1 UMR 7622, CNRS, Université Paris VI, 9 quai Saint Bernard, Bat. C,
75252 Paris, cedex 05, France
2 Institut Jacques Monod, CNRS, Université Paris VII, 2 place Jussieu,
75251 Paris, cedex 05, France
* Author for correspondence (e-mail: marie-helene.verlhac{at}snv.jussieu.fr)
Accepted 10 July 2003
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SUMMARY |
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Key words: DOC1R, MAPK, Mouse meiotic maturation, Microtubules
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Introduction |
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We describe the isolation of another MAPK partner using the same two-hybrid
screen: DOC1R (D19ERTD144E - Mouse Genome Informatics), a murine homologue of
a potential human tumor suppressor gene DOC1R (deleted in oral cancer one
related) (Zhang et al., 1999).
Until now, little was known about this protein that has been found on the
basis of its homology with human DOC1, a human tumor suppressor gene
(Todd et al., 1995
). DOC1R is
related to a coiled-coil region of a kinesin (KIF14) of unknown function. Like
MISS, DOC1R is rich in proline residues in its N terminus. DOC1R has a perfect
consensus site for MAPK phosphorylation, a potential CDK2 binding site and a
potential cyclin/CDK binding site (Zhang
et al., 1999
). We show here that DOC1R has been conserved from
Xenopus laevis to human, which suggests that it performs important
functions in vertebrate species. In mouse oocytes, DOC1R is present at all
stages of meiotic maturation and is regulated by multiple phosphorylations.
Both cyclin B/CDC2 and MAPK are able to phosphorylate DOC1R in vitro, and the
MOS/.../MAPK pathway phosphorylates DOC1R in vivo. This protein is localized
in dots on microtubules especially on metaphase I and II spindles.
Consistently, a DOC1RGFP fusion localizes to the metaphase II spindle. The
depletion of DOC1R by microinjection of antisense (asRNA) or double-stranded
(dsRNA) RNA directed against its endogenous mRNA has a strong effect on the
metaphase II spindle morphology. Injected oocytes harbor spindles with astral
microtubules, as well as numerous asters in the cytoplasm, suggesting that
DOC1R regulates microtubule organization during the CSF arrest of metaphase II
oocytes. We show that this phenotype is specific to DOC1R as it can be rescued
by the overexpression of the Xenopus protein. Thus, we have
discovered a new class of proline-rich proteins, MISS and DOC1R, substrates of
MAPK that regulate microtubule organization during the CSF arrest of mouse
oocytes.
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Materials and methods |
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Two-hybrid screen
The two-hybrid screen was performed as previously described
(Lefebvre et al., 2002).
RT-PCR assay
For preparing RNA from mouse ovaries, total RNA were extracted using the
Rneasy mini Kit (Qiagen). Immature mouse oocytes were lysed in PBS without
total RNA extraction. Then 500 ng of RNA from ovaries, or 30 immature oocytes
in sterile PBS were treated with 2 U of RQ1 DNAse (Promega) for 20 minutes at
37°C and heated for 5 minutes at 85°C. The first strand cDNA synthesis
was performed with 50 U of MmuLV Superscript (Life Technologies) using 2.5
µM random hexamer (pdN6, Pharmacia), 1 mM dNTPs (Promega), in 10 mM
Tris-HCl pH 8.3, 5 mM MgCl2, 50 mM KCl for 1 hour at 37°C,
followed by 5 minutes at 95°C. The PCR amplification was performed using
5'-ATGCCTCGAGATGACGTACAAGCCAATCGC and
5'-ATGCGAATTCCCGTGCGGGCATTGCGTTCT primers, at 55°C for 30
cycles.
Plasmid construction and in vitro synthesis of capped RNA, asRNA and
dsRNA
The pRN3MYC2-DOC1R was constructed by RT-PCR amplification of the DOC1R
open reading frame from RNA isolated from mouse ovaries. Total RNA were
extracted using the Rneasy Mini Kit (Qiagen). The reverse transcription was
performed on 500 ng of RNA. The PCR amplification was done on 50 ng of RNA/DNA
using 5'-GATCGAATTCATGWSNTAYAARCCNATHGCN and
5'-GATCGCGGCCGCTTACGTGCGGGCATTGCGTTC primers. The PCR product was then
cloned into the pRN3MYC2 vector. The pRN3DOC1R-GFP was obtained by PCR
subcloning at XhoI/EcoRI sites using
5'-ATGCCTCGAGATGACGTACAAGCCAATCGC and
5'-ATGCGAATTCCCGTGCGGGCATTGCGTTCT primers. The pET-DOC1R plasmid was
constructed by subcloning at EcoRI/Not1 sites into the
pET30a vector (Novagen). The DOC1R protein expressed from the pET30a vector
contains a 6His repeat inside 51 additional amino acids, which makes it bigger
of about 5 kDa.
The Xenopus cDNA was isolated from the Xenopus oocyte
cDNA library (Iouzalem et al.,
1998) by PCR using the 5'-ATGCGAATTCATGTCGTATAAACCAT and
3'-ATGCGCGGCCGCTCATGTGCGGGCACTGCGTTCTGT primers on 200 ng of library.
The PCR product was further cloned into pRN3 at the
EcoRI/NotI sites.
The in vitro synthesis of capped RNA, asRNA and dsRNA was performed using linearized plasmids with the mMessage mMachine kit for capped RNAs (Ambion) or with the Megascript Kit for as and dsRNA (Ambion). The capped RNAs and asRNA were then purified on RNeasy columns (Qiagen) and eluted in the injection buffer (10 mM Tris, 0.1 mM EDTA, pH 7.4) at a final concentration of 0.5 µg/µl. Aliquots were then stored at -80°C. For the production of dsRNA, each strand of complementary RNA was first precipitated with ethanol then washed in phenol/chloroform and further incubated at 85°C for 5 minutes. After annealing for 3 hours at 37°C, 4 µl aliquots of dsRNA were stored at - 80°C.
Microinjection of synthetic RNA
Microinjection into mouse oocytes was performed as described
(Verlhac et al., 2000).
Co-immunoprecipitation in Xenopus oocyte extracts
Xenopus oocyte microinjection as well as oocyte extraction was
performed as previously described (Gavin et
al., 1999). Samples of 30 oocytes were extracted in 300 µl of
lysis buffer (80 mM ß-glycerophosphate, pH 7.4, 20 mM EGTA, 15 mM
MgCl2, 100 µg/ml leupeptin, 100 µg/ml aprotinin, 1 mM sodium
orthovanadate, 2 mM PMSF). The oocyte extract was then clarified by
centrifugation at 13,000 rpm at 4°C for 20 minutes. The supernatant was
removed from the overlying lipid layer and the yolk protein and was further
clarified by a second centrifugation at 13,000 rpm at 4°C for 10 minutes.
The oocyte extracts were stored at -70°C. For the immunoprecipitation of
endogenous xp42mapk, we used an anti-ERK2 antibody conjugated to
agarose (Santa Cruz Biotechnology). Xenopus oocyte extracts (300
µl) were pre-cleared with 20 µl of protein A coupled to agarose beads
for 30 minutes at 4°C. The cleared extracts were then incubated for 2
hours at 4°C with the antibody coupled to 20 µl of agarose beads. The
beads were washed four times in 1 ml lysis buffer supplemented with 150 mM
NaCl and then processed for immunoblotting.
Dephosphorylation assay
Just after collection and lysis, oocytes were incubated with or without 400
U of -phosphatase (New England Biolabs) in
-phosphatase buffer
at 37°C for 1 hour and then processed for immunoblotting.
Preparation of recombinant DOC1R protein
The DOC1R recombinant protein was prepared from the HMS174 E. coli
transformed with the pET-DOC1R plasmid. The protein was purified from bacteria
under native conditions as described in the QIAexpressionist handbook
(Qiagen).
Kinase assays
The in vitro kinase assays were performed on 0.1 µg of purified DOC1R or
2.5 µg of Histone H1 in 10 µl of Histone H1 kinase buffer [80 mM
ß-glycerophosphate, 20 mM EGTA, 15 mM MgCl2, 2 µg/ml
leupeptin, 2 µg/ml aprotinin, 2.5 mM benzamidine, 1 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride and 1 mM DTT, pH 7.4] with 0.025
µg of commercial recombinant active rat ERK2 (BIOMOL, Ref SE-137) or 0.025
µg of cyclin B/CDC2 (Biolabs) supplemented with 6.25 µCi of
[-32P]-ATP (3000 Ci/mmole) and 50 µM cold ATP, for 30
minutes at 37°C. Reactions were stopped by the addition of sample buffer
(Laemmli, 1970
) and were
analyzed by SDS-PAGE followed by autoradiography.
2D gel electrophoresis
Oocytes microinjected with MYC-DOC1R encoding RNA were collected 14 hours
after GVBD and stored at -80°C in water containing 1 mM PMSF and 10
µg/ml leupeptin/pepstatin/aprotinin. They were then processed as previously
described (Louvet-Vallee et al.,
2001).
Immunocytochemistry
Immunocytochemistry was performed as previously described
(Brunet et al., 1999). To
visualize endogenous DOC1R, oocytes were first fixed for 30 minutes in 3.7%
formaldehyde at 30°C then treated for 10 minutes in 0.25% TritonX-100 in
PBS, washed in 0.1% Tween 20/PBS, incubated for 1 hour with the primary
affinity-purified anti-DOC1R antibody (at 1:50) in 3% BSA/0.1% Tween 20/PBS.
They were then washed in 0.1% Tween 20/PBS and further incubated for 1 hour
with the secondary anti-rabbit FITC (1:80) in 3% BSA/0.1% Tween 20/PBS. After
incubation with the secondary antibody, all samples were washed in 0.1% Tween
20/PBS, incubated for 5 minutes in Propidium Iodide (5 µg/ml in
0.1%Tween20/PBS), then washed three times in PBS before mounting in Citifluor
(Chem. Lab., UCK).
Immunoblotting
Oocytes at the appropriate stage of maturation were collected in sample
buffer (Laemmli, 1970) and
heated for 3 minutes at 100°C. We used the following antibodies: an
affinity-purified anti-DOC1R antibody directed against the LVRECLAETERNART
peptide (Sigma Immunochemicals), the anti-MYC 9E10 monoclonal antibody (sc-40;
Santa Cruz Biotechnology), an anti-Ezrin antibody
(Louvet-Vallee et al., 2001
)
and the anti-Erk antibody (sc-94; Santa Cruz Biotechnology).
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Results |
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By homology searches in databases, we also isolated ESTs encoding homologues of the DOC1R protein (Fig. 1C). The DOC1R and human DOC1R protein are almost identical (they present 95% of identities, Fig. 1D). Interestingly, the protein is highly conserved from Xenopus laevis to human (about 70% of identities, Fig. 1D). This conservation suggests that DOC1R mediates important function(s) in vertebrate species.
DOC1R mRNA and protein are present in mouse oocytes
To investigate if the DOC1R mRNA was expressed in mouse ovaries and
oocytes, we performed RT-PCR using specific primers on total RNA from ovaries
and immature oocytes. The presence of the DOC1R mRNA in ovaries and oocytes is
shown in Fig. 2A (lanes 2 and
3).
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Therefore these experiments show that both DOC1R mRNA and protein are expressed in mouse oocytes.
DOC1R is regulated by phosphorylation during meiotic maturation of
mouse oocytes
Because it required 200 oocytes to see a faint band corresponding to DOC1R
on immunoblot, we followed the behavior of an overexpressed DOC1R protein
during meiotic maturation after microinjection of RNA encoding MYCDOC1R into
immature oocytes. Samples of microinjected oocytes were collected at different
times during meiosis resumption. In contrast to MYC-MISS protein, the MYCDOC1R
protein accumulates in GV oocytes and at all stages of meiotic maturation
(Fig. 3A) (Lefebvre et al., 2002). The
apparent increase in MYC-DOC1R protein amount during meiosis is due to the
progressive translation of injected RNA. In immature oocytes, the protein
migrates to an apparent molecular weight of 26 kDa
(Fig. 3A, lane 1). By contrast,
during meiosis resumption the MYC-DOC1R protein up-shifts, migrating more
slowly at about 30 kDa, suggesting that it is regulated by post-translational
modifications.
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The down-shift is not complete, suggesting that the treatment with
-phosphatase was not totally efficient, or that the protein undergoes
post-translational modifications other than phosphorylation.
Both cyclin B/CDC2 and MAPK phosphorylate DOC1R
As MYC-DOC1R undergoes post-translational modifications in oocytes
collected one hour after GVBD, when MPF is active and MAPK inactive
(Verlhac et al., 1994), we
tested the ability of both kinases to phosphorylate DOC1R in vitro. For that,
we incubated either purified cyclin B/CDC2 or active ERK2 in the presence of
[
-32P]-ATP and purified DOC1R protein. As shown on the
autoradiograph, both purified cyclin B/CDC2 and active MAPK are able to in
vitro phosphorylate the DOC1R protein (Fig.
4A, lanes 1 and 4) to levels close to the histone H1
phosphorylation (Fig. 4A, lanes
2 and 5). This is consistent with the prediction of one MAPK phosphorylation
site and one cyclin/CDK-binding site
(Shintani et al., 2000
) in the
DOC1R protein sequence.
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To check whether the upper migrating forms of MYCDOC1R present both in
wild-type and Mos-/- oocytes share the same
post-translational modifications, we performed 2D gel analysis. We observed
that the upper band from wild-type oocytes resolved into two major isoforms
(Fig. 4C, top panel). To
position the different isoforms of MYC-DOC1R from one sample to the other, we
used Ezrin as an internal control (not shown)
(Louvet-Vallee et al., 2001).
In metaphase II Mos-/- oocytes, the isoforms corresponding
to the upper migrating band were shifted towards the basic pole (OH-,
Fig. 4C, bottom panel). These
results show first that the band migrating in 1D at an apparent molecular
weight of 30 kDa corresponds to different isoforms of DOC1R. Second these
results show that MYC-DOC1R is less phosphorylated in
Mos-/- oocytes, as phosphorylations confer negative
charges to proteins. These experiments suggest that the protein is effectively
phosphorylated by the MOS/.../MAPK pathway and that other kinases are also
responsible for DOC1R phosphorylation.
DOC1R is present at all stages of meiotic maturation and localizes on
metaphase spindles
We followed the endogenous DOC1R localization during meiotic maturation. We
show that DOC1R is present in immature oocytes
(Fig. 5A) and during all stages
of meiotic maturation (Fig.
5B-E), confirming the immunoblotting analysis of the exogenous
MYC-DOC1R protein. DOC1R accumulates in immature oocytes in the nucleus
(Fig. 5A), as previously
described in interphasic human cells
(Zhang et al., 1999). During
the first meiotic division (Fig.
5B-D), DOC1R accumulates in the cytoplasm and localizes in dots in
the vicinity of the chromosomes in a region enriched in microtubules.
Microtubule re-organization has been well described during mouse oocyte
maturation (Brunet et al.,
1999
); microtubules form early during the first division around
the chromosomes and organize into a bipolar spindle about 2 hours after
meiosis resumption. DOC1R localization follows microtubule organization during
metaphase I. In metaphase II (Fig.
5E), DOC1R also accumulates in the cytoplasm and on the spindle.
To prove that the DOC1R protein is associated with microtubules, we treated
metaphase II oocytes with nocodazole, which induces microtubule
depolymerization (Fig. 5F). In
these oocytes, the DOC1R protein was diffusely located in the cytoplasm, which
proves that DOC1R associates specifically with spindle microtubules. As a
control, we performed immunofluorescence after blocking the purified antibody
with the immunogenic peptide and no staining was observed
(Fig. 5G). Furthermore, like
the endogenous DOC1R protein, a DOC1R-GFP fusion localizes in the germinal
vesicle of immature oocytes (Fig.
5H) and on the metaphase II spindle
(Fig. 5I).
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To prove that the phenotype we observed was specific, we injected the dsRNA together with RNA encoding the Xenopus DOC1R protein. Despite the amino acid conservation between the murine and the Xenopus DOC1R proteins, the Xenopus cDNA diverges from the mouse, and thus is corresponding cDNA cannot be targeted by the dsRNA. As shown on Fig. 6F,K, the Xenopus protein complements the microtubule defect induced by the dsRNA. The injection of the RNA coding the Xenopus protein has no effect by itself on microtubule organization (Fig. 6C). So the cytoplasmic asters as well as nucleation of microtubules from the spindle poles is solely due to DOC1R depletion.
Altogether, our experiments demonstrate that DOC1R regulates microtubule organization at least in metaphase II (see Discussion). The depletion of DOC1R induces severe damage to the microtubule cytoskeleton, which may compromise chromosome segregation after fertilization and hence compromise further embryo development.
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Discussion |
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DOC1R has a consensus site for MAPK phosphorylation, a potential CDK2
binding site and a potential cyclin/CDK binding site
(Shintani et al., 2000). We
show that DOC1R is expressed at all stages of meiotic maturation and that it
is regulated by multiple phosphorylations. First, cyclin B/CDC2 is able to
phosphorylate DOC1R in vitro, which is consistent with the presence of one
potential cyclin/CDK binding site in its coding sequence. Furthermore, DOC1R
becomes phosphorylated early in metaphase I, when MPF is active but not MAPK.
Second, MAPK is able to phosphorylate DOC1R in vitro and in vivo, in agreement
with the presence of one consensus site for MAPK phosphorylation in its
sequence.
By 2D gel analysis, we show that the post-translational modifications that affect DOC1R during meiotic maturation are quite complex. Some modifications can be attributed to MPF activation, some to the MOS/.../MAPK pathway and it cannot be excluded that DOC1R is also modified by post-translational modifications other than phosphorylations. The characterization of DOC1R modifications by phosphorylation will be the object of further studies.
DOC1R function during meiotic maturation of mouse oocytes
Endogenous DOC1R is localized in dots on microtubules during all stages of
meiotic maturation in particular on metaphase I and II spindles. These dots
could reflect either an accumulation of DOC1R into vesicular structures
associated with microtubules or macromolecular complexes containing DOC1R
multimers.
Consistent with DOC1R association with microtubules, its depletion leads to drastic phenotypes in metaphase II arrested oocytes: elongated spindles enriched in astral microtubules and numerous asters of microtubules in the cytoplasm. The phenotype is specific to DOC1R depletion (1) because it is observed only when the endogenous protein can no longer be detected on the metaphase II spindle and (2) because overexpression of the Xenopus protein complements the defects observed after dsRNA injection.
The phenotype can be explained by extensive microtubule polymerization from
MTOCs (microtubule organizing centers) that in mouse oocytes are present at
spindle poles as well as foci in the cytoplasm
(Maro et al., 1985). It can be
interpreted as a reduced ability of the chromatin to stabilize microtubules in
its vicinity. Normally, meiotic spindles are devoid of astral microtubules and
cytoplasmic MTOCs do not form microtubule asters. The depletion of DOC1R
promotes microtubule nucleation and/or elongation. This suggests that DOC1R
normally increases microtubule dynamics in metaphase II arrested oocytes. We
cannot exclude that DOC1R has a similar function in metaphase I, because we
could not completely deplete the endogenous pool of DOC1R during the first
meiotic division. The absence of a detectable phenotype in metaphase I could
be explained by a low turnover of the protein and therefore a lack of full
efficiency of the dsRNA during metaphase I.
DOC1R localization and the phenotype observed after its depletion are
consistent with the potential tumor suppressor role of the DOC1R gene
(Zhang et al., 1999). Indeed,
human tumors are characterized by chromosomal instability primary resulting
from spindle organization defects
(Saunders et al., 2000
).
A new vision of the metaphase II arrest of vertebrate oocytes
As for MISS, we could not find obvious invertebrate homologues of DOC1R.
However, the protein sequence of DOC1R has been highly conserved from
Xenopus laevis to human. Moreover, we show here that the
Xenopus protein can functionally complement the mouse DOC1R. So we
believe that both DOC1R and MISS mediate important functions specific to
vertebrate species.
The meiotic metaphase II spindle harbors an unusual location in the cell:
it is closely associated with the cortex, which is enriched in actin
microfilaments. This association allows spindle rotation at fertilization, a
prerequisite to second polar body extrusion that is essential for further
embryo development. It is interesting that DOC1R is related to a coiled-coil
region in a kinesin (KIF14) (for a review, see
Miki et al., 2001) of unknown
function that contains both a kinesin as well as a myosin domain. We can
imagine that DOC1R somehow regulates interactions between spindle microtubules
and microfilaments of the cortex that are necessary for maintaining proper MII
spindle organization.
The discovery of new MAPK substrates, such as DOC1R and MISS, involved in
the regulation of microtubule organization, is of crucial importance for our
understanding of the processes controlling the metaphase II arrest of
vertebrate oocytes. It suggests that the MOS/.../MAPK pathway not only
controls CSF arrest by maintaining a high MPF activity through
p90rsk activity (Bhatt and
Ferrell, 1999; Gross et al.,
1999
; Gross et al.,
2000
) but also ensures that the spindle is properly organized
during the arrest for the success of fertilization. Our findings are
consistent with the unpublished data showing that the maintenance of bipolar
spindles assembled in Xenopus egg extracts requires MAPK activity, but not
p90rsk activity (Horne et al., 2003).
In conclusion, we have discovered two proteins that seem essential for
mouse embryo development as their absence results in severe damage to the
microtubule network of metaphase II mouse oocytes. Indeed C. elegans
mei-1 and mei-2 mutants (proteins that regulate microtubule
dynamics in meiosis and induce severing of microtubules to produce small
meiotic spindles) extrude large polar bodies and show aneuploidy
(Srayko et al., 2000).
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ACKNOWLEDGMENTS |
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