3091
Journal of Cell Science 111, 3091-3100 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
JCS3821
A cell cycle regulated MAP kinase with a possible role in cytokinesis in
tobacco cells
Ornella Calderini1, László Bögre1,*, Oscar Vicente1,‡, Pavla Binarova2, Erwin Heberle-Bors1
and Cathal Wilson1
1Institute
2Institute
of Microbiology and Genetics, University of Vienna, Dr Bohrgasse 9, A-1030 Vienna, Austria
of Microbiology, Czech Acadamy of Sciences, Norman Borlaug Center for Plant Science, Videnska 1083, Prague 4,
14200, Czech Republic
*Author for correspondence (e-mail: [email protected])
‡Present address: Instituto de Biologia Molecular y Celular de Plantas, Universidad Politécnica de Valencia, Camino de Vera s/n, E-46022 Valencia, Spain
Accepted 6 August; published on WWW 23 September 1998
SUMMARY
Mitogen-activated protein (MAP) kinases have been
demonstrated to have a role in meiosis but their
involvement in mitotic events is less clear. Using a
peptide antibody raised against the tobacco MAP kinase
p43Ntf6 and extracts from synchronized tobacco cell
suspension cultures, we show that this kinase is activated
specifically during mitosis. Entry into mitosis appears to
be necessary for the activation of the kinase, which
occurs as a post-translational event. The activation of the
kinase occurs in late anaphase/early telophase. The p43Ntf6
protein shows a transient localization to the cell plate in
anaphase cells, in the middle of the two microtubule arrays
characteristic of the phragmoplast, a plant-specific
structure involved in laying down the new cell wall. The
combined data support a role for the MAP kinase p43Ntf6
in cytokinesis in tobacco cells.
INTRODUCTION
1997; Bowser and Reddy, 1997). Microtubule-associated
proteins (MAPs) are also found associated with the
phragmoplast microtubules during cell division (Chan et al.,
1996). Phragmoplastin, a dynamin-like protein, associates with
the phragmoplast in late anaphase (Gu and Verma, 1996). The
properties and dynamic behaviour of phragmoplastin during
cytokinesis, as measured by a phragmoplastin-GFP fusion
protein in living cells, clearly indicate a role in cell plate
formation, possibly in membrane fusion events (Gu and Verma,
1997). The KNOLLE gene product is also involved in
cytokinesis, and shows similarities to syntaxins, a protein
family involved in vesicular trafficking (Lukowitz et al., 1996).
The KNOLLE protein may be involved in vesicle fusion
(Lauber et al., 1997). Both of these proteins localize to the cell
plate in dividing plant cells (Gu and Verma, 1997; Lauber et
al., 1997).
Mitogen-activated protein (MAP) kinases are involved in
diverse eukaryotic signal transduction pathways responding to
mitogens, differentiation factors and stress (Levin and Errede,
1995; Robinson and Cobb, 1997). One of the many functions
of MAP kinases appears to be in the reorganization of the
cytoskeleton. A significant fraction of the MAP kinase pool is
found in the cytoplasm associated with the microtubule
cytoskeleton (Reszka et al., 1995; Morishima-Kawashima and
Kosik, 1996) and has been shown to regulate cytoskeletal
organization (Reszka et al., 1997). Microtubule-associated
proteins such as MAP2 and MAP4 (Ray and Sturgill, 1987;
A unique feature of cytokinesis in plant cells is the formation
of a phragmoplast, which is involved in the deposition of
material required to form the cell plate. The phragmoplast
forms from the late anaphase to the late telophase stages of the
cell cycle. Many of the structural and dynamic features of
phragmoplast formation have been elucidated using electron
microscopy and immunofluorescence methods (Schopfer and
Hepler, 1991; Samuels et al., 1995). Cell plate formation starts
in the cell centre and then progresses towards the cell
periphery. The early stages are marked by the presence of two
microtubular arrays with opposite polarity. These microtubules
transport Golgi-derived vesicles to the newly forming cell
plate, where they fuse to form a tubulo-vesicular network that
later consolidates into a smooth tubular sheet in which callose
is deposited. As the forming cell plate progresses towards the
periphery of the cell, microtubules are displaced from the
centre and follow the leading edges of the expanding
phragmoplast.
Microtubule organization and vesicle transport/fusion events
are central to the formation of the the new cell wall. A number
of proteins which appear to be involved in these events have
been identified, such as kinesin-like microtubule motor
proteins (Mitsui et al., 1996; Asada and Collings, 1997), some
of which have been shown to localize to the phragmoplast
microtubules during cytokinesis (Liu et al., 1996; Asada et al.,
Key words: Cytokinesis, MAP kinase, Mitosis, Phragmoplast,
Tobacco
3092 O. Calderini and others
Hoshi et al., 1992), p220 (Shiina et al., 1992), and tau (Drewes
et al., 1992), are substrates for MAP kinases. Vesicle
trafficking/fusion events may also be regulated by MAP
kinases, as they can phosphorylate the GTPase dynamin I
(Earnest et al., 1996), and Rab4 (Cormont et al., 1994), which
are involved in membrane traffic in eukaryotic cells. Activated
MAP kinases are epitopes for the MPM-2 antibody (Kuang and
Ashorn, 1993; Taagepera et al., 1994), which recognises
phosphoepitopes predominantly in mitotically dividing cells,
and labels mitotic microtubules in plant cells (Traas et al.,
1992; Binarova et al., 1994; Smirnova et al., 1995).
Many MAP kinase cDNA clones have been isolated from
plants (for a recent review, see Machida et al., 1997), and
numerous signals leading to their activation have been
described. Wounding (Seo et al., 1995; Usami et al., 1995;
Bögre et al., 1997a), elicitors (Suzuki and Shinshi, 1995;
Stratmann and Ryan, 1997; Zhang et al., 1998), drought
(Jonak et al., 1996), pollen hydration (Wilson et al., 1997),
and hormones (Zhang and Klessig, 1997), all result in the
activation of MAP kinases or MAP kinase-like activities.
However, little is known about the possible functions and the
downstream targets of these kinases following stimulation.
Treatment of parsley cells with a pathogen-derived
oligopeptide elicitor leads to MAP kinase activation and
translocation of the kinase to the nucleus (Ligterink et al.,
1997), where it might function in the stimulation of expression
of plant defense genes. A recombinant MAP kinase from
alfalfa, MMK2, could phosphorylate a 39 kDa protein in
detergent-resistent cytoskeletal preparations from carrot cells
(Jonak et al., 1995).
We previously reported on the isolation of three MAP
kinases from tobacco (Wilson et al., 1995). One of these,
p45Ntf4, has been shown to be activated by hydration of dry
pollen grains (Wilson et al., 1997). In the present study we have
used a peptide antibody raised against one of the other MAP
kinases, p43Ntf6, to investigate its role during the cell cycle in
synchronized tobacco cultures, and show that it is activated in
mitosis and possibly has a function in cytokinesis.
MATERIALS AND METHODS
Plant tissue culture, synchronization, and measurement of
cell cycle parameters
Tobacco cells (cell line BY-2) were maintained in modified Linsmaier
& Skoog medium according to the method of Nagata et al. (1992).
Cells were synchronized by diluting a 7-day-old culture 1:5, and after
incubation for 6 hours, 10 µg/l aphidicolin was added to the medium.
The cells were incubated for a further 18 hours, and the drug was then
removed by five washes with medium, before resuspension in the
original volume. Flow cytometric analysis and measurement of the
mitotic index were as described previously (Bögre et al., 1997b).
Treatment of cells with roscovitine was performed by taking
synchronized cultures 6 hours after the release from the aphidicolin
block and adding 100 µM roscovitine. Washing out of the roscovitine
was as described for aphidicolin.
Treatment of suspension cultures with microtubule drugs
The microtubule disrupting drugs APM (amiprophos-methyl) and
propyzamide were added at the concentrations and for the times
described in the text and figure legends. Taxol was added at a
concentration of 50 µM. For the propyzamide blocking and release
experiment, cells were synchronized with aphidicolin as described
above, and 7 hours after the drug release propyzamide (10 µM) was
added for a further 7 hours. Washing out of the propyzamide was as
described for aphidicolin.
Antibodies and recombinant proteins
The anti-p43Ntf6 MAP kinase antibody (AbP6) was raised in rabbits
against a synthetic peptide corresponding to the last 12 amino acids
of the p43Ntf6 protein (Wilson et al., 1995). The AbP6 polyclonal
serum was purified on Protein A-Sepharose (Pharmacia). The
PSTAIRE antibody was raised against a synthetic peptide representing
the conserved region of the cdc2 protein, and has been described
previously (Bögre et al., 1997b). The secondary antibody used in
immunoblotting was horseradish peroxidase-conjugated anti-rabbit
IgG (Promega, Madison, WI, USA) used at a dilution of 1:9,000. As
required, the AbP6 antibody was blocked by incubation with 100 µg
peptide for 2 hours at 4°C, before immunoblotting or
immunoprecipitation.
For immunofluorescence studies, the anti-tubulin antibody used
was monoclonal anti-α-tubulin (mouse IgG1 isotype) (Sigma
BioSciences, St Louis, MO, USA). Secondary antibodies were antimouse IgG FITC-conjugate for the anti-tubulin antibody, and antirabbit IgG CY3-conjugate for AbP6, both from Sigma BioSciences.
The tobacco MAP kinase proteins p43Ntf3, p45Ntf4 and p43Ntf6 used
in this study have been described previously (Wilson et al., 1995).
Approximately 40 ng of each protein was used in immunoblots.
Preparation of cell extracts
Frozen cells were ground in extraction buffer (25 mM Tris-HCl, pH
7.5, 15 mM MgCl2, 15 mM EGTA, 75 mM NaCl, 1 mM DTT, 0.1%
Nonidet P-40, 5 mM p-nitrophenylphosphate, 60 mM βglycerophosphate, 0.1 mM Na3VO4, 1 mM NaF, 1 mM PMSF, 10
µg/ml leupeptin, aprotinin, and soybean trypsin inhibitor, 5 µg/ml
antipain, chymostatin, and pepstatin), and then centrifuged for 40
minutes at 20,000 g. The supernatants were taken and the protein
concentration was determined by the Bio-Rad protein assay system.
Immunoblotting
Proteins were run on 12% (unless otherwise stated) SDSpolyacrylamide gels and blotted to polyvinylfluoride membranes
(Immobilon-P, Millipore). The membrane was blocked in PBS-T (137
mM NaCl, 2.7 mM KCl, 4.3 mM disodium hydrogen phosphate, 1.4
mM potassium dihydrogen phosphate, and 0.05% Tween-20, pH 7.2)
containing 3% non-fat dried milk overnight and then incubated in the
same solution containing the primary antibody for 2 hours. The
membranes were then washed in five changes of PBS-T, 10 minutes
each wash, followed by incubation for 1 hour in the secondary
antibody diluted in PBS-T containing 3% non-fat dried milk. After an
additional five washes in PBS-T, the immunoreactivity was visualized
using the SuperSignal Substrate western blotting detection system
from Pierce (Rockford, IL, USA).
Measurements of protein kinase activities
p13suc1 beads were prepared as described previously (Bögre et al.,
1997b). For immunokinase assays to measure MAP kinase activity,
the anti-p43Ntf6 antibody, AbP6, was bound to Protein A-Sepharose
beads (Pharmacia) by incubation of the crude antibody with the beads
for 2 hours at 4°C with agitation, and then washing the slurry three
times with immunoprecipitation buffer (10 mM Tris-HCl, pH 7.4, 150
mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5%
Nonidet P-40). The p13suc1-bound or AbP6-bound beads were added
to cell extracts containing 100 µg of protein and incubated for 2 hours
at 4°C with agitation. The beads were washed three times with
immunoprecipitation buffer, and twice with kinase buffer (30 mM
Hepes, pH 7.4, 50 mM KCl, and 4 mM MgCl2). Kinase reactions were
performed in 10 µl of kinase buffer containing 20 µM cold ATP, 2
µCi [γ-32P]ATP (3,000 Ci/mmol), and 2 µg myelin basic protein for
AbP6 or 5 µg histone HI for p13suc1, as substrate at 30°C for 15
MAP kinase in plant cytokinesis 3093
minutes and then stopped by the addition of Laemmli sample buffer.
The reactions were run on 15% SDS-polyacrylamide gels, dried, and
exposed to Kodak X-OMAT AR films.
Indirect immunofluorescence microscopy
Cells were fixed in 3.7% formaldehyde in PBS with 0.1% Triton X100 for 1 hour. After washing in PBS, the cells were treated with an
enzyme solution (0.5% cellulase R10 (Onozuka), 0.25% macerozyme
(Calbiochem), 0.25% pectinase, 0.25% driselase, 5 mM CaCl2, 0.5 M
mannitol) for 30 minutes at 37°C. After washing in PBS the cells were
attached to slides coated with poly-L-Lysine (Sigma) and extracted
with 100% methanol (−20°C) for 10 minutes. After washing with PBS
and BPBS (PBS containing 1% BSA), the samples were
immunostained. The primary antibody was applied and left for 1 hour
at 37°C. After washing with PBS and BPBS, the samples were
incubated with the secondary antibody for 45 minutes at 37°C.
Samples were washed with PBS, and DNA was stained with 1 µg/ml
DAPI in PBS. The slides were mounted in an antifade mounting
medium (Dako, Glostrup, Denmark). Preparations were examined
using an Olympus epifluorescence microscope equipped with
standard fluorescence filters. Photographs were taken on T-MAX 400
film (Kodak).
Fig. 1. Specificity of the anti-p43Ntf6 antibody, AbP6. (A) The three
tobacco recombinant MAP kinases, p43Ntf3 (3), p45Ntf4 (4), and
p43Ntf6 (6) were separated by SDS-PAGE and stained with
Coomassie Brilliant Blue R 250 (top panel), or blotted to membranes
and probed with the AbP6 antibody by immunoblotting (bottom
panel). (B) The AbP6 antibody was used in immunoblots on cell
suspension protein extracts either with (+) or without (−) prior
blocking of the antibody with the peptide used to raise the antibody.
(C) Cell extracts were prepared from synchronized cell suspension
cultures 12 hours after the release from an aphidicolin-induced
block, and immunoprecipitated with AbP6, either with (+) or without
(−) prior blocking of the antibody with the peptide used to raise the
antibody. The immunoprecipitates were then used for kinase assays
using MBP as a substrate, followed by SDS-PAGE and
autoradiography.
RESULTS
Specificity of the anti-p43Ntf6 antibody
A polyclonal antibody (called AbP6) was raised against a
peptide corresponding to the last 12 amino acids of the tobacco
MAP kinase p43Ntf6 (Wilson et al., 1995). The specificity of
the antibody was tested using immunoblot analysis with the
three recombinant tobacco MAP kinases p43Ntf3, p45Ntf4 and
p43Ntf6 (Wilson et al., 1995). AbP6 specifically recognised the
p43Ntf6 protein (Fig. 1A). The antibody was blocked by
preincubation with the peptide that had been used to raise the
antibody, and then compared with the unblocked antibody in
immunoblots of tobacco cell suspension culture extracts. No
band could be detected using the blocked antibody (Fig. 1B).
Similarly, in immunokinase assays, the MBP-phosphorylating
activity in immunoprecipitates from synchronized cells (see
below) could be blocked by preincubation of the antibody with
the peptide (Fig. 1C). Together, these data demonstrate the
specificity of the antibody.
Activation of a MAP kinase during mitosis
Tobacco cell suspension cultures were synchronized by
incubation of the cells with aphidicolin for 18 hours, which
blocks cells in the G1/S transition. After washing out the
drug, and incubation in drug-free medium, cells were taken
at different time points and the cell cycle stages were
analysed by flow cytometry. The measurement made
immediately after washing out the drug showed that the cells
were blocked at the G1/S boundary (Fig. 2A, 0 h). After 3
hours in drug-free medium the cells passed through S phase,
and by 8 hours a single peak corresponding to G2 cells was
seen. From 10 to 12 hours, the cells passed through mitosis.
To determine the number of cells passing through mitosis, the
mitotic index was measured at the same time points. The
highest percentage of metaphase/anaphase/telophase cells
was found 10-12 hours after the release from the aphidicolin
block (Fig. 2B).
Protein extracts were prepared from cell aliquots taken at all
time points, and immunoprecipitated using the antibody AbP6.
The immunoprecipitates were tested for kinase activity using
myelin basic protein (MBP), a standard substrate for MAP
kinases, as a substrate in the presence of [γ-32P]ATP, and the
immunokinase assays were then resolved by SDS-PAGE and
autoradiographed. MAP kinase activity increased dramatically
10 hours after the release of the cells from the aphidicolin
block. The activity peaked at 12 hours and then declined to
background levels at approximately 18 hours (Fig. 2C). Thus,
the activation, and peak of activity, of the MAP kinase
corresponded to the time when the cells were passing through
mitosis.
Immunoblotting was performed using the protein extracts
from the aphidicolin synchronized cells and the antibody
AbP6. A single band with the expected molecular mass of 43
kDa was detected in all samples (Fig. 2D). No cell cyclerelated changes occurred in the protein levels over the time
course of the experiment, suggesting that the protein is
expressed constitutively and that the changes seen in the kinase
activity are due to post-translational events. The identification
of a single band in immunoblots further demonstrates the
specificity of the antibody.
For comparison, the same samples were incubated with
p13suc1-Sepharose beads and the bound histone H1 kinase
activity was measured. The p13suc1 protein is a yeast protein
that is associated in a complex with the cyclin dependent kinase
(CDK) cdc2 in Schizosaccharomyces pombe (Brizuela et al.,
1987). The p13suc1-Sepharose beads bind several members of
the plant CDK family, and can be used to measure CDKassociated kinase activity, with a major peak of activity during
G2 and mitosis (Bögre et al., 1997b). Histone HI kinase activity
were observed at all stages from the synchronized tobacco cell
extracts used in this study, since p13suc1 binds the different
members of the CDK family which are expressed at different
stages of the cell cycle (Magyar et al., 1997). However, a major
peak of activity was observed at 10 hours, when the cells were
passing through mitosis (Fig. 2C). The amount of material
from each sample bound by the p13suc1 beads was controlled
by western analysis of the complexes using a PSTAIRE
3094 O. Calderini and others
antibody which recognises CDKs (Bögre et al., 1997b).
Approximately an equal amount of PSTAIRE-reactive protein
was detectable in each sample (Fig. 2C).
Fig. 2. Activation of the p43Ntf6 MAP kinase in mitosis.
(A) Synchronization of tobacco cell suspension cultures. Flow
cytometric analysis of cultures after the release from the aphidicolininduced block and culture in drug-free medium. The relative DNA
content of 201 and 401 on the abscissa correspond to cells in G1 and
G2, respectively. Cells are in G1 after the release (0 h) and progress
synchronously through the cell cycle. (B) Mitotic indices of the same
cells shows the passage of cells through M phase at 10-12 hours.
(C) Protein extracts from synchronized cells were prepared at the
indicated time points and tested for p43Ntf6 kinase activity (using
MBP as a substrate) or p13suc1-associated kinase activity (using
histone HI as a substrate) by kinase assays, followed by SDS-PAGE
and autoradiography. The amount of material bound by the p13suc1
beads was controlled by immunoblotting using a PSTAIRE antibody.
(D) The p43Ntf6 protein is expressed constitutively throughout the
cell cycle. Protein extracts from cells taken at the indicated time
points were separated by SDS-PAGE, and immunoblotted with the
AbP6 antibody. The numbers to the right indicate the molecular
masses of the marker proteins in kDa.
To test whether entry into M-phase might be necessary for
MAP kinase activation, synchronized cells were taken 6 hours
after the release from the aphidicolin block, when they had
accumulated in G2 (Fig. 2A), and incubated in the presence of
the drug roscovitine for a further 13 hours. Roscovitine blocks
cells in the G2 phase of the cell cycle through inhibition of
CDKs (Meijer et al., 1997; Planchais et al., 1997). This was
confirmed by flow cytometry and measuring the mitotic index
of roscovitine-treated cells (Fig. 3A,B). Measurement of MAP
kinase activity in protein extracts from cells taken during this
time showed that no activation occurred over the entire 13
hours in roscovitine (Fig. 3C). Even though roscovitine acts as
an inhibitor of CDK complexes, some p13suc1-associated
kinase activity was detected in extracts from roscovitinetreated cells (Fig. 3C). This is most probably because the
inhibitory action of roscovitine can be reversed after protein
extraction and bead affinity purification (Planchais et al.,
1997). Following 8 hours incubation in roscovitine, cell
aliquots were washed and incubated in drug-free medium.
Analysis of the cell cycle progression after the washout, by
flow cytometry and measurement of the mitotic index, showed
that the cells entered M-phase (Fig. 3A,B). Measurements of
kinase activities showed that the entry into M phase was
accompanied by an increase in MAP kinase activity and
p13suc1-associated kinase activity (Fig. 3C), which was not
seen in cells incubated in roscovitine for the same time. It
cannot be excluded that the concentration of roscovitine used
(100 µM) had an effect on protein kinases other than CDKs
(Meijer et al., 1997). However, in tobacco BY-2 suspension
cultures, 50 µM roscovitine was shown to be necessary to
cause an efficient G2 arrest (Planchais et al., 1997), and
roscovitine is a selective inhibitor of CDKs, requiring
approximately 100 times higher concentrations to inhibit plant
MAP kinases than plant CDKs, and is not very stable within
plant cells (P. Binarova et al., unpublished). Therefore, it seems
probable that the absence of MAP kinase activity in roscovitine
treated cells resulted from a G2 arrest caused by inhibition of
CDKs, and entry into mitosis is necessary for the activation of
the MAP kinase.
MAP kinase activity is not present in metaphase
arrested cells
Mitosis and cytokinesis in plant cells are marked by two
microtubule arrays, the mitotic spindle which is involved in
chromosome alignment and chromosome separation, and the
phragmoplast which is involved in deposition of the cell plate
(Samuels et al., 1995). We tested whether there might be some
link between the mitotic MAP kinase activity which we
detected in synchronized cultures and the organization of the
microtubules. Cells which contained activated MAP kinase (12
hours after the release from the aphidicolin block) were treated
with various concentrations of the microtubule destabilising
drugs APM (amiprophos-methyl) and propyzamide for 2
hours. No MAP kinase activity was detected in the cells at
higher drug concentrations (Fig. 4A). APM was more effective
in the elimination of the kinase activity. A significant decrease
in kinase activity occurred at 0.01 µM APM, and was
completely absent at 0.1 µM (Fig. 4A). Instead, 1 µM
propyzamide was required to eliminate the kinase activity (Fig.
4A). The mitotic index of each cell culture was determined,
and showed that both microtubule destabilizing drugs led to
MAP kinase in plant cytokinesis 3095
metaphase arrest of the cells (Fig. 4B). (Depolymerization of
the microtubules results in condensed chromosomes which are
not aligned on the cell equator because the mitotic spindle is
not yet reformed (Samuels et al., 1998). Such prometaphase
cells are herein referred to as metaphase arrested cells). Again,
APM was more effective at arresting the cells than
propyzamide. The decrease in kinase activity broadly mirrored
the degree of metaphase arrest. Kinase assays after binding of
the same samples to p13suc1-Sepharose beads showed that the
histone HI activity increased in the drug-treated cells (Fig. 4A),
as expected for cells blocked in metaphase. Either intact
microtubules are required for MAP kinase activity, or the
kinase is activated after metaphase when the cells pass to
anaphase.
Intact microtubules are not sufficient for MAP kinase
activity
To try and understand further the relationship between
microtubules and MAP kinase activity, cells were taken 12
hours after the release from the aphidicolin block (when
mitosis is occurring and the kinase is activated), treated with
APM and propyzamide for shorter time intervals, and protein
extracts were assayed for p43Ntf6 kinase activity. Within as
little as 10 minutes, APM led to a significant reduction in MAP
kinase activity, while both drugs eliminated MAP kinase
activity from these cells by 30 minutes (Fig. 5A, left panels).
Cells were also treated with the microtubule stabilizing drug
taxol, and extracts were tested for kinase activity. The taxoltreated cells also showed a reduction in kinase activity, similar
to that seen with the microtubule destabilizing drugs (Fig. 5A,
left panels). The amount of p43Ntf6 protein bound by the
antibody was controlled by immunoblot analysis of the
immunoprecipitates of extracts from the taxol treated cells. A
band of approximately equal intensity was observed in all
samples, running below the IgG (Fig. 5B, left panel). The
recombinant p43Ntf6 protein was also immunoprecipitated and
run in parallel with these samples to verify the position of the
p43Ntf6 protein (data not shown).
Assaying p13suc1-associated histone HI kinase activity
showed a decrease in activity in the control samples, as cells
progressed from G2 into M phase (Fig. 5A, right panels). In
the APM and propyzamide treated cells, there was an increase
in histone HI kinase activity, as noted above due to the
metaphase arrest. In taxol treated cells, an increase in histone
HI kinase activity was observed, which could also be due to
metaphase arrest of these cells (Fig. 5A, right panels). The
amount of material bound by the p13suc1 beads from extracts
of taxol treated cells was controlled by western analysis of the
complexes using a PSTAIRE antibody, and showed that the
increase in activity was not due to differences in the amounts
of CDKs complexed by the p13suc1 beads (Fig. 5B, right panel).
Mammalian cells arrested with taxol maintain high levels of
p34cdc2 activity (Andreassen and Margolis, 1994), while
treatment of cells with taxol can cause them to arrest in mitosis
(Jordan et al., 1993).
Fig. 3. Entry into mitosis is required for MAP kinase activation.
Cells were taken 6 hours after the release from the aphidicolin block
and incubated with roscovitine, and then sampled at the time points
indicated. Roscovitine blocks cells in G2, as shown by flow
cytometry (A, roscovitine) and measurement of the mitotic index (B,
roscovitine), and prevents activation of the MAP kinase, as measured
by kinase assays of the same samples (C, roscovitine) for MAP
kinase activity (MBP) and p13suc1-associated kinase activity (HI).
Cell aliquots were taken 8 hours after the addition of roscovitine, the
roscovitine was washed out, and the cells were incubated in drug-free
medium for a further 7 hours. Removal of the roscovitine releases
cells from the G2 block, resulting in entry into M phase (A, B,
roscovitine wash out), and leads to activation of the MAP kinase
(MBP) and p13suc1-associated kinase (HI) (C, roscovitine wash out).
3096 O. Calderini and others
Fig. 4. Microtubule-disrupting drugs abolish MAP kinase activity.
(A) Cells were taken 12 hours after the release from the aphidicolin
block and treated with increasing concentrations (shown on the top in
µM concentrations) of the microtubule-disrupting drugs APM or
propyzamide for 2 hours. Cell extracts were tested for p43Ntf6 kinase
activity (using MBP as a substrate) or p13suc1-associated kinase
activity (using histone HI as a substrate) by kinase assays, followed
by SDS-PAGE and autoradiography. (B) Microtubule-disrupting
drugs cause metaphase arrest. The mitotic indices of the same cells
used in (A) were measured.
Cells treated with APM, propyzamide and taxol were
inspected microscopically by DAPI and anti-tubulin staining to
investigate the state of the cells after the respective drug
treatments. APM caused a rapid depolymerization of
microtubules in as little as 10 minutes, and complete
elimination of microtubules by 60 minutes (Fig. 5C, middle
panels). Depolymerization of the microtubules by propyzamide
occurred in approximately 30 minutes, and, as with APM, no
tubulin staining was observed at 60 minutes (Fig. 5B, right
panels). In taxol treated cells the microtubules were
maintained, although there was some disordering of the
Fig. 5. Intact microtubules are not sufficient for MAP kinase activity.
Cells were treated with APM (1 µM), propyzamide (prop., 1 µM), or
taxol (50 µM), and protein extracts were prepared at the time points
indicated. (A) The extracts were immunoprecipitaed with the antip43Ntf6 antibody, AbP6, or bound to p13suc1 beads, and used in
kinase assays followed by SDS-PAGE and autoradiography. The left
panels show phosphorylation of MBP by p43Ntf6, while the right
panels show phosphorylation of histone HI by CDKs. Both the
microtubule-disrupting drugs (APM and propyzamide) and the
microtubule stabilizing drug (taxol) abolish MAP kinase activity.
(B) Loading control of extracts from taxol treated cells. The AbP6
immunoprecipitates and p13suc1-bound material were separated by
10% SDS-PAGE, blotted to membranes and immunodetected with
AbP6 (for AbP6 immunoprecipitates) or a PSTAIRE antibody (for
p13suc1-bound material). (C) Effects of the different drugs on cells.
Cells were labelled with an anti-tubulin antibody (Anti-tub), or DAPI
to visualize the chromatin. The numbers after each drug represent the
time (in minutes) of each treatment at which staining was performed
(see text for details).
MAP kinase in plant cytokinesis 3097
A
Fig. 6. Activation of the MAP kinase occurs in late anaphase/early
telophase. (A) The number of metaphase cells and
anaphase/telophase cells after release from a propyzamide-induced
metaphase arrest was determined after staining with DAPI. (B) Cells
were immunostained with an anti-tubulin antibody and the number of
mitotic spindles and phragmoplasts were counted. (C) Kinase assays
of cell extracts using p13suc1 beads (HI) or the AbP6 antibody (MBP)
show that the MAP kinase is activated after the inactivation of
p13suc1-associated kinase, at a time when cells are in late
anaphase/early telophase.
B
C
spindles compared to the untreated control cells (Fig. 5C, left
panels). After 60 minutes in taxol, all mitotic cells had the
appearance shown in Fig. 5C, which appear to be blocked in
metaphase, similarly to the APM and propyzamide treated
cells. The loss of MAP kinase activity at earlier time points
may be due to the exit of cells from the phase in which the
kinase is active, while at the same time the entry of other cells
into this phase is blocked by the drug. Since the loss of MAP
kinase activity was observed within 10 minutes after APMinduced metaphase arrest, the active state of p43Ntf6 appears to
be very transient. These data suggest that normal microtubule
dynamics and/or progression into anaphase is necessary for
MAP kinase activation.
MAP kinase activation occurs in late anaphase/early
telophase
A more direct determination of the timing of MAP kinase
activation was performed by taking cells at frequent time
intervals after the release from a metaphase arrest and
measuring cell cycle progression, spindle and phragmoplast
formation, and kinase activities. Cells were synchronized by
incubation with aphidicolin, as described above. After washing
out the aphidicolin, the cells were incubated in drug-free
medium for 7 hours, at which stage they are passing through
the G2 stage. The cells were then incubated in the presence of
propyzamide for 7 hours, which, as shown above, causes
metaphase arrest. The propyzamide was washed out and the
cells were cultured in drug-free medium and sampled at half
hourly intervals.
To follow the cell cycle progression after the drug release,
the number of metaphase and anaphase/telophase cells were
counted at each time point. As shown in Fig. 6A, propyzamide
induced a metaphase arrest. By 2 hours after the propyzamide
release, the majority of metaphase cells had progressed to
anaphase, and by 3 hours most of these latter cells were in late
anaphase/early telophase (Fig. 6A). Staining with an antitubulin antibody showed that the microtubules were indeed
depolymerized by the propyzamide treatment, but that mitotic
microtubule arrays were fully recoverable after washing out the
drug (Fig. 6B). This anti-tubulin staining also revealed a
correlation between the number of anaphase/telophase cells
Fig. 7. Immunolocalization of p43Ntf6 by
indirect immunofluorescence.
(A) Immunostaining with AbP6. A distinct
band is seen to traverse the width of the cell.
This band is located in between the separating
chromosomes in anaphase, as evidenced by
DAPI staining (B) of the same cell.
Immunostaining with the AbP6 antibody (C)
and an anti-tubulin antibody (D) shows
labelling by the AbP6 antibody of the cell plate
in between the two microtubular arrays of the
phragmoplast. (E) DAPI staining of the same
cell. Bar, 10 µm.
3098 O. Calderini and others
and the formation of the phragmoplast (Fig. 6B), which forms
from late anaphase to late telophase.
Cell extracts were measured for CDK-associated activity
using p13suc1 beads and MAP kinase activity by immunokinase
assays with the AbP6 antibody. As shown in Fig. 6C, the
p13suc1-associated activity is high in the metaphase arrested
cells, but then declines as cells pass through anaphase. CDK
activity declines as mitotic cyclins are degraded at the
metaphase to anaphase transition (Glotzer et al., 1991). Some
histone HI kinase activity was observed at later time points,
which may be due to the multiple CDKs in plants which can
bind p13suc1, and some of which are activated in mitosis
(Magyar et al., 1997). By contrast, the AbP6-precipitated
kinase activity shows an increase 2.5 hours after the release
from the propyzamide-induced metaphase arrest, when CDK
activity had already decreased. Therefore, the MAP kinase
appears to have a function later in the cell cycle than the mitotic
cyclin dependent kinase. From the analysis of the cell cycle
progression discussed above, this corresponds to late
anaphase/early telophase.
Intracellular localization of p43Ntf6
Indirect immuofluorescence studies were undertaken to try and
determine the cellular localization of the MAP kinase at
different stages of the cell cycle. Cells were doubly labelled
with the AbP6 antibody and DAPI. With AbP6, a generally
diffuse staining pattern was observed in cells at different stages
of the cell cycle, while the chromatin was not stained (data not
shown). A distinct labelling pattern was found only in anaphase
cells, where, as well as the diffuse staining, a band traversing
the width of the cell was observed (Fig. 7A). This band
appeared to be located in the middle of the separating
chromosomes (Fig. 7B). This AbP6 labelling pattern was not
observed in all anaphase cells, and may represent a transient
accumulation of the protein in this location. Triple labelling
with the AbP6 antibody (Fig. 7C), an anti-tubulin antibody
(Fig. 7D) and DAPI (Fig. 7E) showed that the band is localized
in the middle of the two tubulin arrays that are characteristic
of the phragmoplast in anaphase/telophase.
DISCUSSION
Activation of the tobacco p43Ntf6 MAP kinase in
mitosis
The involvement of MAP kinases in meiosis is well
documented. MAP kinase activity is required for the
progression of G2-arrested Xenopus oocytes into the first and
second meiosis, and for the arrest of mature oocytes in
metaphase II, possibly through the activation and stabilization
of M-phase promoting factor, a complex of Cdc2 kinase and
cyclin B (Sagata, 1997). Release of clam oocytes from a G2/M
cell cycle arrest by fertilization is accompanied by MAP kinase
activation, but this MAP kinase is not active in succeeding
mitotic cell cycles (Shibuya et al., 1992). In fact, a role for
MAP kinases in mitosis is still unclear (Shibuya et al., 1992).
MAP kinases (Tamemoto et al., 1992) and a MAP kinase-like
molecule (Heider et al., 1994) have been reported to be
activated during M phase in mammalian cells. Inactivation of
MAP kinase is required for entry into M phase of the first
mitotic cycle after fertilization of oocytes, but not in the
subsequent rapid cell cycles (Abrieu et al., 1997), and has also
been reported to be necessary for the G1/S-phase transition in
starfish eggs (Tachibana et al., 1997). In Xenopus cell cycle
extracts, which are thought to mimic mitosis, MAP kinase is
required for the spindle assembly checkpoint, but not for
normal M phase entry and exit (Minshull et al., 1994; Takenaka
et al., 1997). The lack of kinase activity in metaphase arrested
cells described in this study indicates that this MAP kinase is
functionally distinct from the MAP kinase described in
Xenopus, whose activity is required for metaphase arrest
(Minshull et al., 1994; Takenaka et al., 1997). The absence of
activity was not due solely to depolymerization of the
metaphase microtubules, since a reduction in activity also
occurred in cells treated with taxol, which stabilized the
microtubules, but rather may depend on a transient cell cycle
event after metaphase.
In the present study we provide clear evidence that a MAP
kinase is activated during mitosis. Immunokinase assays of
protein extracts from synchronized tobacco cells using a
peptide antibody raised against the tobacco MAP kinase
p43Ntf6 showed that the kinase is activated specifically as cells
pass through mitosis. Further, immunokinase assays after
blocking cells in the G2 phase of the cell cycle, and then
releasing the block, allowing cells to enter into mitosis,
indicated that entry into mitosis is necessary for the activation
of the kinase. Immunoblotting and antibody blocking
experiments showed that p43Ntf6 is responsible for the
observed activity.
Four main groups of plant MAP kinases exist, and the so far
identified functions of two of these groups suggest that they
respond to different forms of stress. No functions have yet been
ascribed to the other two groups, one of which includes p43Ntf6.
The amino acid sequence of p43Ntf6 (Wilson et al., 1995) shows
highest identity to a recently identified alfalfa MAP kinase
which appears to perform a similar function (L. Bögre et al.,
unpublished). Together, these two MAP kinases may define a
new functional group of plant MAP kinases.
MAP kinase activation occurs in late anaphase/early
telophase
Phosphorylation/dephosphorylation events are important for a
number of mitotic events. As in other organisms, plant cyclindependent kinases (CDKs) control the passage of cells through
the G2/M transition (Doonan and Fobert, 1997). Multiple
CDKs exist in plants, and are activated at different stages of
the cell cycle (Bögre et al., 1997b). Immunoprecipitation of
CDK activity using p13suc1-Sepharose beads has been shown
to result in a major peak of activity during mitosis (Bögre et
al., 1997b). The CDK activity decreases in the
metaphase/anaphase transition due to the degradation of
cyclins (Glotzer et al., 1991). In the synchronized tobacco cell
suspension cultures used in this study, the p13suc1-associated
histone HI kinase activity also showed a major peak of activity
during mitosis. The kinetics of p43Ntf6 kinase activation
showed a peak of activity overlapping histone HI kinase
activity in cells released from a G1/S phase arrest. The timing
of MAP kinase activation was further refined by following
kinase activities in cells released from metaphase arrest due to
propyzamide-induced microtubule depolymerization. The
MAP kinase was activated later in the cell cycle than the
p13suc1-associated CDK activity that, according to the analysis
MAP kinase in plant cytokinesis 3099
of the cell cycle stages, corresponded to the G2/M-phase CDK.
This suggested that the MAP kinase has a role later in the cell
cycle than the G2/M CDK. Further, the activation of p43Ntf6 is
not solely dependent on the recovery of microtubule structures,
which could already be found after drug removal, but rather
depends on the transit through a cell cycle stage in late
anaphase/early telophase.
A role for the p43Ntf6 MAP kinase in cytokinesis
In late anaphase/early telophase a plant-specific cytoskeletal
structure, the phragmoplast, is found, that is required to build
the cell wall between the daughter cells. An intimate
relationship exists between microtubules and other molecules
necessary for phragmoplast and cell plate formation. Motor
proteins, such as kinesins (for review see Asada and Collings,
1997), and membranes (Schopfer and Hepler, 1991; Samuels
et al., 1995) are associated with the phragmoplast microtubular
arrays, and the transport of vesicles along microtubules by
motor proteins may be involved in the delivery of these vesicles
to the nascent cell plate. Indirect immunofluorescence studies
with the anti-p43Ntf6 antibody and double labelling with the
anti-p43Ntf6 antibody and an anti-tubulin antibody identified a
band in between the interdigitating phragmoplast arrays.
Immunofluorescence studies have shown the co-localization of
kinesin-like proteins (Liu et al., 1996; Asada et al., 1997;
Bowser and Reddy, 1997) and MAPs (Chan et al., 1996) with
the phragmoplast microtubules, while the cell plate in the
middle of the two microtubular arrays was not labelled. By
contrast, the anti-p43Ntf6 antibody showed labelling in the
midzone between the microtubule arrays. Similar labelling
patterns have been observed for the dynamin-like proteins
phragmoplastin (Gu and Verma, 1996, 1997) and ADL1
(Lauber et al., 1997). Dynamins are a family of GTPases
involved in endocytosis and Golgi membrane protein retention
(Robinson et al., 1993; Wilsbach and Payne, 1993). The
localization of plant dynamin to the cell plate is in accordance
with a general role for dynamins in vesicular trafficking/fusion
events, and phragmoplastin appears to be associated with small
vesicles on the cell plate (Gu and Verma, 1997). Although
mammalian dynamin can be phosphorylated by MAP kinases
(Earnest et al., 1996), and phragmoplastin may be
phosphorylated (Gu and Verma, 1997), the plant dynamins do
not contain the consensus MAP kinase phosphorylation site(s).
The KNOLLE protein in Arabidopsis, which is related to
vesicle-docking syntaxins, also localizes to the cell plate in
dividing cells, and appears to be necessary for vesicle fusion
rather than vesicle transport (Lauber et al., 1997).
The detailed analysis of the timing of the activation of the
tobacco MAP kinase p43Ntf6 presented in this study indicates
that it is a late event in the cell cycle, with activation occurring
later than the G2/M CDK, during late anaphase/early telophase.
This together with the localization of the kinase to the cell plate
suggests that p43Ntf6 may have a role in phragmoplast
formation and synthesis of the new cell wall.
O.C. was a recipient of an Italian National Research Council
fellowship, Progetto Finalizzato RAISA, Bando N.203.12.20/1,
codice N.12.03.02, and a European Union research fellowship BIO4
CT96 5070. This work was supported by grants from the Austrian
Academic Exchange Programme to E.H.B and P.B., the Academy of
Sciences of the Czech Republic to P.B., grant number GA AVCR
5020803/1998, the Austrian Fonds zur Förderung der
wissenschaftlichen Forschung project P11481-GEN, and the
European Union BIOTECH project SIME, project number BIO4
CT96 0275.
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