From the Divisions of Yeast Genetics and § Protein Structure, National Institute for Medical Research, Mill Hill, London NW7 1AA, Great Britain
Received for publication, February 10, 2003, and in revised form, March 6, 2003
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ABSTRACT |
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The Cdc5 protein of budding yeast is a polo-like
kinase that has multiple roles in mitosis including control of the
mitotic exit network (MEN). MEN activity brings about loss of mitotic kinase activity so that the mitotic spindle is disassembled and cytokinesis can proceed. Activity of the MEN is regulated by a small
GTPase, Tem1, which in turn is controlled by a two-component GTPase-activating protein (GAP) formed by Bfa1 and Bub2. Bfa1 has been
identified as a regulatory target of Cdc5 but there are conflicting
deductions from indirect in vivo assays as to whether phosphorylation inhibits or stimulates Bfa1 activity. To resolve this
question, we have used direct in vitro assays to observe the effects of phosphorylation on Bfa1 activity. We show that when Bfa1
is phosphorylated by Cdc5, its GAP activity with Bub2 is inhibited
although its ability to interact with Tem1 is unaffected. Thus,
in vivo inactivation of Bfa1-Bub2 by Cdc5 would have a
positive regulatory effect by increasing levels of Tem1-GTP so
stimulating exit from mitosis.
In budding yeast, the final stages of mitosis are controlled by
the mitotic exit network
(MEN).1 Once the chromosomes
are partitioned equally between mother and daughter cells, the MEN
eliminates mitotic kinase activity, a prerequisite for spindle
disassembly and cytokinesis (reviewed in Refs. 1 and 2). In the MEN,
the key element controlling the pathway is Tem1 (3), a
GTPase with biochemical properties similar to those of the Rho family
of small GTPases (4). The MEN is negatively regulated by Bub2 and Bfa1
(5-8), which together act as a two-component GTPase-activating protein
(GAP) for Tem1. Presumably, Bfa1/Bub2 reduce MEN activation in
vivo by reducing Tem-GTP levels (4). The positive regulation of
Tem1, and thus the MEN, has been linked to the activity of Lte1 (9,
10), a putative guanosine nucleotide exchange factor for Tem1
(11).
Following Tem1 activation, the functional sequence of the MEN involves
activities of Cdc15 kinase followed by Dbf2 kinase with its
associated binding protein, Mob1 (12). Ultimately, the MEN triggers the
long term release of Cdc14 from the nucleolus, which, through a series
of dephosphorylation events, reduces mitotic kinase activity (13).
Recently, the Cdc14 early anaphase release (FEAR) pathway has been
described which controls an initial release of Cdc14 from the
nucleolus, which may contribute to optimal activity of the MEN although
the precise role of the FEAR remains unclear (14-16).
An important regulator of these activities is the polo-like kinase,
Cdc5. Cdc5 has several essential roles in mitosis (12, 17-19) as well
as regulation of the MEN and FEAR pathways. Even in the MEN there is
evidence for more than one role for Cdc5. Net1, the protein that
sequesters Cdc14 to the nucleolus, is phosphorylated in
vitro and in vivo by Cdc5 so facilitating release of
Cdc14 during anaphase (20, 21). Cdc5 also has a positive but
unidentified role at some stage between Tem1 and activation of
Dbf2 (12). Furthermore, Cdc5 phosphorylates Bfa1 both in
vitro and in vivo during mitosis in normally growing
cells (15, 22, 23). Two opposite interpretations have been made on the
role of this Bfa1 phosphorylation. Hu and co-workers (23) observed that
Bfa1 was unphosphorylated in a nocodazole arrest, a condition where the MEN is kept inactive. For this and several other reasons, they equated
phosphorylation of Bfa1 with an active MEN and hence proposed that
phosphorylation inhibited Bfa1 GAP activity. However, Lee et
al. (12, 22) reached the opposite conclusion. They observed that Bfa1 was highly phosphorylated in a nocodazole arrest and that
overexpression of Cdc5 inhibited the Dbf2 activity that normally signifies an active MEN (12,
22).2 By linking
phosphorylation of Bfa1 with arrest of the MEN, they concluded that
phosphorylation of Bfa1 increased GAP activity to negatively control
mitotic exit.
It is important to note that these opposite conclusions on the effects
of phosphorylation on Bfa1 GAP activity were based on downstream assays
of the MEN and not on direct biochemical analyses of Bfa1 behavior. To
resolve this important question of how Cdc5 and Bfa1 regulate the MEN,
we have directly examined how phosphorylation affects the in
vitro properties of Bfa1 in combination with Tem1 and Bub2. This
purely in vitro approach was possible as the sites of
phosphorylation in Bfa1 by Cdc5 in vivo are largely
equivalent to those produced in vitro (23). In
vitro, Bfa1 binds to Tem1 but has different activities depending on whether or not Bub2 is also present. Without Bub2, Bfa1 inhibits GTP
hydrolysis and GTP dissociation from Tem, while Bfa1 acts as a
two-component GAP in the presence of Bub2 (4). Here we show that in the
absence of Bub2, phosphorylation of Bfa1 by Cdc5 does not affect
dissociation and hydrolysis of GTP bound to Tem1. However, in the
presence of Bub2, phosphorylation of Bfa1 by Cdc5 reduces GAP activity.
We therefore conclude that one role of Cdc5 in the MEN is to reduce the
hydrolysis of GTP by Bfa1/Bub2, so increasing Tem1-GTP levels and
promoting exit from mitosis. However, Bfa1 phosphorylation cannot
account for the complete control of GAP activity and so other
regulatory mechanisms for the MEN are also discussed.
Plasmid and Strain Construction--
Protein Expression and Purification--
Tem1, MBP-Bfa1, and
GST-Bub2 were prepared as described earlier (4). Yeast harboring
plasmids encoding GST-Cdc5 and GST-Cdc5KD were grown to a cell density
of 107/ml in medium supplemented with 2% sucrose as carbon
source. After addition of 2% galactose, the cultures were grown for
6 h at 30 °C and harvested. For GST-Cdc5, growth medium was YEP
(1% yeast extract, 2% bacto-peptone), while cells expressing
GST-Cdc5KD were in YNB selective minimal medium. Frozen pellets of
cells were thawed, resuspended in cold lysis buffer (50 mM
Tris, pH 7.5, 250 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 1 mM dithiothreitol, 10 mM
NaF, 50 mM Phosphorylation of Bfa1--
Phosphorylation reactions using
radiolabeled ATP were carried out in 30 µl of kinase buffer (50 mM Tris/HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol) containing 1-10 µg of substrate.
10-50 ng of kinase were added followed by 50 µM ATP and
0.1 µl of [ Nucleotide Exchange and GTPase Assays--
Conditions for
[ Phosphorylation of Bfa1 by Cdc5--
The aim of this work was to
characterize the effects of Cdc5-dependent phosphorylation
on Bfa1. A major technical requirement was to produce Cdc5 in
sufficient quantities and with sufficient activity to fully
phosphorylate Bfa1 so that any effects of phosphorylation would be
reproducible and have their maximal impact. Cdc5 was initially prepared
from Escherichia coli and Baculovirus expression systems but
was unable to convert Bfa1 completely to an electrophoretically slower,
phosphorylated form (data not shown). Eventually, we developed a
yeast-based expression system for a GST-Cdc5 derivative, which is
activated by S165D and T238D mutations and stabilized by deletion of 70 N-terminal residues (24, 25). In vivo, the activity of this
GST-Cdc5 fusion protein was indicated by complementation of a
cdc5 deletion mutant under low expression conditions (use of
glucose with a GAL promoter) (data not shown). GST-Cdc5KD provided a
kinase-dead control and has a N209A mutation (23) and an equivalent 70-residue N-terminal deletion. GST-Cdc5 and GST-Cdc5KD were readily purified (Fig. 1A). The
differential activities of affinity-purified GST-Cdc5 and GST-Cdc5KD
were confirmed using myelin basic protein as a substrate (Fig.
1B). Conditions for efficient phosphorylation of Bfa1 were
established using increasing amounts of GST-Cdc5 (Fig. 1C).
With suboptimal levels of Cdc5, multiple isoforms of Bfa1 were visible
by protein staining in agreement with the presence of multiple
phosphorylation sites in the molecule. However, with 5 ng or more
GST-Cdc5 per assay, a single slower migrating form of phosphorylated
Bfa1 was detected. No change in the mobility of Bfa1 was produced by
treatment with equivalent amounts of GST-Cdc5KD. The efficient and
specific phosphorylation of Bfa1 GST-Cdc5 was further confirmed by the
differential radiolabeling produced by GST-Cdc5 or GST-Cdc5KD (Fig.
1C). Note that in the following examination of the effects
of phosphorylation on Bfa1, optimally phosphorylated Bfa1 was prepared
with GST-Cdc5 as described above, while non-phosphorylated Bfa1 was
prepared by mock-treatment with catalytically inactive GST-Cdc5KD.
Thus, our data are specific to Cdc5 kinase and cannot be attributed to
a passive Cdc5-Bfa1 interaction or to unspecified contaminating
material in the kinase preparation.
Phosphorylation of Bfa1 by Cdc5 Has No Effect on Inhibition of Tem1
GTP Hydrolysis and Dissociation--
The effects of phosphorylation of
Bfa1 on Tem1 activity were first examined in the absence of Bub2. Under
these conditions unphosphorylated Bfa1 inhibits both the intrinsic GTP
hydrolysis and GTP dissociation activities of Tem1 (4). The inhibitory effects of Bfa1 on GTP hydrolysis and GTP dissociation were monitored by loading Tem1 with [ Phosphorylation of Bfa1 by Cdc5 Reduces GAP Activity with
Bub2--
The effect of phosphorylation of Bfa1 was next examined in
assays of GAP activity where Bub2 is also present. These assays were
performed by loading Tem1 with [
To further characterize the influence of Cdc5 phosphorylation on Bfa1,
GAP activity was assayed after a fixed time with increasing amounts of
Bub2 (Fig. 3B). As seen above, no loss of
[ We have used an in vitro approach to understand the
effects of Cdc5 kinase on the regulation of Tem1 and the MEN. We
demonstrate that when Bfa1 is phosphorylated by Cdc5, its GAP activity
with Bub2 for Tem1 is reduced. The equivalent result of this activity in vivo would be for Cdc5 to increase Tem1-GTP levels so
bringing about positive regulation of the MEN. Our in vitro
observation that phosphorylation reduces the GAP activity of Bfa1 is
consistent with in vivo results showing that cells with a
non-phosphorylatable form of Bfa1 have reduced ability to activate the
MEN (23). Nevertheless, it is still somewhat unclear why Hu et
al. (23) and Lee et al. (12, 22) reached opposite
conclusions on the in vivo effects of phosphorylation on
Bfa1 activity. One possibility is that the two groups observed
different extents of phosphorylation and only complete phosphorylation
was inhibitory. This idea stems from more recent observations of
Pereira and co-workers (15) who showed that a phosphorylated
form of Bfa1 is already present in a metaphase arrest. Upon progression
into anaphase, Bfa1 is then further phosphorylated. Although it is not
clear whether both changes are caused by Cdc5, there does appear to be
two phosphorylation events on Bfa1: one before metaphase that does not
inhibit the Bfa1/Bub2 GAP activity and a second during anaphase that
could participate in activation of MEN by inhibiting its GAP activity.
In considering how phosphorylation affects Bfa1 and GAP activity, Hu
et al. (23)proposed that phosphorylated Bfa1 has a lower affinity for Tem1 as judged by co-immune precipitation. However, our
in vitro results show that phosphorylated and
non-phosphorylated forms of Bfa1 have equal affinities for Tem1 as
judged by their similar abilities to inhibit GTP hydrolysis and
dissociation. An alternative mechanism may involve interference in the
interaction of phosphorylated Bfa1with Bub2 rather than with Tem1. It
is notable that a C-terminal deletion form of Bfa1 lacking the domain
responsible for Bub2 interaction still has partial in vitro
GAP activity in combination with Bub2 even though the two proteins can
no longer interact (4). This might explain why we observe only a
partial inhibition of Bfa1/Bub2 GAP activity after Bfa1
phosphorylation. Moreover, it has been recently shown that Bub2 is also
phosphorylated in a cell cycle-dependent manner, and it was
proposed that this modification could affect GAP activity (26). Since
GAP activity is reduced after phosphorylation of the mammalian GAPs,
RGS16 and p120-GAP (27, 28), the complete inhibition of Bfa1/Bub2 GAP
activity might only be achieved by phosphorylation of both components.
The partial inhibitory effect of Bfa1 phosphorylation on GAP activity
might also reflect the multiple levels of regulatory mechanisms
controlling mitotic exit. Indeed, cell cycle progression is apparently
normal in There are numerous indications that MEN-like regulatory pathways are
evolutionarily conserved. The MEN is closely related to the SIN
(septation initiation network) pathway of Schizosaccharomyces pombe, where the Tem1-like small GTPase, Spg1, is again regulated by a two component GAP comprising Cdc16 and Byr4, which are homologous to Bub2 and Bfa1 (reviewed in Refs. 1 and 2). As with the MEN of
budding yeast, the activity of the SIN depends on a polo kinase, Plo1,
which is the S. pombe homologue of Cdc5. It is not yet known
whether Byr4 is phosphorylated by Plo1, although considerable genetic
evidence points to Cdc16/Byr4 being affected by Plo1 (31). In fission
and budding yeasts the polo-like kinases may also stimulate SIN/MEN
activity downstream of their respective two-component GAPs, although in
neither species is it clear how this stimulation occurs (12, 32, 33).
Polo kinase homologues extend from yeasts to humans and are
ubiquitously implicated in mitotic regulation. Similarly, homologues of
many other components of the MEN have been recognized in higher
eukaryotes (summarized in Ref. 1) and roles for Rho-like GTPases, of
which Tem1 is an example, have also been seen in late mitosis and
cytokinesis in higher eukaryotes (34, 35). Together, these observations
suggest conservation in the MEN-like pathways and their regulation by
polo-like kinases by mechanisms such as the one described here.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
70N-CDC5 was
amplified by PCR using as template a plasmid containing a
CDC5 allele harboring two point mutations, S165D and T238D
(kindly provided by F. Uhlmann and M. Sullivan). The amplified sequence
starting at codon 71 of CDC5 was cloned in-frame with GST in
a URA3-based plasmid under the control of the
GAL1 promoter. The URA3 was then replaced by a
TRP1 marker, and the plasmid was introduced into a strain
deleted for CDC5 and harboring a CDC5-URA3 plasmid (KKY902-2BTa). The CDC5-URA3 plasmid was eliminated
by growth on 5-fluoro-orotic acid to generate strain MGY29. A
70N-CDC5 kinase-dead plasmid expressing GST-Cdc5KD was
constructed using a similar strategy but the template was a
CDC5 allele harboring an N209A point mutation that
inactivates the kinase (23).
-glycerophosphate, 1 mM sodium
orthovanadate and Complete protease inhibitor mixture (Roche
Diagnostics Ltd.)), and passed twice through a French pressure cell.
Crude protein extract was centrifuged twice at 18,000 × g at 4 °C for 20 min. The supernatant was gently mixed
with glutathione-agarose beads at 4 °C. After 1 h, the beads
were packed in a column and washed with washing buffer (50 mM Tris, pH 7.5, 250 mM NaCl, 1% Nonidet P-40,
and 1 mM dithiothreitol). GST-Cdc5 protein was eluted in 50 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Nonidet
P-40, 1 mM dithiothreitol, and 20 mM reduced
glutathione. Elution of protein was monitored by SDS-PAGE. Peak
fractions were pooled and dialyzed in 50 mM Tris/HCl, pH
7.5, 100 mM NaCl, 2 mM dithiothreitol, and 25%
glycerol. Aliquots were frozen at
80 °C.
-32P]ATP (Amersham Biosciences,
370 MBq/ml, 3000 Ci/mmol). After incubation for 15 min at 30 °C,
reactions were stopped by addition of 10 µl of 4× Laemli buffer and
heating at 100 °C for 2 min. After SDS-PAGE electrophoresis,
radiolabeling was visualized by autoradiography. For preparative
reactions of phosphorylated Bfa1, the protein was incubated in the same
reaction mixture with 10 mM non-radioactive ATP instead of
[
-32P]ATP. Reaction volumes were increased to
accommodate up to 100 µg of Bfa1. Incubation was for 4 h. After
dialysis against 50 mM Tris-HCl, pH 7.5, 100 mM
NaCl, 2 mM dithiothreitol, 25% glycerol, the
phosphorylated Bfa1 (1 µg/µl) was stored at
80 °C. An
identical reaction on Bfa1 was carried out using kinase dead GST-Cdc5KD to provide non-phosphorylated control material.
-32P]GTP exchange and hydrolysis assays were as
described elsewhere (4).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Purification of Cdc5 kinase and
phosphorylation of Bfa1 by Cdc5. A, increasing amounts
of affinity purified GST-Cdc5 and GST-Cdc5KD revealed by Coomassie blue
staining after SDS-PAGE. Molecular weight markers are indicated.
B, phosphorylation of MBP. Coomassie Blue shows staining of
input material. C, phosphorylation of Bfa1 after treatment
with increasing amounts of the kinases indicated. Phosphorylation was
detected by reduced electrophoretic mobility of Coomassie Blue-stained
or -32P-labeled material.
-32P]GTP in the presence of
varying amounts of non-phosphorylated or phosphorylated Bfa1. Amounts
of [
-32P]GTP bound to Tem1 were assayed before and
after 5-min incubation at 30 °C. In the absence of any Bfa1,
50-55% of bound [
-32P]GTP was lost from Tem1 in 5 min (Fig. 2). This reflects the loss of
[
-32P]GTP by the intrinsic GTP hydrolysis and GTP
dissociation activities of Tem1 (4). In the presence of
non-phosphorylated Bfa1 there was a progressive increase in the amount
of [
-32P]GTP remaining bound to Tem1 resulting from
the inhibition of GTP hydrolysis and GTP dissociation as seen
previously (4). When phosphorylated Bfa1 was used, there was a similar
inhibition of GTP loss from Tem1 (Fig. 2). Thus, phosphorylation by
Cdc5 does not impair the ability of Bfa1 to interact with Tem1 and inhibit GTP dissociation and hydrolysis.
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Fig. 2.
Inhibition of Tem1 GTP dissociation and GTP
hydrolysis by Bfa1. Tem1 was loaded with
[ -32P]GTP in the presence of the concentrations of
Bfa1 indicated. After addition of excess cold nucleotide, radioactive
label bound to Tem1 was assayed at time 0 and after 5 min.
,
phosphorylated Bfa1;
, non-phosphorylated Bfa1. The data represent
the percentage of initial radioactivity remaining after incubation.
Data points are the average of at least two assays, which typically
differed by less than 5%.
-32P]GTP in the
presence of phosphorylated or non-phosphorylated Bfa1. Reactions were
then incubated with or without Bub2. In a kinetics experiment,
Tem-[
-32P]GTP and Bfa1 were incubated with a single
amount of Bub2, and the amount of radioactive GTP bound to Tem1 was
assayed at frequent intervals during incubation at 30 °C. The
addition of Bub2 with non-phosphorylated Bfa1 caused the expected loss
of radioactivity (Fig. 3A),
which is due to stimulation of Tem1 GTP hydrolysis by Bfa1/Bub2 GAP
activity (4). When phosphorylated Bfa1 was used in parallel assays with
Bub2, the kinetics of the GAP activity were clearly reduced by ~50%.
This level of inhibition was repeatedly detected in multiple assays of
GAP activity where levels of filter binding were highly reproducible
and varied by no more than 5%. The possibility that the
phosphorylation status of Bfa1 was affected by the GAP assay itself was
excluded by the observation that there was no change in the level of
Bfa1 phosphorylation during the GAP reaction (data not shown). In the
absence of any Bub2, ~85-90% of the radioactive
[
-32P]GTP remained bound to Tem1 throughout (Fig.
3A). This high level of Tem-[
-32P]GTP
results from the inhibition of the intrinsic GTP dissociation and
GTPase activities of Tem1 by Bfa1 as described above (Fig. 2).
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Fig. 3.
Phosphorylated Bfa1 has reduced GAP
activity. A, Tem1 was incubated in the presence of 1.6 µM phosphorylated ( ,
) or non-phosphorylated Bfa1
(
,
). Then Bub2 (100 ng) was added (
,
) or not (
,
),
and the time course of GTP hydrolysis was followed in filter binding
assays. B, increasing amounts of Bub2 were added to
Tem1-[
-32P]GTP in the presence of 1.6 µM
phosphorylated (
) and non-phosphorylated Bfa1 (
). Amounts of
radioactive label bound to Tem1 were assayed before and after 15-min
incubation at 30 °C. The data represent the percentage of initial
radioactivity remaining after incubation. Data points are the average
of at least two assays, which typically differed by less than
5%.
-32P]GTP from Tem1 occurred from control reactions
incubated with no Bub2, whether or not Bfa1 was phosphorylated.
However, addition of Bub2 resulted in the expected GAP activity as
judged by the progressive loss of [
-32P]GTP from Tem1
as Bub2 concentrations increased. Significantly, when Bfa1 had been
phosphorylated by Cdc5 approximately twice the amount of Bub2 was
required to produce the same GAP activity compared with
non-phosphorylated Bfa1. Thus, both kinetic and a concentration
dependence assays demonstrate that phosphorylation of Bfa1 by Cdc5
reduces the ability of Bfa1 to function as a GAP in association with Bub2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
bfa1 mutants, at least without spindle perturbations. Therefore, timely MEN activity is not only provided by
inhibition of Bfa1 as
bfa1 cells have a permanent rather
than a temporal loss of GAP activity. Further regulatory input comes from Lte1, which is a putative positive regulator for Tem1 (11). Although activity of Lte1 is dispensable under normal growth conditions (11, 29), disruptions elsewhere in the overall activation of the MEN
can make Lte1 activity essential (14). Furthermore, recent data also
implicate PAK (p21-activated kinase)-like kinases in the
positive regulation of mitotic exit (30). Thus, there seems to be a
high degree of redundancy in the overall control of mitotic exit which
is governed by the antagonistic and opposing effects of a number of
regulatory events, of which phosphorylation of Bfa1 is just one example.
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ACKNOWLEDGEMENTS |
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We thank Sanne Jensen and Geoffroy de Bettignies for advice and encouragement and Katrin Rittinger and Edward Wheatley for help in Tem1 preparation. We are grateful to Matthew Sullivan and Frank Uhlmann for plasmids and advice.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by Association for International Cancer Research Grant
99-008.
¶ To whom correspondence should be addressed: Division of Yeast Genetics, National Inst. for Medical Research, Mill Hill, London NW7 1AA, Great Britain. E-mail: ssedgwi@nimr.mrc.ac.uk.
Published, JBC Papers in Press, March 10, 2003, DOI 10.1074/jbc.C300059200
2 L. H. Johnston, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: MEN, mitotic exit network; GAP, GTPase-activating protein; FEAR, Cdc14 early anaphase release; GST, glutathione S-transferase; MBP, myelin basic protein; PAK, p21-activated kinase; SIN, septation initiation network.
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