 |
INTRODUCTION |
Cell cycle events are regulated by the
cyclin-dependent kinases
(CDKs)1 (for reviews, see
Refs. 1-5). Cyclins are responsible for kinase activation, substrate
specificity, and intracellular localization (6). The key regulator of
the G2/M transition is the M phase-promoting factor (MPF),
a complex constituted of the catalytic subunit p34cdc2 (7-9)
and the regulatory subunit cyclin Bcdc13 (6, 10-11).
Activation of MPF at the onset of mitosis is associated with
modifications of phosphorylation of its two subunits (11-13). In late
prophase, Cdc2 is phosphorylated on three residues: Thr-14, Tyr-15, and
Thr-161. Thr-161 phosphorylation is catalyzed by the Cdc2-activating
kinase identified as a complex between Cdk7 (MO15), cyclin H, and MAT1
(14). This phosphorylation is necessary for Cdc2 activity (15, 16). The
Thr-14 and Tyr-15 residues of Cdc2 are located in the ATP-binding
pocket of the kinase (17). Following cyclin B binding to Cdc2 (6, 12,
18), Cdc2 becomes phosphorylated on Thr-14 by the Myt1 kinase (19, 20)
and on Tyr-15 by the Wee1/Mik1 or Myt1 kinases (20-22). In yeast, only Tyr-15 is phosphorylated in G2 (23). Cdc2 activation at
prophase/metaphase transition requires dephosphorylation of both Thr-14
and Tyr-15. These dephosphorylations occur in two successive steps:
first Thr-14 and then Tyr-15 (24). The Cdc25 dual-specificity
phosphatase dephosphorylates both residues (reviewed in Ref. 25). The
Pyp3 tyrosine phosphatase acts on Tyr-15 in fission yeast (26). A positive feedback loop originating from Cdc2 leads to the autocatalytic amplification of the complex (27, 28). This is possibly partially generated by the singly phosphorylated form of Cdc2, dephosphorylated on Thr-14/phosphorylated on Tyr-15 (24).
Simultaneously with Cdc2 dephosphorylation, cyclin B becomes
phosphorylated as described in a variety of models such as yeast (6),
starfish oocytes (13), sea urchin eggs (11), goldfish oocytes (29),
Xenopus oocytes (30), and human cells (31). The residues
phosphorylated in cyclin B1 have been identified in
Xenopus: Ser-2, Ser-94, Ser-96, Ser-101, and Ser-113 (32, 33). Mutational studies (29, 32, 33) have suggested that cyclin B
phosphorylation is required neither for activity of the Cdc2 kinase,
for Cdc2 binding, nor for cyclin B destruction in anaphase. More
recently, Li et al. (34) showed that if the residues were
mutated to nonphosphorylatable residues, the Cdc2-cyclin B complex does
not migrate from the cytoplasm to the nucleus and therefore loses its
MPF activity. The kinase(s) responsible for cyclin B phosphorylations
has not been identified so far. Autophosphorylation of the cyclin B by
the Cdc2 kinase itself has been suggested in sea urchin eggs (11) and
in Xenopus oocytes (35). Furthermore, mitogen-activated
protein kinase is able to phosphorylate Ser-94 or Ser-96 of
Xenopus cyclin B1 (32). Cyclin B2
has also been found to be a substrate for the Xenopus
c-mos proto-oncogene product in vitro (36), but
not in vivo (37). Recently, a kinase (Cyk) phosphorylating
only one cyclin B2 residue (Ser-53) in vitro has been partially characterized in Xenopus (38).
Taking advantage of the high and natural synchrony of starfish oocytes,
we have investigated the regulation of cyclin B phosphorylation. Cyclin
B phosphorylation is easily detected on an immunoblot following SDS-PAGE; compared with the unphosphorylated isoform, phosphorylated cyclin B displays a retarded migration ("shift"). We first clearly show in vitro that cyclin B phosphorylation is not required
for Cdc2 kinase activity. Second, in vivo as well as
in vitro, the cyclin B phosphorylation shift requires Cdc2
activation (dephosphorylation of both Thr-14 and Tyr-15 inhibitory
residues). Then in vitro experiments using chemical
inhibitors of CDKs, like olomoucine (39), roscovitine (40), and
purvalanol (41), and cyclin B phosphopeptide mapping demonstrate that
Cdc2 is the kinase responsible for the cyclin B phosphorylation shift
observed at metaphase in starfish oocytes. Furthermore, two experiments
clearly show that Cdc2 phosphorylates its own associated cyclin B
subunit, rather than the cyclin B subunit from another complex. Cyclin
B phosphorylation thus occurs in an intra-MPF complex fashion.
 |
EXPERIMENTAL PROCEDURES |
Chemicals and Reagents
Sodium orthovanadate, 1-methyladenine (1-MeAde), EGTA, EDTA,
MOPS,
-glycerophosphate, dithiothreitol, sodium fluoride,
p-nitrophenylphosphate, leupeptin, aprotinin, soybean
trypsin inhibitor, benzamidine, vitamin K3, ATP, and Tween
20 were obtained from Sigma. Nonidet-P40 was purchased from Fluka.
Green A-agarose beads were obtained from Amicon. Goat anti-mouse IgG
(horseradish peroxidase-coupled) was purchased from Bio-Rad.
[
-32P]ATP (PB168; 3000 Ci/mmol; 1 mCi/ml),
32P (40 mCi/ml), ACS scintillation fluid, hyperfilm MP, and
ECL detection reagents were purchased from Amersham Pharmacia Biotech. Purified protein phosphatase 2A (PP2A1) for Cdc2 treatment
was generously donated by Dr. R. W. MacKintosh (University of
Dundee, UK), and another PP2A1 for cyclin B treatment was
kindly donated by Dr. H. Y. L. Tung (University of Dundee,
UK). The Escherichia coli strains expressing GST-Cdc25A and
GST-Pyp3 were provided by Dr. K. Galaktionov and Dr. D. Beach (Cold
Spring Harbor Laboratory, Cold Spring Harbor, NY), and Dr. J. Millar
and Dr. P. Russell (La Jolla), respectively. Cdk2-cyclin E was kindly
provided by Dr. W. Harper (Houston). The inhibitor, purvalanol, was
generously donated by Dr. N. Gray (Berkeley). Monoclonal anti-PSTAIRE
antibodies (raised against the NH2-EGVPSTAIRESLLKEGGC-COOH
peptide) and monoclonal anti-cyclin Bcdc13 (Bufo)
antibodies were generously donated by Dr. M. Yamashita (Sapporo).
Buffers
Calcium-free artificial sea water (CaFASW) contained 452.2 mM NaCl, 10.08 mM KCl, 29.8 mM
MgCl2 (6H2O), 17.2 mM
MgSO4 (7H2O), 10 mM Tris-HCl, pH 8 (42).
High salt Barth (HSB) medium contained 110 mM NaCl, 2 mM KCl, 1 mM MgSO4, 0.5 mM Na2HPO4, 2 mM
NaHCO3, 15 mM Tris, buffered at pH 7.4 with HCl.
Homogenization buffer contained 60 mM
-glycerophosphate,
15 mM p-nitrophenylphosphate, 25 mM
MOPS, pH 7.2, 15 mM EGTA, 15 mM
MgCl2, 2 mM dithiothreitol, 1 mM
sodium orthovanadate, 1 mM sodium fluoride, 1 mM disodium phenylphosphate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, 100 µM benzamidine.
Bead buffer contained 50 mM Tris-HCl, pH 7.4, 5 mM NaF, 250 mM NaCl, 5 mM EDTA,
0.1% Nonidet P-40, 5 mM EGTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, 100 µM benzamidine.
Buffer C contained 60 mM
-glycerophosphate, 30 mM p-nitrophenylphosphate, 25 mM
MOPS, pH 7.0, 5 mM EGTA, 15 mM
MgCl2, 1 mM dithiothreitol, 0.1 mM
sodium orthovanadate.
Three transfer buffers were used: anode buffer 1 (0.3 M
Tris, 20% methanol, pH 10.4), anode buffer 2 (0.025 M
Tris, 20% methanol, pH 10.4), and cathode buffer (0.025 M
Tris, 0.04 M glycine, 20% methanol, pH 9.4).
Tris-buffered saline/Tween 20 (TBST) contained 50 mM Tris,
pH 7.4, 150 mM NaCl, 0.1% Tween 20.
Preparation of Starfish Oocytes
Starfish Oocyte Maturation--
The starfish Marthasterias
glacialis were collected in Northern Brittany and kept under
running sea water until use. The gonads were dissected out and gently
torn open in ice-cold CaFASW. Oocytes were then filtered through
cheesecloth and washed four times in CaFASW to remove the
1-MeAde-producing follicle cells. They were resuspended in the same
medium as a 10% (v/v) suspension. Oocyte maturation was triggered by
the addition of 1-MeAde to a final concentration of 1 µM.
During maturation time course experiments, 1-ml aliquots of the oocyte
suspension were withdrawn at regular intervals after hormonal
stimulation and centrifuged in microtubes, and the oocyte pellets were
frozen in liquid nitrogen.
Vitamin K3 Treatment--
Vitamin K3 is
a powerful inhibitor of the Cdc25 phosphatase (24, 43). It inhibits
hormone-induced oocyte maturation (24). Oocytes were treated with
vitamin K3 (0-250 µM final concentration) for 15 min prior to the 1-MeAde addition. After a 30-min incubation, aliquots were centrifuged, and oocyte pellets were frozen in liquid nitrogen.
Preparation of Prophase and Metaphase Oocytes--
1-ml aliquots
of an oocyte suspension before (prophase oocytes) or 20 min after the
1-MeAde addition (metaphase oocytes) were rapidly centrifuged, the
supernatant was removed, and the oocyte pellets were frozen in liquid nitrogen.
Preparation of Xenopus Oocytes
Adult Xenopus laevis females were purchased from CRBM
(Centre de Recherche en Biologie Moléculaire, Montpellier,
France). Frogs were primed with 500 international units of human
chorionic gonadotropin and stored overnight in laying tanks containing
HSB. Mature oviposited eggs were dejellied in 2% cysteine in HSB
medium during 10 min under agitation. They were washed three times in HSB medium and frozen immediately at
80 °C until use.
Purification of Cdc2-cyclin B on p9CKShs1-Sepharose
Beads
The Cdc2-cyclin B complex was purified from starfish oocytes by
affinity chromatography on p9CKShs1-Sepharose beads,
prepared as described in Azzi et al. (44). 400 µl of
homogenization buffer were added per 100 µl of prophase or metaphase
oocyte pellets. After sonication, extracts were centrifuged at
14,000 × g for 10 min at 4 °C. The supernatant was
then incubated at 4 °C for 30 min and under constant rotation, with
10 µl of p9CKShs1-Sepharose beads in the presence of 400 µl of bead buffer. After removal of the supernatant, the beads were
washed three times with ice-cold bead buffer. The complex was eluted or
not eluted by free p9CKShs1 (2 mg/ml) for 30 min under
constant rotation and used for further analysis. The same protocol was
used for the preparation of Xenopus MPF.
Microsequencing of Starfish M. glacialis Cyclin B
Starfish cyclin B from prophase and metaphase oocytes were
prepurified first on Green A-agarose beads (loading in 10-fold diluted
homogenization buffer, extensive washing with the same buffer, and
elution with 0.2 M NaCl in the same buffer). This procedure
leads to rapid concentration and approximately 20-fold purification of
Cdc2-cyclin B.2 The
concentrated kinase was then affinity-purified on
p9CKShs1-Sepharose beads and analyzed by SDS-PAGE and Amido
Black staining. The prophase and metaphase cyclin B bands were digested
and partially microsequenced at the Institut Pasteur (Paris).
Preparation and Purification of GST-Pyp3 and GST-Cdc25A
Fusion Proteins
Bacterial growth and purification of the fusion proteins were as
described by Borgne and Meijer (24).
In Vitro Dephosphorylation of Cdc2 by Purified Phosphatases
p9CKShs1-Sepharose beads, loaded with prophase
oocyte extracts, were prepared as described above. Following the bead
buffer step, the beads were washed three times with Tris buffer A prior
to incubation for 30 min at 30 °C with 100 µl of recombinant
phosphatases and/or 2 µl of PP2A1. The dephosphorylation
reaction was stopped by the addition of 1 ml of bead buffer. The beads
were washed three times with bead buffer before cyclin B
phosphorylation assays.
In Vitro Cyclin B Phosphorylation Assay
The prophase Cdc2-cyclin B complex purified on
p9CKShs1-Sepharose beads and treated or not treated with
different phosphatases was incubated for 10 min at 30 °C with 15 µM ATP in a final volume of 30 µl. The phosphorylation
reaction was stopped by the addition of 1 ml of bead buffer. The beads
were washed three times with bead buffer before the addition of 50 µl
of 2× Laemmli sample buffer and analysis by SDS-PAGE and Western blotting.
In Vitro Cyclin B Dephosphorylation
The metaphase Cdc2-cyclin B complex was treated with 100 µl of
GST-Pyp3 or 10 µl of PP2A1, after washes with Tris buffer
A, for 30 min at 30 °C. The beads were then washed with bead buffer and recovered with 50 µl of 2× Laemmli sample buffer prior to analysis by SDS-PAGE and Western blotting.
Cyclin B Phosphopeptide Mapping
For in vivo cyclin B labeling, 30 ml of prophase
starfish oocytes (a 10% suspension in CaFASW) were incubated at room
temperature with [32P]phosphate (40 mCi/ml) for 3 h.
1-MeAde was then added for a 20-min incubation. After washes of the
oocytes with CaFASW, an extract was prepared for Cdc2-cyclin B
purification on p9CKShs1-beads. In vitro labeled
cyclin B was obtained in the conditions of the phosphorylation assay
(see above) in the presence of [
-32P]ATP. In
vivo and in vitro 32P-labeled cyclin B were
resolved by SDS-PAGE. Labeled cyclin B was detected by autoradiography,
and the bands were excised from the gel. Cyclin B was subjected to
complete digestion with trypsin or thermolysin as described by Walaas
et al. (45). The phosphopeptides were separated by
electrophoresis at pH 3.5 (first dimension) and chromatography on
silica plates (second dimension).
Histone H1 Kinase Activity Assay
The kinase activity of Cdc2-cyclin B was measured after its
purification on p9CKShs1-Sepharose beads and various
treatments. Assays were performed by incubation of 10 µl of packed
p9CKShs1-Sepharose beads for 5 min at 30 °C with 1 mg/ml
histone H1 and 15 µM [
-32P]ATP in a
final volume of 30 µl. Assays were terminated by transferring the
tubes into ice. After a brief centrifugation, 25 µl of supernatant were spotted on 2.5 × 3-cm pieces of Whatman p81 phosphocellulose paper. Filters were washed five times in 1% phosphoric acid, dried, and transferred in plastic scintillation vials with 1 ml of ACS (Amersham Pharmacia Biotech) scintillation fluid.
[32P]Phosphate incorporation in the histone H1 substrate
was measured in a Packard counter. 50 µl of 2× Laemmli sample buffer
were added to the remaining beads and supernatant prior to SDS-PAGE and
analysis by autoradiography.
Inhibition of Cyclin B Phosphorylation and of Histone H1
Kinase Activity Assay
We have used three chemical inhibitors of CDKs, olomoucine (39),
roscovitine (40, 46), and purvalanol (41) which are purine derivatives
and act as competitive inhibitors for ATP binding. Inhibition
experiments were performed on prophase Cdc2-cyclin B complex,
previously dephosphorylated by GST-Cdc25A, and eluted from
p9CKShs1-Sepharose beads by free p9CKShs1 (2 mg/ml). Histone H1 kinase activity was assayed by incubation of 2.5 µl of soluble Cdc2-cyclin B for 10 min at 30 °C with 15 µM [
-32P]ATP and various concentrations
of inhibitors, in the presence or absence of 1 mg/ml histone H1, in a
final volume of 30 µl. The histone H1 kinase activity of Cdk2-cyclin
E was assayed using 0.5 µl of purified kinase incubated for 5 min at
30 °C with 1 mg/ml histone H1, 15 µM
[
-32P]ATP, and various concentrations (0-100
nM) of p27Kip1 (a GST fusion protein provided
by Dr. B. Ducommun) in a final volume of 30 µl. The histone H1 kinase
activities were measured in a Packard counter. The cyclin B
phosphorylation assay was monitored as described above in the presence
of ATP and increasing concentrations of inhibitors and analyzed by
autoradiography or Western blotting.
Electrophoresis and Western Blotting
Proteins bound to p9CKShs1-Sepharose beads were
recovered with 2× Laemmli sample buffer. Samples were run in 10%
SDS-polyacrylamide gels. For detection of 32P-labeled
proteins, gels were stained with Coomassie Blue and exposed overnight
to Hyperfilm MP. For Western blotting, proteins were transferred from
the gel to a 0.1-µm nitrocellulose sheet (Schleicher and Schuell) in
a milliblot-graphite electroblotter system (Millipore Corp.) for 30 min
at 1.5 mA/cm2 in transfer buffers. Subsequently, the filter
was blocked with 5% low fat milk in TBST for 1 h. The filter was
then washed with TBST and incubated for 1 h with the first
antibodies (anti-PSTAIRE (1:2000) or anti-cyclin B (1:1000)). After
four washes (1 × 20 min, 3 × 5 min) with TBST, the
nitrocellulose sheet was treated for 1 h with horseradish
peroxidase-coupled secondary antibodies diluted in TBST (1:1000). The
filter was then washed five times (1 time for 20 min, 4 times for 5 min) with TBST and analyzed by enhanced chemiluminescence with ECL
detection reagents and hyperfilm MP.
 |
RESULTS |
In Vivo Cyclin B Phosphorylation at the Prophase/Metaphase
Transition in Starfish Oocytes--
The addition of 1-methyladenine to
prophase-arrested starfish oocytes triggers Cdc2-cyclin B kinase
activation, nuclear envelope breakdown, and entry into meiotic division
(reviewed in Ref. 47). The complex is inactive in prophase, Cdc2 being
phosphorylated on Thr-14 and Tyr-15 and cyclin B being
unphosphorylated. When cells enter in metaphase, Cdc2 is sequentially
dephosphorylated first on Thr-14 and then on Tyr-15 (24), while cyclin
B becomes phosphorylated.
Cyclin B phosphorylation results in a change in mobility (shift) on
SDS-PAGE as detected by silver staining (Fig.
1) and with monoclonal anti-cyclin B
antibodies (Fig. 2). The two bands
corresponding to prophase and metaphase cyclin B were partially
microsequenced and compared with the M. glacialis cyclin B
known sequence (10). This comparison shows that our prophase and
shifted metaphase bands correspond to bona fide M. glacialis
cyclin B (Fig. 1).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 1.
Microsequencing of prophase and metaphase
M. glacialis cyclin B. Starfish Cdc2-cyclin B
complex was purified from prophase (P) and metaphase
(M) oocytes by affinity chromatography first on Green
A-agarose beads and then on p9CKShs1-Sepharose beads and
analyzed by SDS-PAGE and silver staining (A). Cyclin B bands
were partially microsequenced and compared with the M. glacialis cyclin B known sequence (B).
|
|

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 2.
In vivo dephosphorylation of
Cdc2/phosphorylation of cyclin B during starfish oocyte
maturation. Prophase oocytes were treated with 1 µM
1-MeAde. At regular intervals, aliquots of the oocyte suspension were
withdrawn and frozen in liquid nitrogen. The Cdc2-cyclin B kinase was
purified from oocyte extracts by affinity chromatography on
p9CKShs1-Sepharose beads and was analyzed by SDS-PAGE and
Western blotting with anti-cyclin B (A) and anti-PSTAIRE
(B) antibodies. Nuclear envelope breakdown occurred 20 min
after the 1-MeAde addition, in this experiment.
|
|
The changes in electrophoretic mobility of the two MPF subunits can be
monitored during the oocyte maturation time course following
stimulation by 1-MeAde (Fig. 2). The phosphorylation state of the
complex was analyzed after purification of Cdc2-cyclin B on
p9CKShs1-Sepharose beads, SDS-PAGE, and Western blotting
with anti-PSTAIRE antibodies for Cdc2 and with anti-cyclin B antibodies
for cyclin B. At metaphase entry, cyclin B phosphorylation occurs
simultaneously with Cdc2 dephosphorylation (Fig. 2).
To ensure that the observed cyclin B shift is due to a phosphorylation
reaction, we have treated the purified metaphase cyclin B with
different phosphatases (Fig. 3,
A, B, and C). Treatment with
PP2A1 leads to cyclin B dephosphorylation, while treatment with the tyrosine phosphatase GST-Pyp3 does not (Fig. 3A).
After treatment with PP2A1, cyclin B presents the same
electrophoretic mobility as the prophase cyclin B.

View larger version (72K):
[in this window]
[in a new window]
|
Fig. 3.
Histone H1 kinase activity of different
phosphorylation states of the Cdc2-cyclin B complex. First, the
Cdc2-cyclin B kinase was purified from prophase (P) or
metaphase oocyte extracts (M) by affinity chromatography on
p9CKShs1-Sepharose beads and treated or not treated with
different phosphatases. The complex was next assayed for its
histone H1 kinase activity. [32P]phosphate
incorporation in histone H1 was measured by direct counting
(C) or by autoradiography (F). The
p9CKShs1-bound proteins were then analyzed by SDS-PAGE and
Western blotting with anti-cyclin B (A and D) and
anti-PSTAIRE (B and E) antibodies.
|
|
Cyclin B Phosphorylation Is Not Required for the Catalytic Activity
of Cdc2-Cyclin B--
We next investigated the importance of cyclin B
phosphorylation for the catalytic activity of the Cdc2-cyclin B
complex. The dephosphorylated Cdc2-unphosphorylated cyclin B complex
can be obtained in vitro by dephosphorylation of metaphase
cyclin B by PP2A1 (Fig. 3, A, B, and
C) or by dephosphorylation of prophase Cdc2 by recombinant
GST-Cdc25A (Fig. 3, D, E, and F). This
complex displays the same histone H1 kinase activity as the metaphase complex (dephosphorylated Cdc2-phosphorylated cyclin B) (Fig. 3,
C and F). This directly demonstrates that cyclin
B phosphorylation is not necessary for Cdc2 kinase activity.
Cyclin B Phosphorylation Requires Cdc2 Activation--
Prior to
the 1-methyladenine addition, oocytes were treated with increasing
concentrations of vitamin K3, an inhibitor of Cdc25
phosphatase (24). A dose-dependent inhibition of germinal vesicle breakdown (GVBD; Fig.
4A) and of Cdc2
dephosphorylation was observed (Fig. 4C). A
dose-dependent inhibition of cyclin B phosphorylation was
also observed (Fig. 4B). These results suggest that Cdc2
activation (by dephosphorylation of Thr-14 and Tyr-15) is necessary for
cyclin B phosphorylation in vivo.

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 4.
Vitamin K3 inhibits oocyte
maturation, Cdc2 dephosphorylation, and cyclin B phosphorylation.
Prophase oocytes (P) were treated with various
concentrations of an inhibitor of Cdc25, vitamin K3 (0-250
µM) prior to exposure to 1 µM 1-MeAde. The
rate of germinal vesicle breakdown (GVBD) was recorded
(A) after 30 min, and aliquots of the oocyte suspension were
withdrawn. The Cdc2-cyclin B kinase was purified from oocyte extracts
by affinity chromatography on p9CKShs1-Sepharose beads and
was analyzed by SDS-PAGE and Western blotting with anti-cyclin B
(B) and anti-PSTAIRE (C) antibodies
(P, control Cdc2-cyclin B, prophase).
|
|
We next set up an in vitro cyclin B kinase assay (Fig.
5, A and B).
Prophase oocyte Cdc2-cyclin B was first purified and immobilized on
p9CKShs1-Sepharose beads. The Cdc2 subunit was then
dephosphorylated with GST-Cdc25A phosphatase. Cyclin B phosphorylation
was initiated by the addition of 15 µM ATP and incubation
at 30 °C. Incubation with ATP without prior Cdc25A phosphatase
treatment did not lead to cyclin B phosphorylation (Fig. 5,
A and B). Pretreatment with Cdc25A and incubation
without ATP did not lead to cyclin B phosphorylation either. When the
complex was dephosphorylated by Cdc25A and then incubated with ATP,
cyclin B phosphorylation occurred. The phosphorylated cyclin B obtained
in vitro could be dephosphorylated by phosphatase 2A (data
not shown).

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 5.
In vitro cyclin B shift
assay. The Cdc2-cyclin B kinase was purified from prophase
(P) oocyte extracts by affinity chromatography on
p9CKShs1-Sepharose beads and treated or not treated by
different phosphatases. The cyclin B phosphorylation reaction was
performed in the presence of 15 µM ATP. The complex was
analyzed by SDS-PAGE and Western blotting with anti-cyclin B
(A and C) and anti-PSTAIRE (B and
D) antibodies (control Cdc2-cyclin B, metaphase
(M)). The intensities of the cyclin B and phosphocyclin B
signals are not directly comparable, because the antibodies do not
exactly display the same sensitivity toward unphosphorylated and
phosphorylated cyclin B. In addition, phosphatase treatment is often
accompanied by a reduction of the cyclin B signal.
|
|
The Cdc25 phosphatase effect is mimicked by the successive treatments
of Cdc2-cyclin B with the Ser/Thr phosphatase 2A and the tyrosine
phosphatase Pyp3, which together completely dephosphorylate Cdc2 (Fig.
5, C and D). Treatment with only one of these
enzymes does not allow cyclin B phosphorylation to occur upon exposure to ATP. These results show that both Thr-14 and Tyr-15 residues of Cdc2
should be dephosphorylated, allowing full Cdc2 activation (24) for
cyclin B phosphorylation. Therefore, Cdc2 activation is also necessary
for cyclin B phosphorylation in vitro.
Cyclin B Phosphorylation Is Sensitive to Chemical Inhibitors of
CDKs--
To further analyze the role of Cdc2 in cyclin B
phosphorylation, we have performed (Fig.
6) the cyclin B shift assay in the presence of roscovitine, a selective inhibitor of CDKs (40, 46). The
soluble Cdc2-cyclin B complex from prophase oocytes was previously
dephosphorylated by Cdc25A and incubated with ATP in the presence or
absence of 10 µM of roscovitine, a concentration that
inhibits the histone H1 kinase activity of Cdc2 (Fig. 6A). The presence of roscovitine inhibits cyclin B phosphorylation, as shown
by anti-cyclin B immunoblotting (Fig. 6B). These results further suggest that cyclin B phosphorylation is dependent on Cdc2
kinase activity.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 6.
Sensitivity of the in vitro
cyclin B shift to roscovitine. The Cdc2-cyclin B kinase was
purified from prophase (P) oocyte extracts by affinity
chromatography on p9CKShs1-Sepharose beads, treated with
GST-Cdc25A, and eluted by free p9. The histone H1 kinase activity assay
was performed in the presence of 15 µM ATP, 1 mg/ml
histone H1 with or without 10 µM roscovitine. The complex
was assayed for its histone H1 kinase activity by direct counting
(A). The cyclin B phosphorylation was performed in the
presence of 15 µM ATP with or without 10 µM
roscovitine. The complex was analyzed by SDS-PAGE and Western blotting
with anti-cyclin B antibodies (B) (control Cdc2-cyclin B,
metaphase (M)).
|
|
We next tested other specific inhibitors of CDKs on the in
vitro phosphorylation of cyclin B. Soluble Cdc2-cyclin B was
prepared as in Fig. 6, and both Cdc2-cyclin B kinase activity and
cyclin B phosphorylation were simultaneously assayed in the presence of
increasing concentrations of purvalanol, roscovitine, or olomoucine (Fig. 7). Histone H1 kinase activities
were measured by direct counting (Fig. 7A). Cyclin B
phosphorylation was detected by autoradiography (Fig. 7B).
The same dose-dependent inhibitions of histone H1 kinase activity and of cyclin B phosphorylation were observed with the three
inhibitors, purvalanol being more efficient than roscovitine, itself
being more efficient than olomoucine. These results show that both
activities are inhibited in a very similar fashion by the three
inhibitors of CDKs. Most if not all of the histone H1 kinase activity
recovered on p9CKShs1-Sepharose beads can be attributed to
Cdc2-cyclin B (24, 44). The p9CKShs1-Sepharose beads also
present affinity for Cdk2. We therefore tested the effect of
p27kip1, a powerful inhibitor of Cdk2 but not of Cdc2, on
cyclin B phosphorylation (Fig. 8). The
Cdk2 inhibitor was unable to inhibit in vitro cyclin B
phosphorylation, excluding a possible involvement of Cdk2 in cyclin B
phosphorylation. The histone H1 and cyclin B kinase activities measured
in our assays (Figs. 6 and 7) can thus be attributed to the same
kinase, Cdc2-cyclin B, i.e. MPF.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 7.
Inhibition of cyclin B phosphorylation and
histone H1 kinase activity by chemical inhibitors of Cdc2. The
Cdc2-cyclin B kinase was purified from prophase oocyte extracts
(P) by affinity chromatography on
p9CKShs1-Sepharose beads, treated with GST-Cdc25A, and
eluted by free p9. Histone H1 kinase activity assay was performed by
incubation of the soluble complex with 15 µM ATP, 1 mg/ml
histone H1, and increasing concentrations of purvalanol, roscovitine,
or olomoucine. The complex was assayed for its histone H1 kinase
activity by direct counting (A). Cyclin B phosphorylation
was assayed similarly in the presence of 15 µM ATP and
increasing concentrations of purvalanol (1), roscovitine
(2), or olomoucine (3) and detected by
autoradiography following SDS-PAGE (B).
|
|

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 8.
p27kip1 does not inhibit cyclin B
phosphorylation. Purified cdk2-cyclin E was assayed for its
histone H1 kinase activity in the presence of increasing concentrations
of p27kip1 (0-100 nM).
[32P]phosphate incorporation in histone H1 was measured
by direct counting (A). The Cdc2-cyclin B kinase was
purified from prophase oocyte extracts (P) by affinity
chromatography on p9CKShs1-Sepharose beads and pretreated
with GST-Cdc25A. The cyclin B phosphorylation reaction was performed in
the presence of 15 µM [ -32P]ATP with
increasing concentrations of p27kip1 (0-100 nM).
Cyclin B phosphorylation was detected after SDS-PAGE and Western
blotting with anti-cyclin B antibodies (B) (control
Cdc2-cyclin B, prophase (P)). Signal intensities were
measured to evaluate the cyclin B phosphorylation (A).
|
|
We next compared in vivo and in vitro cyclin B
phosphorylation by phosphopeptide mapping. Cyclin B was labeled
in vivo, by incubating maturing starfish oocytes with
[32P]phosphate, or in vitro under the
conditions of the shift assay in the presence of
[
-32P]ATP. Both in vivo and in
vitro phosphorylated cyclin B were purified on
p9CKShs1-Sepharose beads and on SDS-PAGE. The labeled
cyclins were then digested by trypsin (Fig.
9, A, B, and
C) or thermolysin (Fig. 9, D-F). The
two-dimensional phosphopeptide maps of in vivo and in
vitro phosphorylated cyclin B were similar, but not entirely identical, in each experimental procedure (Fig. 9, compare
panels A and B and panels
D and E). This shows first that most of the sites
phosphorylated in our shift assay and most sites phosphorylated during
maturation are the same. Second, these results are consistent with the
fact that Cdc2, which phosphorylates cyclin B in our in
vitro kinase assay, is the major physiological cyclin B kinase. Third, other kinases may additionally phosphorylate cyclin B in vivo.

View larger version (91K):
[in this window]
[in a new window]
|
Fig. 9.
Phosphopeptide maps of in vivo
and in vitro phosphorylated cyclin B. In vivo phosphorylated cyclin B was obtained after
incubation of the maturing oocytes with [32P]phosphate.
Cdc2-cyclin B was then purified from metaphase oocyte extracts by
affinity chromatography on p9CKShs1-Sepharose beads.
In vitro phosphorylated cyclin B was obtained under the
conditions of the shift assay in the presence of
[ -32P]ATP. The in vivo (A and
D) and in vitro (B and E)
labeled cyclin B were purified by SDS-PAGE and extensively digested by
trypsin (A, B, and C) or thermolysin
(D, E, and F). The phosphopeptides
were then resolved by chromatography and electrophoresis at pH 3.5 and
detected by autoradiography. In C and F, the
phosphopeptides derived from the in vivo and in
vitro cyclin B were mixed.
|
|
Cyclin B Phosphorylation Occurs as an Intra-MPF Complex
Event--
We next wondered if Cdc2-cyclin B was phosphorylating
itself on cyclin B (intra-MPF phosphorylation) or phosphorylating the cyclin B subunit of other complexes (extra-MPF phosphorylation). In a
first experiment, we prepared soluble and active Cdc2-cyclin B complex
from both starfish and Xenopus metaphase oocytes. Each of
these MPF was added to soluble prophase complex dephosphorylated or not
dephosphorylated by Cdc25, in the presence or absence of ATP (Fig.
10). In the intraspecific experiment
using active MPF from starfish (Fig. 10A), the prophase
cyclin B does not shift in the presence of ATP. It shifts only after
Cdc25 treatment, which activates prophase Cdc2. Therefore, the addition
of active starfish MPF is unable to induce phosphorylation of the
prophase cyclin B in the presence of ATP. The signals are similar in
the presence or absence of ATP; prophase cyclin B remains in its lower position on the immunoblot. The same result was obtained in the interspecific experiment using active Xenopus MPF, which is
unable to induce starfish prophase cyclin B phosphorylation (Fig.
10B). The absence of effect is plainly seen in the
interspecific experiment, because Xenopus cyclin B is not
recognized by our anti-cyclin B antibodies. These results demonstrate
that Cdc2 is able to phosphorylate its own associated cyclin B subunit
but not cyclin B from another complex.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 10.
Cdc2 is unable to phosphorylate cyclin B
from another complex. Soluble prophase Cdc2-cyclin B complex
(P) (from starfish oocytes) and soluble metaphase
Cdc2-cyclin B complex (from starfish or Xenopus oocytes)
were prepared as described under "Experimental Procedures." 10 µl
of prophase MPF (starfish), dephosphorylated or not with GST-Cdc25A,
was incubated with or without 10 µl of active MPF from starfish
(A) or Xenopus (B) metaphase oocytes
in the presence or absence of 15 µM ATP for 10 min at
30 °C. The reaction was stopped by the addition of 50 µl of 2×
Laemmli sample buffer prior to SDS-PAGE and Western blot analysis with
anti-cyclin B antibodies (control starfish Cdc2-cyclin B, metaphase
(M)).
|
|
We have performed another type of experiment to demonstrate the
intra-MPF nature of cyclin B phosphorylation. We reasoned that if
cyclin B phosphorylation results from intra-MPF rather than
extracomplex phosphorylation, it should be insensitive to dilution,
i.e. independent of enzyme/substrate concentration, or in
other words independent of the probability of enzyme/substrate encounter. We incubated a constant quantity of soluble and activated prophase Cdc2-cyclin B with [
-32P]ATP in increasing
reaction volumes from 15 to 150 µl (Fig.
11). The ability of Cdc2 to
phosphorylate cyclin B was not affected by volume changes; the level of
phosphorylated cyclin B remained constant independently of the dilution
(Fig. 11A). The same experiment was made in the presence of
a low histone H1 concentration (Fig. 11B). Despite the
presence of competing histone H1, cyclin B phosphorylation remained
unaffected by the dilution. In contrast, phosphorylation of the
exogenous substrate decreased according to the reaction volume. This
result clearly demonstrates the intracomplex nature of the
phosphorylation reaction.

View larger version (62K):
[in this window]
[in a new window]
|
Fig. 11.
Intra-MPF phosphorylation of cyclin B. Soluble and in vitro dephosphorylated prophase Cdc2-cyclin B
complex was prepared as described under "Experimental Procedures."
2.5 µl of the complex was incubated at 30 °C for 5 min. with 15 µM [ -32P]ATP in the presence
(B) or absence (A) of 2.5 µl of histone H1 (0.1 mg/ml) in increasing reaction volumes (15-150 µl). The reaction was
stopped by the addition of 50 µl of 4× Laemmli sample buffer prior
to SDS-PAGE and autoradiography.
|
|
 |
DISCUSSION |
In Vivo Cyclin B Phosphorylation at the Prophase/Metaphase
Transition--
Cyclin B phosphorylation at the G2/M
transition of the cell cycle has been described in numerous models from
yeast to human cells (6, 11, 13, 29-31). We have characterized the
electrophoretic shift of cyclin B occurring at entry in metaphase in
starfish oocytes as a phosphorylation reaction (Fig. 3A).
During the maturation time course (Fig. 2), cyclin B phosphorylation
occurs simultaneously with the Cdc2 dephosphorylation. The
dephosphorylated Cdc2-dephosphorylated cyclin B complex (obtained
either by treatment of the prophase Cdc2 with Cdc25 or by treatment of
the metaphase cyclin B with PP2A1) displays a kinase
activity equivalent to that of the metaphase complex (dephosphorylated
Cdc2-phosphorylated cyclin B) (Fig. 3). This shows that cyclin B
phosphorylation is not essential for the catalytic activity of the
complex and confirms similar data obtained indirectly (mutation of
phosphorylation sites) in goldfish (29) and Xenopus (33)
oocytes. Studies from Li et al. (33, 34) have shown the
requirement of cyclin B1 phosphorylation for nuclear
localization and Xenopus oocyte maturation. Nuclear localization of cyclin B1 is regulated by its
phosphorylation at sites within the cytoplasmic retention signal domain
(34, 48). Phosphorylation of cyclin B1 masking the
cytoplasmic retention signal would therefore permit migration into the
nucleus, allowing phosphorylation of nuclear substrates by MPF. Ookata
et al. (49) have demonstrated that in starfish oocytes,
Cdc2-cyclin B complex is indeed activated in the cytoplasm prior to its
translocation to the nucleus.
Cyclin B Phosphorylation Is Carried Out by the Cdc2-Cyclin B
Complex--
We have shown by various types of experiments that cyclin
B phosphorylation is only possible when Cdc2 is dephosphorylated. First, when Cdc25 is inhibited in vivo by vitamin
K3, no cyclin B phosphorylation is observed (Fig. 4).
Second, the in vitro cyclin B kinase assay consisting in
incubation of the prophase complex with ATP only works when Cdc2 has
previously been treated with Cdc25 or with PP2A1 plus Pyp3
successively (Fig. 5). It clearly appears that Cdc2 activation by
dephosphorylation of its inhibitory Thr-14 and Tyr-15 residues is
required for both in vivo and in vitro
phosphorylation of cyclin B.
Knowing the importance of Cdc2 activation, we next examined the
involvement of Cdc2 kinase activity in in vitro cyclin B
phosphorylation. The cyclin B kinase is sensitive to chemical
inhibitors of CDKs (Figs. 6 and 7). The level of sensitivity of cyclin
B phosphorylation to purvalanol, roscovitine, and olomoucine has been
tested (Fig. 7). Both histone H1 kinase activity and cyclin B
phosphorylation, assayed simultaneously, were similarly and
dose-dependently inhibited by the three inhibitors.
Phosphorylation is also inhibited by the presence of histone H1, a
competitive substrate (Fig. 3). Although p9CKShs1-Sepharose
beads bind Cdc2, Cdk2, and Cdk3 in mammalian cell extracts (50, 51), in
starfish oocytes the extreme and natural synchrony eliminates potential
contaminations by CDKs from other cell cycle phases. Furthermore, an
unusually large amount of p34cdc2-cyclin B kinase is
accumulated in oocytes (8, 13) and the histone H1 kinase activity
associated to the p9CKShs1-bound proteins essentially
corresponds to p34cdc2-cyclin B (24, 44). As a precaution, we
tested p27kip1, an inhibitor of Cdk2 (Fig. 8). The
noninhibition of cyclin B phosphorylation by p27kip1 eliminates
Cdk2 as a candidate for the cyclin B kinase. Therefore, in
vitro, the cyclin B kinase could be nothing other than Cdc2 itself. Furthermore, according to the phosphopeptide map analysis (Fig.
9), in vivo and in vitro cyclin B phosphorylated
sites are very similar. Since Cdc2 phosphorylates cyclin B in
vitro, Cdc2 seems to be the major physiological cyclin B kinase
responsible for the cyclin B shift observed in vivo. The
consensus sequence for a phosphorylation site by Cdc2 is
(S/T)PX(K/R) as reviewed by Nigg (52) and Kishimoto (53),
but other distantly related sites are probably equally phosphorylated.
The M. glacialis cyclin B contains the sequence
87SPEP as a potential candidate for phosphorylation by
Cdc2. From sequence alignments, Ser-87 corresponds to the
phosphorylated Ser-94 from X. laevis cyclin B1.
The sites corresponding to X. laevis cyclin B1
Ser-2, Ser-96, and Ser-113 are not conserved in starfish. Ser-94,
corresponding to X. laevis Ser-101, is not a consensus Cdc2
phosphorylation site, and it might be phosphorylated by another kinase.
In this study, we have used cyclin B complexed to Cdc2 as a substrate
for the cyclin B kinase, presumably the most physiological substrate.
Other studies using recombinant cyclin B or cyclin B peptides have
proposed other kinases as potential cyclin B1 kinases, such
as mitogen-activated protein kinase (32), and cyclin B2
kinases, such as c-Mos (36) and Cyk (38). The physiological relevance
of these kinases remains unknown. However the incomplete identity of
the in vivo and in vitro cyclin B phosphopeptide
maps suggests the existence of additional kinases, besides Cdc2, which may be active in vivo on cyclin B. Nevertheless, even the
identification of the individual phosphorylation sites would not allow
the definitive identification of the kinases, since some kinase sites
overlap (mitogen-activated protein kinase and Cdc2, for example).
Existence of an Intra-MPF Phosphorylation of Cyclin
B--
Autophosphorylation of cyclin B by the Cdc2 kinase itself was
suggested, but not demonstrated, several years ago, in sea urchin eggs
(11) and in Xenopus oocytes (35). Our results now
demonstrate the existence of an intra-MPF phosphorylation of cyclin B
(Fig. 10). Active MPFs purified from starfish and Xenopus
metaphase oocytes are unable to induce phosphorylation of the prophase
cyclin B associated with prophase (inactive) Cdc2. We cannot rule out
the possibility that the Xenopus MPF does not recognize
starfish cyclin B. This is certainly not the case in the intraspecific
experiment. The dilution experiment (Fig. 11) clearly demonstrates that
cyclin B phosphorylation occurs in an intracomplex fashion; dilution of
the enzyme/substrate has no consequence on the phosphorylation reaction. Since cyclin B phosphorylation is only intracomplex, phosphorylation of prophase cyclin B by the active complex is obviously
impossible, as shown in Fig. 10. This intracomplex mechanism may have a
physiological significance in immediately coupling cyclin B
phosphorylation to Cdc2 dephosphorylation (and activation). In this
case, enzyme and its substrate are close to each other and present in
an equimolar ratio. Such a mechanism explains why cyclin B
phosphorylation is concomitant with Cdc2 dephosphorylation preceding
nuclear translocation in response to 1-methyladenine (Fig. 2). We
propose that activated Cdc2 rapidly phosphorylates its own cyclin B
subunit in the cytoplasmic retention signal domain, allowing nuclear
translocation of the complex as discussed above. Furthermore, intra-MPF
phosphorylation of cyclin B is possible because the complex is highly
active in its unphosphorylated cyclin B form (Fig. 3).
Conclusion--
This study has allowed us to confirm directly that
1) cyclin B phosphorylation is not necessary for the kinase activity of the MPF, 2) Cdc2 is the main kinase responsible for the cyclin B
phosphorylation-associated shift observed at the prophase/metaphase transition in starfish oocytes, and 3) an intra-MPF reaction is the
mechanism of starfish cyclin B phosphorylation. This unique intracomplex cyclin B phosphorylation belongs to the regulatory pathway
controlling MPF function.