From the Centre de Recherche de Biochimie Macromoléculaire, CNRS UPR 1086, 1919 Route de Mende, 34293 Montpellier cedex 5, France and § IFREMER, 17390 La Tremblade, France
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ABSTRACT |
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X-PAKs are involved in negative control of the
process of oocyte maturation in Xenopus (1). In the present
study, we define more precisely the events targetted by the kinase in
the inhibition of the G2/M transition. We show that
microinjection of recombinant X-PAK1-Cter active kinase into
progesterone-treated oocytes prevents c-Mos accumulation and activation
of both MAPK and maturation-promoting factor (MPF). In conditions
permissive for MAPK activation, MPF activation still fails. We
demonstrate that a constitutive truncated version of X-PAK1
(X-PAK1-Cter) does not prevent the association of cyclin B with
p34cdc2 but rather prevents the activation of the inactive
complexes present in the oocyte. Proteins participating in the MPF
amplification loop, including the Cdc25-activating Polo-like kinase are
all blocked. Indeed, using active MPF, the amplification loop is not turned on in the presence of X-PAK1. Our results indicate that X-PAK
and protein kinase A targets in the control of oocyte maturation are
similar and furthermore that this negative regulation is not restricted
to meiosis, because we demonstrate that G2/M progression is
also prevented in Xenopus cycling extracts in the presence of active X-PAK1.
In Xenopus laevis, oocytes are naturally arrested at
the G2/M transition of meiosis I. Upon release of
progesterone by the follicle cells surrounding the oocytes, maturation
occurs and the cell cycle resumes. Translation of the proto-oncogene
c-Mos (a MAPK1 kinase
kinase), is an essential preliminary event (2, 3) in turning on the
MAPK cascade, which eventually results in almost simultaneous
activation of MAPK and maturation-promoting factor (MPF), a heterodimer
composed of p34cdc2 kinase and cyclin B (4, 5). Eggs eventually
arrest again at metaphase of second meiosis with high MPF and
cytostatic factor (in which c-Mos participates (6)) activities until
fertilization. Conversion of inactive pre-MPF complexes, stored in
oocytes, into active MPF requires dephosphorylation of residues
Thr14 and Tyr15 on p34cdc2 kinase (7).
Dephosphorylation of these amino acids is crucial for MPF activation,
and this process is highly regulated by an activating phosphatase,
Cdc25 (for review see Ref. 8), and two inhibitory kinases, Myt1 (9) and
Wee1 (10).
Although oocyte maturation has been extensively studied over the past
10 years as a model to understand the mechanisms involved in reentry
into the cell cycle, the signal transduction pathway between
progesterone binding to its receptor and the activation of the MAPK
cascade and MPF is ill understood. Protein kinase A (PKA) appears to be
a crucial player in these events, because activation of progesterone
receptor is followed by a sudden drop in cAMP concentration (11) and
likely a subsequent inactivation of protein kinase A. Indeed,
microinjection of the PKA catalytic subunit prevents
progesterone-induced maturation in oocytes, whereas the expression
of the PKA regulatory subunit is sufficient to induce maturation (12).
Matten et al. (13) reported that PKA negatively regulates
maturation by controlling both c-Mos translation and Cdc25 activation.
How PKA acts on c-Mos de novo translation is, however,
unclear, and we have demonstrated that in conditions in oocytes in
which activation of MAPK is allowed, PKA does not inhibit the c-Mos
translation (14), raising the possibility that PKA could act on a
single target in the negative regulation of maturation.
A number of studies demonstrate the importance of the MAPK cascade in
MPF activation (6, 15) during Xenopus oocyte maturation. Indeed, microinjection of recombinant c-Mos is itself sufficient to
induce oocyte maturation in the absence of hormonal treatment (16, 17),
whereas CL100 (MAPK phosphatase-1) inhibits progesterone-induced maturation (18). Important work in Saccharomyces cerevisiae toward the understanding of the control of the MAPK cascade has been
undertaken in the last few years. The response to the mating pheromone
signal is mediated by the direct interaction of the G p21-activated kinases (PAKs) are Ste20 homologues. Understanding of
their functional importance in signal transduction and in regulation of
the actin cytoskeleton is rapidly growing (see Refs. 22-24). We have
cloned Xenopus PAKs and recently demonstrated that a member
of this family is involved in regulation of the oocyte maturation
process (1). Indeed, we showed that microinjection of a constitutive
truncated version of X-PAK1 (X-PAK1-Cter) into oocytes completely
blocks both insulin and progesterone mediated maturation, and
conversely, the dominant negative version of the kinase facilitated
this process. In this present report, we focus on how X-PAK1 negatively
regulates maturation. Our results demonstrate that X-PAK1-Cter prevents
c-Mos synthesis and MAPK activation in progesterone-treated oocytes.
Even if MAPK is activated by injection of recombinant c-Mos, no H1
kinase activity can be detected. All the enzymes involved in regulation
of p34cdc2 kinase are affected by X-PAK1-Cter expression in
progesterone-treated oocytes. We show that in the presence of X-PAK1
kinase activity, pre-MPF complexes can efficiently form but activation
of pre-MPF fails. Furthermore, in the presence of active MPF, X-PAK1
kinase activity prevents the amplification loop from being switched on. We have analyzed a possible link of transduction pathways used by PKA
and X-PAK in controlling maturation and show that X-PAK1-Cter is still
capable of inhibiting maturation induced by the regulatory domain of
PKA. Finally, using cell-free Xenopus egg cycling extracts, which mimic the alternation of S and M phases of the first embryonic cycles, we can reproduce the inhibition of entry into mitosis with the
catalytic domain of X-PAK1. Thus the involvement of Xenopus PAK in G2/M transition is not restricted to oocyte meiotic
maturation and can be extended to the first embryonic cell cycles.
Preparation and Handling of Oocytes--
Xenopus
females were obtained from the CNRS breeding center located in
Montpellier, France. Fully grown oocytes were prepared, free from
follicle cells, by collagenase treatment in Ca2+-free
OR2 (12), then transferred in MMR (100 mM NaCl,
2 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 0, 1 mM EGTA, and 5 mM HEPES, pH 7.7) in which all experiments were performed.
The usual volume microinjected was 40 nl; injections were performed
near the equator of the oocytes. Progesterone was used with a final
concentration of 1 mM; cycloheximide (100 µg/ml) was
added 1 h before further treatment (progesterone or microinjection).
Oocytes were homogenized (10 µl/oocyte) in XB buffer (50 mM sucrose, 100 mM KCl, 0, 1 mMCaCl2, 1 mM MgCl2, and 10 mM HEPES, pH 7.8) and then briefly centrifuged for 2 min at
13000 rpm to remove yolk proteins. Supernatants were then used for
Western blots or immunoprecipitations.
Cycling Extracts--
Cycling extracts were prepared essentially
as described (25). Briefly, fresh eggs were dejellied in 2% cysteine
(pH 7.8) washed and parthenogetically activated. Eggs were crushed at
12,000 × g for 15 min, and the cytoplasmic layer was
supplemented with an ATP regenerating system, cytocholasin B and
protease inhibitors, before use. Sperm chromatin (500/µl) and
[35S]methionine (1 mCi/ml) were added to the extract at
time 0. Recombinant proteins (2 µl, at 0.2 mg/ml per 50 µl extract)
were added after 10 min. Nuclei assembled from demembranated sperm
nuclei and NEBD (26) were observed with a fluorescent microscope using
Hoechst 33342 to stain chromosomes. Aliquots were withdrawn at
indicated times and directly frozen in liquid nitrogen. All samples
were thawed together and treated simultaneously, either
immunoprecipated with a mix of anti-Xenopus cyclin B1 and B2
or with anti-Xenopus cyclin A; H1 kinase assays were
performed on the different immunoprecipitates and analyzed by15%
SDS-PAGE. Total extracts were also analyzed on Western blots with
anti-p34cdc2 antibodies directed against the C terminus.
Plasmids and Recombinant Proteins--
Cloning of X-PAK1-Cter in
pMal vector has been reported previously (1). The c-Mos pMal plasmid
was kindly provided by A. Nebreda and T. Hunt (27).
Both recombinant c-Mos-MalBP and X-PAK1-CterMalBP proteins were
produced as soluble proteins and purified on amylose-Sepharose matrix
according to standard proctocols (28). The sea urchin glutathione
S-transferase-cyclin B construct was kindly donated by Dr.
J. Gautier (glutathione S-transferase is fused to sea urchin type B cyclin (from amino acids 13 to 409)); the recombinant protein was expressed in Escherichia coli and affinity purified as
described previously (29).
Construction of pSP64T plasmid encoding histidine-tagged full-length
Xenopus Myt1 is described in Ref. 30. The pSP64 plasmid encoding full-length 5'-His-tagged Wee1 was constructed as follows. The
pET construct encoding full-length Wee1 (kindly provided by W. G. Dunphy) was used (1 ng) in a PCR reaction with both BATG primer
(5'-CCGTCGACGGCATATGAGGACGGCCATGTCATG) containing a
SalI restriction site (underlined) and CTERWEE primer
(5'-CCAAGCTTGCAGTCCCAGCTCCTGAATCCACA-3') containing a HindIII restriction site
(underlined). The resulting PCR product was cloned in frame in pQE30
vector (Qiagen) linearized by SalI and HindIII.
Full-length clone were screened and sequenced and then used in a PCR
reaction (1 ng) with both pQE31 primer (5'-CAATTGTGAGCGGATAACAATT-3')
and CTERWEE primer described above. The resulting PCR product was
EcoRI blunt inserted in pSP64 vector. In vitro
translations of both pSP64 Myt1 and pSP64T Wee1 were performed using
TNT coupled rabbit reticulocyte lysate kit (Promega) and
[35S]methionine-labeled according to the manufacturer's instructions.
Antibodies, Immunoprecipitation, and Western Blot
Analysis--
Polyclonal antibodies directed against
Xenopus c-Mos and MAPK were obtained from Santa Cruz
(Santa Cruz, CA). The c-Mos antibody was raised against peptide
314-335 within the C-terminal domain of Xenopus c-Mos;
the MAPK antibody was raised against peptide 305-327 of rat ERK1 and
recognizes both ERK1 and ERK2 in all vertebrates tested. Anti-Plx1
polyclonal antibody raised against a peptide encoding the 17 C-terminal
amino acids of Plx1, the Xenopus anti-Cdc25 antibody raised
against the recombinant protein, and the X-PAK1-Cter antibodies against
the C-terminal peptide of the protein were previously described (1,
30). Anti-Xenopus cyclin B1, B2, and A polyclonal antibodies
have been made in the laboratory by immunizing rabbits against
recombinant fusion proteins.
The sea urchin cyclin B was recovered by incubating oocyte extracts
with glutathione-Sepharose beads in modified RIPA buffer (10 mM NaH2PO4, pH 7.5, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5%
deoxycholate, 80 mM
Cyclin B and cyclin A immunoprecipitates were performed by incubation
of the extracts with the indicated antibodies linked to protein
A-Sepharose in modified RIPA buffer for 1 h at 4 °C and washed
three times in modified RIPA buffer and twice in kinase buffer (25 mM HEPES, pH 7.5, 25 mM MgCl2, 25 mM Kinase Activities of MPF--
Total Histone H1 kinase activities
were assayed according to the protocol described by Labbé
et al. (31). Immunoprecipitates were incubated for 15 min in
20 µl of kinase buffer containing 1 µl of histone H1 at 5 mg/ml, 1 µl of 1 mM ATP, and 0.5 µl of [
In gel MBP kinase assays were performed exactly as described by
Shibuya et al. (32). Briefly, samples were run in a 15% polyacrylamide gels containing 0.5 mg/ml MBP. After denaturation and
renaturation steps, gels were incubated in the presence of [
For Plx-1 activities, immunoprecipitates were incubated for 15 min in a
mix containing 1 mg/ml of dephosphorylated
Cyclin B-p34cdc2 kinase used for microinjection was prepared
from starfish oocytes by affinity chromatography on p13 suc1-Sepharose and ion exchange chromatography on MonoQ and MonoS columns as described
(31).
X-PAK1-Cter Prevents Progesterone-induced MAPK Activation--
We
previously observed (1) that catalytically active X-PAK1-Cter inhibits
germinal vesicle breakdown (GVBD) in progesterone-treated oocytes. We
have demonstrated the physiological relevance of our results since a
dominant negative (X-PAK1-CterK/R) mutant reproducibly facilitates
hormone-induced maturation. To investigate how X-PAK1-Cter exerts its
inhibitory function, we first examined, as shown in Fig.
1 (A-D), the fate of the well
known events triggered by progesterone treatment or c-Mos
microinjection during oocyte maturation in the presence or in the
absence of active X-PAK1-Cter. Possible effects of X-PAK1-Cter in the
absence of maturation inducing factor were controlled in parallel. H1
kinase activity, reflecting MPF activation, is shown in Fig.
1A. When GVBD occurred in c-Mos microinjected or
progesterone-treated oocytes, a strong activity was measured; by
contrast, when X-PAK1-Cter was injected 1 h before c-Mos
microinjection or progesterone treatment, no GVBD was observed and H1
kinase activation was completely abolished (Fig. 1A).
Because p21-activated kinases are involved in MAPK regulation, we
analyzed the accumulation of the c-Mos proto-oncogene (Fig.
1B) by Western blot, as well as MAPK activity against the
substrate MBP in gel (Fig. 1C) and by immunoblotting using
anti-MAPK antibodies (Fig. 1D). Neither c-Mos
accumulation (Fig. 1B) nor MAPK activation (Fig. 1,
C and D) occurred on injection of X-PAK1-Cter
alone, whereas in both progesterone-treated or c-Mos-microinjected
control oocytes, the endogenous proto-oncogene c-Mos was readily
detected by Western blot (Fig. 1B), MAPK was phosphorylated
resulting in its mobility shift upon activating tyrosine
phosphorylation on Western blot (Fig. 1D) and activated as
observed against the MBP substrate (Fig. 1C). In contrast
X-PAK1-Cter efficiently blocked c-Mos accumulation in
progesterone-treated oocytes, which further prevented any activation of
MAPK (Fig. 1, C and D). However, in c-Mos
microinjected oocytes (Fig. 1B), X-PAK1-Cter failed to
prevent MAPK activation (Fig. 1, C and D), and
because MAPK was activated, endogenous c-Mos accumulation was
observed (Fig. 1B), but GVBD did not occur. Thus X-PAK1-Cter
is likely to target not only MAPK activation but also MPF activation in
progesterone-treated oocytes and therefore mimics the effects of PKAc
on oocyte maturation (13, 14).
Catalytically Active X-PAK1 Does Not Interfere with Pre-MPF Complex
Formation--
Free p34cdc2 is believed to be in large excess
in stage 6 oocytes because only a limited amount of endogenous cyclin B
is available (33), and microinjection of recombinant sea urchin cyclin
B is sufficient to induce GVBD and MPF activation (34). We took advantage of these observations to test whether X-PAK1 is involved in
regulation of cyclin B-p34cdc2 complex formation. To do so, we
examined the association of the exogenous cyclin B to the endogenous
p34cdc2 subunit in the presence of X-PAK1-Cter. X-PAK1-Cter was
injected into oocytes 1 h before cyclin B injection, five oocyte
aliquots were withdrawn at different times after injection as
indicated, and the exogenous cyclin B was recovered from crushed eggs
on glutatione-Sepharose beads. Bound proteins were tested in parallel, for associated H1 kinase activity and by Western blot for the presence
of cyclin B associated p34cdc2 kinase. In control oocytes
injected with recombinant cyclin, H1 kinase was readily detected on
glutathione-beads (Fig. 2, lower panel), but when oocytes were injected with recombinant cyclin B
in the presence of X-PAK1-Cter, no H1 kinase activity (Fig. 2,
lower panel, +X-PAK1-Cter) was obtained, even
upon longer incubation (data not shown). An equivalent amount of Cdc2
subunit was bound to exogenous cyclin B in these oocytes (Fig. 2,
upper panel, +X-PAK1-Cter) compared with controls
(Fig. 2, upper panel, Upon Progesterone Treatment, p34cdc2 Kinase Regulatory
Enzymes Are Affected by X-PAK1-Cter Activity--
Activation of MAPK
by c-Mos microinjection is not sufficient to prevent the
inhibition of maturation by X-PAK1-Cter. Cyclin B and p34cdc2
subunits can associate in the presence of X-PAK1-Cter, but these pre-MPF complexes fail to activate. Thus catalytically active X-PAK1
must target a component involved in the activation/inactivation status
of p34cdc2 upstream regulators. During the first embryonic
cycle, both Myt1 and Wee1 kinases are inactivated by
hyperphosphorylation at the G2/M transition (9, 10), and at
least one of the kinases responsible for Wee1 phosphorylation is
p34cdc2 itself (10), which participates in this way to the MPF
amplification loop. Hyperphosphorylation is easily visualized in gel by
a dramatic mobility shift of the kinases. Murakami and Vande Woude (35) recently showed that Wee1 is not yet present in stage 6 oocytes; Myt1
may thus be the only Cdc2 inhibitory kinase present in
Xenopus oocytes. Nevertheless, microinjection of in
vitro transcribed Xe-Wee1 RNAs in oocytes inhibits
progesterone-induced maturation probably by changing the
kinase/phosphatase ratio, thus impeding activation, due to the limited
amounts of endogenous regulatory proteins available. As shown in Fig.
3A, we injected very small amounts (so as not to interfere with the physiological balance of
proteins present in oocytes) of in vitro translated
[35S]methionine-labeled kinases to assess their
phosphorylation state in progesterone-treated oocytes and in the
presence of active X-PAK1-Cter. As expected, in control
progesterone-treated oocytes, both Myt1 and Wee1 are
hyperphosphorylated; but in contrast, no shift is observed when
progesterone-treated oocytes are microinjected with X-PAK1-Cter,
indicating that neither kinase is inactivated by progesterone treatment
if X-PAK1-Cter is present.
Inactivation of p34cdc2 inhibitory kinases is coincident with
phosphorylation of the p34cdc2 activating Cdc25 phosphatase.
Cdc25 phosphatase is positively regulated by at least two kinases, Plx
(36) and MPF itself. To test whether Cdc25 activation is also targetted
in the X-PAK1 inhibition of maturation, we performed Western blot
analysis using anti-Cdc25 antibodies of stage 6 oocytes and
progesterone-treated oocytes in the presence or absence of X-PAK1-Cter.
As previously shown, Cdc25 phosphatase was shifted in mature
progesterone-treated oocytes (37, 38), compared with control stage 6 oocytes. No shift was obtained when X-PAK1 had been injected in
progesterone-treated oocytes, demonstrating that X-PAK1 prevents
progesterone-induced activation of Cdc25c. Cdc25c is a target of Plx, a
Xenopus Polo-like kinase (36), and our group has reported
that Plx is a component of the MPF amplification loop required for full
MPF activation at the onset of mitosis. Thus our next experiment was to
verify the level of activation of Plx in progesterone-treated oocytes in the presence of catalytically active X-PAK1. As shown on Fig. 3C, no activity is detected on Plx immunoprecipitates in
stage 6 oocytes using casein as a substrate, whereas as early as 3 h after progesterone treatment we detected a very strong Plx activity. In contrast when progesterone-treated oocytes had been injected with
X-PAK1-Cter, no Plx activity is associated with immunoprecipitates. Thus X-PAK1 prevents Plx activation induced by progesterone treatment of the oocytes by an as yet unknown mechanism.
X-PAK1-Cter Prevents the Amplification Loop from Being Turned
On--
Our results indicate that X-PAK1 is likely to exert its
control over the maturation process by negatively regulating an early event required to activate the small amount of MPF necessary to turn on
the positive feedback loop. To further confirm the role of X-PAK1-Cter,
we injected subthreshold concentration of active cyclin
B-p34cdc2 complexes purified from starfish 1 h after a
preliminary injection of MalBP or X-PAK1-Cter (Fig.
4). In the absence of X-PAK1-Cter, 50%
of the oocytes presented a white spot, characteristic of GVBD, as soon
as 1 h after MPF injection, whereas in the presence of X-PAK1-Cter
activity, no maturation spot was observed. We examined the effect of
X-PAK1-Cter injection on the active cyclin B/p34cdc2 kinase
complex in terms of MAPK activation and H1 kinase activity and by
Western blot analysis of XPAK1 and Cdc25. Western blot analysis of
XPAK1 (Fig. 4, upper panel) demonstrates that the recombinant protein is stable throughout the experiment. The zero time
(cyclinB-p34cdc2) represents the subthreshold H1 kinase
activity injected in oocytes and measured immediately after the
injection (Fig. 4, bottom panel). The kinase amplification
loop is rapidly turned on because H1 kinase activity reached a maximum
as early as 1.5 h after MPF injection. This occurred in parallel
with both MAPK activation and a shift of Cdc25 as revealed by Western
blot. In contrast, the injected H1 kinase was not amplified in the
presence of X-PAK1-Cter; the MPF activity is therefore not sufficient
by itself in the presence of X-PAK1-Cter to phosphorylate the first
element of the positive amplification loop.
X-PAK1-Cter Blocks G2/M Progression in the First
Mitotic Cycle--
To test whether X-PAK1-Cter inhibition of MPF
activation is specific for the G2/M transition during
Xenopus oocyte maturation or can be extended to the first
mitotic cell cycles, we took advantage of Xenopus cycling
egg extracts, which spontaneously oscillate between interphase and
mitosis. Cycling extracts were prepared as described previously (25).
Purified recombinant MalBP or X-PAK1-Cter fusion proteins were added 10 min after sperm chromatin addition (t = 0) to the
extract, [35S]methionine was added in parallel to follow
endogenous cyclin synthesis and degradation (Fig. 5). Aliquots were
withdrawn at various times for analyses, and sperm chromatin swelling,
nuclei formation, and nuclear envelope breakdown (NEBD) were monitored microscopically. NEBD was routinely observed in control extracts containing purified MalBP protein at around 60-70 min after the start
of the experiment, and by 80-90 min mitosis had ended (data not
shown). However, NEBD was never observed in extracts supplemented with
X-PAK1-Cter. Instead, nuclei grew larger in size and remained stable
for at least 2 h. We first analyzed cyclin A-Cdc2 activation on
cyclin A immunoprecipitates using histone H1 as a substrate. Fig.
5A shows the kinetic of cyclin
A-p34cdc2 kinase activation. In control extracts with MalBP
added, kinase activity increases during interphase to reach a maximum
at 60 min, just before NEBD, and then disappears due to cyclin A
degradation (Fig. 5A, MAlBP). In contrast, when X-PAK1-Cter
kinase is present in the extract (Fig. 5A, X-PAK1-Cter),
cyclin A-p34cdc2 activity increases to finally stabilize at a
very high level, but no drop in the kinase activity can be detected
during the entire length of the experiment, indicating that cyclin A
degradation did not occur in these extracts.
Identical samples were immunoprecipitated with a mix of antibodies
directed against cyclin B1 and cyclin B2 to analyze cyclin B-p34cdc2 activation (Fig. 5B). As expected, in
control extracts (Fig. 5B, MalBP) cyclin
B-p34cdc2 activity increased to maximum at 70 min, activation
of the kinase (H1) correlated with phosphorylation of the
radiolabeled cyclin B1/B2 synthesized in the extract
(CB1), as visualized by a shift in gel. At 80 min,
cyclin B-p34cdc2 activity decreased dramatically, and the
radiolabeled cyclin disappeared, indicating that cyclin degradation had
occurred. Cyclin synthesis (CB1) was observed again
at the beginning of the next cycle (100 min). When catalytically active
X-PAK1-Cter was added to the extract, no activation of cyclin B-Cdc2
could be detected (Fig. 5B, X-PAK1-Cter,
H1). This result correlates with the absence of shift of
[35S]methionine-labeled newly synthesized cyclin B, as
well as by stability of the labeled immunoprecipitated cyclin B band
over the time of the experiment (Fig. 5B,
X-PAK1-Cter, CB1). We thus confirm the
previous finding (30) that active cyclin A-p34cdc2 complexes
are not sufficient for the activation of cyclin B-p34cdc2 kinase.
We further analyze, using Western blot with anti-Cdc2 antibodies, that
during the course of incubation (between 20 and 90 min) of the cycling
extract with X-PAK-Cter, p34cdc2 is progressively shifted,
reflecting phosphorylation and consequent inactivation on
Tyr15 and Thr14 residues (Fig. 5C,
X-PAK1-Cter). The phosphorylation on tyrosine was confirmed
using anti-phospho-tyrosine antibodies (data not shown). At 90 min, in
the presence of X-PAK-Cter, roughly half of the p34cdc2
subunit, is shifted, and the lower band probably represents
p34cdc2 associated with cyclin A (Fig. 5C,
X-PAK1-Cter). In contrast, in the MalBP supplemented
extract, all p34cdc2 subunit is present as a faster migrating
form upon NEBD, corresponding to full activation of cyclin
B-p34cdc2 complexes (Fig. 5C, MalBP).
Therefore, the inhibitory effect of X-PAK1-Cter on G2/M
progression is not restricted to meiosis.
X-PAK1 Prevents Oocyte Maturation Induced by PKA Regulatory
Subunit--
Protein kinase A is a tetramer composed of two regulatory
and two catalytic subunits (39), and cAMP binding to the regulatory subunit of protein kinase A induces its release from the catalytic subunit and subsequent activation of the kinase. Microinjection into
oocytes of an excess of PKA regulatory subunit inhibits endogenous PKA
activity and induces oocyte maturation (40). Because X-PAK1-Cter targets the same components to bring about the block of the
G2/M transition as those previously reported for the
catalytic subunit of protein kinase A, we next wanted to study a
possible link between protein kinase A and X-PAK transduction pathways
in the control of the G2/M transition. Thus, we injected
oocytes first with X-PAK1-Cter and then with PKA/R. The amount of PKA/R
we injected is itself sufficient to bind all endogenous PKA/C and
inactivate PKA, because we observed a maturation spot on these oocytes
and confirmed that indeed maturation occurred by H1 kinase activation
and c-Mos accumulation (Fig. 6).
However, in the presence of X-PAK1-Cter, PKA/R was no longer able to
induce c-Mos synthesis because no accumulation of the
proto-oncogene c-Mos was detected by Western blot (Fig. 6) and no
activation of pre-MPF occurred, because H1 kinase activity was low and
the oocytes remained morphologically indistinguishable from stage 6 oocytes.
Considering the above result, we addressed the question whether X-PAK
could be an effector downstream of PKA on the same transduction pathway. If this is the case we should measure the increased activity of the endogenous X-PAK1 when PKA is activated. However, because the
three cloned Xenopus PAKs present a high level of homology in their kinase domain (1),2
we tested a possible enhancement of kinase activity by active PKA for
all three X-PAKs. We thus isobutylmethylxanthine-treated or mock
treated oocytes for various lengths of time (1-5 h) and immunoprecipitated the endogenous PAKs with three different specific antibodies. Kinase assays using histone 2B as a substrate were performed on the immunoprecipitates. No significant changes in the
three Xenopus PAKs kinase activity could be measured between the oocytes presenting an increased PKA activity and the control oocytes (data not shown).
We tested yet another potential link between the two pathways, a
potential regulatory phosphorylation of PKA/R and/or PKA/C by
X-PAK1-Cter. X-PAK1-Cter was microinjected in oocytes for either 1 or
2 h, and the active recombinant kinase recovered on amylose-beads was then tested for its ability to use PKA/C and PKA/R subunits as
substrates in in vitro kinase assays. No phosphorylation of PKA/C nor PKA/R was observed under these conditions (data not shown).
Oocyte maturation is a highly regulated process, and a complete
understanding of the many intertwining regulations involved is far from
being a reality. GVBD is always associated with high cyclin
B-p34cdc2 activity, which results from the activation of
stockpiled complexes, occurring by a well described amplification loop.
Although experimental conditions have been described in which GVBD can
occur in the absence of MAPK activity (34), it is widely accepted that
the MAPK cascade participates in normal maturation in the activation of
MPF. Understanding the event required for activation of the first
cyclin B-p34cdc2 complex, which in turn will start the
amplification loop, is crucial to explain the maturation process, and
in this regard identification of new regulatory proteins will certainly
help to reconstitute the sequence of activation of the different pathways.
In this report we investigate how constitutive Xenopus
p21-activated kinase negatively controls the oocyte maturation process. Our results indicate that X-PAK1 mimics the effects previously reported
for the catalytic subunit of protein kinase A by preventing c-Mos
accumulation as well as MAPK and MPF activation (13, 14). We show, in a
context in which MAPK activation is activated by injection of
c-Mos, that accumulation of the endogenous proto-oncogene does
occur, in the presence of X-PAK1. Thus X-PAK1 does not interfere with
the amplification loop in which active MAPK controls c-Mos synthesis (42) but rather prevents the initial MAPK activation. Even on
c-Mos microinjection, no H1 kinase activity is detected, indicating that X-PAK1 also affects MPF activation. We used recombinant cyclin B, and active MPF, to demonstrate that formation of new pre-MPF
complexes is not affected but that they are not activated in the
presence of active X-PAK1.
Indeed, we show that the regulatory kinases and Cdc25 phosphatase
identified in MPF amplification during the maturation process remain in
their initial state. A well accepted concept is that activation of the
first MPF molecules is the result of activation and/or
inactivation of Cdc25 and Myt1, respectively, by an upstream regulating
element different from MPF itself. Indeed, in SDS-PAGE, Cdc25 displays
a complex phosphorylation pattern and can also be phosphorylated and
activated by kinases other than MPF itself, Plx-1 (36), and
possibly Raf-1(43).
Raf-1 may be a Cdc25-activating kinase (43), and it also acts as a MAPK
kinase kinase (44) whose involvement in regulation of maturation is
subject to controversy. Using a dominant negative mutant approach it
has been reported (45) that Raf-1 activation is required for
progesterone-induced maturation, but this result was challenged (46).
It was recently shown that autophosphorylation of Raf-1 on serine 621 (47) is a way to down-regulate its activity, and this same site can
also be phosphorylated by PKA. We discussed in a previous paper a
possible transient activation of Raf-1 in Xenopus oocytes,
upon down-regulation of PKA, during normal maturation induced by
progesterone (14). Because Raf-1 mobility shift observed during
maturation by progesterone is usually equated to the kinase activation,
we verified that Raf-1 is indeed prevented from shifting upon
progesterone treatment of oocytes injected with X-PAK1-Cter (data not
shown). As X-PAK1 belongs to the well known family of PAK proteins that
were first isolated as upstream regulators of MAPK cascades,
involvement of X-PAK in Raf-1 kinase regulation is an interesting hypothesis.
We show that X-PAK1-Cter negatively targets the same events in
inhibition of maturation as PKA/C and moreover that X-PAK1-Cter prevents maturation induced by the regulatory subunit of protein kinase
A, but we have been unable to demonstrate any direct activation of
endogenous X-PAK1 or two other closely related family members X-PAK2
and X-PAK3 by PKA/C. Both pathways could also be linked in a different
manner; for example, X-PAK could interfere with the ability of PKA to
form a tetramer (for example by phosphorylation of PKA/R) and
consequently inhibit its inactivation. Indeed it has previously been
demonstrated that PKA/RII can be phosphorylated in vitro by
different kinases, and recently Keryer et al. (41) reported
that MPF can also phosphorylate it. We verified that neither PKA/R nor
PKA/C are substrates of X-PAK1-Cter. At the moment we cannot rule out
other possible cross-links in the pathways used by the two kinases;
however, another possible explanation of our results would be that both
pathways are parallel and that active X-PAK and PKA target an
identical substrate that allows the MPF amplification loop to be turned on.
Both PKA and X-PAK kinases are also capable of inhibiting the
G2/M transition during the first mitotic cycle, at which
point cyclin B-p34cdc2 is still regulated by
phosphorylation on Tyr15, but control by MAPK activity is
absent. We show that X-PAK1-Cter does not prevent cyclin
A-p34cdc2 activation in cell-free egg cycling extracts, and in
fact the kinase reaches an abnormally high level of activity compared
with a control cycling extract supplemented with MalBP fusion protein. Nevertheless, under these conditions, high cyclin A-p34cdc2
activity does not allow cyclin B-p34cdc2 activation, and as a
consequence the cyclin degradation pathway is not turned on. We show
that cyclin B-p34cdc2 complexes are indeed formed, but no H1
activity is associated with them, and the p34cdc2 subunit
displays a slow electrophoretic mobility indicative of phosphorylation
on Tyr15. We conclude that X-PAK1-Cter activity is very
specific because it does not interfere with the activity of cyclin
A-p34cdc2 complexes, which are not subject to a regulation by
phosphorylation on Tyr15 (30, 48), but it does interfere
specifically with the activation of cyclin B-p34cdc2 complexes
by preventing dephosphorylation of Tyr15. In 1994 Grieco
et al. (49) showed that PKA activity oscillates during the
first embryonic cycles in Xenopus extracts, being high at
interphase and falling at the onset of mitosis. They demonstrated that
sustained PKA activity inhibits cycle progression in a
dose-dependent manner and that extracts eventually arrest
in interphase with a high level of inactive pre-MPF complexes. In this
context, X-PAK1-1Cter and PKA probably act independently of a control
over MAPK activity, because MAPK is inactive in the cycling extract,
and so this cannot be compared with the G2/M transition
block described previously (50), in which the proto-oncogene c-Mos
is added to cycling extracts. The work of Grieco et al. (49)
indicates that PKA could stimulate an okadaic acid-sensitive
serine/threonine phosphatase pathway that dephosphorylates Cdc25.
Plx kinase is another Cdc25 positive regulatory kinase (36) recently
shown (30) to be required for the G2/M transition during
the first mitotic cell cycle in Xenopus eggs. Cyclin
B-p34cdc2 enhances Plx activation, which triggers Cdc25 and MPF
activation in a positive feedback loop. Adding blocking anti-Plx
antibodies to an egg cycling extract suppressed entry into mitosis,
although a strong cyclin A-p34cdc2 activity is detected, as we
observed ourselves in the X-PAK1-Cter block in cycling extracts.
Moreover, pre-MPF complexes also remain inactive and phosphorylated on
Tyr15, if Plx is blocked with these antibodies. We did not
investigate the status of Plx in cycling extracts incubated with
X-PAK1-Cter, but we show that during the maturation process Plx
activation is prevented by active Xenopus p21-activated kinase.
In summary, our data demonstrate that PKA and X-PAK pathways
target the same components in their inhibition of G2/M
progression during meiosis as well as in the first mitotic cycle in
Xenopus and that their transduction pathways are likely to
be connected at the point where the MPF amplification loop is turned
on. We are currently in the process of isolating physiological
substrates of X-PAK active kinase in Xenopus extracts to get
new insights into factors regulating the G2/M transition.
INTRODUCTION
Top
Abstract
Introduction
References
subunit of G
protein heterotrimers to Ste20, a serine threonine kinase (19).
Activation of Ste20 results in activation of the MAPK kinase kinase
Ste11, which allows the MAPK cascade to be turned on and results in
MAPK (Kss1 and Fus3) activation (reviewed in Ref. 20). In
Xenopus oocytes, Gotoh et al. (18) showed that
microinjection of constitutively active Ste11 is able to efficiently
induce the maturation process. Moreover, addition of the N-terminal
regulatory domain of Ste11 to Xenopus extracts prevents a
constitutive form of Ste20 from activating MAPK (21).
EXPERIMENTAL PROCEDURES
-glycerophosphate, 50 nM
NaF, and 1 mM dithiothreitol). The beads were washed
several times, boiled in Laemmli buffer, loaded onto SDS-PAGE, and
analyzed by Western blotting with the anti-Cdc2 antibody.
-glycerophosphate, 2 mM dithiothreitol,
and 0.1 mM orthovanadate).
-32P]ATP, loaded on 15% SDS-PAGE, and visualized by autoradiography.
-32P]ATP. Gels were washed and dried; in gel MBP
kinases were visualized by autoradiography.
-casein, 100 mM ATP, 2 mM MgCl2, and 50 mM Tris, pH 7.5. Immunocomplexes were boiled in Laemmli
buffer and run on SDS-PAGE; the phosphorylation of casein substrate was
visualized by autoradiography.
RESULTS
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Fig. 1.
X-PAK-Cter inhibits meiotic maturation
induced by both progesterone treatment and c-Mos
microinjection. A, oocytes were microinjected
with X-PAK1-Cter for 1 h or injected with buffer before
progesterone treatment or c-Mos microinjection. Oocytes were
monitored for GVBD, and homogenates were prepared 6 h after
hormonal stimulation or last microinjection. Total cell extracts (1.5 oocyte equivalents/reaction) were assayed for H1 kinase activities
(A) or c-Mos accumulation monitored by Western blotting
(B) or MAPK activities measured by both in gel
phosphorylation of MBP substrate (C) and changes in the
electrophoretic mobility of MAPK detected by Western blotting
(D). In this experiment, GVBD occurred 3 h after
progesterone treatment or c-Mos microinjection of oocytes but
never when X-PAK1-Cter was coinjected in oocytes. Similar results were
obtained in four independent experiments.
X-PAK1-Cter), but the
Cdc2 subunit migrated as a slower band, indicating that residues Thr14 and Tyr15 are phosphorylated. Therefore,
pre-MPF complex formation occurs but is not activated in the presence
of X-PAK1-Cter.
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Fig. 2.
X-PAK-Cter prevents recombinant sea urchin
cyclin B from inducing meiotic maturation in Xenopus
oocytes without preventing pre-MPF complex formation.
Xenopus stage 6 oocytes were first microinjected
(+X-PAK1-Cter) or not ( X-PAK1-Cter) with
recombinant X-PAK1-Cter protein before a second injection 1 h
later with recombinant sea urchin cyclin B. After the second injection,
oocytes were homogenized at indicated times, sea urchin cyclin B was
recovered on glutathione-Sepharose beads, and purified complexes were
immunoblotted with an antibody directed against the C-terminal domain
of Xenopus p34cdc2 (upper panel) or
assayed for H1 kinase activities (lower panel).
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Fig. 3.
p34cdc2 kinase regulatory enzymes are
affected by X-PAK1-Cter activity during progesterone-induced meiotic
maturation. A, stage 6 Xenopus oocytes were
divided into three groups. In group 1, they were simultaneously
microinjected with in vitro translated
35S-labeled MYT1 (upper panel) or WEE1 (bottom
panel) and X-PAK1-Cter 1 h before progesterone treatment
(Pg +, X-PAK1-Cter +). In group 2 X-PAK1-Cter
microinjection was omitted (Pg +, X-PAK1-Cter
). The third group of control oocytes were only microinjected
with in vitro translated 35S-labeled MYT1 or
WEE1 (Pg
, X-PAK1-Cter
). 5 h after
progesterone treatment, oocytes were homogenized and monitored for
changes in electrophoretic mobility of WEE1, MYT1 directly by
autoradiography (A). B, oocytes were treated
exactly as in A except microinjection of the in
vitro translation product was omitted. The shift of endogenous
Cdc25 was analyzed by Western blotting. C,
Xenopus stage 6 oocytes were microinjected with X-PAK1-Cter
protein 1 h before progesterone treatment (Pg +,
X-PAK1-Cter +), and control progesterone-treated oocytes
(Pg +, X-PAK1-Cter
) and stage 6 oocytes
(Pg
, X-PAK1-Cter
) were analyzed in
parallel. At the indicated time after progesterone treatment, oocytes
were homogenized, and Plx1 activities were measured on
immunoprecipitates (3 oocyte equivalents/reaction) using casein as a
substrate (upper panel). H1 kinase activities were measured
in parallel on the same homogenates (lower panel).
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Fig. 4.
X-PAK1-Cter prevents the amplification-loop
of Xenopus pre-MPF from being turned on.
Xenopus stage 6 oocytes were microinjected
(X-PAK1-Cter +) or not (X-PAK1-Cter ) with
X-PAK1-Cter protein before a second injection 1 h later with
purified starfish cyclin B-p34cdc2 kinase. At different times
(90, 180, and 270 min) after the second injection, oocytes were
withdrawn, homogenized, and analyzed by in-gel MBP kinase assay for
determination of MAPK activities (B). Xenopus
Cdc25 electrophoretic mobility shift was monitored in parallel by
Western blot (C), and the same blot was dehybridized and
further probed with anti-X-PAK1 antibodies to verify the stability of
the microinjected recombinant protein during the time course of the
experiment (A). H1 kinase activities were measured in total
cell extracts immediately after injection of starfish cyclin
B-p34cdc2 kinase (0) or at different times (90, 180, and 270 min) after this injection (D). Time 0 indicates that the
starfish cyclin B-p34cdc2 kinase activity injected is not
sufficient by itself to induce GVBD and requires the amplification loop
to be turned on, because GVBD50 only occurred at about 45 min after microinjection of the kinase.
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Fig. 5.
X-PAK1-Cter blocks G2/M
progression in the first mitotic cycle.
[35S]Methionine and sperm chromatin were added at time 0 to a Xenopus cycling egg extract. 10 min later, either MalBP
or X-PAK1-Cter recombinant proteins were added to the extract. Samples
were withdrawn at indicated times and observed microscopically for
nuclei formation and NEBD. Later on, aliquots of the samples were
immunoprecipitated with anti-cyclin A antibodies, and the
immunoprecipitates were tested for H1 kinase activity (A).
Other aliquots of the same samples were analyzed for H1 kinase activity
on anti-cyclin B1 and B2 immunoprecipitates (B,
H1). The upper part of the same gel was exposed
longer to visualize endogenous cyclin B synthesis, shift by
phosphorylation, and degradation. Aliquots of the same samples taken
between 20 and 90 min during the time course of the experiment were
directly loaded on SDS-PAGE, and the total p34cdc2 subunits
were analyzed by Western blot. In this particular experiment, NEBD was
detected at 60 min in MalBP supplemented extract, and NEBD never
occurred in X-PAK1-Cter supplemented extracts.
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Fig. 6.
X-PAK1-Cter prevents meiotic maturation
induced by the microinjection of the PKA regulatory subunit.
Xenopus stage VI oocytes were microinjected or not with
X-PAK1-Cter protein and injected again 1 h later with the purified
R1 regulatory subunit of PKA. 5, 7, and 9 h after the second
injection, oocytes were homogenized, and total cell extracts were
analyzed for H1 kinase activities (upper panel,
H1) or c-Mos accumulation monitored by Western blotting
(bottom panel, c-mos). The appearance of a white
spot, indicating that maturation had occurred, was obvious in R1
injected oocytes as soon as 4 h after the injection but was never
observed when X-PAK1-Cter was injected before PKA/R.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We are indebted to Marcel Dorée, Jean
Claude Labbé, and Thierry Lorca for fruitful discussions and the
gift of purified MPF. We acknowledge Dr. Keryer for the gift of
PKARII recombinant subunit. We especially thank Daniel Fisher for
reading the manuscript and Pascal de Santa Barbara for help with artwork.
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FOOTNOTES |
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* This work was supported by grants from the Association pour la Recherche sur le Cancer and La Ligue Nationale contre le Cancer (to S. G. and N. M.).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.
Fellow of La Ligue Nationale contre le Cancer.
¶ To whom correspondence should be addressed. Fax: 33-467521559; E-mail: morin{at}crbm.cnrs-mop.fr.
The abbreviations used are: MAPK, mitogen-activated protein kinase; MPF, maturation-promoting factor; PKA, protein kinase A; PAK, p21-activated kinase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Plx, Polo-like kinase; GVBD, germinal vesicle breakdown; NEBD, nuclear envelope breakdown.
2 S. Faure, J. Cau, S. Vigneron, C. Delsert, and N. Morin, unpublished results.
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REFERENCES |
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