University of Newcastle upon Tyne, Institute of Cell and Molecular Biosciences, Medical School, Framlington Place, Newcastle NE2 4HH, UK
Author for correspondence (e-mail:
michael.whitaker{at}ncl.ac.uk)
Accepted 26 November 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Cell cycle, GFP-PCNA, XCL100, ERK, Sea urchin, DNA replication
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As its name implies, the appearance of PCNA immunofluorescence in the
nucleus has for a long time been used as a marker of S phase
(Takasaki et al., 1981). It
has been shown by immunofluorescence to localise to the replication complex
throughout DNA synthesis in Xenopus cell-free extracts and to
dissociate as DNA synthesis is completed
(Hutchison and Kill, 1989
).
This pattern has been confirmed in mammalian cells
(Leonhardt et al., 2000
;
Somanathan et al., 2001
) using
the GFP chimera employed in the experiments reported here. PCNA forms a trimer
that associates with DNA polymerase
and is the sliding clamp essential
for the processivity of the polymerase. Aphidicolin, a DNA polymerase
,
and
inhibitor (Avkin et al.,
2002
) thus prevents association of PCNA with the replication
complex provided it is present before PCNA is able to bind. By contrast, if
aphidicolin is added once the replication fork has begun, it stalls the
replication fork and prevents the dissociation of PCNA from chromatin
(Dimitrova and Gilbert,
2000
).
We demonstrate that nuclear localization and accumulation of GFP-PCNA is a proxy for S phase, in that it does not occur when DNA synthesis is prevented with the DNA polymerase inhibitor aphidicolin. Nor does it occur when eggs are treated with the recombinant ERK phosphatase (XCL-100) or with U0126. Nuclear localisation of GFP-PCNA is unaffected by U0124, an inactive analogue of U0126. The effects on S phase were confirmed by measuring incorporation of [3H]-thymidine in embryos treated with the MEK inhibitor U0126. Abrogating the ERK pathway with both XCL-100 and U0126 also prevented decondensation of sperm chromatin within the zygote nucleus, as well as nuclear envelope breakdown, and the formation of a bipolar spindle. By contrast, at the concentrations previously used (20 µM), PD98059 did not mimic the effects of either recombinant XCL100 or U0126. Instead, it provoked a very delayed accumulation of GFP-PCNA in the female pronucleus over a period of 2-3 hours and a correspondingly slow incorporation of [3H]-thymidine.
We conclude that a rapid, Ca2+ induced stimulation of the ERK pathway immediately after fertilization is required for proper activation of the sperm head at fertilization and for triggering S phase and cell cycle progression, including nuclear envelope breakdown and mitotic spindle formation. We discuss these findings in the light of the previous data.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Gamete handling
All biochemical experiments were carried out in air conditioned room at
16-17°C during the Lytechinus pictus breeding season
(May-October) to ensure reproducibility. Sea urchin eggs and sperm (L.
pictus) were collected in artificial sea water (ASW) [410 mM NaCl, 39 mM
MgCl2, 15 mM MgSO4, 2.5 mM NaHCO3, 10 mM
CaCl2, 10 mM KCl, 1 mM EDTA (pH 8.0)].
Microinjection procedure and treatment with inhibitors
Proteins or mRNA were microinjected using borosilicate glass micropipettes
(Harvard Apparatus GC150F-10) that were manipulated with an Eppendorf
micromanipulator. Injections comprised 0.1-0.5% egg volume. Concentrations in
the text are final concentrations in the egg. U0126 and U0124 treatments were
applied 10-15 minutes before fertilization or ionophore activation.
Hoechst 33342 dye staining in vivo
The embryos were suspended as normal and allowed to undergo the cell cycle.
Samples of 100 µl cell suspension were regularly taken out and stained in
vivo for 5 minutes before observation.
Expression constructs and purification of recombinant proteins
GFP-PCNA protein
GFP-fused human PCNA sequence was provided in pENeGFPPCNA2mut
(Leonhardt et al., 2000).
BamHI-XbaI GFPPCNA fragment containing SV40 nuclear
localisation signal at the N terminus was subcloned into pBCSK vector and the
BamHI-SacI fragment inserted into pCal-n. The construct was
confirmed by sequencing. The calmodulin tagged protein was purified on
calmodulin affinity resin (Stratagene). For microinjection, the protein was
dissolved in microinjection buffer (500 mM KCl; 20 mM PIPES; 100 µM EGTA;
pH 6.8).
GFPPCNA mRNA
GFP-PCNA BamHI-NotI fragment from pBCSK+ vector was
subcloned into pBluescript RN3 vector
(Lemaire et al., 1995). To
create pBluescript RN3GFP-PCNA the BglII site was destroyed. Ambion
mMessage mMachine kit with T3 RNA polymerase was used for in vitro
transcription.
XCL100 protein phosphatase
The XCL100 gene was provided in pGEX vector. XCL100 was then subcloned into
pBADB/gIII vector. The pBADXCL100 construct was used in the experiments of
Fig. 4. Because of the low
yield, in later experiments (Fig.
2) the NcoI-HindIII fragment from pBADXCL100 was
subcloned into pCal-n vector and the protein was purified as described above.
For microinjection, the XCL100 phosphatase was dissolved in PBS.
|
|
Preparation of sea urchin cell lysates and treatment with U0126
All experiments were carried out in whole sea urchin embryos undergoing the
mitotic cell cycle. Cell lysates from Lytechinus pictus were prepared
as described earlier (Philipova and
Whitaker, 1998). Lysates for MAP kinase assays were prepared in T
buffer after two washes of eggs/embryos in Ca-free ASW and one wash in T
buffer. These for hH1 kinase assays were prepared in H buffer (25 mM MOPS, 15
mM EGTA, 15 mM MgCl2, 1 mM DTT, supplemented with protease and
phosphatase inhibitors, pH 7.2). The final cell suspension contained not more
than 50 µl of eggs in 1 ml ASW. In each experiment, the batch was split in
two: half of them as controls and the other half treated. The percentage of
cells undergoing nuclear envelope breakdown (NEB) was always determined.
Experiments were carried out only with batches of eggs showing at least 95%
fertilization. The length of the cell cycle is slightly different for
different batches of eggs, meaning that the timing of NEB varies from
experiment to experiment. Stock solutions of 50 mM U0126 were always prepared
immediately before use and were gradually diluted to 100 µM and 20 µM in
ASW with vortexing at room temperature. The solution was then cooled down to
16°C.
In vitro protein kinase assays
MAP kinase and hH1 kinase assays were carried out as previously described
(Philipova and Whitaker,
1998). The experiments involving inhibition with U0126 were
carried out in the following way: the egg suspension was split in two aliquots
for control and for U0126 treatment 10 minutes before fertilization
(U-10). All kinase assays were performed at the same time and the
two gels were washed, dried, exposed and analysed together. Each result is
representative of three or more independent experiments.
Immunoprecipitation and western blotting
Immunoprecipitation and western blotting experiments were carried out as
previously described (Philipova and
Whitaker, 1998), except that the immune complexes were not boiled.
Instead, they were left for 15 minutes at room temperature in sample buffer
and then frozen or loaded onto a gel for PAGE and blotting.
Measurement of [3H]-thymidine incorporation
Incorporation of [3H]-thymidine was measured as described
previously (Kawahara et al.,
2000). Pre-incubation in 1 µCi/ml [3H]-thymidine was
carried out for 30 minutes at 16°C.
Fluorescence measurements
In the experiments presented in Fig.
2A and Fig. 3G,
GFP-PCNA fluorescence was observed using a Nikon Diaphot 300 inverted
microscope with narrow band pass filters (Ex 480±15 nm, DM 505, Em 520
nm±10 nm) controlled by the filter wheel (Sutter Instrument). Images
were collected using a charge-coupled device camera (CCD; Photometrix Coolsnap
fx, Roper Scientific) controlled by Metafluor software version 4 (Universal
Imaging, West Chester, PA). Where DNA was stained with UV excited Hoechst
33342, labelled eggs were observed using narrow band-pass filters (Ex
355±50 nm, DM 400, Em 450 nm longpass). Pictures of the fluorescently
labelled DNA and the differential interference contrast (DIC) images in
Fig. 4 were taken using a Nikon
Coolpix 950 digital camera. For CCD camera images the Nikon objective
40x/1.3NA oil was used. In the experiments shown in
Fig. 1,
Fig. 2C,
Fig. 3A and
Fig. 5, confocal inverted
microscope (Leica Lasertechnic GmbH, Heilderberg, Germany) images were
obtained. For single excitation scanning of GFP, the 488 nm line of an
Argon-Krypton laser and 530 nm long-pass emission filter was used.
|
|
|
Image processing and analysis
Image processing was carried out using Metamorph software to obtain the
average pixel intensity of the region of interest of the image. Fluorescence
intensities were normalized to the point of earliest fluorescence detection.
NEB was normalized to 70 minutes after fertilization.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In order to study the pattern of GFP-PCNA accumulation during the first
cell cycle, we microinjected recombinant GFP-PCNA protein to a concentration
of 60 nM. Fig. 1B illustrates
the accumulation of chimera during S phase of the first cell cycle. GFP-PCNA
is detectable in the male pronucleus as early as 5 minutes after fertilization
(not shown) and by 20 minutes it is accumulating in both male and female
pronuclei. After pronuclear fusion, GFP-PCNA associated with sperm chromatin
is gradually distributed evenly throughout the nucleus. By the time of NEB,
there has been a 20-fold accumulation of GFP-PCNA compared with its first
detectable levels; at NEB the chimera is released into the cytoplasm. The DNA
polymerase and
inhibitor aphidicolin, which prevents DNA
synthesis in sea urchin embryos (Ikegami
et al., 1979
; Nishioka et al.,
1984
), completely abolishes accumulation of GFP-PCNA into both
male and female nuclei (Fig.
1B). These data demonstrate that nuclear accumulation of GFP-PCNA
is a proxy for DNA synthesis.
The ERK phosphatase XCL100 prevents nuclear accumulation of GFP-PCNA and decondensation of sperm chromatin
The recombinant ERK phosphatase CL100 inactivates ERK1 in vitro
(Alessi et al., 1993); CL100
was shown to block ERK activation in vitro at a concentration of around 2
µM (Lewis et al., 1995
).
mRNA encoding the Xenopus CL100 homologue, XCL100, injected into G2
arrested Xenopus oocytes has been shown to delay or prevent
progesterone-induced meiotic maturation
(Sohaskey and Ferrell, 2002
).
It has been reported that much lower concentrations (75 nM) of XCL100 have no
apparent effect on mitotic progression
(Wang et al., 1997
).
Microinjection of the recombinant phosphatase to a final concentration of 1.5 µM had a small but not significant effect on nuclear localization of GFP-PCNA, while a concentration of 2.5 µM significantly (P<0.0001, Fig. 2B) reduced nuclear accumulation to around 20% of controls at 30 minutes, 12% at 50 minutes and 20% at the time of NEB, 65 minutes (Fig. 2A,B). GFP-PCNA fluorescence in zygotes injected with 2.5 µM XCL100 was not uniformly distributed (see Fig. 2A, arrow). As in untreated zygotes (Fig. 1B), residual GFP-PCNA fluorescence was brightest in the region of sperm chromatin, presumably reflecting its lesser degree of decondensation relative to the female-derived chromatin in the zygote nucleus. NEB did not take place in embryos injected with 2.5 µM XCL 100.
These data indicate by proxy that the ERK phosphatase prevents DNA synthesis and suggest that inactivation of ERK1 prevents decondensation of sperm chromatin within the zygote nucleus.
XCL 100 prevents nuclear GFP-PCNA accumulation in eggs activated with the Ca2+ ionophore A23187
Treating unfertilized sea urchin eggs with the Ca2+ ionophore
A23187 generates a Ca2+ transient comparable in magnitude to that
at fertilization and leads to the activation of ERK1, onset of S phase and
then nuclear envelope breakdown, though no spindle forms in the absence of the
centrosomes that are donated by the sperm at fertilization
(Philipova and Whitaker, 1998;
Whitaker and Steinhardt,
1982
). When unfertilized eggs injected with GFP-PCNA are activated
using 20 µM A23187, GFP-PCNA accumulates in the nucleus as it does in
fertilized eggs, though its onset was advanced slightly relative to fertilized
eggs (Fig. 2C). The extent of
accumulation by the time of NEB in A23187-treated eggs is comparable with that
in fertilized eggs.
Microinjection of XCL-100 to a concentration of 2.5 µM prevented A23187-induced nuclear accumulation of GFP-PCNA (Fig. 2C).
These results indicate that Ca2+-triggered ERK1 activation is required for accumulation of GFP-PCNA in the nucleus.
Nuclear accumulation of GFP-PCNA, [3H]-thymidine incorporation and MAP kinase activation are all prevented by U0126
The soluble MEK inhibitor U0126 has been shown to act specifically to block
ERK activation at concentrations of 50-100 µM
(Gross et al., 2000;
Walter et al., 2000
).
Inhibition of ERK activation during mitosis has been reported to require
100-400 µM U0126 (Chung and Cheng,
2003
; Walter et al.,
2000
). We added U0126 to unfertilized eggs suspended in ASW and
fertilized the eggs 10 minutes later. Fig.
3A,B shows that a concentration of 20 µM U0126 did not
significantly alter the progressive localization of GFP-PCNA in the male and
female pronuclei and the zygote nucleus. By contrast, 100 µM U0126
completely abolished the localization, while the inactive analogue at the same
concentration was without effect. Neither 20 µM nor 100 µM U0126 when
alone added to unfertilized eggs led to nuclear accumulation of GFP-PCNA
(Fig. 3B). U0126 (100 µM)
also completely blocked the incorporation of [3H]-thymidine into
DNA in fertilized eggs (Fig.
3C). This concentration of U0126 did not stimulate
[3H]-thymidine incorporation when added to unfertilized eggs in the
absence of sperm (Fig. 3D).
U0126 (100 µM) prevented the post-fertilization increase in MAP-kinase
activity (Fig. 3E). We have
previously shown that the increase in MAP kinase activity immediately after
fertilization is due to activation of a sea urchin ERK1
(Philipova and Whitaker,
1998
). Fig. 3F
confirms that the phosphorylation of ERK1 dimers
(Cobb and Goldsmith, 2000
;
Khoklatchev et al., 1998
)
correlates with measured MAP kinase activity, being absent in eggs treated
with 100 µM U0126.
These data demonstrate that MEK inhibition by U0126 prevents activation of ERK1, the onset of DNA synthesis and nuclear localization of GFP-PCNA after fertilization, suggesting that activation of the ERK signalling pathway is necessary for DNA synthesis. They also independently confirm that the nuclear localization and accumulation of GFP-PCNA correlates with the presence or absence of DNA synthesis in sea urchin embryos. We found no evidence that inhibition of the MEK pathway in unfertilized eggs led to DNA synthesis measured either as nuclear accumulation of GFP-PCNA or [3H]-thymidine incorporation.
U0126 prevents nuclear GFP-PCNA accumulation and [3H]-thymidine incorporation in eggs activated with Ca2+ ionophore
Treatment with the Ca2+ ionophore A23187 leads to GFP-PCNA
accumulation in the nucleus. Accumulation in the nucleus induced by A23187 is
prevented by incubation with 100 µM U0126
(Fig. 3G). A23187 stimulates
incorporation of [3H]-thymidine into DNA
(Nishioka and Magagna, 1981)
(Fig. 3H); as with GFP-PCNA
accumulation, the onset of incorporation appears to be slightly earlier than
in fertilized eggs, though the reasons for this are unknown. U0126 (100 µM)
also prevents A23187-induced incorporation of [3H]-thymidine into
DNA (Fig. 3H).
These data are consistent with our finding that ERK1 activation at
fertilization can be triggered by A23187
(Philipova and Whitaker, 1998)
and confirm that the ERK pathway is essential for the initiation of DNA
synthesis.
XCL100 and U0126 have identical effects on sperm chromatin decondensation within the zygote nucleus
We investigated the effects of ERK pathway inhibition on sperm chromatin
decondensation using both U0126 and XCL100.
Fig. 4 shows that sperm
chromatin is readily visible as a region of accentuated staining with the DNA
dye Hoechst 33342 at 25 minutes after fertilization and that by 55 minutes
sperm chromatin has dispersed within the nucleus, giving uniform staining with
H33342. By contrast, both 2.5 µM XCL100 and 100 µM U0126 remarkably
delay sperm chromatin dispersal with the zygote nucleus. At 70 minutes, when,
in control embryos, chromatin is beginning to condense throughout the nucleus
just before NEB, treated embryos still maintain sperm chromatin in an
undispersed state. Treated embryos fail to undergo NEB and show evidence of
continued failure to disperse sperm chromatin even at 120 minutes, when
control embryos are undergoing first cleavage and are in S phase of the second
embryonic cell cycle. Evidence of a degree of chromatin condensation
comparable with control embryos at 70 minutes is seen only at 170 minutes
inside a still intact nucleus, when control embryos are in S phase of the
third cell cycle. No mitotic spindle formed in the embryos treated with U0126
or microinjected with XCL100, an observation that we confirmed using embryos
injected with rhodamine-labelled tubulin (data not shown).
These data confirm that inhibition of the ERK pathway prevents dispersal of the sperm chromatin within the zygote nucleus and demonstrate that the low molecular weight U0126 and the recombinant protein XCL100 give rise to an identical phenotype with identical timing. They also indicate that inhibition of the ERK pathway arrests the cell cycle at some point before nuclear envelope breakdown and spindle formation.
Activation of Cdk1 at mitosis is prevented by treatment with U0126
It has been established that preventing DNA synthesis using aphidicolin in
sea urchin embryos prevents nuclear envelope breakdown and formation of
bipolar mitotic spindles (Nishioka et al.,
1984). Full activation of Cdk1 at the time of mitosis does not
occur in the presence of aphidicolin
(Patel et al., 1997
), though
active Cdk1 gradually accumulates to levels seen during mitosis over a period
of 2 hours [(Geneviere-Garrigues et al.,
1995
; Hinchcliffe et al.,
1999
); our unpublished data]. We therefore measured both ERK1 and
Cdk1 activities in embryos treated with U0126 at the time that control embryos
were undergoing mitosis. We found, as expected, that ERK1 activation at the
time of mitosis (Philipova and Whitaker,
1998
) was prevented by incubation with 100 µM U0126, as was
Cdk1 activity (see Fig. S1 in the supplementary material).
These observations indicate that inhibition of the ERK pathway immediately
after fertilization prevents DNA synthesis and that, as expected, blocking DNA
synthesis prevents the timely activation of Cdk1
(Geneviere-Garrigues et al.,
1995).
The effects of the ERK pathway inhibitor PD98059 do not mimic those of the recombinant ERK phosphatase, in marked contrast to U0126
PD98059 is an inhibitor of MEK at concentrations of 20 µM. It has been
shown to induce DNA synthesis in unfertilized sea urchin eggs using a
fluorescence method involving the use of bromo-deoxyuridine
(Carroll et al., 2000). We
added 20 µM PD98059 to unfertilized eggs microinjected with GFP-PCNA. We
found a very slow, aphidicolin-sensitive accumulation of GFP-PCNA in the egg
pronucleus. Accumulation at 55 minutes, the time at which accumulation had
reached 20-fold in fertilized controls, was comparable with that detected at
15 minutes in these controls (Fig.
5A,B). Nuclear levels then rose to around 15-fold by 130 minutes
after addition, before levelling off. These results were confirmed by
measurements of [3H]-thymidine incorporation in unfertilized eggs
treated with 20 µM PD98059 (Fig.
5C).
Aphidicolin prevented PD98059-induced nuclear accumulation of GFP-PCNA in unfertilized eggs (Fig. 5B), demonstrating once more that this accumulation is a reliable proxy for DNA synthesis.
Microinjection of 2.5 µM XCL100 into unfertilized eggs did not lead to nuclear accumulation of GFP-PCNA (Fig. 2B), unlike PD98059. Neither did U0126 cause nuclear accumulation of GFP-PCNA (Fig. 3B).
PD98059 at the same concentration (20 µM) was not able to prevent DNA synthesis in fertilized eggs, as judged by nuclear accumulation of GFP-PCNA (Fig. 5D).
These results demonstrate that the effects of PD98059 do not in any way resemble those of the MAP kinase phosphatase XCL-100, unlike the effects of U0126, which are identical to those of XCL-100.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We did not observe discrete foci of GFP-PCNA within the nucleus that might
correspond to spatially clustered replicons with coordinated replication
timing, such as were seen with GFP-PCNA in mammalian cell nuclei
(Leonhardt et al., 2000;
Somanathan et al., 2001
). In
non-mammalian early embryos (Xenopus, Drosophila), replication
origins are closely spaced at 10 kb
(DePamphilis, 1999
) and
replicate simultaneously. This might explain the lack of focal sites in sea
urchin embryos.
The accepted view in the field is that the onset of DNA synthesis occurs
after syngamy, that is, the fusion of male and female pronuclei
(Longo and Plunkett, 1973).
However, we observe substantial accumulation of GFP-PCNA in the male
pronucleus some time before pronuclear fusion. Accumulation can be prevented
by aphidicolin. This implies that it is the formation of replication complexes
rather than an excess of GFP-PCNA that lead to accumulation of GFP-PCNA in the
male pronucleus. GFP-PCNA fluorescence intensity is fivefold higher in the
male pronucleus compared with the female pronucleus prior to syngamy. This
difference is almost certainly due to the difference in the density of
chromatin in the two pronuclei, as the DNA-staining Hoechst dye 33342 shows a
similar four- to sevenfold higher intensity in the male pronucleus (not
shown).
We were keen to confirm that the distribution of GFP-PCNA after
fertilization mirrored that of the endogenous sea urchin protein. However,
none of three different antibodies raised against mammalian PCNA crossreacts
with the sea urchin protein. The concordance of our data with those obtained
using the same construct in HeLa cells
(Leonhardt et al., 2000) and
the effects of aphidicolin on nuclear accumulation nonetheless gives us
confidence that the PCNA chimera reflects DNA synthetic activity.
It has been previously shown that cyclin E is associated predominantly with
the male pronucleus of the sea urchin zygote; it appears in the female
pronucleus only after syngamy
(Schnackenberg and Marzluff,
2002). However, cyclin A/Cdk2 is found to be predominantly
localized to the female pronucleus (Moreau
et al., 1998
; Schnackenberg
and Marzluff, 2002
). The female pronucleus is able to initiate DNA
synthesis in the absence of sperm, if activated by Ca2+ ionophore.
In other words, it possesses the machinery necessary for activation of DNA
synthesis. Further work is needed to test the possible roles of Cdk2, cyclin E
and cyclin A in the formation of replication complexes and initiation of S
phase after syngamy.
ERK1 activation at fertilization is required for nuclear accumulation of GFP-PCNA, the initiation of DNA synthesis and progress through the cell cycle
Microinjection of recombinant XCL-100, an ERK phosphatase, prevented
accumulation of GFP-PCNA in both male and female pronuclei. This implies that
formation of replication complexes requires the activation of ERK1 after
fertilization and suggests a mechanism in which activation of ERK1 promotes
the accumulation of PCNA and possibly cyclin E in the male pronucleus.
The effects of the MEK inhibitor U0126 are identical to those of the ERK phosphatase XCL-100. Both prevent nuclear accumulation of GFP-PCNA, decondensation of male chromatin within the zygote nucleus and the formation of a bipolar spindle. Experiments with U0126 demonstrate also that blocking activation of the ERK pathway prevents incorporation of [3H]-thymidine into DNA. The increases in ERK1 and Cdk1 activity that would accompany mitosis are absent in embryos treated with the MEK inhibitor U0126. Overall, the results presented here suggest that the first embryonic cell cycle arrests before S phase in the absence of ERK1 activation at fertilization.
The effects of another MEK inhibitor, PD 98059, do not conform to the
effects of the ERK phosphatase XCL-100. The differences observed between the
effects of PD98059 and XCL-100 can be summarised as follows: PD98059 did not
prevent nuclear accumulation of GFP-PCNA after fertilization or ionophore
treatment, unlike XCL-100; PD98059 did not prevent male pronuclear
decondensation and bipolar spindle formation, unlike XCL-100; PD98059 induced
a slow accumulation of GFP-PCNA in the nucleus of unfertilized eggs, unlike
XCL-100. Thus, PD98059 acts on a target distinct from that targeted by XCL-100
and U0126. It has been shown that PD98059 is less effective than U0126 as an
inhibitor of the MEK-ERK pathway (Favata
et al., 1998; Gross et al.,
2000
; Pitts et al.,
1998
).
We confirm the finding (Carroll et al.,
2000) that treatment of unfertilized eggs with PD98059 leads to
the events of DNA synthesis. However, we show that PD98059 does not induce a
physiological S phase: the onset of DNA synthesis and its rate of increase
lags very substantially behind that observed in fertilized (diploid) or
ionophore-activated (haploid) eggs. These observations demonstrate that
PD98059 is acting on a different aspect of the ERK signalling pathway than the
ERK phosphatase XCL-100 and the MEK inhibitor U0126. In fact, it has
previously been shown that 2.5 µM PD98059 was sufficient to induce a fall
in phosphorylated ERK in unfertilized eggs comparable with that measured at
fertilization (Carroll et al.,
2000
). We have determined that this concentration is not
sufficient to induce any detectable replication complex formation over a
period of 2.5 hours. An effect of DNA synthesis has been reported previously
only at 20 µM (Carroll et al.,
2000
), as confirmed in this study. There is thus no correlation
between a fall in measured ERK activity and DNA synthesis induced by
PD98059.
The role of MAP kinase at fertilization in sea urchin eggs: reconciliation and a synthesis
The inhibition of nuclear GFP-PCNA uptake in embryos by an ERK phosphatase
implies that ERK activation is necessary for the onset of DNA synthesis and so
supports the original observation that ERK1 activity increases rapidly and
transiently immediately after fertilization
(Philipova and Whitaker,
1998). This study found that the major in-gel kinase activity was
due to a 44 kDa protein that was identified by peptide sequencing as ERK1. The
post-fertilization increase was accounted for by threefold increase in MBP
phosphorylation that was immunoprecipitable using an anti-ERK1 antibody,
comparable with that seen during mitosis. A contemporary study reported a 50%
post-fertilization increase in MBP activity followed by another peak at
mitosis, though the increase was not reflected in phosphorylation of GST-Myc
(Chiri et al., 1998
). A later
study reported a small though not significant increase at 5 minutes after
fertilization using an anti-phosphoERK antibody
(Carroll et al., 2000
).
Statistical analysis of the data from this study indicates that it would be
predicted to detect a threefold increase with statistical significance with
80% power, that is, four out of five times. In parallel experiments, no
increase in activity was detected using immunoprecipitation with a pan
anti-ERK monoclonal and Elk1 as substrate. A further study
(Kumano et al., 2001
)
confirmed the report of a 43 kDa MAP kinase active in unfertilized eggs and
falling after fertilization or Ca2+ ionophore treatment. The
activity was sensitive to low (0.5 µM) doses of U0126 and did not reappear
during mitosis. The authors conclude that this activity is distinct from the
activity we reported previously (Philipova
and Whitaker, 1998
) and measure in the present study. We concur
and also conclude that this distinct activity does not control DNA synthesis,
as we show here that treatment of unfertilized eggs with U0126 induces neither
GFP-PCNA accumulation nor incorporation of [3H]-thymidine.
Our current cell physiology data point to an increase in ERK1 activity as
essential at fertilization. Can the differences in the biochemical data be
explained? All three studies used different antibodies to identify and/or
immunoprecipitate a 43-44 kDa protein with ERK substrate specificity. Only one
study determined that the major component of MBP activity was ERK1 by peptide
sequencing (Philipova and Whitaker,
1998). This study measured soluble ERK1 activity, while the other
two used whole cell samples. It appears therefore that it is soluble ERK1
activity that correlates with activation of DNA synthesis after
fertilization.
In mammalian somatic cells, it has been shown that activation of Cdk2 is
accompanied by the formation of MAP kinase-Cdk2 complexes and that
inactivation of MAP kinase prevents activation and nuclear localization of
Cdk2 and entry into S phase (Blanchard et
al., 2000). Cdk2 association with cyclins E and A are essential
for entry and progression through S phase
(Coverley et al., 2002
) and
ERK activation controls the activating phosphorylation of Cdk2 Thr-160
(Chiariello et al., 2000
). Sea
urchin eggs are arrested in a G1-like stage before fertilization, unlike most
other oocytes (Whitaker,
1996
). So although a fall in ERK activity is detected in frog,
ascidian and mouse oocytes as they exit a meiotic division, it would be
predicted that sea urchin eggs, by analogy with somatic cells, would exhibit
an increase in ERK activation as they enter S phase after fertilization
(Whitaker, 1996
;
Whitaker and Patel, 1990
;
Whitaker and Steinhardt,
1982
).
The Ca2+ signal is alone sufficient to induce the nuclear accumulation of GFP-PCNA and the onset of DNA synthesis in unfertilized eggs
We have previously demonstrated that treatment of eggs with the
Ca2+ ionophore A23187 activates ERK1
(Philipova and Whitaker,
1998). A23187 generates a Ca2+ transient in eggs
comparable with that recorded at fertilization
(Whitaker and Steinhardt,
1982
). Here, we show that the ionophore causes nuclear
accumulation of GFP-PCNA and incorporation of [3H]-thymidine into
DNA (Steinhardt and Epel,
1974
; Whitaker and Steinhardt,
1982
). Nuclear accumulation of GFP-PCNA in response to A23187 is
prevented by microinjection of the recombinant ERK phosphatase XCL-100 and
also by treatment with the MEK inhibitor U0126, as is
[3H]-thymidine incorporation into DNA. Thus, the activating
Ca2+ signal causes the onset of DNA synthesis because of activation
of the ERK1 pathway.
It has been known for some time that syngamy is not an essential
pre-requisite for DNA synthesis in the female pronucleus
(Longo and Plunkett, 1973).
The role of cyclin E in parthenotes is unclear. It has been suggested that
cyclin E/Cdk2 activity may relicense replication in the male pronucleus and
that the DNA in female pronucleus may have been relicensed during meiosis
(Schnackenberg and Marzluff,
2002
). It may be that in parthenotes, the assembly of replication
complexes and the onset of processive DNA synthesis is under the sole control
of cyclin A/Cdk2 (Schnackenberg and
Marzluff, 2002
).
Conclusion
This study demonstrates that GFP-PCNA can be used as a surrogate marker for
S phase and that the ERK1 pathway activated by the fertilization
Ca2+ transient controls the onset of DNA synthesis and cell cycle
progression in sea urchin embryos.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/3/579/DC1
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: Department of Pathology, School of Molecular and Clinical
Medicine, University of Edinburgh, Edinburgh EH8 9YL, UK
Present address: Department of Anatomy and Developmental Biology,
University College London, Gower Street, London WC1E 6BT, UK
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abrieu, A., Doree, M. and Fisher, D. (2001).
The interplay between cyclin-B-Cdc2 kinase (MPF) and MAP kinase during
maturation of oocytes. J. Cell Sci.
114,257
-267.
Alessi, D. R., Smythe, C. and and Keyse, S. M. (1993). The human CL100 gene encodes a Tyr/Thr-protein phosphatase which potently and specifically inactivates MAP kinase and suppresses its activation by oncogenic ras in Xenopus oocyte extracts. Oncogene 8,2015 -2020.[Medline]
Avkin, S., Adar, S., Lander, G. and Livneh, Z.
(2002). Quantitative measurement of translesion replication in
human cells: evidence for bypass of abasic sites by a replicative DNA
polymerase. Proc. Natl. Acad. Sci. USA
99,3764
-3769.
Blanchard, D., Mouhamad, S., Auffredou, M., Pesty, A., Bertoglio, J., Leca, G. and Vasquez, A. (2000). Cdk2 associates with MAP kinase in vivo and its nuclear translocation is dependent on MAP kinase activation in IL-2-dependent Kit 225 T lymphocytes. Oncogene 19,4184 -4189.[CrossRef][Medline]
Carroll, D. J., Albay, D. T., Hoang, K. M., O'Neil, F. J., Kumano, M. and Foltz, K. R. (2000). The relationship between calcium, MAP kinase, and DNA synthesis in Sea Urchin egg at fertilization. Dev. Biol. 217,179 -191.[CrossRef][Medline]
Chiariello, M., Gomez, E. and Gutkind, J. (2000). Regulation of cyclin-dependent (Cdk) 2 Thr-160 phosphorylation and activity by MAP kinase in late G1 phase. Biochem. J. 349,869 -876.[Medline]
Chiri, S., de Nadai, C. and Ciapa, B. (1998).
Evidence for MAP kinase activation during mitotic division. J. Cell
Sci. 111,2519
-2527.
Chung, E. and Cheng, R. H. (2003). Phosphorylation of Cdc20 is required for its inhibition by the spindle checkpoint. Nat. Cell Biol. 4, 748-753.[CrossRef]
Cobb, M. and Goldsmith, E. (2000). Dimerization in MAP kinase signalling. Trends Biochem. Sci. 25, 7-9.[CrossRef][Medline]
Coverley, D., Laman, H. and Saskey, R. (2002). Distinct roles for cyclins E and A during DNA replication complex assembly and activation. Nat. Cell Biol. 4, 523-528.[CrossRef][Medline]
DePamphilis, M. (1999). Replication origins in metazoan chromosomes: fact or fiction? BioEssays 21, 5-16.[CrossRef][Medline]
Dimitrova, D. and Gilbert, D. (2000). Temporally coordinated assembly and disassembly of replication factories in the absence of DNA synthesis. Nat. Cell Biol. 2, 686-694.[CrossRef][Medline]
Favata, M. F., Horiuchi, K. Y., Manos, E. J., Daulerio, A. J.,
Stradley, D. A., Feeser, W. S., Vandyk, D. E., Pitts, W. J., Earl, R. A.,
Hobbs, F. et al. (1998). Identification of a novel inhibitor
of mitogen-activated protein kinase kinase. J. Biol.
Chem. 273,18623
-18632.
Fisher, D., Abrieu, A., Simon, M. N., Keyse, S., Verge, V., Doree, M. and Picard, A. (1998). MAP kinase inactivation is required only for G2-M phase Transition in early embryogenesis cell cycles of the starfishes Marthasterias glacialis and Astropecten aranciacus. Dev. Biol. 202, 1-13.[CrossRef][Medline]
Geneviere-Garrigues, A. M., Barakat, A., Doree, M., Moreau, J.
L. and Picard, A. (1995). Active cyclin B-cdc2 kinase does
not inhibit DNA replication and cannot drive prematurely fertilized sea urchin
into mitosis. J. Cell Sci.
108,2693
-2703.
Gotoh, Y., Nishida, E., Matsuda, S., Shiina, N., Kosako, H., Shiokawa, K., Akiyama, T., Ohta, K. and Sakai, H. (1991). In vitro effects on microtubule dynamics of purified Xenopus M phase-activated MAP kinase. Nature 349,251 -254.[CrossRef][Medline]
Gross, S. D., Schwab, M. S., Taieb, F. E., Lewellyn, A. L., Qian, Y.-W. and Maller, J. L. (2000). The critical role of the MAP kinase pathway in meiosis II in Xenopus oocytes is mediated by p90(Rsk). Curr. Biol. 10,430 -438.[CrossRef][Medline]
Guadagno, T. M. and Ferrell, J. E. J. (1998).
Requirement for MAPK activation for normal mitotic progression in Xenopus egg
extracts. Science 282,1312
-1315.
Hinchcliffe, E. H., Thompson, E. A., Miller, F. J., Yang, J. and
Sluder, G. (1999). Nucleo-cytoplasmic interactions that
control nuclear envelope breakdown and entry into mitosis in the sea urchin
zygote. J. Cell Sci.
112,1139
-1148.
Hinegardner, R., Rao, B. and Feldman, D. (1964). The DNA synthetic period during early development of the dea urchin egg. Exp. Cell Res. 36, 53-61.[CrossRef][Medline]
Hulleman, E. and Boonstra, J. (2001). Regulation of G1 phase progression by growth factors and the extracellular matrix. Cell Mol. Life Sci. 58, 80-93.[Medline]
Hutchison, C. and Kill, I. (1989). Changes in the nuclear distribution of DNA polymerase alpha and PCNA/cyclin during the progress of the cell cycle, in a cell-free extract of Xenopus eggs. J. Cell Sci. 93,605 -613.[Abstract]
Ikegami, S., Amemiya, S., Oguro, M., Nagano, H. and Mano, Y. (1979). Inhibition by aphidicolin of cell cycle progression and DNA replication in sea urchin embryos. J. Cell Physiol. 100,439 -444.[Medline]
Ito, S., Dan, K. and Goodenough, D. (1981). Ultrastructure and 3H-thymidine incorporation by chromosome vesicles in sea urchin embryos. Chromosoma 83,441 -453.[CrossRef][Medline]
Kawahara, H., Philipova, R., Yokosawa, H., Patel, R., Tanaka, K.
and Whitaker, M. (2000). Inhibiting proteasome activity
causes overreplication of DNA and blocks entry into mitosis in sea urchin
embryos. J. Cell Sci.
113,2659
-2670.
Khoklatchev, A., Canagarajah, B., Wilsbacher, J., Robinson, M., Atkinson, M., Goldsmith, E. and Cobb, M. (1998). Phosphorylation of the MAP kinase ERK2 promotes its homodimerisation and nuclear translocation. Cell 93,605 -615.[Medline]
Kumano, M., Carroll, D. J., Denu, J. M. and Foltz, K. R. (2001). Calcium-mediated inactivation of the MAP kinase pathway in sea urchin eggs at fertilization. Dev. Biol. 236,244 -257.[CrossRef][Medline]
Lemaire, P., Garrett, N. and Gurdon, J. B. (1995). Expression cloning of Siamois, a Xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis. Cell 81,85 -94.[Medline]
Leonhardt, H., Rahn, H. P., Weinzierl, P., Sporbert, A., Cremer,
T., Zink, D. and Cardoso, M. C. (2000). Dynamics of DNA
replication factories in living cells. J. Cell Biol.
149,271
-279.
Lewis, T., Groom, L. A., Sneddon, A. A., Smythe, C. and Keyse,
S. M. (1995). XCL100, an inducible nuclear MAP kinase
phosphatase from Xenopus laevis: its role in MAP kinase inactivation in
differentiated cells and its expression during early development.
J. Cell Sci. 108,2885
-2896.
Longo, F. J. and Plunkett, W. (1973). The onset of DNA synthesis and its relation to morphogenetic events of the pronuclei in activated eggs of the sea urchin, Arbacia punctulata. Dev. Biol. 30,56 -67.[CrossRef][Medline]
Meinecke, B. and Krischek, C. (2003). MAPK/ERK kinase (MEK) signalling is required for resumption of meiosis in cultured cumulus-enclosed pig oocytes. Zygote 11, 7-16.[CrossRef][Medline]
Moreau, J. L., Marques, F., Barakat, A., Schatt, P., Lozano, J. C., Peaucellier, G., Picard, A. and Geneviere, A. M. (1998). Cdk2 activity is dispensable for the onset of DNA replication during the first mitotic cycles of the sea urchin early embryo. Dev. Biol. 200,182 -197.[CrossRef][Medline]
Nebreda, A. R. and Ferby, I. (2000). Regulation of the meiotic cell cycle in oocytes. Curr. Opin. Cell Biol. 12,666 -675.[CrossRef][Medline]
Nishioka, D. and Magagna, L. S. (1981). Increased uptake of thymidine in the activation of sea urchin eggs. Exp. Cell Res. 133,363 -372.[CrossRef][Medline]
Nishioka, D., Balczon, R. and Schatten, G. (1984). Relationships between DNA synthesis and mitotic events in fertilized sea urchin eggs: aphidicolin inhibits DNA synthesis, nuclear breakdown and proliferation of microtubule organizing centers, but not cycles of microtubule assembly. Cell. Biol. Int. Rep. 8, 337-346.[Medline]
Patel, R., Wright, E. M. and Whitaker, M. (1997). Caffeine overrides the S-phase cell cycle block in sea urchin embryos. Zygote 5, 127-138.[Medline]
Pelech, S., Tombes, R., Meijer, L. and Krebs, E. (1988). Activation of myelin basic protein kinases during echinoderm oocyte maturation and egg fertilization. Dev. Biol. 130,28 -36.[Medline]
Philipova, R. and Whitaker, M. (1998). MAP
kinase activity increases during mitosis in early sea urchin embryos.
J. Cell Sci. 111,2497
-2505.
Pitts, W. J., Earl, R. A., Hobbs, F., Copeland, R. A., Magolda,
R. L., Scherle, P. A. and Trzaskos, J. M. (1998).
Identification of a novel inhibitor of mitogen-activated protein kinase
kinase. J. Biol. Chem.
273,18623
-18632.
Roovers, K. and Assoian, R. K. (2000). Integrating the MAP kinase signal into the G1 phase cell cycle machinery. Bioassays 22,818 -826.[CrossRef][Medline]
Ruderman, J. V. (1993). MAP kinase and the activation of quiescent cells. Curr. Opin. Cell Biol. 5, 207-213.[Medline]
Schnackenberg, B. J. and Marzluff, W. F.
(2002). Novel localization and possible functions of cyclin E in
early sea urchin development. J. Cell Sci.
115,113
-121.
Sohaskey, M. L. and Ferrell, J. E., Jr (2002).
Activation of p42 mitogen-activated protein kinase (MAPK), but not c-Jun
NH2-terminal kinase, induces phosphorylation and stabilization of MAPK
phosphatase XCL100 in Xenopus oocytes. Mol. Biol. Cell
13,454
-468.
Somanathan, S., Suchyna, T. M., Siegel, A. J. and Berezney, R. (2001). Targeting of PCNA to sites of DNA replication in the mammalian cell nucleus. J. Cell. Biochem. 81, 56-67.[CrossRef][Medline]
Steinhardt, R. A. and Epel, D. (1974). Activation of sea-urchin eggs by a calcium ionophore. Proc. Natl. Acad. Sci. USA 71,1915 -1919.[Abstract]
Sumerel, J. L., Moore, J. C., Schackenberg, B. J., Nichols, J. A., Canman, J. C., Wessel, G. M. and Marzluff, W. F. (2001). Cyclin E and its associated cdk activity do not cycle during early embryogenesis of the sea urchin. Dev. Biol. 234,425 -440.[CrossRef][Medline]
Sun, Q. Y., Breitbart, H. and Schatten, H. (1999). Role of the MAPK cascade in mammalian germ cells. Reprod. Fert. Dev. 11,443 -450.[Medline]
Takasaki, Y., Deng, J. S. and Tan, E. M.
(1981). A nuclear antigen associated with cell proliferation and
blast transformation. J. Exp. Med.
154,1899
-1909.
Walter, S. A., Guadagno, S. N. and Ferrell, J. E.
(2000). Activation of Wee1 by p42 MAPK in vitro and in cycling
xenopus egg extracts. Mol. Biol. Cell
11,887
-896.
Wang, X. M., Zhai, Y. and Ferrel, J. E. J.
(1997). A role for mitogen-activated protein kinase in the
spindle assembly checkpoint in XTC cells. J. Cell
Biol. 137,433
-443.
Whitaker, M. (1996). Control of meiotic arrest.
Rev. Reprod. 1,127
-135.
Whitaker, M. and Patel, R. (1990). Calcium and cell-cycle control. Development 108,525 -542.[Abstract]
Whitaker, M. and Steinhardt, R. A. (1982). Ionic regulation of egg activation. Q. Rev. Biophys. 15,593 -666.[Medline]