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INTRODUCTION |
The regulation of cell division is complex and tightly controlled.
A number of protein kinases and phosphatases play important roles in
the coordination of this process. Maturation promoting factor, a
complex of the serine/threonine kinase Cdc2 and its partner cyclin B,
has been shown to be essential for successful progression from
G2 to M phase (reviewed in Ref. 1). In turn, a number of
other proteins have important functions in helping regulate the
activity of Cdc2/cyclin B. In Xenopus laevis, the polo-like kinase Plx1 has emerged as potentially impacting this complex
by phosphorylating and thus increasing the activity of Cdc25C, the
phosphatase responsible for activating the Cdc2/B kinase (2). There is
currently debate in the field whether this is an organism-specific
function; experiments with small interfering RNA in mammalian
cells do not point to polo-like kinase (Plk1) having a similar role
(3). However, experiments with Plk1 immunoprecipitated from Jurkat T
leukemia cells suggest that human Plk1 can indeed perform this function
in vitro (4). Furthermore, recent evidence suggests that
Plk1 phosphorylation of Cdc25C leads to nuclear localization of the
mitotic phosphatase (5), thus contributing to its regulation in a much
different manner. Despite this controversy over Cdc25C activation, it
has been well established that Plk1 activity is critical for many other
mitotic processes (for review, see Ref. 6).
Through a number of genetic studies in yeast and Drosophila,
the role that Plk1 plays in regulating numerous aspects of mitosis has
become more clear (7). Monopolar spindles, misaligned chromosomes, and
a failure to undergo cytokinesis are all hallmarks of Plk1 mutants
(cdc5 in budding yeast and polo in Drosophila)
(8, 9). How these steppingstones to successful cytokinesis are dependent upon Plk1 function is more ambiguous and only partially understood. Plk1 has been shown to phosphorylate components of the
anaphase promoting complex
(APC),1 thus activating the
complex and allowing ubiquitin-mediated proteolysis to occur at the end
of anaphase (10-14). In addition, a direct link has also been
demonstrated to exist between Plk1 and Cdc2/cyclin B independent of
Cdc25 activation. Several groups have shown that Plk1 is capable of
phosphorylating key residues on cyclin B that are responsible for its
nuclear relocation during prophase (15, 16). Thus, what is emerging is
that this kinase may be a component of multiple key checkpoints that
must be passed for normal cell division. Just as intriguing are the
number of studies that have recently pointed to the large
overexpression of Plk1 in tumors; correlations have been made between
the relative level of Plk1 expression and long term prognosis/survival
(17-22).
Regulation of Plk1 activity appears to occur through two separate
mechanisms. The cell controls the activity of Plk1 dramatically through
transcriptional repression (23). At G1 phase and in quiescent cells, the level and activity of Plk1 is barely detectable (24, 25). As the cell progresses through the cell cycle and undergoes
replication, the level of Plk1 rises markedly and reaches a peak
between G2 and M phases. Following successful cytokinesis, Plk1 is ubiquitinated and destroyed by the same complex it helped activate, the APC, thereby returning to the low basal level seen in
G1 phase (26). In addition to this tight transcriptional control, the kinase is also up-regulated by phosphorylation (24, 27).
Recent work in X. laevis has provided us with the most information in this regard. Maller and co-workers (28) purified an
activity from Xenopus eggs they termed xPlkk1, for
Xenopus Plk kinase, which they suggest is responsible for
activating Plx during oocyte maturation. Following this discovery, a
mammalian protein, Slk, which shares significant homology to xPlkk1,
was shown to phosphorylate and activate Plk1 in vitro
(29).
Here we show that another protein kinase may contribute to the
regulation of Plk1. The murine gene Lok shows a great deal of sequence
similarity to both xPlkk1 and Slk. We, along with another group,
recently cloned the human homolog of Lok called Stk10 (30). In addition
to sharing significant sequence similarity with both of these polo-like
kinase kinases, we show here that Stk10 is also able to phosphorylate
Plk1. Unlike Lok, Stk10 has a widespread tissue distribution, showing
expression in a number of proliferative tissues as well as in multiple
tumor lines. These data raise the intriguing possibility that Stk10 may
play a role in regulating Plk1 in these specific biological settings.
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EXPERIMENTAL PROCEDURES |
Cloning/Subcloning--
A human Stk10 clone,
AA459448, was first identified using a Smith-Waterman search of the EST
data base with the human Slk gene (GenBankTM
accession number AB002804) as a query. Sequence analysis of the
1286-bp insert identified a 1227-bp open reading frame (409 amino
acids) with the potential to encode the N terminus of a novel human STK
related to the human Slk gene product. An additional Smith-Waterman
search using the C terminus of the Slk gene as a query yielded three
additional EST numbers, AA323687, AA380492, and AA168869, that encode
the C-terminal region of human Stk10. PCR using single-stranded
cDNA from human testis and the H23 tumor cell line enabled a
complete sequence of Stk10 to be assembled. PCR primers were designed
based upon the ESTs recovered as well as murine Lok (m_Lok).
To generate a kinase-dead mutant of Stk10, primers were designed to
convert the conserved lysine (amino acid 66) in the kinase domain to an
isoleucine using the Stratagene QuikChange kit (primers used were
5'-GCTTTGGCTGCGGCCATAGTCATTGAAACCAAG-3' and
5'-CTTGGTTTCAATGACTATGGCCGCAGCCAAAGC-3'). Clones were sequenced to
ensure that only the correct mutation was present. Wild type and
the kinase-dead mutant Stk10, tagged on the C terminus with the HA
epitope, were also subcloned into pBabepuro for stable cell line
generation in NIH-3T3 cells.
Northern Blots--
A Clontech Northern blot
(#7780-1, 12-lane multiple tissue Northern blot) was incubated
with a 659-bp fragment of Stk10 (1263-1922, EcoRI/BamHI) according to the manufacturer's
protocol with slight modifications (200 µCi of
[
-32P]ATP was used to label the probe, which
was incubated with the blot overnight at 65 °C). After multiple
washes, the blot was exposed to a PhosphorImager screen and later read
on a Amersham Biosciences PhosphorImager.
Immunoprecipitations, Kinase Assays, and Western
Analysis--
Cells were lysed in Nonidet P-40 lysis buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol,
1% Nonidet P-40, 2 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, aprotinin (0.15 unit/ml), 20 µM leupeptin, 5 mM sodium vanadate) for 10 min prior to a clarifying centrifugation at 14,000 rpm. Protein
concentrations were determined using a Pierce BCA kit (Rockford, IL).
Equivalent amounts of protein lysate were pre-cleared by incubation
with protein A-agarose for 1 h. The cleared lysates were then
transferred to a fresh tube containing protein A-agarose and 2 µg of
affinity-purified Stk10 antibody and rocked for 5-16 h at 4 °C.
Immune complexes were washed three times in lysis buffer and three
times in cold kinase buffer (3 mm MnCl2, 10 mM
MgCl2, 20 mM Hepes, pH 7.6). 10 µl of kinase
buffer containing 10 µCi of [
-32P]ATP, 200 µM ATP, and 5 µg of histone H2A were then added to each
tube and incubated for 20 min at 30 °C. Kinase assays with Stk10 and
Plk1 were washed an additional three times in a high salt buffer (500 mM LiCl, 100 mM Tris, pH 7.4) before the final washes with cold kinase buffer.
The samples were resolved by SDS-PAGE and transferred to
nitrocellulose; the membranes were blocked with 3% nonfat dry milk (in
TBS-Tween, pH 8.0) and then incubated at 4 °C overnight with appropriate antibody. Anti-Stk10 was used at 0.5 µg/µl, anti-Plk1 (Zymed Laboratories Inc. or Santa Cruz Biotechnology)
was used at 1:1000, anti-phospho-ERK2 (Santa Cruz Biotechnology) was
used at 1:5000, anti-FLAG (Sigma, St. Louis, MO) was used at 1:4000, and anti-HA (12-CA5, BAbCO, Berkeley, CA) was used at 1:1000.
Stk10 Antibody Generation/Affinity
Purification--
A glutathione S-transferase (GST) fusion
protein was constructed by subcloning an internal, poorly conserved
region of Stk10 (amino acids 352-477) into pGEX-4T utilizing
BamHI and SalI restriction sites. BL21 cells were
transformed with this construct, and protein production was induced
with 1 mM
isopropyl-1-thio-
-D-galactopyranoside. The protein was
then purified on glutathione-Sepharose 4B (Amersham Biosciences),
eluted with excess free glutathione and dialyzed overnight with
phosphate-buffered saline. Naïve rabbits were injected every 3 weeks with 0.5 mg of GST fusion protein. The first injection was
mixed 1:1 with Freund's complete adjuvant, and all others were mixed
1:1 with Freund's incomplete adjuvant. To affinity purify the crude
serum, it was incubated with CnBr-activated Sepharose 4B covalently
bound to the GST fusion protein according to manufacturer's
instructions (Amersham Biosciences). After numerous washes, the
antibody was eluted off of the resin with propionic acid, neutralized,
and dialyzed overnight in PBS.
Xenopus Assays and Manipulations--
Xenopus females
were purchased from Nasco (Fort Wilkinson, WI). Oocyte removal and
injection were done as previously described (31). Briefly, oocytes were
removed and defolliculated by incubation in MMR (100 mM
NaCl, 2 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM HEPES, pH 7.5)
containing collagenase A (Roche Applied Science, Indianapolis, IN) (1.5 mg/ml) for 1.5 h. Oocytes were then washed several times in MMR
and cultured overnight in 50% Leibovitz-15 medium (Invitrogen, San
Diego, CA). Eighteen hours after isolation, oocytes were injected with
~30 ng of in vitro transcribed RNA encoding the Stk10
proteins (wild type or kinase-dead). Capped RNA was transcribed using
the Ambion Message Machine kit (Austin, TX). Following injection of RNA, progesterone (Sigma) was added to the Leibovitz medium at 1 µg/ml. GVBD was scored by the appearance of a white dot in the animal
pole of the oocyte.
Oocytes were lysed in Nonidet P-40 buffer by gentle pipetting through a
micropipette tip and spinning briefly at 14,000 to remove insoluble
material. Lysates were then used for Western analysis.
Cell Culture, Transfection, and Synchronization--
HeLa, 293T,
and COS-7 cells were maintained in Dulbecco's minimal essential
medium (Invitrogen) supplemented with 10% fetal bovine serum and
penicillin/streptomycin. HEK-293T and COS-7 cells were transiently
transfected using SuperFect (Qiagen, Valencia, CA) according to the
manufacturer's specifications and incubated for 48 h before being
lysed and analyzed for protein expression. HeLa cells were synchronized
by a thymidine/aphidicolin block and then released into fresh media,
and samples were collected periodically for biochemical and flow
cytometry analysis. Cells were plated at a low density (5 × 105/10-cm plate), allowed to attach, and then incubated
with 2 mM thymidine (Sigma) for 12 h. 8 h after
being washed and released into fresh media, 1 µg/ml aphidicolin
(Sigma) was added to the media and the cells were incubated for another
12 h. The cells were then washed and released to progress through
the cell cycle by incubation with fresh media. NIH-3T3 cells were
synchronized by a sequential serum starvation (0.1% FBS) for 36 h, followed by incubation with fresh media (10% FBS) containing 1 µg/ml aphidicolin for 16 h. The cells were then washed twice
with PBS, and fresh media was added (10% FBS) to allow free
progression through the cell cycle. Cells were periodically collected
for flow cytometry analysis.
Cell Cycle Analysis by Flow Cytometry--
Cells were detached
with trypsin and washed once with PBS. The cell pellets were then
resuspended in 0.5 ml of PBS, fixed by the slow addition of ice-cold
70% ethanol, and stored at
20 °C for at least 2 h. The fixed
cells were centrifuged at 300 × g for 5 min and washed
once in PBS. The cell pellets were then stained in 0.1% Triton X-100,
20 µg/ml propidium iodide (Roche Applied Science), 200 µg/ml RNase
(Sigma) in PBS and incubated for 30 min in the dark.
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RESULTS |
Expression Pattern of Stk10--
The closest characterized
ortholog to Stk10 (sharing 88% identity) is murine lymphocyte-oriented
kinase (Lok). Western analysis has suggested that Lok expression is
relatively restricted to lymphoid tissue (32). To ascertain whether
Stk10 shares a common expression pattern with the mouse homolog we
performed Northern blot analysis with a Clontech 12 tissue array blot and an Stk10-specific probe. As shown in Fig.
1A, high expression was seen
in certain rapidly proliferating tissues (spleen, placenta, and
peripheral blood leukocytes), but transcripts were also detectable in
the other tissues profiled (brain, heart, skeletal muscle, colon, thymus, kidney, liver, small intestine, and lung) indicating that Stk10
has a more widespread pattern of expression than has been reported so
far for its mouse homolog, Lok (32). In all tissues examined, multiple
transcripts were observed, although we saw no evidence that different
splice variants are translated. In addition, TaqMan analysis was
performed on a similar set of tissues; results correlated well with
that seen by Northern analysis (data not shown).

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Fig. 1.
Stk10 expression. A, a
Clontech multiple tissue Northern blot was
incubated with an Stk10-specific probe. Highest expression is seen in
rapidly proliferating tissue (spleen, placenta, and peripheral blood
leukocytes), although multiple transcripts are also seen in the other
tissues profiled. B, expression in tumor lines was examined
by Western analysis. Proteins (50 µg of whole cell lysate) were
resolved by SDS-PAGE, transferred to nitrocellulose, and probed with an
Stk10-specific antibody.
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To detect Stk10 protein expression, a rabbit polyclonal antibody was
generated against the internal domain of Stk10 as described under
"Experimental Procedures." Given the elevated level of Stk10 mRNA in highly proliferative tissue, we examined a set of tumor cell lines for Stk10 protein expression. It is clear from Fig. 1B that Stk10 is expressed in many different tumor lines;
interestingly, the highest level of expression does not occur in the
leukemic lines as one might predict based upon the tissue distribution of the murine ortholog.
Kinase Activity of Stk10 in Vitro--
To verify the kinase
activity of recombinant Stk10, we transiently transfected HEK-293T
cells with DNA encoding either wild type or a kinase-dead mutant of
Stk10. The proteins were immunoprecipitated using an HA-epitope tag
engineered onto the C terminus, and a kinase assay was performed as
described under "Experimental Procedures." The results of this
assay are shown in Fig. 2A.
Clear autophosphorylation of wild type Stk10 as well as robust
phosphorylation of histone H2A was observed. No autophosphorylation
or phosphorylation of substrate by the kinase-dead mutant was detected.
The choice of substrate was based upon earlier published results with
Lok (32). Unlike Lok, however, Stk10 does not appear to phosphorylate
myelin basic protein (data not shown). Western analysis (Fig.
2B) shows the levels of wild type and kinase-dead Stk10
assayed for activity. Slightly less kinase-dead than wild type protein
was assayed in this experiment; however, the difference in histone
phosphorylation between the two samples far outweighs the minor
difference in expression.

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Fig. 2.
Kinase activity of transfected Stk10.
HEK-293T cells were transiently transfected with DNA encoding wild type
or kinase-dead Stk10 or pcDNA3 vector. Proteins were
immunoprecipitated with an HA antibody, and activity was measured
against histone H2A. A, autoradiograph of kinase assay.
B, Western analysis of same samples, probed with an HA
antibody.
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Stk10 as a Potential Polo-like Kinase Kinase--
To understand
the potential role for Stk10 in human cells, we examined two similar
homologs with known functions found in humans and X. laevis
(hSlk and xPlkk1, respectively). As Fig. 3 illustrates, both genes have a domain
structure similar to Stk10, i.e. they contain an N-terminal
kinase domain as well as a coiled-coil domain at the C terminus. Most
importantly, hSlk and xPlkk1 have a common function in cells; they both
act as polo-like kinase kinases, phosphorylating and activating
polo-like kinase. These sequence similarities raised the possibility
that Stk10 might act in a like fashion to regulate the activity of
Plk1.

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Fig. 3.
Domain structure comparison of Stk10, Slk,
and xPlkk1. The three polo-like kinase kinases are shown aligned,
with the kinase domains and the coiled-coil domains shaded.
The identities that Slk and xPlkk1 each share with Stk10 are indicated
both by individual domain and overall.
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Co-association of Plk and Stk10 in Cells--
We next examined
whether Plk and Stk10 can associate in cells; such an interaction would
suggest that Plk is an in vivo substrate for Stk10. We
therefore performed immunoprecipitations from both exponentially
growing and nocodazole-treated HeLa cells using the Stk10-specific
antibody we had generated. The resulting immunoprecipitations were
analyzed by SDS-PAGE, transferred to nitrocellulose, and probed with
both Stk10 and Plk1 antibodies. The association between Plk1 and Stk10
was clearly seen in nocodazole-treated cells (Fig. 4A) but not visible in
exponentially growing cultures. A straightforward explanation for this
may be the difference in Plk1 protein levels between the two samples.
Plk1 expression is low in most phases of the cell cycle, and it reaches
maximal expression between G2 and M phases.

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Fig. 4.
Co-association of Plk1 and Stk10 in
cells. A, an association is seen in nocodazole-treated
HeLa cells. Lysates were incubated with pre-immune (PI) or
Stk10 immune (I) serum, resolved by SDS-PAGE, and
transferred to nitrocellulose; the top half of the membrane
was probed with anti-Stk10, the bottom with anti-Plk1. IgG
is denoted by the asterisk. B, the association is
also seen in synchronized cells during G2/M phases.
Synchronized cells were collected every 2 h and lysed, and Stk10
was precipitated. Stk10 levels are shown in the upper panel,
and co-precipitated Plk1 is shown in the middle panel. The
lower panel shows absolute levels of Plk1 present in the
lysate before immunoprecipitation. ~50 µg of whole cell lysate was
used, and the blot was probed with Plk1 antibody.
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To examine this interaction in non-arrested cells, we synchronized HeLa
cells at the G1/S interface and collected samples every
2 h after their release with fresh media. Stk10 was
immunoprecipitated from lysates containing equal amounts of protein,
and Western analysis was performed. Consistent with the data from
nocodazole-treated cells, Plk1 co-association with Stk10 was most
clearly visible between 10 and 12 h during G2 and M
phases, when Plk1 levels are highest (Fig. 4B, upper
panel). Interestingly, there is a detectable amount of Plk1
present in the lysates at 14 h when little association between
Stk10 and Plk1 is seen. We cannot rule out the possibility, therefore,
that phosphorylation of either protein during M phase may contribute to
their association at this time.
Cell Cycle Expression/Activity Profile of Stk10--
To
examine the cell cycle profile of Stk10, we again synchronized HeLa
cells at the G1/S interface using a thymidine/aphidicolin block. Upon release from this arrest, samples were collected every 2 h to assay for kinase activity and Western analysis. In contrast with the pattern seen with Plk1, the abundance of Stk10 did not vary
significantly during the time course (Figs. 4B and
5B) suggesting that the
expression profile of Stk10 is constant throughout the cell cycle.
Similarly, the ability of Stk10 to both autophosphorylate and
phosphorylate histone H2A was relatively stable (Fig. 5A). This pattern of activity and expression is very similar to that seen
with another human polo-like kinase kinase, hSlk.

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Fig. 5.
The activity and abundance of Stk10 remains
constant throughout the cell cycle. Synchronized HeLa cells were
collected every 2 h and lysed, and Stk10 was precipitated.
A, kinase assay showing both autophosphorylation
(upper panel) and incorporation into histone H2A
(lower panel). PI is a sample incubated with
pre-immune sera, and Ex. is a sample of exponentially
growing cells. Note that the lower amount of activity in this sample
(Ex.) correlates with the smaller amount of Stk10
precipitated. B, Western analysis of assayed samples. After
resolving the kinase assay samples by SDS-PAGE, the proteins were
transferred to nitrocellulose, exposed to film (A), and then
incubated with anti-Stk10 for Western analysis (B).
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Stk10 Can Phosphorylate Plk1 in Vitro--
To confirm the
potential role of Stk10 as a polo-like kinase kinase, we performed
in vitro kinase assays with Plk1 as the substrate. Briefly,
a kinase-dead mutant of Plk1, tagged on the C terminus with an HA
epitope, was transfected into COS-7 cells. Kinase-dead Plk1 was used to
avoid signal generated by autophosphorylation. Separate dishes of cells
were transfected with either a wild type or kinase-dead form of Stk10
tagged on the N terminus with a FLAG epitope. 48 h after
transfection the cells were lysed, clarified lysates were mixed, and
the proteins were immunoprecipitated with their respective tags. After
a stringent set of washes, an in vitro kinase assay was
performed. Fig. 6A
(upper panel) clearly shows the high
incorporation of 32P into the kinase-dead Plk1 only occurs
in the presence of wild type Stk10. A Plk immunoblot indicates that
equal amounts of kinase-dead Plk were present in each sample assayed
(Fig. 6A, lower panel). In addition, to
demonstrate the efficiency of the reaction, the amount of substrate
(kinase-dead Plk1) was titrated and found to directly modulate the
reaction rate (data not shown).

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Fig. 6.
Stk10 can phosphorylate Plk1 in
vitro. A, Stk10 and kinase-dead Plk1 were
expressed in COS-7 cells, lysates were mixed, and the proteins were
immunoprecipitated by their respective FLAG and HA tags. Upper
panel, autoradiograph showing 32P incorporation into
kinase-dead Plk1 and Stk10. Lower panel, Western analysis of
the same samples. The blot was probed with Plk1 antibody to show
equivalent Plk1 expression across the samples. The extra (non-Plk1)
band that is seen in the left two lanes is due to the HA
antibody. B, endogenous Stk10 was immunoprecipitated from
synchronized G1 phase HeLa cells and mixed with kinase-dead
Plk1 precipitated from COS-7 cells through an HA tag. Upper
panel, autoradiograph showing 32P incorporation into
kinase-dead Plk1 and Stk10. Lower panel, Western analysis of
the same samples. The blot was probed with Plk1 antibody.
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To extend these findings, we then assayed endogenous Stk10 precipitated
from aphidicolin/thymidine-arrested HeLa cells for the ability to
phosphorylate kinase-dead Plk1. Synchronized, G1 phase
cells were used to reduce the background from endogenous, active, wild
type Plk1, which could co-precipitate with Stk10 during other phases of
the cell cycle. As shown in Fig. 6B, endogenous Stk10 is
able to phosphorylate Plk1 in vitro, lending support to the
model that Stk10 is an upstream regulator of Plk1 in
vivo.
Overexpression of WT Stk10 in Xenopus Oocytes Accelerates
GVBD--
Microinjection of RNA encoding xPlkk1 into immature, Stage
VI Xenopus oocytes can impinge on the normal maturation
process that occurs after progesterone exposure. Overexpression of wild type xPlkk1 results in an acceleration of germinal vesicle breakdown (GVBD) compared with uninjected controls or expression of a kinase-dead mutant (28). Based upon the high degree of similarity between Stk10 and
xPlkk1, we investigated whether injection of Stk10 RNA could similarly
increase the rate of progesterone-induced maturation. RNA corresponding
to wild type and kinase-dead Stk10 was microinjected into Stage VI
immature oocytes, the oocytes recovered overnight and were then treated
with progesterone. As displayed in Fig. 7A, overexpression of wild
type Stk10 resulted in an acceleration of GVBD. Maturation was scored
by the appearance of a white dot at the animal pole, which is formed
when the nucleus breaks down and pigment granules are displaced.
Oocytes lysed for Western analysis are displayed in Fig. 7B.
Clear expression of both wild type and kinase-dead Stk10 is visible.
The correlation of a biological phenotype between xPlkk1 and Stk10
strongly suggests the two proteins share a common function in
cells.

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Fig. 7.
Increased rate of maturation in
Xenopus oocytes in the presence of wild type
Stk10. Immature Stage VI oocytes were microinjected with wild type
(wt) or kinase dead (kd) Stk10 RNA or left
uninjected as a negative control and then incubated with progesterone.
Groups of oocytes were frozen at the end of the time course (10 h) for
biochemical analysis. A, time course of maturation.
Closed square, wt; open square, kd; open
diamond, uninjected. B, Western analysis of treated
oocytes. Six oocytes each were immunoprecipitated with a FLAG antibody
to show the level of Stk10 expression.
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Overexpression of Kinase-dead Stk10 Results in Abnormal DNA
Content--
Misregulation of Plk expression in cells results in a
number of nuclear defects. We determined whether overexpressing wild type or kinase-dead Stk10 could interfere with normal Plk function and
thus lead to nuclear aberrations. NIH-3T3 cells were engineered to
stably overexpress either a wild type or a kinase-dead mutant of Stk10,
tagged on the C terminus with an HA epitope. We noted that it was much
more difficult to isolate single clones expressing the kinase-dead
mutant than to find wild type-expressing clones. Growth curves
confirmed that cells expressing kinase-dead Stk10 grew more slowly than
those expressing the wild type protein (data not shown).
Given our proliferation data and the potential role that Stk10 plays in
cell cycle regulation, we next examined the ability of these various
clones to cycle in a synchronous manner. The cells were blocked at the
G1/S interface with aphidicolin, then fixed, stained, and
analyzed by flow cytometry. The results from a typical experiment are
shown in Fig. 8. A dramatic effect was observed in the clones expressing kinase-dead Stk10. In a
dose-dependent fashion, expression of the mutant Stk10
resulted in an abnormal DNA content. At the time sampled, when the
majority of each population should be arrested in G1/S
phase with 2N DNA, it is clear that most of the cells expressing the
kinase-dead allele of Stk10 have 4N DNA content (Fig. 8B,
top panels). This effect continued as the cells progressed
through the cell cycle; when control cells had duplicated their DNA to
4N, the kinase-dead expressing cells also increased their DNA content
to ~8N (Fig. 8B, bottom panels). In addition,
these cells were not multinucleate; instead, a process related to
endoreduplication appeared to have occurred. In support of this theory
we found that the nuclear size of the kinase-dead expressing cells was,
on average, larger than those of the vector-transfected cells or wild
type-expressing cells (data not shown). Expression of the kinase-dead
Stk10 clearly interfered with normal cell cycle progression and
cytokinesis.

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Fig. 8.
Expression of a dominant negative Stk10 in
NIH-3T3 cells results in abnormal DNA content. Flow cytometry was
performed on cells treated with aphidicolin to block them before the
start of S phase and 6 h after release. The kinase-dead clones
show varying degrees of DNA content abnormality consistent with their
level of Stk10 expression. A, Western analysis demonstrating
relative levels of Stk10 expression in the clones profiled. Note that
the two blots are separate exposures; the relative amount of
kinase-dead clone 11 is roughly equivalent to that of wild type clone
1. B, cell cycle analysis (propidium iodide staining) of
individual clones. The top panels show the profiles of
aphidicolin-arrested cells; the bottom panels show those
cells 6 h after release from their block.
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DISCUSSION |
Over the past few years it has become clear that Plk1 plays a key
role in controlling the cell cycle, specifically in regulating many
aspects of mitosis. Entrance to mitosis may rely on this kinase;
polo-like kinase has been implicated in indirectly activating the
mitotic kinase complex Cdc2/cyclin B by phosphorylating the activating
phosphatase, Cdc25C (2, 4, 33). Exit from mitosis has also been
associated with Plk function; activation of the APC, the proteasome
responsible for destroying a number of mitotic proteins, has been shown
to occur through Plk1 (10-14). In this study we examined the function
of Stk10, a previously uncharacterized member of the Ste20 family of
serine/threonine kinases. Based upon sequence homology with Slk and
xPlkk1, known polo-like kinase kinases in humans and X. laevis, we posited that Stk10, too, might exert control over the
activity of Plk1. Toward that end, we show here that Stk10 is
expressed, like Plk1, in highly proliferative normal tissue as well as
in a number of tumor cell lines. In addition, Stk10 co-associates with
Plk1 in cells and is capable of phosphorylating a catalytically
inactive mutant version of Plk1 in vitro. More compelling is
the observed phenotype of NIH-3T3 cells engineered to overexpress a
dominant negative form of Stk10. These cells display an abnormal cell
cycle profile and increased DNA content in an Stk10-dose dependent
manner. Such a phenotype is consistent with a partial loss of Plk1
function in these cells. We presume that expression of kinase-dead
Stk10 does not completely inhibit Plk activity; we would expect that
full inhibition of the kinase would result in a cell cycle block.
Experiments in a number of genetic organisms such as
Drosophila and Saccharomyces
cerevisiae have shown convincingly that expression of mutant
forms of Plk1 can lead to monopolar spindles, abnormal chromosome
segregation, and other gross errors in cytokinesis. Furthermore, recent
studies utilizing small interfering RNA in HeLa and U2OS cells
suggest that depletion of Plk1 transcript can also interfere with
sister chromatid separation and normal cytokinesis in mammalian
systems. In fact, additional studies imply that the absolute amount of
Plk1 in the cell is of critical importance; fluctuations in either
direction can lead to abnormalities. Interestingly, Mundt et
al. (34) has shown that overexpression of wild type Plk1 in
HeLa cells can lead to multiple and fragmented nuclei. All of these
studies support the idea that interfering with the normal activity
level of Plk1, whether through the introduction of a dominant negative
mutant or the supplementation of normal activity with exogenous wild
type Plk1, can have deleterious effects on the cell. Perturbation of
this balance results in a host of cell duplication mistakes. Clearly,
the tight regulation of Plk1 that exists on multiple levels has evolved
for a distinct reason.
Our experiments in this current study further reinforce the idea that
the amount of Plk1 activity is of critical importance to successful
cell division. NIH-3T3 cells that express a kinase-dead mutant of Stk10
have a greatly altered cell cycle phenotype. At each phase, the cells
contain a larger amount of DNA than normal, suggesting that a process
similar to endoreduplication has occurred. Not surprisingly, the cells
with up to twice as much DNA as controls also have larger nuclei and
divide more slowly than vector-transduced cells (data not shown). There
are a number of possible mechanisms capable of producing this observed
phenotype. The most straightforward is that the expressed Stk10 is
exerting a dominant negative effect on the endogenous Plk1, preventing
its normal activation. This would result in a condition similar to that
of overexpressing a dominant negative form of Plk1. Alternatively, the
exogenous protein may be binding to Plk1, allowing its normal
activation to occur but slowing down or preventing the necessary
proteolytic destruction that must take place at the end of mitosis. In
this way, the result would mimic that seen when wild type Plk1 is
overexpressed in cells, i.e. fragmented or duplicated nuclei
(34). Additional experiments are needed to fully explain which
mechanism is behind our observed phenotype. It is interesting to note
that wild type Plk1 is overexpressed in a number of tumor cell lines
(35). It has been shown that constitutive expression of Plk1 can
transform cells (36); certainly it is true that the type of genetic
instability described above is a clear hallmark of the cancerous cell.
Given the stringent control that the cell must place on the activity of
Plk1, it is not surprising that there now appear to be multiple kinases
capable of phosphorylating the cell cycle regulator. Although the
upstream activator of Plk1 has not been definitively identified, our
current study demonstrating the ability of Stk10 to phosphorylate Plk1
in vitro suggests it is a candidate, along with Slk (29),
for the physiological activator. Northern analyses of Stk10 and Slk
imply that the expression pattern of the two genes is somewhat
complementary. Highest levels of Slk transcript are observed in heart
and skeletal muscle, whereas Stk10 RNA is less common in these tissues,
showing higher levels instead in spleen and peripheral blood
leukocytes. The two protein kinases may therefore provide the same
function to Plk1 but in different cell types. Further evidence that
Stk10 plays a role in activating Plk1 in vivo is provided by
Erikson and co-workers. In a recent publication they state that
unpublished experiments have demonstrated the ability of Lok, the mouse
ortholog of Stk10, to activate Plk1 in vitro (37).
The expression pattern we observe for Stk10 is somewhat different than
that reported for Lok. Kuramochi et al. (32) showed that the
expression of Lok is quite restricted, detecting transcript and protein
only in hematopoietic tissue. Although we do observe high levels of
Stk10 transcript in lymphocytic organs, a number of other tissues
showed measurable levels of Stk10 RNA as well. It is possible that the
function of Stk10 is more complex in humans than in mice and so
requires expression in additional tissues. A potential area for
divergent complexity between the species lies within the Plk family
itself. Although quite a bit of work has been done to elucidate the
function of Plk1, less is known about Plk2 and Plk3 (also known as Snk
and Fnk/Prk). Both of these genes are immediate-early gene products and
so appear to play a role in regulating earlier phases of the cell
cycle, G1 and S phases, although recent work suggests that
Fnk may also impact on M phase (38-40). Given that the Plks share a
relatively conserved kinase domain, it is possible that Stk10 and/or
Slk may also act as regulators of these kinases in human cells, perhaps
thus requiring the broader expression pattern observed.
We have also shown through Western analysis that there is a detectable
amount of Stk10 present in a panel of tumor cell lines. A number of
investigators have reported elevated levels of Plk1 protein and
transcript in tumors and tumor cell lines. Because there have thus far
been no reports of activating mutations in Plk1 in cancerous cells,
there is clearly a need in these cells for an upstream activator.
Experiments with Plx in X. laevis have demonstrated that
phosphorylation plays an obligate role in the activation of polo-like
kinase. Stk10 may provide this function in human tumors; it is possible
that Slk also contributes to this activity, at this point expression
profiles in tumors for Slk have not been reported. Interestingly, we
found no evidence that the activity of Stk10 is regulated in a cell
cycle-dependent fashion. Both protein level and activity
remained relatively constant in all phases of the cell cycle. It is
still possible that there are more subtle variations than we were able
to measure; we note that the differences in activity reported for Slk
were extremely modest, although investigators did present evidence that
the protein is phosphorylated during M phase.
Our study provides strong evidence that Stk10 is one of the
physiological activators of Plk1. It will be interesting in future studies to examine the specific sites in Plk1 phosphorylated by Stk10.
As noted earlier, Jang et al. (37) stated that Lok, the mouse ortholog of Stk10, phosphorylated Plk1 in vitro on
threonine 210, which they demonstrated was an in vivo site
of phosphorylation during mitosis. It seems logical to hypothesize that
Stk10 will also activate Plk1 in the same fashion; further experiments
will help resolve this question.