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J. Biol. Chem., Vol. 277, Issue 38, 35176-35182, September 20, 2002
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From the LBCMCP-CNRS UMR5088, Université Paul Sabatier, 118 route de Narbonne, 31077 Toulouse, France
Received for publication, May 7, 2002, and in revised form, June 18, 2002
CDC25B phosphatases are essential regulators that
control cyclin-dependent kinases activities at the entry
into mitosis. In this study, we demonstrate that serine 146 is required
for two crucial features of CDC25B1. It is essential for CDC25B1 to
function as a mitotic inducer and to prevent CDC25B1 export from the
nucleus. We also show that serine 146 is phosphorylated in
vitro by CDK1-cyclin B. However, phosphorylation of CDC25B does
not stimulate its phosphatase activity, and mutation of serine 146 had
no effect on its catalytic activity. Serine 146 phosphorylation is
proposed to be a key event in the regulation of the CDC25B function in
the initiation of mammalian mitosis.
The first member of the CDC25 family was identified in the fission
yeast Schizosaccharomyces pombe as a
dose-dependent inducer of mitosis (1). Since then,
homologous regulators have been found in every eukaryotic organism
examined. Meanwhile, the biochemical properties of CDC25 were
deciphered and its essential role in the control of the activation of
CDC2 by dephosphorylation was characterized. CDC25 is a dual
specificity phosphatase that directly activates the CDC2 kinase at the
G2/M transition by dephosphorylating tyrosine 15 and
threonine 14 (2). In yeast, there is only one form of CDC25, whereas in
human cells three CDC25 phosphatase genes have been identified (3-5).
Although they share about 50% similarity at the protein level, they
are involved in specific regulatory processes. CDC25C and CDC25B are
thought to activate CDK1-cyclin complexes at the
G2/M transition, and CDC25A regulates G1/S
complexes, respectively (6-8). In fact, the exact role of CDC25B is
still controversial (9). On the basis of antisense oligonucleotide
studies, it has been proposed that CDC25B is required for progression
in S phase (10). Other reports are more in favor of a role for CDC25B
in late G2 as a regulator of centrosomal microtubule
nucleation (11) and as a starter of mitosis (9, 12). Ablation of CDC25B
by microinjection of specific antibodies blocks cell cycle progression
by inhibition of entry into mitosis (13). Since several different
isoforms of CDC25B have been detected in human cells (14, 15) and since
the above mentioned studies made no distinction between CDC25B
variants, we cannot exclude the possibility that each isoform has a
specific function at a particular stage of the cell cycle or in a
defined subcellular compartment.
The activity of CDC25 phosphatases is regulated both at the
translational and the post-translational levels. CDC25A and CDC25B expression appears to be cell cycle-regulated, whereas CDC25C is fairly
constant. Phosphorylation of CDC25 is an important regulator of its
phosphatase activity. CDC25C undergoes extensive phosphorylation of its
NH2-terminal regulatory domain at mitosis, and this
strongly stimulates its catalytic activity toward CDK1-cyclin B, thus
creating a positive feedback loop (16, 17). Similarly, CDC25A is
phosphorylated and activated by CDK2-cyclin E at the G1/S
transition and in turn dephosphorylates and fully activates that
complex (18). It has been shown that CDC25B is phosphorylated in cell
extracts prepared from cells in S-phase to mitosis, and it has been
proposed that it is consequently activated (13, 19). CDC25B is an
unstable protein, and phosphorylation also participates in the
regulation of its degradation (12); it is degraded in a
proteasome-dependent manner upon phosphorylation by CDK1-cyclin
A (20).
As already mentioned, we and others have identified at least three
splice variants of CDC25B (14, 15). These three isoforms differ by the
presence or the absence of two peptides, peptide A (14 residues) and
peptide B (41 residues), which are both located in the amino terminus
regulatory region of the phosphatase. These two peptides lie in domains
that are fairly well conserved between evolutionarily distant members
of the CDC25 family (14). In this study, we show that serine 146, a
phosphorylation site located within domain B, is essential for the
mitotic inducer activity associated with the nuclear retention of CDC25B1.
DNA Constructs--
pET14b- and pcDNA3-derived
plasmids containing CDC25B and the N-terminal deletion mutants are
described elsewhere (14, 20-22). The serine 146 mutant was generated
by PCR. pGEX-2T containing the entire open reading frame of human
CDC25C (16) was a gift from Dr. G. Draetta (European Institute
of Oncology, Milan, Italy).
Cell Culture Conditions--
HeLa cells and U2OS cells were
grown as previously described (21). To obtain cells arrested in
mitosis, HeLa cells were incubated for 16 h in the presence of 250 ng/ml nocodazole (Sigma). Lovastatine and hydroxyurea were used at a
concentration of 40 µM and 10 mM,
respectively, to obtain cells either in G1 or at the
G1 to S transition (14). HeLa cells were transfected
using Exgen-500 (Euromedex) following the manufacturer's instructions.
The U2OS cell line conditionally expressing HA-tagged CDC25B1 (under
the control of the tet promoter) is described
elsewhere.2 Expression of
CDC25B1 was induced by tetracycline removal from the culture
medium, and cells were harvested 24 h latter.
Cell Lysate and Immunoprecipitation--
Exponentially growing
cells, drug-arrested cells, or transfected HeLa cells were lysed in LB
buffer (50 mM Tris, pH 7.5, 250 mM NaCl, 0.1%
Triton X-100, 5 mM EDTA, 1 mM dithiothreitol)
in presence of protease inhibitors: leupeptin (5 µg/ml), aprotinin (5 µg/ml), trypsin inhibitor (10 µg/ml),
L-1-tosylamido-2-phenylethyl chloromethyl ketone (20 µg/ml), 1-chloro-3-tosylamido-7-amino-2-heptanone (20 µg/ml),
phenylmethylsulfonyl fluoride (17 µg/ml), calpain inhibitor I (20 µg/ml), and phosphatase inhibitors (50 mM NaF and 0.1 mM Na3VO4) (except for phosphatase
assays) for 30 min on ice and centrifuged at 14,000 rpm for 10 min at
4 °C. Protein concentration was determined. In the case of
nocodazole-arrested cells, a total cell extract was directly used for
phosphorylation assay, or in some experiments CDK1-cyclin B was
immunoprecipitated using anti-cyclin B
antibody.3 In the case of
transfected cells, HA-tagged CDC25B1 or S146G-CDC25B1 proteins were
immunoprecipitated using 12CA5 mouse monoclonal antibody raised against
the HA epitope (Roche Molecular Biochemicals) and then used in
phosphatase assay or Western blot analysis. Western blot analyses were
performed with the CDC25B C-terminal peptide antibody (1:2000).
Production of Recombinant Proteins--
In
vitro transcription and translation of CDC25B and mutant proteins
were performed using the TNT-quick system (Promega) in the presence or
absence of [35S]methionine.
Spodoptera frugiperda (Sf9) cells were maintained in
Insect X-press medium (BioWhittaker) supplemented with 10%
heat-inactivated fetal calf serum (Eurobio), gentamicin (250 µg/ml),
amphotericin B (2.5 µg/ml), penicillin (100 units/ml), and
streptomycin (100 µg/ml) at 27 °C. For CDK-cyclin complex
expression, Sf9 cells were seeded at a density of 60,000 cells/cm2 1 day prior to the infection and then co-infected
with recombinant baculoviruses encoding human CDK1 (CDC2) and cyclin B. Insect cell extracts were prepared 72 h postinfection as follows.
Cells were lysed with lysis buffer (10 mM Tris, pH 7.5, 25 mM NaCl, 50 mM NaF, 0.1 mM sodium
orthovanadate) using a Dounce homogenizer and then diluted in 4 volumes
of solubilization buffer (25 mM NaH2PO4, pH 7.5, 250 mM NaCl, 10%
glycerol, 0.02% Tween 20) and centrifuged at 100,000 × g for 1 h at 4 °C. GST-CDC25B1, GST-S146G-CDC25B1, and GST-CDC25C fusion proteins produced in bacteria were purified as
previously described (16).
Phosphorylation Assays--
In vitro translated
protein or recombinant GST fusion proteins were immunoprecipitated with
an anti-CDC25B polyclonal antibody directed against a C-terminal
peptide of CDC25B and incubated with 15 µg of cellular extract from
baculovirus-infected Sf9 cells or 200 µg of different
cellular extracts, respectively, in kinase assay buffer (16, 20)
containing 5µCi of [ Phosphatase Activity Assay--
Recombinant GST fusion proteins
(CDC25B1, S146G-CDC25B1, and CDC25C) were incubated with 400 µg of
nocodazole-arrested HeLa cells extracts in kinase assay buffer in
presence of 1 mM ATP, 10 µg of creatine kinase, and 10 mM creatine phosphate for 30 min at 30 °C. Recombinant
proteins were recovered on glutathione beads, washed three times with
LB buffer in the absence of phosphatase inhibitors (NaF and
Na3VO4), and washed once with phosphatase buffer (30 mM Tris pH 8.2, 75 mM NaCl, 0.67 mM EDTA, 0.033% bovine serum albumin, 1 mM
dithiothreitol). CDC25 B phosphatase activity was determined using
fluorescein diphosphate (Molecular Probes, Inc., Eugene, OR) as
substrate at the final concentration of 20 µM for 30 min
at room temperature as described (24). Fluorescence was monitored using
a multiwell plate reader (Fluoroskan Ascent, Labsystems; excitation
filter, 485 nm; emission filter, 538 nm). The phosphatase activity of
HA-CDC25B1 or HA-S146G-CDC25B1 expressed in HeLa cells was determined
as described above after immunoprecipitation from cell lysates with
anti-HA antibody.
Immunofluorescence--
HeLa cells seeded on glass coverslips
were transfected, and cells were fixed 24 h latter using 3.7%
formaldehyde and then permeabilized with 0.25% Triton X-100 and cold
methanol. HA-CDC25B1 and HA-mutants were detected by incubating first
the coverslips with 12CA5 mouse monoclonal (1:2500) (Roche Molecular
Biochemicals), followed by a second incubation with
Alexa-594-conjugated goat anti-mouse antibody (1:500) (Molecular
Probes). In all cases, DNA was visualized using Hoechst 33258 dye at 1 µg/ml (Sigma). The mitotic index was calculated by monitoring the
cells (transfected or not) presenting a condensed chromatin aspect.
Ability of CDC25B1 to Induce Mitosis Is Abolished by the S146G
Mutation--
In order to study the in vivo activity of
CDC25B1 phosphatase, we expressed an HA epitope-tagged version of the
wild type CDC25B1 in asynchronous HeLa cells under the control of the
cytomegalovirus promoter. The transfected cells were identified and
monitored by immunofluorescence staining with anti-HA antibodies. As
shown in Fig. 1, CDC25B1 acted as an
inducer of mitosis, since, 24 h after transfection, about 20% of
the cells expressing the CDC25B1 phosphatase displayed a condensed
chromatin aspect. In contrast, this mitotic inducing activity was
abolished with CDC25B1-S146G, a mutant protein that was tested in the
course of a systematic study on putative phosphorylation sites. This
serine, located in the alternately spliced B domain, is
reminiscent of a CDK consensus phosphorylation site and is conserved in
most of the CDC25 sequences from various eukaryotic species (14).
Serine 146 Is a Major Phosphorylation Site--
Using a cell line
conditionally expressing HA-CDC25B1, we show that, as inferred from its
electrophoretic mobility shift, CDC25B1 was strongly phosphorylated in
cells that were arrested at mitosis by a nocodazole treatment (Fig.
2A, upper
panel). This effect correlated with a high level of
CDK1-cyclin B kinase activity (Fig. 2A, lower
panel). We next investigated whether serine 146 was a
phosphorylation site for the CDK1/CDC2 protein kinase. Using a set of
amino-terminally truncated CDC25B1 proteins generated by site-directed
mutagenesis (Fig. 2B), we found that deletion of the first
274 residues fully abolished the phosphorylation of the protein,
indicating that major phosphorylation sites for CDK1-cyclin B are all
located in that amino-terminal region. Deletion of 186 residues of
CDC25B already had a dramatic effect on its phosphorylation, but a
shorter deletion (
We next examined whether the synthetic 42-residue peptide containing
serine 146 that is located between residues 108 and 186 was
phosphorylated in vitro by CDK1-cyclin B. As shown in Fig. 3A, CDK1-cyclin B
immunoprecipitate readily phosphorylated it (lanes
1 and 2), whereas control immunoprecipitates did
not (lanes 3 and 4). The serine 146 residue was then mutated to a nonphosphorylatable residue, and the
kinetics of phosphorylation of both S146G-CDC25B1 mutant and wild type
CDC25B1 by CDK1-cyclin B were analyzed. As shown in the autoradiography
(Fig. 3B, left panels), the
S146G-CDC25B1 mutant did not display the electrophoretic mobility shift
that rapidly occurs upon phosphorylation of wild-type CDC25B1 by
CDK1-cyclin B and was less efficiently phosphorylated. Consonant with
this observation, quantification of the level of 32P
incorporation (Fig. 3B, right panel)
indicated that the overall phosphorylation of S146G-CDC25B1 was about
50% of that observed in the wild-type CDC25B1 protein. To confirm this
observation, an in vitro phosphorylation assay with
recombinant GST-CDC25B1 proteins and mitotic HeLa cell extract obtained
by nocodazole treatment was performed. As shown in Fig. 3C,
upon incubation, wild type CDC25B1 displayed changes in its
electrophoretic mobility that were only partially observed in the case
of the S146G mutant protein, the band of highest electrophoretic
mobility and the additional smear being absent. When the HeLa cell
extract was depleted from CDK1-cyclin B activity by cyclin B
immunoprecipitation (Fig. 3E) prior to the incubation with
CDC25B1, the electrophoretic mobility shift of the protein was fully
abolished (Fig. 3D, lane 3). Taken
together, these data indicate that serine 146 is phosphorylated in vitro by the CDK1-cyclin B complex.
CDC25B1 Is Not Activated upon Phosphorylation by CDK1-Cyclin
B--
Several reports have described the activation of CDC25A and
CDC25C phosphatases by CDC2-dependent phosphorylation (16,
18). On the basis of indirect evidence, it was also proposed that
CDC25B is similarly regulated (13). We therefore examined this
hypothesis and the possibility that serine 146 participates in the
activation of CDC25B1. To address this question, we prepared both
wild-type and S146G mutant recombinant proteins in fusion with GST by
expression in Escherichia coli and affinity purification on
glutathione-Sepharose. A GST-CDC25C protein was also produced in the
same way. These purified proteins were incubated in the presence of
mitotic cell extracts prepared from nocodazole-treated HeLa cells to
allow their phosphorylation by CDK-cyclin complexes (Fig.
4A). The GST fusion proteins
were recovered from the incubation mix by affinity purification, and
the phosphatase activity of CDC25B was then quantified. As shown in
Fig. 4B, we found that the specific activities of unmodified
CDC25B and S146G-CDC25B1 recombinant proteins were identical (271 and
259 units, respectively) and about 9-fold higher than the activity of
CDC25C (28 units). As expected, the activity of recombinant CDC25C was
amplified 6-7-fold upon phosphorylation by a mitotic extract. However,
in contrast and in disagreement with what has been proposed by others
(13), the phosphatase activity of recombinant CDC25B was not increased
following incubation and phosphorylation with a mitotic extract. Thus,
CDC25B activity is not up-regulated upon phosphorylation by CDK1, and
mutation of serine 146 has no effect on the catalytic activity of this phosphatase.
We also next examined whether a down-regulation of the catalytic
activity of CDC25B1 in vivo could account for the
observation that the S146G mutation abolished the mitotic inducing
effect. The phosphatase activity of wild type CDC25B1 and S146G-CDC25B1 was monitored in HeLa cells transfected with the corresponding HA-tagged constructs. After immunoprecipitation with anti-HA
antibodies, the phosphatase activity was measured. As shown in Fig.
4C, the phosphatase activities of these two enzymes were
found to be very similar. Thus, the role of serine 146 in the mitotic
inducer effect of CDC25B1 does not appear to depend on a change of its
catalytic activity.
Serine 146 Regulates the Intracellular Localization of
CDC25B1--
The intracellular localization of CDC25B1 was
investigated by immunofluorescence staining in exponentially growing
HeLa cells transfected with constructs allowing the expression of the
wild-type and the S146G-CDC25B1 mutant proteins. As we previously
reported (21), in the case of wild type CDC25B1 expression, the
percentage of cells displaying either a strictly nuclear or cytoplasmic
localization pattern was 15 and 20%, respectively (Fig.
5A). In contrast, in cells
expressing the S146G-CDC25B1 mutant, the percentage of exclusive nuclear localization was only about 5%, whereas in about 45% of the
cells, the localization of the protein was entirely cytoplasmic. Thus,
it appears that the mutation of serine 146 impairs the ability of
CDC25B1 protein to be located within the nucleus and favors its
retention in the cytoplasmic compartment.
Since it is likely that CDC25 proteins shuttle between cytoplasmic and
nuclear compartments, this defect in nuclear localization may be due
either to altered nuclear import or to deficient nuclear retention. We
further examined this issue using leptomycin B (LMB), a potent CRM1
exportin inhibitor (25). Wild-type CDC25B1 was almost totally retained
in the nucleus in HeLa cells treated with LMB (Fig. 5B).
Similarly, the mutant protein CDC25B1-S146G was also detected in the
nucleus in 86% of the cells, indicating that it was correctly targeted
to the nucleus but not exported because of the presence of leptomycin
B. In contrast, a CDC25B1 mutant protein with an inactivating mutation
of the nuclear localization signal (NLS) (i.e. the
replacement of the KRR motif by AGA (21)) was incapable of being
targeted to the nucleus (Fig. 5B). Thus, we can conclude
that the S146G mutation does not impair entry of CDC25B1 into the
nucleus but alters its retention.
CDC25B1 Mitotic Inducing Effect Is Dependent on Its Nuclear
Localization and Is Abolished by the S146G Mutation--
The effect of
the subcellular localization of CDC25B1 on its activity as an inducer
of mitosis was further examined. This effect was found to be dependent
on the nuclear localization of CDC25B1, since when an NLS-mutant was
expressed, the percentage of cells in mitosis was decreased to 7.7%, a
level similar to that observed in control pcDNA-transfected cells
(Fig. 6). Furthermore, in the presence of
LMB, about 20% of the cells expressing the wild-type CDC25B1 entered
mitosis, whereas only about 8% of the cells expressing the
CDC25B1-S146G mutant did so (Fig. 6).
These results indicate that localization of CDC25B1 into the nucleus is
required for its mitotic inducer function. However, when CDC25B1-S146G
is artificially tethered within the nucleus by LMB, it fails to act as
a mitotic inducer, indicating that this function probably requires an
additional nuclear event that is also dependent on serine 146 (see
"Discussion").
The work presented here stems from the observation that a mutation
of serine 146 glycine abolishes the mitotic inducing effect of the
CDC25B1 phosphatase and reports the experiments that have been
undertaken to identify the molecular basis of that observation.
We have found that serine 146 is one of the major sites of CDC25B1
phosphorylation in vitro by CDK1-cyclin B. Serine 146 is included in the alternately spliced domain B, a 41-residue peptide that
is present in the CDC25B1 and -B3 variants but absent in CDC25B2. As we
reported when CDC25B splicing variants were identified (14), this
domain is conserved between species. In some cases, a threonine residue
replaces the serine, but this phosphorylatable amino acid is always
followed by a proline. Furthermore, this residue is conserved between
the three human CDC25s, and it has recently been shown that alternative
splicing of this domain also exists in the other human CDC25
phosphatase (26). Such a conservation of the genomic organization of
the CDC25 locus is likely to reflect a crucial functional role for this
region of the protein. However, structural data are still missing to
help in clarifying this issue.
We have shown that CDC25B catalytic activity is not up-regulated upon
phosphorylation by CDK1-cyclin B, and its specific activity is already
high when compared with CDC25C. Furthermore, using recombinant
CDK-cyclin complexes produced and purified from baculovirus-infected Sf9 cells (CDK2/E, CDK2/A, CDK1/A), we also did not observe any increase of the catalytic activity of
CDC25B1.4 Thus, in contrast
to what has been reported for CDC25A and CDC25C (16, 18), CDC25B
activity appears to be constitutively high and not up-regulated by
CDK-cyclin complexes.
We demonstrate here that nuclear targeting of CDC25B1 is essential to
activate entry into mitosis. The loss of function of the S146G-CDC25B1
mutant together with its predominant cytoplasmic localization led us to
examine the effect of a nuclear localization signal mutation. We have
shown that the NLS-mutant is fully retained in the cytoplasm and that
it is not active either as a mitotic inducer. Together, these results
indicated that cytoplasmic accumulation of the CDC25B1 phosphatase had
no detectable effect on the triggering of mitotic events. At least as
far as CDC25B1 is concerned, our data do not support the proposal that
cytoplasmic accumulation of CDC25B phosphatase at mitosis triggers
centrosomal microtubule nucleation, whereas CDC25C regulates nuclear
G2/M events (11). In this model, the activity of CDC25B is
suggested to be required to activate the cytoplasmic pool of
CDK1-cyclin B, which is responsible for the earliest changes in
microtubule dynamics. Of course, we cannot exclude the possibility that
another variant of CDC25B may be specifically targeted to CDK1-cyclin B
complexes associated with the cytoskeleton and therefore involved in
the regulation of its dynamics. In fact, the CDC25B3 variant was used
in Gabrielli's work, where it was shown to cause the formation of
abnormal minispindles upon overexpression (11). Under our experimental
conditions (i.e. with CDC25B1) we did not observe these
minispindles; however, we found that the CDC25B3 variant is a less
potent inducer of mitosis than CDC25B1, with only about 10% of cells
in mitosis 24 h after transfection (data not shown).
The data reported in this study demonstrate that CDC25B1 must be
targeted to the nucleus to exert its mitotic inducer effect. This
localization is dependent both on the integrity of its nuclear localization signal and on the integrity of the phosphorylatable serine
146. Nevertheless, the nuclear localization of CDC25B1 is not
sufficient in itself to trigger mitosis, as demonstrated by the lack of
induction of mitosis when the S146G-CDC25B1 mutant is retained in the
nucleus by leptomycin B. This observation indicates the existence of an
additional level of regulation that is essential for access to the
nuclear substrates and that is likely to be dependent on serine 146 phosphorylation. Furthermore, only about 20% of the cells expressing
CDC25B1 entered mitosis even when it was fully retained in the nucleus
in the presence of LMB. This observation strongly suggests that only
a given population of cells is sensitive to the mitotic inducer
effect of the phosphatase. One can suggest that the only cells
targeted are those that have reached a late cell cycle window where
rate-limiting regulatory partners or substrates are available.
As depicted in Fig. 7, we currently have
two working hypotheses to explain our observations. The first is that
the nuclear export sequence of CDC25B1 is masked or altered when serine
146 is phosphorylated by CDK1-cyclin B. A change in the steric
conformation of CDC25B1 would then prevent its interaction with the
export machinery and retain it in the nucleus (Fig. 7A).
Phosphorylation-dependent interaction between cyclin D1 and
the CRM1 exportin (27) and between Pho4 and the Msn5 exportin (28) have
already been reported, suggesting that site-specific phosphorylation
events could either positively or negatively regulate nuclear export.
In the case of CDC25B1, phosphorylation of serine 146 would prevent the
interaction between the nuclear export signal that is located within
the first 40 residues and CRM1. Alternatively, phosphorylation of
serine 146 may induce an interaction between CDC25B1 and an associated protein (Fig. 7B). This interaction would be required for
the phosphatase to remain within the nucleus and would be essential to
perform its function as demonstrated by the observation that the
nuclear retention of S146G-CDC25B1 by LMB is not sufficient to restore
its activity. The identity of the protein that putatively associates
with CDC25B1 remains to be established.
This is the first report of a regulatory mechanism that is specific for
one of the three CDC25B splice variants. Although the exact function of
these variants in cell cycle control remains to be elucidated, one can
speculate that specific phosphorylation events may regulate the
interactions with partners, modulate the subcellular localization, and
therefore have important implications for the regulation of CDC25B
activity. Whether these specific properties are involved in the
oncogenic properties of CDC25B remains to be investigated. However,
since an association with CDC25B2 overexpression has been reported in
several types of tumors (29,
30),5 it is tempting to
speculate that the alteration of CDC25B variant-specific properties
participates in such a process.
We gratefully acknowledge M. Yashida
for the gift of leptomycin B; H. Mazarguil for peptide B synthesis; and
J. M. Darbon, J. P. Javerzat, and J. Smith for comments and suggestions.
*
This work was initiated with support from l'Association
pour la Recherche contre le Cancer and la Fondation pour la Recherche Médicale and is currently funded by la Ligue Nationale Contre le
Cancer.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.
§
Recipient of a postdoctoral fellowship from the Société
de Secours des Amis des Sciences.
Published, JBC Papers in Press, July 9, 2002, DOI 10.1074/jbc.M204430200
2
N. Theis-Febvre, B. Ducommun, and V. Baldin,
submitted for publication.
3
V. Baldin, unpublished data.
4
V. Baldin and C. Cans, unpublished data.
5
C. Toulas and V. Baldin, unpublished results.
The abbreviations used are:
CDK, cyclin-dependent kinase;
HA, hemagglutinin;
GST, glutathione S-transferase;
LMB, leptomycin B;
NLS, nuclear
localization signal.
Nuclear Localization of CDC25B1 and Serine 146 Integrity Are
Required for Induction of Mitosis*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP for 60 min at 30 °C
or cold ATP. The precipitates were subjected to SDS-8% PAGE
electrophoresis. Peptide phosphorylation was performed in the same
kinase assay condition except that we used immunoprecipitated CDK1-cyclin B instead of total cell lysate, and the reaction was carried out in 30 min at 30 °C. In the indicated experiments, histone H1 kinase activity was determined on an
immunoprecipitation performed with anti-cyclin B antibodies on
200 µg of cell extracts (23).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (14K):
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Fig. 1.
Serine 146 modulates CDC25B1 activity
in vivo. Exponentially growing HeLa cells were
transfected with pcDNA3 (vector) or constructs allowing
the expression of an HA-tagged version of wild-type CDC25B1
(WT) and S146G-CDC25B1 mutant. The cells were fixed 24 h after transfection and stained to detect the expression of CDC25B1 by
immunofluorescence using monoclonal anti-HA antibodies. The number of
CDC25B1-positive cells displaying a condensed chromatin aspect was
determined. The results presented are the average of three independent
experiments.
108) had no detectable impact (Fig.
2C). These results indicate that the region ranging from
amino acids 108 to 186 comprises one or several residues that is (are)
directly phosphorylated. Alternatively, this region may be required for
the phosphorylation of a site located elsewhere in the protein.
View larger version (24K):
[in a new window]
Fig. 2.
CDC25B1 is phosphorylated in vivo
and in vitro by CDK1-cyclin B. A,
upper panel, analysis of CDC25B1 expression and
mobility in U2OS cells conditionally expressing CDC25B1. 100 µg of
total cell extract from exponentially growing (Expo.)-,
lovastatine (Lova.)-, hydroxyurea (HU)-, and
nocodazole (Noco.)-treated cells induced or not
(NI) to produce CDC25B1 were analyzed by Western blot using
an anti-CDC25B antibody. Lower panel, CDK1-cyclin
B kinase activity. 200 µg of total cell extract, used in
A, were immunoprecipitated with an anti-cyclin B antibody,
and phosphorylation of histone H1 was monitored. B,
schematic representation of the CDC25-B1 amino-terminal deletion
mutants. The N terminus poly-His tag is indicated (gray
box). Numbering indicates the number of residues
that have been deleted at the N terminus of the protein. The position
of the B domain is indicated with an arrowhead.
C, in vitro translated wild-type CDC25B1 and
amino-terminal truncated mutants were incubated for 60 min at 30 °C
with CDK1-cyclin B. Proteins were visualized after electrophoresis, and
the incorporation of 32P was quantified. Phosphorylation
was normalized to the concentration of each CDC25B protein estimated on
a parallel Western blot (data not shown). Background phosphorylation
obtained with a control cell lysate was subtracted from the data that
are presented. The S.E. from three independent experiments is
indicated.
View larger version (37K):
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Fig. 3.
Serine 146 is phosphorylated by CDK1-cyclin
B. A, phosphorylation of domain B peptide (41 residues) by a
cyclin B immunoprecipitate obtained from Sf9 insect cell lysates
producing (lanes 1 and 2) or not
producing (lanes 3 and 4) CDK1-cyclin
B complexes. The assay was performed using 2 µg (lanes
1 and 3) and 4 µg (lanes
2 and 4) of domain B peptide for 30 min at
30 °C. The samples were run on a 20% gel and autoradiographed.
B, kinetics of phosphorylation of identical amounts of
unlabeled in vitro translated wild-type and S146G CDC25B1 by
CDK1-cyclin B-expressing Sf9 cell lysates. The samples were run
on an 8% gel and autoradiographed (left panel).
The quantification of the incorporation is shown in the
right panel. C, recombinant GST-wild
type (WT) and GST-S146G mutant CDC25B1 were incubated for 0, 15, or 30 min at 30 °C in the presence of mitotic HeLa cell extract
(200 µg) obtained from nocodazole-treated cells. The samples were run
on an 8% acrylamide gel (30:0.2) and transferred, and Western blot
detection was performed using anti-CDC25B polyclonal antibody.
D, recombinant GST-wild type CDC25B1 was incubated
(lanes 2 and 3) or not
(lane 1) for 60 min at 30 °C in the presence
of mitotic HeLa cell extract (200 µg) obtained from
nocodazole-treated cells. In lane 3, the extract
was subjected to a cyclin B immunodepletion prior to the incubation
with CDC25B1. E, histone H1 kinase activity was determined
by immunoprecipitation using anti-cyclin B antibodies on mitotic HeLa
cell extracts (200 µg) immunodepleted (lane 2)
or not (lane 1) in CDK1-cyclin B complexes.
View larger version (21K):
[in a new window]
Fig. 4.
CDC25B1 is not activated by phosphorylation.
A and B, purified GST-CDC25B1, GST-CDC25B1-S146G,
and GST-CDC25C recombinant proteins were incubated (+) or not ( 
) with
400 µg of mitotic HeLa cell extract for 30 min at 30 °C obtained
from nocodazole-treated cells and then recovered on
glutathione-Sepharose beads. The beads were washed as indicated under
"Experimental Procedures," and then CDC25 phosphatase activity was
determined using fluorescein diphosphate as substrate (B).
In A, an aliquot of each sample was run on 8% SDS-PAGE and
then processed for immunodetection of CDC25B or -C to visualize the
molecular weight shift induced by phosphorylation. C, HeLa
cells were transfected as described in the legend to Fig. 1 and
harvested after 24 h. The CDC25B1 wild type or mutant phosphatase
was immunoprecipitated using monoclonal anti-HA antibodies, and its
activity was determined using fluorescein diphosphate as substrate. The
phosphatase activity was normalized to the same amount of CDC25B
proteins, measured by Western blot analysis after immunoprecipitation.
The S.E. from three independent experiments is indicated.
View larger version (14K):
[in a new window]
Fig. 5.
Serine 146 regulates intracellular
localization of CDC25B1. A, asynchronous HeLa cells were
transiently transfected with pcDNA3 vector, allowing
the expression of an HA epitope-tagged version of wild-type or S146G
CDC25B1 proteins. Cells were fixed 24 h after transfection and
stained with anti-HA antibodies. Quantification of the intracellular
localization is shown. The percentages of transfected cells expressing
wild type CDC25B1 (white bars) or S146G CDC25B1
(black bars) and displaying exclusive nuclear
staining or cytoplasmic staining were obtained from five independent
experiments with at least 200 transfected cells examined per
experiment. The S.E. is indicated. Microphotographs of cells displaying
typical cytoplasmic or nuclear staining are presented. B,
asynchronous HeLa cells were transiently transfected with
pcDNA3 vector, allowing the expression of wild type
CDC25B1 or NLS-CDC25B1 (mutation of the KRR motif to AGA) or
S146G-CDC25B1 mutant proteins. Cells were either untreated or treated
with 20 ng/ml LMB for 2 h prior to fixation 24 h after
transfection. Cells were stained with anti-HA antibodies, and the
intracellular localization of each protein was determined. The
percentage of cells displaying an exclusive nuclear staining is
indicated. The values indicated are the mean of three independent
experiments.
View larger version (16K):
[in a new window]
Fig. 6.
CDC25B1 activity is dependent on its nuclear
localization. Asynchronous HeLa cells were transiently transfected
with pcDNA3 vector, allowing the expression of wild
type CDC25B1, NLS-CDC25B1, or S146G-CDC25B1 mutant proteins. Cells were
either untreated or treated with 20 ng/ml LMB for 2 h prior to
fixation 24 h after transfection and stained with anti-HA
antibodies and Hoechst. The percentage of CDC25B1-positive cells (wild
type or mutants) displaying condensed chromatin aspect was determined.
The data are derived from three independent experiments. The S.E. is
indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (14K):
[in a new window]
Fig. 7.
Current working model. CDC25B shuttling
between the cytoplasm and the nucleus is dependent on an NLS and a
nuclear export signal (NES) (21). A, following
phosphorylation of Ser146 by CDK1-cyclin B, the nuclear
export signal sequence is masked, and CDC25B1 is therefore retained in
the nucleus. B, phosphorylation of Ser146 by
CDK1-cyclin B allows the interaction of CDC25B1 with a regulatory
partner that sequesters it in the nucleus.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.:
33-5-61-55-81-10; Fax: 33-5-61-55-81-09; E-mail: ducommun@cict.fr.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Russell, P.,
and Nurse, P.
(1986)
Cell
45,
781-782[CrossRef][Medline]
[Order article via Infotrieve]
2.
Morgan, D. O.
(1995)
Nature
374,
131-134[CrossRef][Medline]
[Order article via Infotrieve]
3.
Galaktionov, K.,
and Beach, D.
(1991)
Cell
67,
1181-1194[CrossRef][Medline]
[Order article via Infotrieve]
4.
Nagata, A.,
Igarashi, M.,
Jinno, S.,
Suto, K.,
and Okayama, H.
(1991)
New Biol.
3,
959-968[Medline]
[Order article via Infotrieve]
5.
Sadhu, K.,
Reed, S. I.,
Richardson, H.,
and Russell, P.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
5139-5143 6.
Hoffmann, I.,
and Karsenti, E.
(1994)
J. Cell Sci.
18,
75-79
7.
Jessus, C.,
and Ozon, R.
(1995)
in
Progress in Cell Cycle Research
(Meijer, L.
, Guidet, S.
, and Tung, H. Y. L., eds), Vol. 1
, pp. 215-228, Plenum Press, New York
8.
Nilsson, I.,
and Hoffman, I.
(2000)
in
Prog. Cell Cycle Res.
(Meijer, L.
, Jézéquel, A.
, and Ducommun, B., eds), Vol. 4
, pp. 107-116, Plenum Press, New York
9.
Karlsson, C.,
Katich, S.,
Hagting, A.,
Hoffmann, I.,
and Pines, J.
(1999)
J. Cell Biol.
146,
573-584 10.
Garner-Hamrick, P. A.,
and Fisher, C.
(1998)
Int. J. Cancer.
76,
720-728[CrossRef][Medline]
[Order article via Infotrieve]
11.
Gabrielli, B. G., De,
Souza, C. P. C.,
Tonks, I. D.,
Clarck, J. M.,
Hatward, N. K.,
and Ellem, K. A. O.
(1996)
J. Cell. Sci.
109,
1081-1093[Abstract]
12.
Nishijima, H.,
Nishitani, H.,
Seki, T.,
and Nishimoto, T.
(1997)
J. Cell Biol.
138,
1105-1116 13.
Lammer, C.,
Wagerer, S.,
Saffrich, R.,
Mertens, D.,
Ansorge, W.,
and Hoffman, I.
(1998)
J. Cell Sci.
111,
2445-2453[Abstract]
14.
Baldin, V.,
Cans, C.,
Superti-Furga, G.,
and Ducommun, B.
(1997)
Oncogene
14,
2485-2495[CrossRef][Medline]
[Order article via Infotrieve]
15.
Forrest, A. R.,
McCormack, A. K.,
DeSouza, C. P.,
Sinnamon, J. M.,
Tonks, I. D.,
Hayward, N. K.,
Ellem, K. A.,
and Gabrielli, B. G.
(1999)
Biochem. Biophys. Res. Commun.
260,
510-515[CrossRef][Medline]
[Order article via Infotrieve]
16.
Hoffmann, I.,
Clarke, P.,
Marcote, M. J.,
Karsenti, E.,
and Draetta, G.
(1993)
EMBO J.
12,
53-63[Medline]
[Order article via Infotrieve]
17.
Izumi, T.,
and Maller, J. L.
(1993)
Mol. Cell. Biol.
4,
1337-1350
18.
Hoffmann, I.,
Draetta, G. F.,
and Karsenti, E.
(1994)
EMBO J.
13,
4302-4310[Medline]
[Order article via Infotrieve]
19.
Gabrielli, B. G.,
Clarck, J. M.,
McCormack, A.,
and Ellem, K. A. O.
(1997)
J. Biol. Chem.
272,
28607-28614 20.
Baldin, V.,
Cans, C.,
Knibiehler, M.,
and Ducommun, B.
(1997)
J. Biol. Chem.
272,
32731-32735 21.
Davezac, N.,
Baldin, V.,
Gabrielli, B.,
Forrest, A.,
Theis-Febvre, N.,
Yashida, M.,
and Ducommun, B.
(2000)
Oncogene
19,
2179-2185[CrossRef][Medline]
[Order article via Infotrieve]
22.
Mils, V.,
Baldin, V.,
Pinta, I.,
Goubin, F.,
Papin, C.,
Waye, M.,
Eychene, A.,
and Ducommun, B.
(2000)
Oncogene
19,
1257-1265[CrossRef][Medline]
[Order article via Infotrieve]
23.
Pagano, M.,
Pepperkok, R.,
Lukas, J.,
Baldin, V.,
Bartek, J.,
and Draetta, G.
(1993)
J. Cell Biol.
121,
101-111 24.
Rice, R.,
Rusnak, J. M.,
Yokokawa, F.,
Yokokawa, S.,
Messner, D. J.,
Boynton, A. L.,
Wipf, P.,
and Lazo, J. S.
(1997)
Biochemistry
36,
15965-15974[CrossRef][Medline]
[Order article via Infotrieve]
25.
Kudo, N.,
Wolff, B.,
Sekimoto, T.,
Schreiner, E. P.,
Yoneda, Y.,
Yanagida, M.,
Horinouchi, S.,
and Yoshida, M.
(1998)
Exp. Cell Res.
242,
540-547[CrossRef][Medline]
[Order article via Infotrieve]
26.
Wegener, S.,
Hampe, W.,
Herrmann, D.,
and Schaller, H. C.
(2000)
Eur J. Cell Biol.
79,
810-815[CrossRef][Medline]
[Order article via Infotrieve]
27.
Alt, J. R.,
Cleveland, J. L.,
Hannink, M.,
and Diehl, J. A.
(2000)
Genes Dev.
14,
3102-3114 28.
Kaffman, A.,
Rank, N. M.,
O'Neill, E. M.,
Huang, L. S.,
and O'Shea, E. K.
(1998)
Nature
396,
482-486[CrossRef][Medline]
[Order article via Infotrieve]
29.
Hernandez, S.,
Hernandez, L.,
Bea, S.,
Pinyol, M.,
Nayach, I.,
Bellosillo, B.,
Nadal, A.,
Ferrer, A.,
Fernandez, P. L.,
Montserrat, E.,
Cardesa, A.,
and Campo, E.
(2000)
Int. J. Cancer
89,
148-152[CrossRef][Medline]
[Order article via Infotrieve]
30.
Takemasa, I.,
Yamamoto, H.,
Sekimoto, M.,
Ohue, M.,
Noura, S.,
Miyake, Y.,
Matsumoto, T.,
Aihara, T.,
Tomita, N.,
Tamaki, Y.,
Sakita, I.,
Kikkawa, N.,
Matsuura, N.,
Shiozaki, H.,
and Monden, M.
(2000)
Cancer Res.
60,
3043-3050
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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