Department of Genetics and Molecular Biology, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan
In Xenopus laevis egg cell cycle extracts that mimic early embryonic cell cycles, activation of MAP kinase and MAP kinase kinase occurs in M phase, slightly behind that of maturation promoting factor. To examine the possible role of MAP kinase in the in vitro cell cycle, we depleted the extracts of MAP kinase by using anti-Xenopus MAP kinase antibody. Like in the mock-treated extracts, the periodic activation and deactivation of MPF occurred normally in the MAP kinase-depleted extracts, suggesting that MAP kinase is dispensable for the normal M phase entry and exit in vitro. It has recently been reported that microtubule depolymerization by nocodazole treatment can block exit from mitosis in the extracts if enough sperm nuclei are present, and that the addition of MAP kinase- specific phosphatase MKP-1 overcomes this spindle assembly checkpoint, suggesting the involvement of MAP kinase in the checkpoint signal transduction. We show here that the spindle assembly checkpoint mechanism cannot operate in the MAP kinase-depleted extracts. But, adding recombinant Xenopus MAP kinase to the MAP kinase-depleted extracts restored the spindle assembly checkpoint. These results indicate unambiguously that classical MAP kinase is required for the spindle assembly checkpoint in the cell cycle extracts. In addition, we show that strong activation of MAP kinase by the addition of a constitutively active MAP kinase kinase kinase in the absence of sperm nuclei and nocodazole, induced mitotic arrest in the extracts. Therefore, activation of MAP kinase alone is sufficient for inducing the mitotic arrest in vitro.
The mitogen-activated protein kinase kinase (MAPKK)1/
MAP kinase cascade is thought to be important for
a wide variety of signal transduction pathways (2, 4,
24, 25, 29, 30). In Xenopus laevis, for example, this classical MAP kinase cascade has been shown to play a crucial
role in meiotic cell cycles during oocyte maturation,
metaphase arrest of unfertilized eggs, and early embryonic
development (5, 7, 9, 15, 26, 32). However, the role
of the MAP kinase cascade in mitotic cell cycles has been
poorly understood. In Xenopus unfertilized eggs which are
arrested at the second meiotic metaphase, MAP kinase is
fully activated, whereas in somatic or early embryonic cell
cycles marked activation of MAP kinase at M phase has
not been detected, although several reports observed the
existence of an activated MAP kinase or an activated
MAP kinase-like molecule in M phase (14, 31).
Recent work of Minshull et al. (20) has identified MAP
kinase or a related molecule(s) as a component of the embryonic spindle assembly checkpoint in Xenopus egg cell
cycle extracts. The Xenopus egg cell cycle extracts are cellfree extracts made from parthenogenetically activated Xenopus eggs (21, 23). They undergo multiple cell cycles,
which are thought to mimic mitosis rather than meiosis, since Mos is degraded upon activation, and show many aspects of cell cycle in vitro. The cell cycle extracts have several advantages: well-synchronized cell cycles, availability
of large quantities of materials, and easy biochemical operation. Minshull et al. (20) showed that the cell cycle extracts are arrested by spindle depolymerization if filled
with high concentrations of sperm nuclei, and that addition of a MAP kinase phosphatase MKP-1 prevents these extracts from being arrested in M phase and drives the extracts previously arrested by the spindle assembly checkpoint into interphase. Cell cycle checkpoints are the systems that monitor the progress of the cell cycle and block
further transitions in the cell cycle until certain events
have been completed (22). The spindle assembly checkpoint is the mechanism that prevents metaphase cells from initiating anaphase until a bipolar spindle is formed and all chromosomes are attached to the spindle (33). From these
results, Minshull et al. (20) suggested the involvement of
MAP kinase in the spindle assembly checkpoint.
Although their elegant work clearly demonstrated the
requirement of a substrate(s) for MKP-1 in the spindle assembly checkpoint, whether or not classical MAP kinase is
required remains to be determined. This is because MKP-1
can dephosphorylate not only classical MAP kinase but
also other substrates, including MAP kinase superfamily
molecules such as SAPK/JNK and p38/HOG1 (19, 27, 28).
To examine directly the requirement of classical MAP
kinase in the system, we produced a highly specific antiMAP kinase antibody and depleted the extracts of MAP
kinase by using it. The results showed that the spindle assembly checkpoint did not work in the MAP kinase-depleted
extracts, and that adding recombinant MAP kinase restored the checkpoint mechanism, indicating unambiguously the requirement of classical MAP kinase in the spindle assembly checkpoint in the Xenopus cell cycle extracts.
Rather surprisingly, however, periodic activation and deactivation of maturation promoting factor (MPF) occurred
normally in the MAP kinase-depleted extracts, suggesting
that MAP kinase may not be required for normal cell cycle
progress.
Preparation of Xenopus Egg Extracts
Xenopus egg cell cycle extracts and oocyte extracts were prepared essentially according to the method as described previously (21, 23). All cell cycle extracts were prepared from parthenogenetically activated eggs. Xenopus sperm nuclei were prepared as described (21, 23). Preparation of the
extracts was done at 4°C. After adding sperm nuclei and/or nocodazole,
we initiated the in vitro cell cycle by warming the extracts to room temperature (~22°C). The time at which we shifted the temperature was t = 0.
Immunodepletion
The cell cycle extracts were centrifuged at 15,000 g for 15 min. As a result,
they were divided into two fractions: one fifth portion is the cloudier
lower layer and the rest is the clearer upper layer. The beads of AF-protein A-Toyopearl (Tosoh, Tokyo, Japan) resin were mixed and incubated
with an equivalent volume of anti-MAP kinase antiserum which was
raised against recombinant Xenopus MAP kinase or preimmune serum at
4°C for 1 h. The beads were washed three times with XB (100 mM KCl,
0.1 mM CaCl2, 1 mM MgCl2, 10 mM K-Hepes, pH 7.7, 50 mM sucrose).
The upper layer was then incubated with the beads on ice for 10 min, occasionally gently shaken by hand, and then centrifuged. The supernatant
was gently mixed with the lower layer, which had been laid on ice. The
mixture was centrifuged at 15,000 g for 10 min, the resulting lower layer
was saved, and the resulting upper layer was again incubated with the
beads for immunodepletion. Then, the beads-treated upper layer and the
saved lower layer were mixed and used as MAP kinase-depleted extracts.
We adopted this two-step immunodepletion procedure to avoid shaking
of the lower layer with beads, because the lower layer is especially fragile
to mechanical shock. All the steps should be done very gently not to perturb the cytoplasm; otherwise the immunodepletion procedure would
have inhibited the ability of the extracts to produce cell cycles.
Preparation of Recombinant Proteins
Glutathione-S-transferase (GST)-conjugated kinase negative Xenopus
MAP kinase (GST-KNMAPK) was expressed in Escherichia coli strain
NM522 by incubating with 0.1 mM IPTG for 12 h at 37°C. Histidinetagged Xenopus MAPKK (His-MAPKK) was expressed in E. coli strain
BL21 (DE3) pLysS by incubating with 0.4 mM IPTG for 12 h at 25°C. The
region containing the kinase domain of Saccharomyces cerevisiae STE11
(residues 341-717) fused to GST (GST-Ste11 Kinase Assays
Histone H1 kinase activity was measured essentially according to the
method described (21), except that the reaction mixture contained 5 µg
histone H1 and 0.5 µCi [ Immunoblotting
Samples were run on a 12% SDS-PAGE gel. The electrophoresed proteins were transferred to a polyvinylidene difluoride membrane (Immobilon; Millipore Corp., Milford, MA). After blocking with 5% skim milk
in TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl), the membrane was incubated with primary antibody followed by horseradish peroxidase-conjugated secondary antibody. Reacted proteins were detected by enhanced
chemiluminescence (Amersham Corp., Arlington Heights, IL).
Activation of MAP Kinase and MAP Kinase Kinase at
M Phase in Cell Cycle Extracts
To see whether the activity of MAP kinase varies during
cell cycle, we made several assays using Xenopus egg cell
cycle extracts, taking advantage of their well-synchronized
cell cycle. MPF activity, which is high at M phase, was assayed as histone H1 kinase activity (Fig. 1 A). It peaked at
40 min (M phase) and then suddenly decreased and returned to the basal level (interphase, I). MBP kinase activity was high in 40-50 min (data not shown; see Fig. 7 B,
left). In-gel kinase assay for MAP kinase (data not shown
and Fig. 7 C, left) and immunoblotting with anti-phosphotyrosine antibody (Fig. 1 C) revealed that 42-kD MAP kinase was tyrosine phosphorylated and activated during M
phase. The level of the MAP kinase activity in M phase of
this in vitro cell cycle was ~30% of that seen in mature oocytes. Activities to phosphorylate MAP kinase (Fig. 1 B,
closed circles) and to activate MAP kinase (Fig. 1 B, open circles) were also high at 40-50 min. These activities could
be attributed to 45-kD MAP kinase kinase (MAPKK), as
shown by the enhanced activity of immunoprecipitated
MAPKK from M phase (Fig. 1, D and E, right). The
amount of MAPKK did not vary during the cell cycle (Fig.
1 F). These data indicated that both MAPKK and MAP
kinase are activated around M phase in the cell cycle extracts. It should be noted that the activation and deactivation of MAPKK and MAP kinase lagged slightly behind
those of MPF (Fig. 1; see also Fig. 7, left). This is consistent
with the data of Minshull et al. (20) that the transient activation of MAP kinase occurred just after the activation
of MPF.
Effect of MAP Kinase Depletion on the Normal Cell
Cycle Progress In Vitro
To examine the role of MAP kinase in the in vitro cell cycle, we made MAP kinase-depleted extracts by immunodepletion. We first produced anti-Xenopus MAP kinase
antiserum by immunizing rabbits with recombinant Xenopus MAP kinase. The obtained antiserum was highly specific for 42-kD Xenopus MAP kinase (Fig. 2 A). Immunodepletion procedures with this antiserum were done very
gently and quickly, as described in Materials and Methods.
Otherwise, the extracts lost viscosity and freshness and
ceased to produce cell cycles.
MAP kinase-depleted extracts (anti-MAP kinase antiserum-treated extracts) and mock-treated extracts (preimmune serum-treated extracts) contained the majority of
the total protein of untreated extracts as demonstrated by
Coomassie brilliant blue staining (Fig. 2 A, lanes 1-3). In
mock-treated extracts, the amount of MAP kinase was equivalent to the amount present in untreated extracts as
judged by immunoblotting (Fig. 2 A, lanes 6 and 7). More
than 95% of MAP kinase was removed from the extracts
by the immunodepletion (Fig. 2 A, lane 8), with a recovery
of MAP kinase in the immunoprecipitated pellet (Fig. 2 A,
lane 10).
We monitored histone H1 kinase activity in these extracts. In both mock-treated and MAP kinase-depleted
extracts, periodic activation of histone H1 kinase was observed (Fig. 2 B). All the extracts were in the first M phase
at around 20-30 min in this series of experiments. They returned to interphase at 40 min followed by the second M
phase at around 90-110 min. The concentration of sperm
nuclei did not affect the kinetics of the cell cycle progress
markedly (Fig. 2 B). In more than five series of these experiments, MAP kinase-depleted extracts were indistinguishable from mock-treated extracts in the kinetics of histone H1 kinase activity. Therefore, we can suggest that
MAP kinase is not necessary for periodic activation of
MPF in the cell cycle extracts.
Requirement of MAP Kinase for the Spindle
Assembly Checkpoint
A previous report has shown that microtubule depolymerization by nocodazole treatment arrests cell cycle extracts
in M phase if containing very high densities of sperm nuclei. The use of MKP-1 further suggested that MAP kinase, the most likely target of MKP-1, may have a role in
the spindle assembly checkpoint (20). However, whether
classical MAP kinase is involved in the mitotic arrest remains to be determined. To address this question, we used
MAP kinase-depleted extracts. We monitored the level of
MPF as histone H1 kinase activity in the extracts supplemented with 9,000 sperm nuclei/µl. In the absence of nocodazole, the extracts returned to interphase after the first
M phase, irrespective of whether MAP kinase was depleted or not (Fig. 3 A). In the presence of nocodazole,
mock-treated extracts were arrested at the first M phase
and the high MPF activity was sustained (Fig. 3 B, mock), whereas MAP kinase-depleted extracts returned to interphase after the first M phase (Fig. 3 B,
When about the half of MAP kinase was left due to insufficient immunodepletion (Fig. 4 B, lane 2), mitotic arrest was achieved by adding a high density of sperm nuclei
and nocodazole to the extracts (Fig. 4 A). This supports
the importance of MAP kinase in the spindle assembly
checkpoint and may suggest the existence of the threshold
of MAP kinase required for the mitotic arrest.
To confirm further that the observed defect in the establishment of mitotic arrest is specifically due to the removal
of MAP kinase, we examined whether purified MAP kinase could rescue the defect in the MAP kinase-depleted
extracts. We expressed histidine-tagged Xenopus MAP kinase in E. coli and purified it. Adding the purified recombinant Xenopus MAP kinase to the MAP kinase-depleted
extracts restored the spindle assembly checkpoint; the extracts became arrested in M phase by the addition of a high
concentration of sperm nuclei and nocodazole (Fig. 5 B).
This result confirmed that MAP kinase is a component of
the spindle assembly checkpoint.
Mitotic Arrest by Ste11 To see whether MAP kinase activity is sufficient to arrest
cell cycle in M phase, we attempted to activate the MAP
kinase cascade in cell cycle extracts. S. cerevisiae STE11
gene codes for an MAPKK-K family molecule, and the
NH2-terminally truncated form is thought to be a constitutively active form (3). We expressed this form of recombinant Ste11 (Ste11
We monitored the MPF level in the cell cycle extracts
with or without Ste11 The mitotic arrest in cell cycle extracts by microtubule
depolymerization requires the presence of a high density of
sperm nuclei. To see whether the mitotic arrest by Ste11
To examine whether Ste11
In this study we used the technique of MAP kinase immunodepletion to examine the role of MAP kinase in the cell
cycle progress and the spindle assembly checkpoint in the
Xenopus cell cycle extracts. The result showed unambiguously that classical MAP kinase is required for the spindle
assembly checkpoint. This confirms and extends the previous study of Minshull et al. (20). Furthermore, rather surprisingly, our present result showed that MAP kinase is
unnecessary for periodic activation of MPF in the normal
cell cycle progress. In addition, we have shown that MAP
kinase activation alone is sufficient for inducing the mitotic arrest in vitro.
In the extracts, activation of MAPKK and MAP kinase
during the cell cycle occurred slightly behind that of MPF
(Figs. 1 and 7). In contrast, when the mitotic arrest was induced by microtubule depolymerization, activation of MAP
kinase occurred before MPF began to be inactivated (Fig. 3).
It can be speculated that activation of MAP kinase prior to
the metaphase/anaphase transition is crucial for inducing
the mitotic arrest. This is consistent with the recent report
of Abrieu et al. (1) suggesting that MAP kinase does not
inactivate but rather prevents the cyclin degradation pathway from being turned on in Xenopus egg extracts. In fact,
in the case of the Ste11 We depleted cell cycle extracts of MAP kinase by using
anti-MAP kinase antibody. The immunodepletion procedure sometimes deteriorated the extracts; in such extracts
periodic activation of MPF did not occur. Then, every experiment was done only when mock-treated extracts behaved as untreated extracts. Even when >95% of MAP kinase could be removed, periodic activation of MPF as
assayed by histone H1 kinase activity occurred normally,
and the kinetics of MPF activation in the MAP kinase-
depleted extracts could be indistinguishable from that in
the mock-treated extracts. Therefore, we may conclude
that MAP kinase is not required for the activation and inactivation of MPF during the normal cell cycle progress. We cannot, however, rule out the possibility that only 5%
of endogenous MAP kinase is sufficient to play a role in
the cell cycle progress.
We have shown here that classical MAP kinase is essential for the spindle assembly checkpoint in the cell cycle
extracts. This conclusion was derived from the following
results. First, when almost all classical MAP kinase was removed by immunodepletion, microtubule depolymerization did not arrest the extracts in M phase. But, when immunodepletion was insufficient and about half of the MAP
kinase was left, the mitotic arrest could be induced. Second, adding purified recombinant Xenopus MAP kinase to
the MAP kinase-depleted extracts rescued the defect in
mitotic arrest. As the previous pioneering work of Minshull et al. (20) demonstrated that a target of MKP-1 is required for the spindle assembly checkpoint, we can conclude that classical MAP kinase was the primary target of
MKP-1 in these experiments.
We have shown further that an active form of MAPKK-K,
Ste11
Materials and Methods
N) was expressed in E. coli
strain XLIBlue by incubating with 0.3 mM IPTG for 4 h at 25°C. To purify
GST fusion proteins, cells were pelleted and homogenized in STE (50 mM
Tris-HCl, pH 8.5, 200 mM NaCl, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 1% aprotinin). The homogenates
were frozen, thawed, and sonicated. After clarification by centrifugation, the lysates were applied to GSH-agarose beads column. The proteins were
eluted with 10 mM GSH in STE. His-MAPKK was purified as described (8). All the proteins were dialyzed against 20 mM Na-Hepes, pH 7.5.
-32P]ATP. To assay myelin basic protein (MBP)
kinase activity, extracts were diluted 40 times with HB (20 mM Na-Hepes,
pH 7.5, 2 mM MgCl2, and 2 mM EGTA). 5 µl of the diluted extract was incubated with 10 µg MBP in a final volume of 15 µl in the presence of 100 µM ATP, 0.5 µCi [
-32P]ATP, and 15 mM MgCl2 for 10 min at 30°C. Activity
to phosphorylate MAP kinase was measured by incubating samples with 5 µg
GST-KNMAPK as described for MBP kinase assay. To measure MAPKK-K
activity of GST-Ste11
N, immunoprecipitated samples were incubated with 2.5 µg His-MAPKK and 5 µg GST-KNMAPK in the presence of 100 µM ATP, 5 µCi [
-32P]ATP, and 15 mM MgCl2 for 20 min at 30°C. All the reactions were terminated by adding the SDS-PAGE sample buffer. After
boiling, samples were subjected to SDS-PAGE. Gels were analyzed by an
image analyzer (FUJIX BAS2000; Fuji Photo Film, Tokyo, Japan). The
kinase detection assay within MBP-containing gels was performed as described (6). To assay an activity to activate MAP kinase, samples were incubated with wild-type recombinant MAP kinase for 15 min at 25°C in the
presence of 200 µM ATP and 15 mM MgCl2. The mixtures were then subjected to the kinase detection assay within MBP-containing gels, and activities of activated MAP kinase were measured. In some cases, MAPKK
was immunoprecipitated with protein A-Sepharose (Pharmacia Fine
Chemicals, Piscataway, NJ) and anti-MAPKK antibody as described (17).
Results
Fig. 1.
Activation of MAP kinase and MAPKK in M phase of
cell cycle extracts. Xenopus egg cell cycle extracts were incubated
with 100 sperm nuclei/µl. Samples were taken and assayed for histone H1 kinase activity (A). MAPKK activities were assayed as
the activity to phosphorylate kinase-negative recombinant MAP
kinase (B, closed circle), and the activity to activate the ability of
wild-type recombinant MAP kinase to phosphorylate MBP (B, open
circle). Interphase (I) and mitotic (M) extracts were immunoblotted with anti-phosphotyrosine antibody PY20 (C). An arrow indicates MAP kinase. MAPKK activities of the same extracts were
assayed as the activity to phosphorylate kinase-negative MAP kinase (KNMAPK; D) and the activity to activate MAP kinase (E).
Assays were done in crude extracts (left) or with anti-MAPKK
antibody immunoprecipitates (right). The same extracts were immunoblotted with anti-MAPKK antibody (F).
[View Larger Versions of these Images (37 + 16K GIF file)]
Fig. 7.
Mitotic arrest by Ste11N in cell cycle extracts. Cell cycle
extracts were incubated with 5,000 sperm nuclei/µl with or without Ste11
N (100 µg/ml). Samples were withdrawn at 10 min intervals and assayed for histone H1 kinase activity (A), MBP kinase activity (B), and MAP kinase activity in MBP-containing
gels (C).
[View Larger Version of this Image (51K GIF file)]
Fig. 2.
Effect of MAP kinase depletion on the normal cell cycle
progress. Xenopus egg cell cycle extracts were subjected to immunodepletion with anti-MAP kinase antiserum or with preimmune
serum (mock treatment), as described in Materials and Methods.
(A) Total protein and MAP kinase were visualized by Coomassie
brilliant blue staining and immunoblotting with anti-MAP kinase
antibody, respectively. Extracts were untreated (lanes 1 and 6),
mock treated (lanes 2 and 7), or treated with anti-MAP kinase
antiserum (lanes 3 and 8), and the remaining extracts were analyzed. The precipitate with the protein A-Toyopearl beads in
mock treatment (lanes 4 and 9) or in anti-MAP kinase antiserum
treatment (lanes 5 and 10) was analyzed. An arrowhead indicates
MAP kinase. (B) MPF activities were monitored as histone H1
kinase activities. Mock-treated extracts (mock) or MAP kinasedepleted extracts (MAPK) were incubated with indicated concentrations of sperm nuclei (Sp). Samples were withdrawn at 10 min intervals. Assayed reactions were subjected to SDS-PAGE and autoradiographed.
[View Larger Versions of these Images (70 + 40K GIF file)]
MAPK). In the
mock-treated extracts with nocodazole, MBP kinase activity became activated at 30 min, and the high activity was
sustained (Fig. 3 C, mock). Consistent with this, immunoblotting with anti-MAP kinase antibody showed that
about half of MAP kinase was activated at 30 min, and almost full activation of MAP kinase was achieved at 40 min
and sustained thereafter (Fig. 3 D, mock). The MAP kinase-depleted extracts had markedly decreased levels of
MBP kinase activity throughout (Fig. 3 C), which was consistent with almost complete loss of MAP kinase in the extracts (Fig. 3 D,
MAPK). The low levels of MBP kinase
activity may be due to the trace amount of MAP kinase left and other kinases that can phosphorylate MBP. These
results clearly indicated the requirement of MAP kinase in
the spindle assembly checkpoint.
Fig. 3.
Effect of MAP kinase depletion on microtubule depolymerization-induced mitotic arrest. Mock-treated extracts (mock)
or MAP kinase-depleted extracts (MAPK) were incubated with
9,000 sperm nuclei/µl in the presence or absence of 10 µg/ml nocodazole (Noc). Samples were withdrawn at 10 min intervals, assayed for histone H1 kinase activity (A and B), MBP kinase activity (C), or immunoblotted with anti-MAP kinase antibody (D).
[View Larger Version of this Image (45K GIF file)]
Fig. 4.
Mitotic arrest by microtubule depolymerization in extracts insufficiently depleted of MAP kinase. (A) Cell cycle extracts which had been insufficiently immunodepleted of MAP kinase (B, lane 2) were incubated with 9,000 sperm nuclei/µl and
nocodazole. Samples were withdrawn at 10 min intervals and assayed for histone H1 kinase activity. (B) Extracts which had been
untreated (lane 1), insufficiently depleted of MAP kinase (lane 2),
and depleted sufficiently of MAP kinase (lane 3) were immunoblotted with anti-MAP kinase antibody. The samples for lanes 2 and 3 were from the extracts assayed in A and Fig. 3 (MAPK),
respectively.
[View Larger Version of this Image (19K GIF file)]
Fig. 5.
Rescue of mitotic arrest by microtubule depolymerization by addition of purified recombinant Xenopus MAP kinase
in the MAP kinase-depleted extracts. MAP kinase-depleted extracts were incubated with 9,000 sperm nuclei/µl and 10 µg/ml
nocodazole without (A) or with (B) histidine-tagged recombinant
wild-type Xenopus MAP kinase. Samples were withdrawn at 10 min intervals and assayed for histone H1 kinase activity.
[View Larger Version of this Image (31K GIF file)]
N
N) fused to GST in E. coli and purified
it. As expected, addition of purified Ste11
N to Xenopus
oocyte extracts resulted in activation of MAP kinase (Fig.
6 A) and MAPKK (Fig. 6 B). Immunoprecipitated Ste11
N showed high MAPKK-K activity (Fig. 6 C). This indicated
that Ste11
N can act as an activator for MAP kinase in
Xenopus egg cell-free systems.
Fig. 6.
Ste11N is a constitutively active MAPKK-K. (A) Xenopus oocyte extracts were incubated with or without 100 µg/ml recombinant Ste11
N. Samples were withdrawn at the indicated times and subjected to the kinase detection assay within MBP-containing
gels. An arrowhead indicates MAP kinase. (B) Xenopus oocyte extracts were incubated with various concentrations of Ste11
N for 2 h.
And then, MAPKK was immunoprecipitated and assayed for the activity to phosphorylate recombinant kinase negative MAP kinase.
(C) Xenopus oocyte extracts were incubated with 100 µg/ml Ste11
N. Ste11
N was then immunoprecipitated from samples withdrawn
at the indicated times and assayed for the activity to activate the ability of recombinant MAPKK to phosphorylate KNMAPK.
[View Larger Versions of these Images (76 + 42 + 14K GIF file)]
N. As already shown, in the control
extracts (without Ste11
N) periodic activation of MPF occurred; in this series of experiments the first peak was at 40 min and the second at 100 min (Fig. 7 A, no addition). In
contrast, in the presence of Ste11
N the high level of MPF
was sustained (Fig. 7 A, +Ste11
N). In this case, strong activation of MAP kinase occurred at 20-30 min, and this
high activity was maintained (Fig. 7, B and C, +Ste11
N). Thus, Ste11
N induced strong activation of MAP kinase
before the metaphase/anaphase transition of the first M
phase and arrested the cell cycle extracts in M phase.
N
requires a certain density of sperm nuclei, we incubated
extracts containing various concentrations of sperm nuclei
with Ste11
N and assayed for histone H1 kinase. The extracts without Ste11
N returned to interphase after the
first M phase and then entered the second M phase (Fig. 8,
top). However, all the extracts with Ste11
N maintained
high levels of histone H1 kinase activity and never returned to interphase from the first M phase, independent
of the concentration of sperm nuclei (Fig. 8). This result
indicated that the mitotic arrest induced by Ste11
N does
not require sperm nuclei.
Fig. 8.
Effect of sperm nuclei concentration on the Ste11Ninduced mitotic arrest. Cell cycle extracts were incubated without
or with various concentrations of sperm nuclei (Sp) in the presence or absence of Ste11
N (100 µg/ml). Samples were withdrawn
at 10 min intervals and assayed for histone H1 kinase activity.
[View Larger Version of this Image (64K GIF file)]
N induces the mitotic arrest
through MAP kinase activation, we incubated the MAP
kinase-depleted extracts and the mock-treated extracts
with or without Ste11
N. In the absence of Ste11
N, both
the mock-treated extracts and the MAP kinase-depleted
extracts returned to interphase after the first M phase and
entered the second M phase (Fig. 9 A, no addition). Almost complete absence of MBP kinase activity in the MAP
kinase-depleted extracts confirmed the removal of MAP
kinase (Fig. 9 B,
MAPK). These data not only represented another example of the data indicating that MAP
kinase is unnecessary for normal cell cycle progress, as
previously shown in Fig. 2 B, but also suggested that both
the extracts were functionally intact. In the presence of
Ste11
N, the mock-treated extracts were arrested in M
phase (Fig. 9 A, +Ste11
N, mock), whereas the MAP kinase-depleted extracts were not arrested in M phase at all
(Fig. 9 A, +Ste11
N,
MAPK). In the MAP kinase-depleted
extracts, Ste11
N did not induce marked activation of
MBP kinase activity (Fig. 9 B, +Ste11
N,
MAPK), as
was consistent with the removal of MAP kinase, but did
activate endogenous MAPKK (Fig. 9 C). These data clearly
indicated that Ste11
N induces the mitotic arrest in the
extracts through activation of MAP kinase, and suggested
that activation of MAP kinase alone is sufficient for inducing the mitotic arrest in the cell cycle extracts.
Fig. 9.
Ste11N induces mitotic arrest through activation of MAP kinase.
Mock-treated extracts (mock) or
MAP kinase-depleted extracts (
MAPK) were incubated with 5,000 sperm nuclei/µl in the presence (+Ste11
N) or absence (no addition)
of Ste11
N (100 µg/ml). Samples were
withdrawn at 10 min intervals and assayed for histone H1 kinase activity (A) and MBP kinase activity
(B). MAPKK was immunoprecipitated from the extracts in the
presence of Ste11
N at 0 and 60 min, and the activity to phosphorylate KNMAPK was measured (C).
[View Larger Versions of these Images (38 + 31K GIF file)]
Discussion
N-induced mitotic arrest, activation of MAP kinase occurred earlier than the peak of MPF
activity (Fig. 7).
N, can induce mitotic arrest in the extracts through
the activation of endogenous MAP kinase. The Ste11
Ninduced mitotic arrest did not require the presence of exogenously added sperm nuclei, unlike microtubule depolymerization-induced mitotic arrest. Although there have
been reports demonstrating that active MAP kinase is sufficient to induce mitotic arrest in cleaving embryos (11), the present study is the first demonstration of the mitotic
arrest by activation of MAP kinase alone in vitro. Thus,
we can suggest that once MAP kinase is activated by the
spindle assembly defect, mitotic apparatus, chromosomes,
or other cellular organelles are not required for inducing
mitotic arrest, and the activated MAP kinase alone is sufficient for the mitotic arrest. We are now examining upstream
pathways and downstream effectors of the MAP kinase
cascade in the spindle assembly checkpoint mechanism.
Received for publication 7 November 1996 and in revised form 18 December 1996.
K. Takenaka is a Research Fellow of the Japan Society for the Promotion of Science. This work was supported by grants in aid from the Ministry of Education, Science, and Culture of Japan to E. Nishida.We thank Dr. Andrew Murray for discussion, many helpful comments, and critical reading of the manuscript. We also thank F. Itoh for the production of anti-MAP kinase antiserum.
GST, gluthathione-S-transferase; MAP, mitogen activated protein; MAPKK, MAP kinase kinase; MAPKK-K, MAP kinase kinase kinase; MBP, myelin basic protein; MKP, Map kinase phosphatase; MPF, maturation promoting factor.