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First published online 15 April 2009
doi: 10.1242/dev.035261
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1 Laboratory of Cell and Developmental Biology, Graduate School of
BioscienceTokyo Institute of Technology, Nagatsuta, Midoriku, Yokohama
226-8501, Japan.
2 Integrated Research Institute, Tokyo Institute of Technology, Nagatsuta,
Midoriku, Yokohama 226-8501, Japan.
Author for correspondence (e-mail:
tkishimo{at}bio.titech.ac.jp)
Accepted 16 March 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Cell cycle, Cyclin, Fertilization, MAP kinase, p90Rsk, Starfish eggs
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Sagata, 1996). All of these
meiotic arrests are released by fertilization, resulting in the initiation of
the embryonic cell cycle. However, the relationship between the release from
the meiotic arrest and the start of the embryonic cell cycle remains unclear.
In particular, release from the meiotic arrest at stages other than G1 is
complicated because it involves both completion of the remaining part of the
meiotic cell cycle and initiation of the embryonic cell cycle. Thus, species
using a G1 arrest are advantageous for elucidating how the embryonic cell
cycle is initiated.
MAP kinase (MAPK) is likely to be generally required for many of the
diverse meiotic arrests in various organisms (for reviews, see
Sagata, 1996;
Masui, 2000;
Kishimoto, 2003). MAPK was
first identified to be essential for meta-II arrest in frog eggs
(Haccard et al., 1993;
Kosako et al., 1994).
Thereafter, involvement of MAPK was demonstrated in meta-II arrest of mouse
eggs (Verlhac et al., 1996);
G1 arrest of unfertilized eggs in starfish
(Tachibana et al., 1997), sea
urchin (Kumano et al., 2001)
and jellyfish (Kondoh et al.,
2006); and in meta-I arrest of sawfly eggs
(Yamamoto et al., 2008).
Meta-I arrest in tunicate eggs may also be regulated by MAPK
(Russo et al., 1998). In every
case, inactivation of MAPK by fertilization results in release from meiotic
arrest and the subsequent entry into the embryonic cell cycle. Thus, a key
question is how a decrease in MAPK activity functions as a general initiator
of the embryonic cell cycle, despite the diversity of meiotic arrest points.
Identification of the mediators and effectors downstream of MAPK would help to
elucidate this question.
In starfish (Asterina pectinifera; renamed Patiria
pectinifera in 2007 – NCBI Taxonomy Browser), unfertilized mature
eggs arrest at pronuclear stage (G1 phase) after completion of meiosis II.
Fertilization releases the G1 arrest to initiate S phase and the following M
phase, leading to the embryonic cell cycle. The starfish G1 arrest depends on
the Mos-MAPK-Rsk (p90 ribosomal S6 kinase) pathway, in which Mos functions as
a MAPK kinase kinase (Tachibana et al.,
2000) and Rsk functions as a mediator immediately downstream of
MAPK (Mori et al., 2006).
During meiotic maturation, MAPK and Rsk are initially activated around
metaphase of meiosis I, depending on new synthesis of Mos, and, unless
fertilization occurs, MAPK and Rsk activities remain elevated until G1 arrest
after completion of meiosis II. Although the physiological substrate of Rsk
for the G1 arrest remains unclear, suppression of Rsk in unfertilized G1 eggs
is necessary and sufficient for release from G1 arrest and entry into S phase
(Mori et al., 2006). However,
it is unknown whether these Rsk-suppressed eggs further progress into M phase
and undergo embryonic cell cycling. Thus it remains unclear whether loss of
Rsk activity is sufficient for starting the embryonic cell cycle.
Entry into M phase in fertilized starfish eggs is regulated by both cyclin
A-Cdk1 and cyclin B-Cdk1 (Okano-Uchida et
al., 1998). In G1-arrested starfish eggs, protein levels of cyclin
A and cyclin B remain low, and fertilization triggers their accumulation.
While cyclin B-Cdk1 remains inactive due to inhibitory phosphorylation of Cdk1
by Wee1 and Myt1, cyclin A-Cdk1 is activated solely by the accumulation of
cyclin A. The active cyclin A-Cdk1 inactivates Wee1 and Myt1 via Plk1,
resulting in the activation of cyclin B-Cdk1 and thus entry into M phase
(Okano-Uchida et al., 2003;
Tachibana et al., 2008).
Thereafter, both cyclin A and cyclin B proteins are destroyed, leading to exit
from M phase. Thus, accumulation of cyclin A and cyclin B is indispensable for
starting the embryonic cell cycle.
In the present study, we found that in unfertilized G1 starfish eggs suppression of Rsk alone is not sufficient for cell cycle progression into M phase, even though S phase occurs, implying that a mechanism other than a decrease in Rsk activity functions to regulate the start of the embryonic cell cycle. We then investigated this, and showed that MAPK prevents entry into M phase through a pathway that is not mediated by Rsk but that leads to repression of protein synthesis of cyclin A and cyclin B. To block the start of the embryonic cell cycle in unfertilized starfish eggs, we propose a dual-lock mechanism in which there are two separate pathways downstream of MAPK: one is a Rsk-dependent pathway that leads to prevention of entry into S phase and the other is a Rsk-independent pathway that leads to prevention of entry into M phase.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The oocytes were treated with 1 µM 1-methyladenine
(1-MeAde) to induce meiotic maturation at 20°C in artificial seawater
(Jamarin Laboratory). After completion of meiosis, mature eggs with female
pronuclei were inseminated. In some experiments, eggs were treated with 10
µM U0126 (Promega), 10 µg/ml aphidicolin (Wako) or 100 µM emetine
(Tokyo Kasei Kogyo). Egg extracts were prepared according to Okano-Uchida et
al. (Okano-Uchida et al.,
2003). Microinjection was performed as described
(Kishimoto, 1986). Immature
oocytes or unfertilized mature eggs were injected with 50 pg GST or GST-Mos
(Tachibana et al., 2000); 25
pg of a fusion protein of GST, importin-β binding domain of
importin-
(IBB), and GFP (Tachibana
et al., 2008); 3 ng purified anti-starfish Rsk antibody
(Mori et al., 2006) or normal
rabbit IgG (Sigma-Aldrich); 0.6 ng of a GST-tagged constitutively active form
of mouse Rsk2 (CA-Rsk-EE) or its kinase negative form (KD-Rsk-EE)
(Perdiguero et al., 2003;
Mori et al., 2006); 20 fmol of
morpholino antisense oligonucleotides (Gene Tools) against either cyclin A
(5'-GGTTTTCACTGAAGGCGAAGGACAT-3') or cyclin B
(5'-ATTGTAACCAATGCGAGTTCTGAGG-3'); or 5 ng of peptide containing
the D-box of starfish cyclin B (KSTLGTRGALENISNVAKNNVQAAAKKEIYC) and a D-box
mutant (KSTLGTAGAAENISNVANNVQAAAKKEIYC)
(Kraft et al., 2005).
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),
anti-MAPK (Upstate), anti-PSTAIR for Cdk1 (gift from Drs M. Yamashita and Y.
Nagahama, National Institute for Basic Biology, Okazaki, Japan),
anti-Phospho-Cdc2 (Tyr15; Cell Signaling Technology) and anti-GST (Nacalai
Tesque). Secondary antibodies were HRP-conjugated anti-rabbit IgG (GE
Healthcare) or anti-mouse IgG (Dako), AP-conjugated anti-rabbit IgG (Dako) or
anti-mouse IgG (Dako), or ExactaCruzF (Santa Cruz Biotechnology). Reacted
proteins were detected by the BCIP/NBT phosphatase substrate system (KPL), or
ECL plus (GE Healthcare) and visualized with LAS1000-plus (Fuji Film). To
reprobe with another antibody, immunoblotted membranes were stripped with WB
Stripping Solution Strong (Nacalai Tesque).
Kinase assay
Histone H1 kinase activity of whole egg extracts, or cyclin A- or cyclin
B-associated Cdk1, was assayed as described
(Okano-Uchida et al., 2003).
Rsk activity was measured as total S6 kinase activity: 1 µl of egg extract
equivalent to half of an egg was added to 9 µl of assay dilution buffer (25
mM Na-β-glycerophosphate, 5 mM EGTA, 1 mM Na-orthovanadate, 1 mM DTT, 20
mM MOPS, pH 7.2), containing 16.9 µM MgCl2, 0.1 mg/ml GST-S6
(Mori et al., 2006), Inhibitor
Cocktail (20-116; Upstate) and 0.2 mCi/ml [
-32P]ATP (GE
Healthcare). The mixture was incubated for 10 minutes at 30°C, then 10
µl of 2x Laemmli sample buffer (LSB) was added, and samples were
heated. Samples of histone H1 or total S6 kinase assay were separated with
12.5% or 15% SDS-PAGE, respectively, stained with Coomassie Brilliant Blue,
and visualized with BAS2000 (Fuji Film).
Immunofluorescence and live cell imaging
To assess DNA replication, eggs were incubated in seawater containing 1 mM
BrdU. The vitelline coat was removed from eggs with Ca2+-free
seawater, pH 9.0, containing 1% thioglycolate and 5 mM EGTA, and the eggs were
treated with an extraction buffer (25 mM imidazole, 10 mM KCl, 10 mM EGTA, 1%
Triton X-100, 15% glycerol, pH 6.9). The extracted eggs were attached to
Biobond-coated (British BioCell) coverslips and fixed with cold methanol.
After DNA denaturation with 1 M HCl, BrdU was stained with anti-BrdU (BD
Biosciences) and Alexa Fluor 568 goat anti-mouse IgG (Invitrogen). DNA was
stained with DAPI. For assaying nuclear envelope breakdown (NEBD), eggs were
injected with GST-IBB-GFP. Fluorescence images were taken using a Zeiss
AxioPlan2 microscope with Plan-Apo 40x/0.95 Korr, Plan-Apo
20x/0.60, or Plan-Apo 10x/0.32 Ph1 objectives and an AxioCam
camera at room temperature (
20°C). The images were acquired with
AxioVision (Zeiss) and processed in Photoshop (Adobe).
35S Pulse-labeling of eggs
Ten eggs were pulse-labeled with 360 µCi/ml of Redivue Pro-mix
L-[35S] in vitro Cell Labeling Mix (GE Healthcare) in seawater for
5 minutes, recovered in 3 µl seawater and immediately frozen in liquid
nitrogen. To measure label uptake into eggs, ten pulse-labeled eggs were
washed three times with seawater and dissolved in LSB. Radioactivity was
measured by liquid scintillation counter
(Lapasset et al., 2008). To
examine the label incorporation into cyclin A and B proteins, 7 µl of lysis
buffer (160 mM Na-β-glycerophosphate, 40 mM EGTA, 30 mM MgCl2,
200 mM KCl, 200 mM sucrose, 1 mM DTT, 0.5% NP-40, 1 mM Na-orthovanadate, 25 mM
NaF, pH 7.3) was added, and egg extracts were prepared according to
Okano-Uchida et al. (Okano-Uchida et al.,
2003). Cyclins A and B were immunoprecipitated with 10 µl of a
50% slurry of Protein A Sepharose CL-4B (GE Healthcare) conjugated with
anti-cyclin A or anti-cyclin B antibody. The immunoprecipitates were washed
three times with lysis buffer and dissolved in 10 µl of 2x LSB. To
examine the overall protein synthesis, recovered eggs were dissolved directly
in LSB. The samples were separated with 10% SDS-PAGE, and transferred to
Immobilon polyvinylidene fluoride membranes (Millipore). The membranes were
dried and incorporation of radioactivity was visualized with BAS2000 (Fuji
Film).
PAT assay
To measure poly(A) tail length, total RNA of immature oocytes and eggs was
isolated with Sepasol-RNA I Super (Nacalai Tesque). The PAT [poly(A) test]
assay was performed with 300 ng of RNA as described
(Salles and Strickland, 1999).
PCR was performed with mRNA specific primer. PCR products were separated with
agarose gel, stained with ethidium bromide and visualized with LAS4000 (Fuji
Film).
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RESULTS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) into
unfertilized eggs that had completed meiosis and were arrested at G1 phase. S
phase was evaluated by BrdU incorporation into newly synthesized DNA and M
phase was evaluated by nuclear envelope breakdown (NEBD), as detected by
whether a fluorescent protein with a nuclear localization sequence (IBB-GFP;
see Materials and methods) was localized in the nucleus or dispersed within
the cytoplasm. As shown in Fig.
1, inhibition of Rsk initiated DNA synthesis, as we reported
previously (Mori et al.,
2006), but did not stimulate NEBD. Thus, in G1 phase-arrested
eggs, loss of Rsk activity is sufficient for entry into S phase, but not for
further cell cycle progression into M phase.
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) (see also
Fig. 4B), MAPK inactivation by
U0126 caused entry into M phase but not S phase
(Fig. 2A,B). Thus, loss of MAPK
activity induces M phase in unfertilized eggs independent of Rsk activity.
Conversely, when MAPK activity was maintained at an elevated level by prior
injection of constitutively active GST-Mos, entry into M phase was prevented
in fertilized eggs (Fig. 2C,D;
Fig. 4C)
(Mori et al., 2006;
Tachibana et al., 2008).
Although these eggs did not enter into S phase owing to the presence of active
Rsk, the prevention of M phase did not appear to be caused by inhibition of S
phase, as prevention of S phase by aphidicolin
(Tachibana et al., 2008) did
not inhibit entry into M phase, although some delay was observed
(Fig. 2D).
Taken together, the above observations indicate that inactivation of MAPK, but not Rsk, is necessary and sufficient for induction of M phase in unfertilized eggs, regardless of whether S phase occurs. This implies that in unfertilized mature starfish eggs arrested at G1 phase, MAPK prevents entry into M phase, independently of the Rsk-mediated prevention of entry into S phase.
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; Tachibana et al.,
2008; Okano-Uchida et al.,
1998). Fertilization led to inactivation of MAPK, accumulation of
cyclin A and cyclin B proteins and activation of their associated Cdk1,
peaking at 70-80 minutes, immediately followed by NEBD
(Fig. 3A;
Fig. 2D). Then, cyclin A and
cyclin B proteins underwent degradation, and their associated Cdk1 became
inactive, resulting in exit from M phase. Thereafter, the levels of cyclin A
and B proteins and their associated Cdk1 activity oscillated along with the
cleavage cycles.
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). This explains why, on the whole, Cdk1 was inhibited by
phosphorylation on Tyr-15 and remained inactive in Rsk-inhibited eggs
(Fig. 3B,C). These behaviors of
Cdk1 are consistent with the above observation that inhibition of Rsk did not
trigger M phase in unfertilized eggs (Fig.
1B). By contrast, when MAPK was inactivated by U0126 in unfertilized mature eggs, both cyclin A and cyclin B accumulated to levels higher than those induced by inactivation of Rsk alone, resulting in activation of their associated Cdk1 (Fig. 4A). In these U0126-treated unfertilized eggs, cyclin A-Cdk1 was first activated, the inhibitory phosphorylation and the following dephosphorylation occurred on Cdk1-Tyr15, and finally cyclin B-Cdk1 became active. Thereafter, protein levels of cyclins A and B and their associated Cdk1 activity oscillated, as observed in fertilized eggs (compare Fig. 4A with Fig. 3A). Furthermore, even when Rsk activity was maintained at elevated levels by CA-Rsk-EE, MAPK inhibition by U0126 caused accumulation of cyclins A and B, resulting in activation of their associated Cdk1 (Fig. 4B; also compare with Fig. 3C). Thus, independently of Rsk, loss of MAPK activity is sufficient for accumulation of cyclins A and B and the subsequent activation of Cdk1.
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Taken together, the above observations indicate that inactivation of MAPK, but not Rsk, is necessary and sufficient to cause accumulation of both cyclin A and cyclin B and the resulting activation of both cyclin A- and cyclin B-Cdk1 in unfertilized eggs. This implies that, independently of Rsk, MAPK inhibits accumulation of cyclin A and cyclin B proteins in unfertilized mature eggs, thus preventing entry into M phase.
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However, since a significant increase in pulse-label incorporation was also observed along with incubation in unfertilized eggs (Fig. 5A, UF lanes), we were suspicious of the changes in uptake of label into eggs. Indeed, a huge increase in the rate of uptake of pulse-labeling occurred along with incubation in unfertilized eggs (at present we do not know the reason), and its rate was similar in fertilized eggs (Fig. 5C, left and middle). Although it remains unclear how much of the total Met/Cys pool in eggs is formed by the labeled amino acids, comparison between the rate of label uptake into eggs and the rate of label incorporation into proteins (compare Fig. 5B with 5C) indicates that the rate of synthesis of cyclins A and B increased following fertilization.
By contrast, when MAPK was kept active by injection of GST-Mos, the increase in label incorporation into cyclins A and B following fertilization was significantly suppressed (Fig. 5D). Conversely, when MAPK was inactivated with U0126 in unfertilized eggs, a significant increase in the rate of label incorporation into cyclins A and B was observed (Fig. 5E), whereas the rate of label uptake was similar between U0126-treated and control DMSO-treated eggs (Fig. 5C, right). Furthermore, this increase caused by MAPK inactivation was detectable even in eggs in which Rsk activity was maintained at an elevated level by injection of CA-Rsk-EE (Fig. 5F). The increase in label incorporation was slightly less than in uninjected eggs, but a similar reduction was also observed in eggs injected with kinase-dead Rsk (KD-Rsk-EE) (Fig. 5F), indicating that the reduction might be caused by CA-Rsk-EE protein itself, but not by its activity. These observations indicate that MAPK, but not Rsk, suppresses the synthesis rate of both cyclin A and cyclin B in unfertilized mature eggs.
To investigate how the rate of synthesis of cyclins A and B is regulated,
we performed the PAT assay (Salles and
Strickland, 1999) to measure poly(A) tail length.
Fig. 6 clearly shows that for
both cyclin A and B mRNAs, poly(A) tail length increased
during meiotic maturation but did not alter during the first cell cycle after
fertilization or U0126 addition. Thus, the increase in the rate of synthesis
of cyclins A and B on release from the G1 phase arrest does not appear to be
regulated by elongation of poly(A) tail.
Taken together, MAPK inhibition of accumulation of cyclin A and cyclin B in unfertilized eggs is most likely to be due to poly(A)-independent repression of their synthesis rate by MAPK.
Rsk promotes proteolysis of cyclin A and cyclin B in unfertilized mature eggs
In unfertilized mature eggs, a low but significant incorporation of
[35S]Met/Cys into cyclin A and cyclin B was detectable
(Fig. 5A,D,E). This
incorporation was abolished by emetine, an inhibitor of protein synthesis
(Fig. 7A), indicating that
cyclin A and cyclin B are continuously synthesized in unfertilized eggs (even
though MAPK represses the rate of synthesis). However, cyclin B protein levels
were largely unaltered and cyclin A protein levels were slightly decreased in
unfertilized eggs (Fig. 3A;
Fig. 4A), implying that both
proteins are continuously degraded in unfertilized eggs.
To confirm this, protein synthesis of cyclin A and cyclin B was specifically inhibited in unfertilized eggs. Injection of morpholino oligonucleotides (MOs) targeting cyclin A and B mRNAs resulted in the disappearance of cyclin A and cyclin B proteins (Fig. 7B, lane 1). This disappearance was suppressed when a peptide containing the cyclin B destruction box (D-box) sequence was co-injected with the MOs, whereas co-injection of a mutant D-box peptide was not effective (Fig. 7B, lanes 2 and 3). These observations indicate that cyclin A and cyclin B undergo proteolysis in a D-box dependent manner in unfertilized eggs arrested at G1 phase.
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Taken together, Rsk appears to positively regulate the D-box dependent proteolysis of cyclin A and cyclin B in unfertilized eggs arrested at G1 phase. It is unclear, however, why inhibition of Rsk resulted in accumulation of cyclin B, but not cyclin A, in unfertilized eggs (Fig. 3B,C; MAPK remained active). Possibly, the protein synthesis rate of cyclin A is lower than that of cyclin B (Fig. 5A,D,E), and hence accumulation of cyclin A might be below the limit of detection, even when its proteolysis is turned off by inhibition of Rsk. Alternatively, under conditions whereby Rsk activity is inhibited but MAPK remains active, an Rsk-independent pathway might support proteolysis of cyclin A in unfertilized eggs.
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DISCUSSION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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). The present observations indicate that there are two
separate pathways downstream of MAPK. One is the previously known pathway that
is mediated by Rsk and leads to prevention of entry into S phase
(Mori et al., 2006); the other
is a novel pathway that is not mediated by Rsk and leads to prevention of
entry into M phase through repression of protein synthesis of cyclin A and
cyclin B (Fig. 8). Such a
dual-lock downstream of MAPK is required for blocking the start of the
embryonic cell cycle, as M phase can be initiated even when S phase is
blocked, and vice versa (Figs 1
and 2). Upon fertilization,
degradation of Mos shuts down both of the dual-lock mechanisms, resulting in
the cell cycle progression into S phase and M phase. To our knowledge, this is
the first demonstration that the Mos-MAPK cascade separates into Rsk-dependent
and Rsk-independent pathways, thereby arresting the cell cycle prior to
fertilization.
Control of the embryonic cell cycle order without checkpoints
In somatic cells, the orderly progression of the cell cycle is ensured by
checkpoint controls (Hartwell and Weinert,
1989). For example, the DNA replication checkpoint monitors
progression of S phase (DNA replication) and allows entry into M phase only
after completion of S phase. Unlike the ordinary somatic cell cycle, however,
a functional cell cycle checkpoint is lacking in the early embryonic cell
cycle of some organisms, including frog
(Newport and Dasso, 1989),
starfish and some sea urchins (Yamada et
al., 1985). In starfish, when DNA replication is inhibited, the
early embryonic cell cycle can progress with M phase cycling without S phase
(Nagano et al., 1981)
(Fig. 2). Thus, to block the
start of the embryonic cell cycle in unfertilized starfish eggs arrested at G1
phase, entry into M phase must be suppressed independently of prevention of S
phase, as revealed by the present study
(Fig. 8).
How then is the order of S phase and M phase in the first embryonic cell
cycle ensured in the absence of the DNA replication checkpoint? Since entry
into M phase is controlled by the M phase cyclin-Cdk1 complex
(Nurse, 1990), there are in
general two ways to maintain low Cdk1 activity until the start of M phase: one
is prevention of synthesis of M phase cyclin, and the other is prevention of
activation of the M phase cyclin-Cdk1 complex after synthesis of M phase
cyclin. In the first case, when the arrest is released, there should be a
period required to accumulate M phase cyclin to a critical level necessary for
entry into M phase, thereby allowing time for S phase to occur before M phase.
Thus in the dual-lock mechanism (Fig.
8), the Rsk-independent pathway, which prevents Cdk1 activation
before, but not after, synthesis of M phase cyclins, contributes to the
ordering of the first cell cycle events after fertilization by delaying M
phase initiation and thereby allowing S phase.
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;
Bitangcol et al., 1998). In
this arrest, accumulation of cyclin A and cyclin B occurs
(Murakami and Vande Woude,
1998), but MAPK directly phosphorylates and activates Wee1 to
inhibit Cdk1 (Murakami et al.,
1999; Walter et al.,
2000). Thus, MAPK-dependent repression of M phase cyclin
synthesis, which is seen in starfish, might not be the only way to ensure the
order of the first embryonic cycle.
MAPK-, but not Rsk-, dependent repression of synthesis of M phase cyclins
In maturing oocytes and early embryos, protein synthesis is generally
regulated through control of translational activity of maternal mRNAs because
these cells are transcriptionally inert. During oocyte maturation in starfish,
synthesis of cyclins A and B is likely to be regulated in a poly(A)-dependent
manner (Standart et al., 1987)
(Fig. 6A). Consistently, mRNAs
of cyclin A and cyclin B contain cytoplasmic polyadenylation elements [CPEs
(reviewed by Mendez and Richter,
2001; Richter,
2007)] in their 3' untranslated region (see
Okano-Uchida et al., 1998;
Miyake et al., 2001), and the
involvement of CPE-binding protein (CPEB) is suggested in cyclin translation
(Lapasset et al., 2005;
Lapasset et al., 2008).
By contrast, after completion of meiosis II, synthesis of cyclins A and B
is likely to be regulated in a poly(A)-independent manner in starfish eggs
(Fig. 6). How then does MAPK
repress translation of cyclin A and B mRNAs independently of
poly(A) tail elongation? In Drosophila, translational regulation of
maternal mRNAs is well-studied before and after fertilization (reviewed by
Vardy and Orr-Weaver, 2007b).
It should be noted that in the PAN GU (PNG) kinase complex mutants, cyclin B
synthesis is downregulated in the absence of shortening of poly(A) tail length
(Vardy and Orr-Weaver, 2007a).
In the wild type, however, elongation of the poly(A) tail occurs at egg
activation, indicating that the MAPK-dependent translational repression in
starfish G1 phase eggs is different from the repression seen in the PNG
mutants. Instead, considering the constant poly(A) tail length, the 5'
cap-dependent regulation (see Vardy and
Orr-Weaver, 2007b) should be implicated in the MAPK-dependent
repression. In the case of somatic cells of mammals, MAPK-interacting kinase
(Mnk), which is a direct target of MAPK, phosphorylates eIF4E (eukaryotic
initiation factor 4E) (Waskiewicz et al.,
1997). This phosphorylation is thought to negatively regulate
5' cap-dependent translation (Knauf
et al., 2001). Mnk is thus an interesting candidate for the
Rsk-independent target of MAPK in repressing synthesis of cyclins A and B in
unfertilized mature starfish eggs.
In any case, it is curious that cyclin A and cyclin B proteins accumulate
at entry into meiosis II (Okano-Uchida et
al., 1998) but their synthesis is repressed after completion of
meiosis II and in unfertilized mature eggs
(Fig. 5), whereas MAPK is fully
active in every case. In starfish, the overall rate of protein synthesis is
low in immature oocytes, increases upon meiosis reinitiation, remains at
elevated levels until the meiosis I/II transition and then diminishes in
unfertilized mature eggs (Lapasset et al.,
2005; Lapasset et al.,
2008). In particular, cyclin B synthesis leading up to entry into
meiosis II appears to depend on MAPK in starfish
[Fig. 3C in Tachibana et al.
(Tachibana et al., 2000)] and
on the MAPK-Rsk pathway in Xenopus as well
(Taieb et al., 2001). It is
thus likely that the MAPK-dependent repression of cyclin synthesis might be
established at the end of meiosis II.
Previously, we demonstrated that parthenogenetic development into
bipinnaria larvae can be induced when Rsk activation is prevented at meiosis
reinitiation, and hence meiosis II is skipped
(Mori et al., 2006). By
contrast, Fig. 1 indicates that
if the timing of Rsk inhibition is delayed until G1 phase after completion of
meiosis II, entry into the first M phase does not occur, and no
parthenogenesis is observed. This difference in parthenogenetic capacity might
be explained by the above consideration that the MAPK-dependent suppression of
protein synthesis should be established during meiosis II, although loss of
centrioles, that can be duplicated, during meiosis II
(Tamura and Nemoto, 2001)
might also be a factor.
Rsk-dependent M phase cyclin destruction in unfertilized starfish eggs
Meta-II arrest in unfertilized Xenopus eggs is accomplished by
Mos-MAPK-Rsk (Sagata et al.,
1989; Haccard et al.,
1993; Kosako et al.,
1994; Bhatt and Ferrell,
1999; Gross et al.,
1999) and Erp1/Emi2 (Schmidt
et al., 2005; Tung et al.,
2005). Erp1 is an inhibitor of the anaphase-promoting
complex/cyclosome (APC/C), which is an E3 ligase for the destruction of
mitotic cyclins (Peters,
2002). Direct phosphorylation of Erp1 by Rsk stabilizes Erp1 and
also promotes its APC/C-inhibiting activity, resulting in meta-II arrest
(Inoue et al., 2007;
Nishiyama et al., 2007). On
release from meta-II arrest, Erp1 undergoes degradation that depends on
Ca2+-CaMKII, Plk1 and SCFβ-TrCP, and hence the APC/C becomes
active to cause degradation of cyclin B
(Liu and Maller, 2005;
Rauh et al., 2005). Thus, Rsk
negatively regulates cyclin B destruction in Xenopus eggs.
By contrast, Rsk positively regulates destruction of cyclin A and cyclin B
in unfertilized starfish eggs arrested at G1
(Fig. 7). How can Rsk exert
opposing effects on M phase cyclin destruction in Xenopus and
starfish eggs? Cyclin B ubiquitylation is performed by APC/C associated either
with Cdc20 in M phase or Cdh1 in G1 phase
(Peters, 2002). As Cdh1
protein is undetectable in unfertilized eggs and until mid-blastula stage in
Xenopus (Lorca et al.,
1998), Cdc20 is probably a major activator of APC/C in meiotic
maturation and early development. The activity of APC/C associated with Cdc20
is positively regulated by Cdk1 (Kramer et
al., 2000). Consistently, low but significant levels of Cdk1
activity were detectable in unfertilized starfish eggs in which Rsk was
active, whereas Cdk1 became inactive after abolishment of Rsk activity
(Fig. 3B)
(Tachibana et al., 2008). This
Cdk1 activity should be supported by Rsk, as Rsk can phosphorylate Myt1 and
downregulate its inhibitory activity on cyclin B-Cdk1
(Palmer et al., 1998). Thus,
Rsk could maintain APC/C-Cdc20 activity and hence promote destruction
box-dependent proteolysis of M phase cyclins in unfertilized starfish eggs,
assuming that Erp1 is absent or not functional in these eggs.
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Footnotes |
|---|
* Present address: Gene Expression Unit, European Molecular Biology
Laboratory (EMBL), Meyerhofstrasse 1, D-69117 Heidelberg, Germany 
|
REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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