-Mad2 or pS-BubR1. (A) After release from thymidine (T) block for 20 h, the nonselected total cell population was lysed and
immunoblotted with the indicated antibodies. (B) After release from thymidine block for the indicated time, cells were harvested and fixed. PI
staining was combined with staining for phospho-histone H3 to identify mitotic cells by flow cytometry. Transfected cells were identified through
spectrin-GFP cotransfection. (C) See legend to panel B. PI profiles of Plk1-T210D cells transfected with control siRNA vector pS or pS-BubR1
were harvested at the indicated times after thymidine release.
2040
VAN DE WEERDT ET AL. MOL.CELL.BIOL.
Page 10
maintain APC/C activation under control of the spindle assem-
bly checkpoint.
By using a phospho-specific antibody for Ser-137, we could
immunoprecipitate Plk1 only after release from the nocoda-
zole block, but not during the arrest, providing the first evi-
dence that Ser-137 is actually phosphorylated in vivo. So far, a
putative kinase for Ser-137 has not been found (25, 28, 44).
Since we show that phosphorylation of this residue may occur
during only a very limited period during mitosis, it may as such
have gone undetected. With a Thr-210 phospho-specific anti-
body, we found an up-regulation of Thr-210 phosphorylation at
the same time as the increase in total Plk1 expression. This
finding suggests that in contrast to phosphorylation of Ser-137,
phosphorylation of Thr-210 in the activation loop of Plk1 may
be needed for an earlier, more general Plk1 activation.
Induced expression of Plk1 mutants, containing phospho-
mimicking mutations on Ser-137 and/or Thr-210 (S137D,
T210D, and S137D/T210D) all caused severe proliferation de-
fects in U2OS-derived cells. In contrast, cell growth was en-
hanced in cells expressing wt Plk1 compared to noninduced
control cells, demonstrating an important role for Plk1 in cel-
lular proliferation. In this respect, it is interesting that Plk1 is
overexpressed in many cancer types and that Plk1 overexpres-
sion can oncogenically transform NIH 3T3 cells (20, 49). Ex-
pression of the active mutants resulted in premature mitotic
entry that was most prominent in cells expressing Plk1-S137D
FIG. 7. Phosphorylation of Plk1 Ser-137 and Thr-210 both contribute to proper mitotic progression. (A) Cells were transfected with control
siRNA vector pS or pS-Plk1 together with the indicated amounts of nontargetable wt Plk1 and pBabepuro to allow selection of transfected cells.
Cells were lysed at 48 h after transfection to analyze Plk1 expression by immunoblotting. (B) Cells were transfected with pS or pS-Plk1 together
with the indicated nontargetable Plk1 mutants. Cells were lysed 48 h after transfection and subjected to Myc-IP kinase assays using -casein as a
substrate (upper panel). The amount of Plk1 that was immunoprecipitated was assayed by Myc-Plk1 immunoblotting (lower panel). (C) Cells were
transfected with pS or pS-Plk1 in combination with spectrin-GFP and reconstituted with the indicated nontargetable (mutant) Plk1, released from
thymidine block, and collected at the next G
1
/S transition after 24 h by the readdition of thymidine. After fixation, DNA profiles were analyzed
by flow cytometry. (D) Average duration of mitosis from prophase until anaphase onset or cell death in wt Plk1- versus Plk1-S137A-reconstituted
cells. The P value comparing both populations is indicated. (E) Cells were transfected with pS-Plk1, H2B-GFP, and nontargetable wt Plk1 to
monitor by time-lapse imaging after synchronization with a single thymidine block. The arrows point out chromosomes detaching from the
metaphase plate. Numbers in the upper left corners of the panels refer to the elapsed time (h:min). (F) See legend to panel E. Cells were
reconstituted with nontargetable Plk1-S137A instead of wt Plk1.
VOL. 25, 2005 SEPARATING THE ROLES OF Plk1 IN MITOSIS 2041
Page 11
or Plk1-S137D/T210D. Expression of Plk1-T210D also re-
sulted in acceleration of mitotic entry, similar to what was
recently described by Jackman et al. (23). However, the effect
of a T210D mutation on mitotic entry appeared to be subtle
compared to the effect of the Plk1-S137D or double mutant,
indicating that the S137D mutation leads to a greater gain of
function for this particular aspect of Plk1 functioning.
The observed premature mitotic entry could be caused by
early activation of Cdc25C and cyclin B1/Cdk1 since Plk1 is
known to directly regulate these proteins. Indeed, injection of
the corresponding Plx1 double mutant S128D/T201D mRNA
into Xenopus oocytes induces premature activation of both
Cdc25C and cyclin B1/Cdk1 (44). Injection of the less active
Plx1-S128D or Plx1-T210D single mutants or wt Plx1 mRNA
did not cause premature entry into mitosis. In contrast, we did
observe early mitotic entry using single mutants. This differ-
ence may reflect different regulation of Plk1 function in human
cells versus Xenopus oocytes. Possibly, Xenopus oocytes require
higher Plk1 activity to allow mitotic entry, given that the kinase
activity of the Plx1 double mutant is about 10 times higher than
that of either single mutant (44).
Besides regulating mitotic entry, Plks are suggested to reg-
ulate mitotic exit and cytokinesis. At least in budding yeast, the
initiation of mitotic exit is known to require Plk activity,
whereas Plk needs to be degraded for the completion of cyto-
kinesis (41). Here, we observed major cytokinesis defects in
U2OS cells induced to express Plk1-S137D or S137D/T210D,
while cells expressing wt Plk1 or Plk1-T210D underwent
proper cytokinesis. This result suggests that dephosphorylation
of Ser-137 may be required for proper execution of cytokinesis,
or that the S137D mutation interferes with normal Plk1 deg-
radation. Alternatively, the observed cytokinesis defects may
be an indirect consequence of improper chromosome segrega-
tion. Our results with induced expression of wt Plk1 are incon-
sistent with experiments performed with HeLa cells transiently
expressing wt Plk1. These cells were temporarily delayed in
mitosis (25, 38) and become multinucleated (38). These more
dramatic effects could be due to higher levels of Plk1 expres-
sion or could reflect a difference in p53 status, since HeLa cells
are p53 deficient and may therefore more easily accumulate
multinucleated cells.
In contrast to what we find with S137D mutants, T210D-
expressing cells were transiently delayed in mitosis. Consistent
with this finding, others have shown an accumulation of mitotic
HeLa cells 2 days after transfection with Plk1-T210D (25).
Concomitantly, a delayed mitosis before anaphase and a small
but significant delay in mitotic exit was shown with Plk1-
T210D-expressing HeLa cells, with mitotic exit measured as
the time from sister chromatid separation until the completion
of cleavage (35). Our data suggest that phosphorylation of
Thr-210 delays inactivation of the spindle assembly checkpoint
through a yet-unidentified mechanism. Recently, a human ki-
nase of the Plk1 family, Plk2/Snk, has also been suggested to
function in the mitotic spindle assembly checkpoint. Depletion
of Plk2 in the presence of nocodazole or paclitaxel gives a
significant increase in apoptosis in mitosis, which suggests that
Plk2 may prevent mitotic catastrophe following spindle dam-
age (4).
Using time-lapse microscopy, we found that expression of
Plk1-S137D or Plk1-S137D/T210D resulted in a substantial
induction of cell death. Cells died either after a defective
cytokinesis or in mitosis. Cell death was not apparent when the
Plk1-S137D or Plk1-S137D/T210D mutants were expressed in
cells continuously arrested in G
1
/S by thymidine, indicating
that the observed cell death requires cell cycle progression and
cannot be attributed simply to excess kinase activity. Instead,
we propose that the observed cell death occurs only after
mitotic entry. The exact mechanism of this mitotic catastrophe
remains to be elucidated, but in this respect it is interesting
that premature cyclin B1/Cdk1 activation before completion of
SorG
2
can induce premature chromatin condensation and
apoptosis (6). Since cells expressing Plk1-S137D and Plk1-
S137D/T210D enter mitosis 6 h after thymidine release, S or
G
2
phase may not have been completed, and this may result in
mitotic catastrophe. Alternatively, premature inactivation of
the spindle assembly checkpoint has also been shown to result
in mitotic catastrophe (37). Indeed, we found that Plk1-S137D
or Plk1-S137D/T210D-expressing cells could not be arrested in
mitosis through actions of the spindle assembly checkpoint.
Upon entry into mitosis, Plk1-S137D-expressing cells activated
the APC/C even in the presence of spindle poisons, such as
nocodazole or paclitaxel. The addition of a proteasome inhib-
itor was able to arrest these cells in mitosis, indicating that the
override of the spindle assembly checkpoint requires protea-
some-dependent protein degradation. Taken together, these
data demonstrate that the expression of S137D mutants can
uncouple APC/C activation from spindle assembly checkpoint
control. This uncoupling could be the result of direct inactiva-
tion of the spindle assembly checkpoint by Plk1-S137D, or
Plk1-S137D could somehow render APC/C activation nonre-
sponsive to the inhibitory signals generated by the spindle
assembly checkpoint. Alternatively, failure of the spindle as-
sembly checkpoint could arise as a secondary consequence of
the premature mitotic entry, as certain checkpoint components
may not have accumulated to sufficiently high levels to enforce
a normal checkpoint arrest. We also studied the importance of
Ser-137 phosphorylation by reconstituting Plk1-depleted cells
with a Plk1-S137A mutant, and we observed a marked delay in
metaphase. Together with the in vivo data with the phospho-
specific antibody, these results indicate that Ser-137 is phos-
phorylated in late mitosis after the spindle assembly check-
point has been silenced, possibly contributing to functions of
Plk1 at the later stages of mitosis.
Taken together, our data show that different activating mu-
tations in Plk1 at putative phosphorylation sites have distinct
and partly opposite effects on spindle assembly checkpoint
activity. Our data indicate that consecutive phosphorylation
events on Plk1 are important to maintain timed execution of
distinct mitotic events and to prevent mitotic catastrophe. As
also suggested by our results with phospho-specific antibodies
in vivo, phosphorylation of Thr-210 may regulate early mitotic
roles of Plk1, whereas later mitotic roles may be brought about
by Ser-137 phosphorylation. Our data with a Plk1-S137D/
T210D mutant indicate that phosphorylation of Ser-137 has a
dominant effect over Thr-210 phosphorylation, suggesting that
consequent Ser-137 phosphorylation may somehow alter the
effect of Thr-210 phosphorylation. Ser-137 phosphorylation
may then promote mitotic progression past a satisfied spindle
assembly checkpoint. Further investigation on other residues
2042 VAN DE WEERDT ET AL. MOL.CELL.BIOL.
Page 12
may shed more light on how other Plk1 functions are sepa-
rated.
ACKNOWLEDGMENTS
We thank members of the Medema lab and J. Pines for helpful
discussions, L. Oomen and L. Brocks for support with time-lapse
microscopy, M. Browning and K. Nixon for help in generating and
testing the phospho-Thr-210 antibody, M. Brandeis for the nondegrad-
able cyclin B1 plasmid, and H. and J. van de Weerdt for logistic
support.
This work was supported by grant 901-28-145 from The Netherlands
Organization for Scientific Research (NWO) and grant NKI 2000-2191
from the Dutch Cancer Society (KWF).
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2044 VAN DE WEERDT ET AL. MOL.CELL.BIOL.
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  • Source
    • "PLK2 can reduce ROS levels by increasing NRF2 antioxidant activity, thereby relieving oxidative stress and cell death. PLK2 can also phosphorylate Ser-137 of PLK1, permitting the bypass of the DNA damage checkpoint and promoting cell cycle progression (Fig. 5D) [4,9,10,33]. Our observations on PLK2 regulation complement previous reports showing that another PLK family member, PLK3, can respond to oxidative stress and interact with p53 for tumor suppression [34,35]. "
    [Show abstract] [Hide abstract] ABSTRACT: We previously reported that Polo-like kinase 2 (PLK2) is highly expressed in cells with defective mitochondrial respiration and is essential for their survival. Although PLK2 has been widely studied as a cell cycle regulator, we have uncovered an antioxidant function for this kinase that activates the GSK3-NRF2 signaling pathway. Here, we report that the expression of PLK2 is responsive to oxidative stress and that PLK2 mediates antioxidant signaling by phosphorylating GSK3, thereby promoting the nuclear translocation of NRF2. We further show that the antioxidant activity of PLK2 is essential for preventing p53-dependent necrotic cell death. Thus, the regulation of redox homeostasis by PLK2 promotes the survival of cells with dysfunctional mitochondria, which may have therapeutic implications for cancer and neurodegenerative diseases.
    Full-text · Article · May 2014 · Free Radical Biology and Medicine
  • Source
    • "Microtubule density was scored as the fraction of pixels with intensity >2× above background . Error bars indicate SEM cell cycle profile of Plk1 RNAi-depleted cells with similar efficiency to wild-type Plk1 (van de Weerdt et al. 2005). This discrepancy is likely explained by technical differences in the rate and extent of Plk1 inactivation. "
    [Show abstract] [Hide abstract] ABSTRACT: Polo-like kinase 1 (Plk1) is an essential mitotic regulator and undergoes periodic phosphorylation on threonine 210, a conserved residue in the kinase's activation loop. While phosphate-mimicking alterations of T210 stimulate Plk1's kinase activity in vitro, their effects on cell cycle regulation in vivo remain controversial. Using gene targeting, we replaced the native PLK1 locus in human cells with either PLK1 (T210A) or PLK1 (T210D) in both dominant and recessive settings. In contrast to previous reports, PLK1 (T210D) did not accelerate cells prematurely into mitosis, nor could it fulfill the kinase's essential role in chromosome congression. The latter was traced to an unexpected defect in Plk1-dependent phosphorylation of BubR1, a key mediator of stable kinetochore-microtubule attachment. Using chemical genetics to bypass this defect, we found that Plk1(T210D) is nonetheless able to induce equatorial RhoA zones and cleavage furrows during mitotic exit. Collectively, our data indicate that K-fibers are sensitive to even subtle perturbations in T210 phosphorylation and caution against relying on Plk1(T210D) as an in vivo surrogate for the natively activated kinase.
    Full-text · Article · May 2012 · Chromosoma
  • Source
    • "The siRNA is directed against the PBD region of Plk1, where all three swap mutants have between six and seven mismatches; thus all three domain swap mutants are not targeted by the siRNA vector used to target Plk1. The full length Plk1(Plk1-fl) and the Kin1–PBD1 vectors were constructed with two silent point mutations in the target sequence recognized by the RNAi-vector, rendering them resistant to knock-down [30,31]. Human osteosarcoma U2OS cells depleted of endogenous Plk1 were co-transfected with expression vectors for the various PBDs or the PBD-swap mutants in combination with a plasmid carrying a selectable marker gene to obtain a pure population of transfected cells. "
    [Show abstract] [Hide abstract] ABSTRACT: Polo-like kinases (Plks) contain a conserved Polo-box domain, shown to bind to phosphorylated Ser-pSer/pThr-Pro motifs. The Polo-box domain of Plk-1 mediates substrate interaction and plays an important role in subcellular localization. Intriguingly, the major interactions between the PBD and the optimal recognition peptide are mediated by highly conserved residues in the PBD, suggesting there is little target specificity conveyed by the various PBDs. However, here we show that the affinity of the purified Plk1-3 PBDs to both a physiological Cdc25C derived phospho-peptide and an optimal recognition phospho-peptide differs significantly among family members. To decipher the role of the PBDs and kinase domains in inferring Plk specificity, we exchanged the PBD of Plk1 (PBD1) with the PBD of Plk2, 3, or 4 (PBD2-4). The resulting hybrid proteins can restore bipolar spindle formation and centrosome maturation in Plk1-depleted U2OS cells to various degrees. In these experiments PBD2 was most efficient in complementing PBD-function. Using the MPM2 antibody that recognizes a large set of mitotic phospho-proteins, we could show that PBD1 and PBD2 display some limited overlap in target recognition. Thus, PBDs convey a significant deal of target specificity, indicating that there is only a limited amount of functional redundancy possible within the Plk family.
    Full-text · Article · Jul 2008 · Biochimica et Biophysica Acta
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