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Volume 272, Number 52, Issue of December 26, 1997
pp. 32731-32734
(Received for publication, October 10, 1997, and in revised form, October 29, 1997)
From the Institut de Pharmacologie et de Biologie Structurale du
CNRS, Université Paul Sabatier, 205 route de Narbonne, 31077 Toulouse cedex, France
ABSTRACT In eukaryotes the activity of CDK1 (CDC2), a
cyclin-dependent kinase that initiates the structural
changes that culminate in the segregation of chromosomes at mitosis, is
regulated by the synergistic and opposing activities of a cascade of
kinases and phosphatases. Dephosphorylation of threonine 14 and
tyrosine 15 of CDK1 by the CDC25 phosphatases is a key step in the
activation of the CDK1-cyclin B protein kinase. Little is currently
known about the role and the regulation of CDC25B. Here we report
in vitro and in vivo data that indicate that
CDC25B is degraded by the proteasome. This degradation is dependent
upon phosphorylation by the CDK1-cyclin A complex but not by
CDK1-cyclin B. These results indicate that CDK1-cyclin A
phosphorylation targets CDC25B for degradation and that this might be
an important component of cell cycle regulation at the G2/M
transition.
INTRODUCTION The eukaryotic cell cycle is controlled by a family of
cyclin-dependent kinases that regulate its key transitions.
The precise timing of the activation of these enzymes is a central
issue of cell cycle regulation. A major regulatory mechanism is
provided by the wee1/mik1- and
myt1-dependent phosphorylation on threonine 14 and tyrosine 15 of CDK1 (cdc2). This inhibitory
phosphorylation keeps the kinase inactive until it is dephosphorylated
by the dual specificity CDC25 phosphatase at the G2/M
transition (1). In human cells, three homologues of CDC25 called
CDC25A, CDC25B, and CDC25C have been identified (2-4). In HeLa cells,
expression of CDC25B is low throughout the cell cycle with an increase
in G2, CDC25C is also predominantly expressed in
G2 (2, 3), and CDC25A is abundant both at the mRNA and
protein levels in late G1 (5). Phosphorylation of CDC25C by
CDK1-cyclin B was shown and proposed to be part of the
self-amplification mechanism of CDK1-cyclin B at mitosis (6).
CDK2-cyclin E-dependent phosphorylation of CDC25A was also
demonstrated, indicating that a similar feedback loop might regulate
the progression in S phase (7). The mechanisms that regulate CDC25B
activity and the precise role of that phosphatase and its splicing
variants (8) in the control of entry into mitosis remain unclear.
Here we report in vitro and in vivo evidences
indicating that CDC25B is degraded by the proteasome and that this
process is dependent on the phosphorylation by the CDK1-cyclin A
kinase. We propose that the rapid degradation of CDC25B is an important regulatory mechanism that ensures the timely coordinated activation of
CDK-cyclin complexes.
MATERIALS AND METHODS The CDC25B coding
sequence corresponding to the B1 splicing variant (8) was cloned in the
pET14b (Novagen) vector. In vitro transcription and
translation were performed using the TNT system (Promega) in the
presence or the absence of [35S]methionine.
Sf9 insect cells were co-infected with recombinant baculovirus encoding for human CDK1 (CDC2) or CDK2 and cyclin B or
cyclin A. Insect cell extracts were prepared 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) in a Dounce homogenizer, then diluted into 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. The supernatants were assayed for
their specific activity toward histone H1. Recombinant GST-p21Cip1 was
produced and purified as described previously (22).
HeLa cells and human lung primary fibroblasts
IMR90 were cultured as already described (23) and treated for 18 h
with 250 and 50 ng/ml of nocodazole respectively. Cells were harvested and lysed as described (23).
To assay the
phosphorylation of CDC25B, in vitro translated protein was
immunoprecipitated with an anti-CDC25B polyclonal antibody directed
against recombinant CDC25B protein and incubated either with 15 µg of
cellular extract or with recombinant cyclin-dependent kinase extract (3 pmol/min/µg of histone H1/µl lysate) in kinase assay buffer (6) containing 5 µCi of [ Human primary fibroblasts IMR90 were treated for 2 and 4 h with 50 µM LLnL1 or
with the same concentration of Me2SO as described (10). Cell extracts were subjected to Western blot analysis using polyclonal anti-CDC25B antibodies. Extracts from Sf9 cells expressing
recombinant CDK1, cyclin A, and CDC25B were prepared as in the case of
IMR90 cells (23) and subjected to Western blot analysis. All Enhanced ChemiFluorescence (ECF) and radioactive isotope detection and quantification were performed using a STORM 840 imager (Molecular Dynamics). Digitalized images were used for artwork.
FSBA, LLnL, LLM, MG132, lactacystin, chloroquine,
and E64D were solubilized in Me2SO.
RESULTS AND DISCUSSION To analyze the
phosphorylation of CDC25B, we produced this protein by in
vitro translation in rabbit reticulocyte lysate. As detected both
by 32P incorporation (Fig.
1a, upper panel) or
by change in electrophoretic mobility of the
[35S]methionine-labeled protein (Fig. 1a,
lower panel), CDC25B was readily phosphorylated after
incubation with cellular extracts from nocodazole-arrested human IMR90
primary fibroblasts and HeLa cells. We then determined the ability of
different human CDK-cyclin complexes produced in baculovirus
co-infected Sf9 cells to phosphorylate CDC25B. Equal
specific activities (determined by histone H1 phosphorylation) were
used to assay the in vitro phosphorylation of CDC25B. As seen in Fig. 1b (upper panel), CDK1-cyclin B,
CDK1-cyclin A, and CDK2-cyclin A complexes efficiently phosphorylated
CDC25B, although CDK1-cyclin B led to higher 32P
incorporation (1.5-fold greater than CDK1-cyclin A). The number and
location of the residues phosphorylated by the three kinase complexes
are probably different because distinct changes in electrophoretic mobility of the 35S-labeled CDC25B protein were observed
(Fig. 1b, lower panel).
[View Larger Version of this Image (50K GIF file)]
When the calpain I inhibitor LLnL (9)
was omitted from the reaction buffer, a rapid degradation of the CDC25B
phosphatase was observed following incubation with CDK1-cyclin A (Fig.
2a). After 30 min of
incubation about 50% of CDC25B was degraded (Fig. 2b).
Control incubations with the same amount of control Sf9 cell lysate had little effect on CDC25B stability, as did incubation with
CDK1-cyclin B. p27kip1, a cell cycle inhibitor that has been
shown to be degraded by the proteasome (10-12), was unstable when
incubated with all tested Sf9 cell lysates (Fig.
2c). By contrast, an unrelated control protein
(i.e. luciferase) was not degraded upon incubation with CDK1-cyclin A (data not shown). When Sf9 cells were
simultaneously co-infected with CDC25B, CDK1, and cyclin A encoding
baculoviruses as shown in Fig.
3c (upper panel), a
major 53-kDa CDC25B degradation product accumulated, similar to that is
observed upon incubation of in vitro translated CDC25B with
CDK1-cyclin A lysate (Fig. 4a). Together, these results
indicate that the instability of CDC25B in both in vitro and
in vivo assays reflects a specific degradation that is
dependent upon the presence of the CDK1-cyclin A complex.
[View Larger Version of this Image (26K GIF file)]
[View Larger Version of this Image (48K GIF file)]
[View Larger Version of this Image (27K GIF file)]
Proteasome inhibitors (13) were used to investigated whether
the proteasome pathway was involved in CDC25B degradation. First, as
shown above, the presence of LLnL was necessary to avoid CDC25B
degradation in vitro (Fig. 2a). Second, when
proteasome inhibitors such as LLnL, MG132 (14) (Fig. 3a), or
especially lactacystin (15) (Fig. 3b) were added to the
phosphorylation assay, the degradation of CDC25B was inhibited but not
the phosphorylation, and the protein was stabilized as its lower
migrating phosphorylated form (Fig. 4a). Chloroquine, a
lysosomal inhibitor, and E64D, a cysteine protease inhibitor, had no
effect on CDC25B degradation (Fig. 3a). Similarly, LLM, an
inhibitor of calpain II that is a less potent proteasome inhibitor, had
a minor protective effect (Fig. 3a). Third, as shown in Fig.
3c (lower panel), in Sf9 insect cells
that were simultaneously co-infected with CDC25B, CDK1, and cyclin A
encoding baculoviruses, addition of lactacystin led to the inhibition
of the degradation and the accumulation of CDC25B. Fourth, human
primary fibroblasts IMR90 express only the CDC25B3 variant (8). CDC25B
protein accumulated in fibroblasts treated for 2 and 4 h with 50 µM LLnL (Fig. 3d) or 30 µM
lactacystin (not shown), although its level remained unchanged in mock
cells treated with the same concentration of Me2SO.
Altogether, this set of in vitro and in vivo
observations indicate that degradation of human CDC25B, as also
suggested in fission yeast (16), is indeed dependent on the proteasome
pathway.
Finally we assayed whether the degradation of CDC25B
was dependent upon phosphorylation by CDK1-cyclin A. Both the ATP
analogue FSBA (17) (Fig. 4a), a broad kinase inhibitor, and
p21Cip1 (Fig. 4b), a specific cyclin-dependent
kinase inhibitor (18), fully abolished CDC25B phosphorylation and
inhibited its degradation. A similar inhibition of the degradation was
observed when CDK1-cyclin A was depleted (H1 kinase activity reduced by
97%) from the lysate by anti-cyclin A immunoprecipitation (Fig.
4c).
Taken together, our results provide evidence that human CDC25B is
degraded by the proteasome in a CDK1-cyclin A
phosphorylation-dependent manner. As suggested by the
inability of CDK1-cyclin B to trigger CDC25B degradation, a specific
and a timely coordinated phosphorylation by the CDK1-cyclin A kinase is
probably a key feature of this process. This observation is in
agreement with the reported accumulation of CDC25B in late
G2 and its rapid disappearance in mitosis (19). CDC25B
activity appears therefore to be regulated at multiple levels including
a targeted degradation that might take place in the fine tuning of
CDK1-cyclin A activity at early stages of the G2/M
transition. Together with recent findings, this report indicates that
degradation of cyclin-dependent kinase inhibitors (11, 12,
20) and activators (this work) that is triggered by
CDK-dependent phosphorylation is a key event in the control of cell proliferation. Abnormal regulation of this mechanism may participate in the oncogenic properties of the CDC25B phosphatase (21).
FOOTNOTES ACKNOWLEDGEMENTS We gratefully acknowledge J. M. Darbon,
G. Draetta, I. Jariel-Encontre, J. Hyams, B. B. Rudkin, A. Valette, and G. Villani for advice and critical reading of the
manuscript.
REFERENCES
COMMUNICATION:
Phosphorylation of Human CDC25B Phosphatase by CDK1-Cyclin A
Triggers Its Proteasome-dependent Degradation*
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-32P]ATP for
60 min at 30 °C. The precipitates were subjected to SDS-PAGE 8%
electrophoresis. When CDC25B phosphorylation and/or level was monitored
by the change in electrophoretic mobility, the assay was performed with
1 mM cold ATP, and the samples were analyzed without
immunoprecipitation.
Fig. 1.
Phosphorylation of CDC25B. a,
in vitro translated CDC25B was incubated with extracts from
human primary fibroblasts (IMR90) or HeLa cells arrested at
mitosis by nocodazole treatment. b, in vitro
translated CDC25B was incubated for 1 h in the presence of 50 µM LLnL with the indicated recombinant CDK-cyclin
lysates. Upper panels, CDC25B protein was immunoprecipitated
with anti-CDC25B and then incubated with cell extract or CDK-cyclin
lysate in the presence of [-32P]ATP (note that the
faint labeling of CDC25B at time 0 with HeLa extract corresponds to the
use of 35S-labeled protein in that specific experiment).
Lower panels, [35S]methionine-labeled CDC25B
was directly incubated with cell extract or CDK-cyclin lysate in the
presence of cold ATP. Proteins were visualized after SDS-PAGE
electrophoresis. CDK2-cyclin A (K2/A), CDK1-cyclin A
(K1/A), and CDK1-cyclin B (K1/B).
Fig. 2.
In vitro degradation of CDC25B.
[35S]Methionine-labeled in vitro translated
CDC25B (a and b) or p27kip1
(c) was incubated or not for the indicated time (1 h for
panel c) with Sf9 cell lysate either uninfected
or expressing the recombinant CDK1-cyclin A or CDK1-cyclin B complexes.
Samples were SDS-PAGE electrophoresed (8%) (a) and the
level of CDC25B (b) or p27kip1 (c) was
quantitated and expressed as a percentage of the amount at time 0. A
log scale is used in b for half-life determination. These
results are representatives of several independent experiments.
Fig. 3.
Cdc25B is degraded by the proteasome in
vitro and in vivo.
[35S]Methionine-labeled in vitro translated
CDC25B was incubated for 1 h with CDK1-cyclin A expressing
Sf9 lysate. a, LLnL, LLM, MG132, chloroquine, and
E64D were added to the assay at the indicated concentrations.
b, lactacystin was added at increasing concentrations. Me2SO used to solubilize lactacystin was also tested as
control. c, Sf9 cells were infected with
the indicated recombinant baculovirus. Cells were collected at the
indicated time or at 48 h after 4 h of treatment with
lactacystin or Me2SO. 20 µg of protein extract were
analyzed by Western blot with anti-CDC25B antibodies. The 53-kDa
degradation product is indicated with an arrowhead. In the
lower panel, cells infected with CDC25B, cyclin A, and CDK1 recombinant viruses were treated as indicated. Quantification of CDC25B
level was performed twice, and the values are expressed in arbitrary
units. d, accumulation of CDC25B3 in asynchronous human
fibroblasts IMR90 treated for 2 or 4 h with 50 µM
LLnL or Me2SO. Cells were recovered, and 100 µg of total
protein extract were subjected to Western blot analysis using
polyclonal antibodies raised against CDC25B. The CDC25B3 protein
migrates at 88 kDa. ECF quantification of CDC25B3 level was performed
and is expressed in arbitrary units.
Fig. 4.
Phosphorylation of CDC25B is required for
degradation by the proteasome. In vitro translated CDC25B
(Control) was incubated with CDK1-cyclin A (K1/A)
in the absence or in the presence of 10 mM FSBA
(a) or in the presence of increasing amounts of recombinant GST-p21Cip1 protein (b). The arrowheads indicate
the CDC25B translational product, and the bracket indicates
the phosphorylated forms. The major degradation product (53 kDa) is
indicated with a asterisk. In c, in
vitro translated CDC25B was incubated with uninfected Sf9 cell lysates (Control) or CDK1-cyclin A
cell lysates that were left alone or subjected to immunodepletion with
anti-cyclin A polyclonal antibodies (less than 3% of residual kinase
activity). Electrophoresis of the sample is shown on the
top, and quantification of the experiment on the
bottom.
To whom correspondence should be addressed. Tel.:
33-5-61-17-59-31; Fax: 33-5-61-17-59-05; E-mail:
ducommun{at}ipbs.fr.
1
The abbreviations used are: LLnL,
N-acetyl-Leu-Leu-norleucinal; FSBA,
p-fluorosulfonylbenzoyl adenosine; LLM,
N-acetyl-Leu-Leu-methioninal; MG132,
Z-Leu-Leu-Leu-CHO; E64D,
(2S,3S)-trans-epoxysuccimyl-L-leucylamido-3-methyl-butane ethyl ester; PAGE, polyacrylamide gel electrophoresis.
Volume 272, Number 52,
Issue of December 26, 1997
pp. 32731-32734
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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