1715
Development 129, 1715-1727 (2002)
Printed in Great Britain © The Company of Biologists Limited 2002
DEV3598
The MAPK pathway triggers activation of Nek2 during chromosome
condensation in mouse spermatocytes
Silvia Di Agostino, Pellegrino Rossi, Raffaele Geremia and Claudio Sette*
Dipartimento di Sanità Pubblica e Biologia Cellulare, Facoltà di Medicina e Chirurgia, Università di Roma ‘Tor Vergata’,
Via O. Raimondo 8, 00173 Rome, Italy
*Author for correspondence (e-mail: [email protected])
Accepted 1 January 2002
SUMMARY
Chromosome condensation during the G2/M progression
of mouse pachytene spermatocytes induced by the
phosphatase inhibitor okadaic acid (OA) requires the
activation of the MAPK Erk1. In many cell systems,
p90Rsks are the main effectors of Erk1/2 function. We have
identified p90Rsk2 as the isoform that is specifically
expressed in mouse spermatocytes and have shown that
it is activated during the OA-triggered meiotic G2/M
progression. By using the MEK inhibitor U0126, we have
demonstrated that activation of p90Rsk2 during meiotic
progression requires activation of the MAPK pathway.
Immunofluorescence analysis indicates that activated Erks
and p90Rsk2 are tightly associated with condensed
chromosomes during the G2/M transition in meiotic cells.
We also found that active p90Rsk2 was able to
phosphorylate histone H3 at Ser10 in vitro, but that the
activation of the Erk1/p90Rsk2 pathway was not necessary
for phosphorylation of H3 in vivo. Furthermore,
phosphorylation of H3 was not sufficient to cause
condensation of meiotic chromosomes in mouse
spermatocytes. Other proteins known to associate with
chromatin may represent effectors of Erk1 and p90Rsk2
during chromosome condensation. Nek2 (NIMA-related
kinase 2), which associates with chromosomes, plays an
active role in chromatin condensation and is stimulated by
treatment of pachytene spermatocytes with okadaic acid.
We show that inhibition of the MAPK pathway by
preincubation of spermatocytes with U0126 suppresses
Nek2 activation, and that incubation of spermatocyte cell
extracts with activated p90Rsk2 causes stimulation of Nek2
kinase activity. Furthermore, we show that the Nek2 kinase
domain is a substrate for p90Rsk2 phosphorylation in vitro.
These data establish a connection between the Erk1/
p90Rsk2 pathway, Nek2 activation and chromosome
condensation during the G2/M transition of the first
meiotic prophase.
INTRODUCTION
from studies performed on Xenopus oocytes, which are
synchronized at the prophase of the first meiotic division and
can be induced to progress through the cycle by stimulation
with progesterone (reviewed by Sagata, 1997). The hormonal
treatment leads to several biochemical changes that result in
maturation of the oocyte: a decrease in cAMP-dependent
protein kinase activity; the synthesis of the protein kinase Mos
and activation of the MAPK pathway; activation of the Pololike-kinase (Plx1) pathway; dephosphorylation of Cdc2 by the
dual specificity phosphatase Cdc25C; and the consequent
activation of the CyclinB/Cdc2 complex, also known as
maturation promoting factor (MPF) (Sagata et al., 1988; Sagata
et al., 1989a; Sagata et al., 1989b; Qian et al., 1998; Qian et
al., 2001). Although MPF activation and oocyte maturation can
be obtained in vivo by microinjection of constitutively active
forms of MAPKs (Gotoh et al., 1995; Haccard et al., 1995),
recent evidence suggests that physiologically activation of the
MAPK pathway is not sufficient for progression of oocytes
through the first meiotic cycle. Indeed, inhibition of MAPKs
by preincubation of oocytes with the specific MEK inhibitor
Sexual reproduction requires the fusion of two haploid cells to
form a diploid zygote. In higher eukaryotes, this implies that
specialized diploid cells, the germ cells, reduce their
chromosome content during meiosis. A single round of DNA
replication followed by two cell divisions characterizes this
process, such that a single diploid cell gives rise to four haploid
cells. In the first division, homologs are initially held together
by the synaptonemal complex, which dissolves at the end of
the pachytene stage, and subsequently by chiasmata produced
during homologous recombination (Roeder, 1997). At
anaphase, sister chromatids do not separate and homologous
chromosomes segregate to the opposite poles of the spindle
into the two daughter cells (reviewed by Roeder, 1997; Handel
and Eppig, 1998; Biggins and Murray, 1999). The second
meiotic division resembles mitosis and sister chromatids are
separated in the daughter cells to give rise to haploid
spermatids (Handel and Eppig, 1998).
Most of the knowledge on meiotic cell cycle events derives
Key words: Meiosis, Spermatocytes, MAPK, Nek2, Chromosome,
Mouse
1716 S. Di Agostino and others
U0126 did not block maturation induced by progesterone
(Gross et al., 2000). However, Qian and collaborators have
demonstrated that immunodepletion of Plx1 from Xenopus
oocyte extracts completely prevents activation of Cdc25C and
MPF, indicating a crucial role of this kinase in the control of
MPF function (Qian et al., 2001).
Maintenance of chromatin condensation between the two
meiotic divisions contributes to prevent inappropriate DNA
replication and allows progression between two metaphases
without the interphase of a normal cell cycle (Gross et al.,
2000). Suppression of DNA replication requires the incomplete
inactivation of MPF at anaphase of the first meiotic cycle
(Furuno et al., 1994). In this regard, both incomplete
degradation and new synthesis of cyclin B contribute to
maintain MPF activity during the exit from metaphase I and to
allow progression to meiosis II without interphase (Kobayashi
et al., 1991; Roy et al., 1996; Ohsumi et al., 1994; Gross et al.,
2000). A fundamental role in this process is played by the
MAPK pathway that, through the concerted action of Mos,
Erks and p90Rsk, maintains the inhibition of the anaphasepromoting complex (APC), thereby reducing cyclin B
degradation (Gross et al., 2000; Taieb et al., 2001). Although
inhibition of Erks activation does not prevent cell cycle
progression and activation of MPF, when the MAPK pathway
is blocked, the metaphase I spindle cannot form and the
complete destruction of cyclin B by the APC allows entry into
interphase and DNA duplication between metaphase I and II
(Gross et al., 2000). Therefore, even though it is not directly
involved in regulation of the activity of cell-cycle dependent
kinases, the MAPK pathway plays a crucial role in meiosis by
allowing the reduction of the genome to a haploid content.
Later in meiosis, Mos and MAPKs are also necessary for the
arrest of oocytes at the metaphase of the second meiotic
division (Sagata, 1997), and this cytostatic role is mediated by
the MAPK effector p90Rsk2 (Gross et al., 1999; Bath and
Ferrell, 1999). However, such a role is restricted to female
meiosis, as spermatocytes do not arrest at metaphase and the
male meiotic cycle is continuous.
Not much is known on meiotic events in mammals. Meiotic
resumption in mammalian oocytes is under the hormonal
control of intracellular cAMP levels (Handel and Eppig, 1998).
A drop in intracellular cAMP triggers meiotic resumption that
temporally correlates with activation of both MAPK and MPF.
Experiments performed with Mos knockout mice have shown
that Mos and the MAPK pathway do not play a role in meiotic
resumption but are required as cytostatic factors that arrest the
cell cycle at metaphase II in ovulated oocytes (Colledge et al.,
1994; Hashimoto et al., 1994). In male mouse spermatocytes,
the prophase of the first meiotic division is a slow and
continuous process that ensures DNA repair after crossing over
and cell growth before the two subsequent divisions that will
give rise to four haploid spermatids. It has been reported that
mid-pachytene spermatocytes can be induced to complete the
prophase of the first meiotic division and enter metaphase by
incubation for 4-6 hours with the phosphatase inhibitor okadaic
acid (OA) (Wiltshire et al., 1995). OA is able to bypass the
checkpoints that physiologically delay the entry into
metaphase, without perturbing the steps involved in this
progression (Wiltshire et al., 1995; Cobb et al., 1999a). Indeed,
metaphase chromosomes obtained by treatment of
spermatocytes with OA are normal bivalents in which the
synaptonemal complex has dissolved, crossing over has been
completed and chiasmata are present (Wiltshire et al., 1995).
Spermatocyte G2/M progression is accompanied by activation
of MPF and the MAPK Erk1 (Wiltshire et al., 1995; Sette
et al., 1999), and it has been shown that inhibition of the
MAPK pathway prevents efficient chromosome condensation
independently of MPF during transition from prophase to
metaphase (Sette et al., 1999).
Pathways that trigger chromosome condensation and
alternative to MPF have been described in mitotic cells. In
cycling extracts obtained from Xenopus eggs, phosphorylation
of histone H3, which correlates with chromatin condensation,
is mediated by the Aurora kinases (Murnion et al., 2001;
Scrittori et al., 2001). Activation of the NIMA-like kinases
(never-in-mitosis in Aspergillus nidulans) Nek1, Nek2 and
Nek3 has also been reported to induce chromatin condensation
independently of MPF (Lu and Hunter, 1995). This observation
is particularly interesting because Nek2 is expressed in
mouse meiotic cells and it is associated with condensing
chromosomes during the prophase of the first division (Tanaka
et al., 1997; Rhee and Wogelmuth, 1997). Furthermore, it has
been observed that Nek2 is activated during OA-induced G2/M
progression, and that the time course of its activation correlates
with that of chromosome condensation (Rhee and Wolgemuth,
1997). Nek2 is known to phoshorylate the centrosomeassociated protein C-Nap1 (Fry et al., 1998a) and it may play
a role in centrosome separation and chromosome dynamics
(Fry et al., 1998b; Uto and Sagata, 2000). However, it is
currently non known what mechanisms are involved in
activation of Nek2 and what role the kinase plays during
meiotic progression of mouse spermatocytes.
In this study, we have investigated the role played by the
MAPK pathway in chromosome condensation during the
G2/M progression induced by OA in mouse pachytene
spermatocytes. We found that p90Rsk2 was the main p90Rsk
isoform expressed in these cells and that it was activated by the
MAPK pathway during meiotic progression. Inhibition of Erks
and p90Rsk2 activation blocks condensation of metaphase
chromosomes. Furthermore, activated Erks and p90Rsk2
were found in tight association with the condensed meiotic
chromosomes. Although p90Rsk2 is able to phosphorylate H3
in vitro, in vivo phosphorylation of the histone induced by OA
treatment of spermatocytes was not affected by inhibition of
p90Rsk2 activity. Finally, we found that Erks and p90Rsk2
were required for the activation of Nek2 during the G2/M
transition in vivo, and that p90Rsk2 is able to activate and
phosphorylate Nek2 in vitro. These data suggest that
chromosome condensation in meiotic cells requires the
activation of the NIMA-like kinase Nek2 by the MAPK
pathway.
MATERIALS AND METHODS
Preparation of testicular cells
Testes from adult CD1 mice (Charles River Italia) were used to prepare
germ cells. After dissection of the albuginea membrane, testes were
digested for 15 minutes in 0.25% (w/v) collagenase (type IX, Sigma)
at room temperature with constant shaking. Digestion was followed by
two washes in minimum essential medium (MEM, Gibco BRL), then
seminiferous tubules were minced using a sterile blade and further
Nek2 activation 1717
digested in MEM containing 1 mg/ml trypsin (Sigma) for 30 minutes
at 30°C. Digestion was stopped by addition of 10% fetal calf serum and
the released germ cells were collected after sedimentation (10 minutes
at room temperature) of tissue debris. Germ cells were centrifuged for
10 minutes at 1000 g at 4°C and the pellet resuspended in 20 ml of
elutriation medium [120.1 mM NaCl, 4.8 mM KCl, 25.2 mM NaHCO3,
1.2 mM KH2PO4, 1.2 mM MgSO4(7H2O), 1.3 mM CaCl2, 11 mM
glucose, 1× essential amino acid (Gibco BRL), penicillin, streptomycin,
0.5% bovine serum albumin (BSA)]. Germ cells at pachytene
spermatocyte and round spermatid steps were obtained by elutriation of
the unfractionated single cell suspension as previously described
(Meistrich, 1977). Homogeneity of cell populations was 80-85%
(pachytene spermatocytes) and 95% (round spermatids), and was
routinely monitored morphologically. Spermatogonia were obtained
from prepuberal mice as previously described (Rossi et al., 1993).
Cell culture and treatments
After elutriation, pachytene spermatocytes were cultured in MEM,
supplemented with 0.5% bovine serum albumin (BSA), 1 mM sodium
pyruvate, 2 mM sodium lactate, in six-well dishes at a density of 106
cells/ml at 32°C in a humified atmosphere containing 95% air and 5%
CO2. After 12 hours, cells were treated with either 0.5 µM okadaic
acid (OA) (Calbiochem) or equal volumes of the solvent DMSO, and
culture was continued for up to 6 hours. In order to suppress the
MAPK cascade, cells were preincubated for 12 hours prior to OA
treatment with the specific inhibitor of MEK1/2 kinases U0126
(Calbiochem) at a concentration of 10 µM or with equal volumes of
the solvent DMSO. For cytological and immunofluorescence analyses
and kinase assays, aliquots of the same samples were taken and
processed as described below.
Plasmid construction
cDNAs for N-terminal hemoagglutinin (HA)-tagged rat Rsk1, murine
Rsk2 and human Rsk3 in pMT2 (Zhao et al., 1996) were kindly
provided by Christian Bjørbaek (Beth Israel Hospital, Boston, MA).
pGEX-3X-Nek21-272 and pGEX-3X-Nek2273-444, which carry the
catalytic and regulatory domains of Nek2, respectively, were
generated by RT-PCR of total RNA of spermatocytes and subsequent
PCR of the full-length cDNA (GI 6754817), using appropriate
primers. pGEX-3X-Nek21-272 contains the initial methionine of Nek2
and a new stop codon introduced at the end of the catalytic domain.
pGEX-3X-Nek2273-444 contains a new methionine introduced
upstream of the regulatory domain of Nek2 and the stop codon of
Nek2. BamHI sites were introduced at both ends for subcloning into
the BamHI site of pGEX-3X (Pharmacia).
Glutathione S-Transferase (GST)-Nek2 regulatory domain
fusion protein synthesis and purification
Escherichia coli cells (BL21) transformed with pGEX-3X-Nek2
constructs were grown at 30°C in LB medium to an optical density
(O.D. 600nm) of 0.5. Expression of recombinant proteins was induced
by the addition of 0.5 mM isopropyl-1-thio-β-galactopyranoside for
4 hours at the same temperature. Cells were pelleted and lysed in
phosphate-buffered saline (PBS) containing 0.1% Triton X-100, 1
mM DTT, protease inhibitors, by probe sonication (three cycles of 1
minute each). Bacterial extracts were clarified by centrifugation at
12000 g and supernatant fractions were incubated with glutathioneSepharose beads (Sigma, G 4510) for 1 hour at 4°C with constant
shaking. After several washes in PBS, proteins were eluted with 50
mM Tris-HCl pH 8, containing 10 mM glutathione (Sigma, G 4251)
and 1 mM DTT. Purified proteins were stored at –80°C in the same
buffer containing 10% glycerol.
RNA extraction and Northern blot analysis
Total RNA was extracted from cells and tissues using the Trizol
Reagent (Gibco BRL) and following the manufacturer’s instructions.
Total RNA (20 µg) was extracted and separated on a 1.2%
agarose/formaldehyde gel and blotted onto nylon membrane
(Hybond-N, Amersham, UK) in 10× saline sodium citrate (SSC)
buffer. The membrane was pre-hybridized at 42°C for 4 hours in a
phosphate buffer solution (60 mM, pH 6.8) containing 50%
formamide, 3×SSC, 10 mM EDTA (pH 7.2), 0.2% SDS and
5×Denhardt solution. Hybridization was carried out overnight under
the same conditions with p90Rsk1, p90Rsk2 and p90Rsk3-cDNA
clones radiolabeled with [α32 P]dATP by random examer labeling.
The membrane was washed once with 1×SSC, 0.1% SDS at room
temperature and three times with 0.2×SSC, 0.1% SDS at 42°C before
autoradiography.
Western blot analysis
Solubilized proteins were boiled for 5 minutes in SDS-PAGE sample
buffer [62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% (w/v) SDS, 0.7
M 2-mercaptoethanol, and 0.0025% (w/v) bromophenol blue], and
resolved on 10% or 15% SDS-polyacrylamide gel electrophoresis
after. Resolved proteins were transferred onto polyvinylidene
difluoride membranes (Millipore). Membranes were then saturated
with 5% nonfat dry milk in PBS for 1 hour at room temperature and
incubated with the primary antibody overnight at 4°C [mouse
monoclonal anti-p-Erk (NEB, 1:500 dilution), rabbit anti-Erk1/2
(Santa Cruz, 1:1000 dilution), or goat polyclonal anti-p90Rsk1, antip90Rsk2 or anti-p90Rsk3 (Santa Cruz, 1:1000 dilution)]. Secondary
antibody conjugated to horseradish peroxidase was incubated with the
membranes for 1 hour at room temperature. Immunostained bands
were detected by chemiluminescent method (Santa Cruz Biotech.).
Chromatin staining
Slides for chromatin analysis were prepared by modifying the
procedures described by Meredith (Meredith, 1969). About 5×105
spermatocytes were lysated in 1 ml of 75 mM KCl solution and left
30 minutes at 37°C. About 0.5 ml of a fixative solution (one part acetic
acid and three parts methanol) were added to the solution and
incubated for 20 minutes at 4°C. Cells were then collected by
centrifugation at 4000 g, resuspended in 0.5 ml fixative solution,
incubated for 20 minutes at room temperature, collected by
centrifugation at 4000 g and resuspended again in 50 µl of the fixative
solution. Two or three drops of the cell suspension were squashed on
a clean slide. DNA was stained with 5% Giemsa.
Immunofluorescence analysis
Slices of frozen adult testis were prepared using a microtome and
placed on glass slides. Control or OA-treated spermatocytes were
spotted on poly-L-lysine coated glass slides and fixed at room
temperature for 15 minutes in 4% paraformaldehyde. Cells were
permeabilized for 10 minutes in 0.1% TritonX-100 and blocked for 1
hour in PBS with 5% BSA. After three washes in PBS, cells were
incubated over night at 4°C with mouse monoclonal anti-p-Erk (NEB,
1:100 dilution), or goat polyclonal anti-p90Rsk1, or anti-p90Rsk2, or
anti-p90Rsk3 (Santa Cruz, 1:400 dilution), as primary antibodies.
After five washes in PBS, cells were incubated for 1 hour at 37°C with
rhodamine-conjugate goat anti-mouse IgG (Calbiochem, catalog
number 401217, 1:30 dilution) and rhodamine-conjugate donkey antigoat IgG (Santa Cruz, catalog number sc-2094, 1:400 dilution). To
stain DNA, Hoechst dye (Sigma) was added for the last 10 minutes
at a final concentration of 0.1 mg/ml. Cells were washed extensively
in PBS and slides were mounted in 50% glycerol in PBS.
Immunofluorescence staining of meiotic nuclei spreads
The procedures used for immunofluorescence analysis of meiotic
prophase chromosomes were a slight modification of the technique
reported by Dobson et al. (Dobson et al., 1994). In brief, cells were
lysed in hypotonic salt (140 mM, pH 8.0), nuclei were attached to
glass multiwell slides and fixed in 2% paraformaldehyde for 6
minutes. After three washes, nuclei were blocked for 1 hour in PBS
with 5% BSA. Fluorescence was performed as described above.
1718 S. Di Agostino and others
Fig. 1. Chromosome condensation requires
MAPK activation in mouse spermatocytes. The
specific inhibitor U0126, which blocks activation
of MAPK signaling, inhibits chromosome
condensation during meiotic progression induced
by okadaic acid. (A) Control cells; (B) cells
treated for 6 hours with 0.5 µM OA, where
condensation of chromatin into metaphase is
observed; (C) cells preincubated with U0126 for
12 hours are no different from control cells;
(D) cells preincubated for 12 hours with 10 µM
U0126 before treatment with OA as described
above, and in which chromatin condensation was
strongly inhibited; cell nuclei were isolated and
stained using 5% Giemsa. (E) Immunoblot
analysis using anti-phospho-Erks monoclonal
antibody (top) or anti-Erks polyclonal antibody
(bottom) of protein extracts (30 µg/lane) from
cells treated as described in A-D.
Immunoprecipitation experiments
Control or treated spermatocytes (approximately 2×106 cell/sample)
were collected by centrifugation at 2000 g for 10 minutes, and washed
twice in ice-cold PBS. Cells were homogenized in lysis buffer (25
mM Hepes, pH 7.5, 100 mM NaCl, 20 mM β-glicerophosphate, 15
mM EGTA, 15 mM MgCl2, 0.1 mM sodium orthovanadate, 1 mM
DTT, 10 µg/ml leupeptin and 10 µg/ml aprotinin, 1 mM PMSF) and
cytosolic fractions were collected after centrifugation at 15,000 g for
10 minutes at 4°C. For immunoprecipitation, 1 µg of goat polyclonal
anti-p90Rsk1, anti-p90Rsk2, anti-p90Rsk3, anti-Nek2 antibodies or
rabbit polyclonal anti-Erk1 (Santa Cruz Biotechnology) were
preincubated for 60 minutes with a mixture of protein A- and protein
G-Sepharose beads (Sigma) or only protein A with PBS containing
0.05% BSA, under constant shaking at 4°C. At the end of the
incubation, the beads were washed twice with PBS and 0.05%BSA,
twice with lysis buffer, and then incubated for 90 minutes at 4°C with
the soluble spermatocyte cell-extracts (0.5 mg protein) under constant
shaking. Sepharose beads-bound immunocomplexes were rinsed three
times with PBS and eluted in SDS-sample buffer for western blot
analysis, or washed twice with the appropriate kinase buffer for
immunokinase assays (see below).
Immunokinase assays
Immunocomplexes bound to sepharose beads obtained from
immunoprecipitation of cell extracts were rinsed twice with either
p90Rsks/Erk1-kinase buffer (50 mM Hepes, pH7.5, 5 mM βglicerophosphate, 2 mM EGTA, 15 mM MgCl2, 0.1 mM sodium
orthovanadate, 1 mM DTT, 10 µg/ml leupeptin and 10 µg/ml
aprotinin) or Nek2 kinase buffer (20 mM Hepes, pH7.5, 5 mM βglicerophosphate, 5 mM MnCl2, 5 mM NaF, 0.1 mM sodium
orthovanadate, 1 mM DTT, 10 µg/ml leupeptin and 10 µg/ml
aprotinin). Pellets were then incubated in the same kinase buffer with
the addition of 10 µM 32P-γ-ATP (0.2 µCi/µl), 1 µg cAMP-dependent
protein kinase inhibitor and the appropriate substrate (500 µM MBPderived peptide (Santa Cruz, sc-3011) for Erk1; 100 µM S6 peptide
(Calbiochem), 1 µg of histone H1 (SIGMA, Type III-S, H-5505) or
0.1 mg/ml Histone H3 (Boehringer) for p90Rsks; or 1 µg full-length
MBP for Nek2. Reactions were carried on in a total volume of 50 µl
for 20 minutes at 30°C, and were stopped either by adding SDSsample buffer and boiling or by spotting 20 µl onto phosphocellulose
paper (Whatman P-81) and immediately immersing it into 0.1%
phosphoric acid. Paper squares were washed five times for 10 minutes
each and air-dried. Radioactivity incorporated was determined by
scintillation counting. Values were normalized for protein content,
determined according to Bradford (Bradford, 1976). Samples diluted
in SDS-sample buffer were separated on SDS-PAGE and the dried gel
exposed to autoradiography.
RESULTS
Chromosome condensation requires MAPK
activation
Incubation of mouse pachytene spermatocytes with OA
induces a rapid condensation of chromatin and G2/M
progression. We have previously shown that these two events
can be separated: inhibition of MAPK pathway by the MEK1/2
inhibitor PD98005 suppresses chromatin condensation,
whereas it does not completely suppress activation of MPF
(Sette et al., 1999). We have used the specific inhibitor U0126
(Favata et al., 1998) to block activation of MAPK signaling in
spermatocytes. This inhibitor is more specific than PD98059
and it can be used at one fifth the concentration (Fig. 1E). In
control cells, only 4% of spermatocytes are in metaphase of the
Nek2 activation 1719
Table 1. OA-induced chromatin condensation during
meiotic G2/M progression in mouse spermatocytes
Sample
Control
OA
U0126
OA+U0126
Spermatocytes in
metaphase
(%±s.d.)
4.0±1.4
40±3.2
3.0±2.3
11±5.7
After 6 hours of treatment with 0.5 µM OA approximately 40% of cells
shows condensation of chromatin into metaphase chromosomes. The effect of
OA is strongly inhibited by 12 hours pretreatment with 10 µM U0126 with
only 11% of cells show metaphase chromosomes. Preincubation of
spermatocytes with U0126 alone had no effect on the number of metaphases
in the cell population. Approximately 300 cells were counted from
representative microscope fields of each sample. Slides from three
independent experiments were analyzed.
first meiotic cycle (Fig. 1A and Table 1). After 6 hours
treatment with 0.5 µM OA, condensation of chromatin into
metaphase chromosomes occurs in approximately 40% of cells
(Fig. 1B and Table 1), indicating that they have progressed
through meiosis. However, pretreatment of cells for 12 hours
with 10 µM U0126 strongly inhibits MAPK activation (Fig.
1E) and chromatin condensation (Fig. 1D), with only 11% of
cells showing metaphase chromosomes (Table 1). U0126 alone
had no effect on the number of metaphases in the cell
population of pachytene spermatocytes, suggesting that, in the
absence of OA, these cells are unable to
progress through meiosis under the culture
condition used (Fig. 1 and Table 1). In all
experiments performed, we noticed that
treatment of spermatocytes with U0126 caused
a small but detectable increase in the basal
activity of MAPKs, as measured by western
blot analysis using the anti-phoshorylated Erks
antibody (Fig. 1E) or by immunokinase assay
using a specific MAPK substrate (data not
shown). This small effect on MAPK activity,
however, was not able to trigger chromatin
condensation (Fig. 1C) and it was not
investigated further.
Expression of p90Rsks in mouse
spermatogenesis
In Xenopus oocytes, the MAPK pathway is
required for spindle formation, suppression of
S phase, and to regulate the activity of APC and
the intracellular levels of cyclin B. All these
effects during oocyte maturation are mediated
by the MAPK effector p90Rsk (Gross et al.,
2000). In order to investigate the role of p90Rsk
in male meiosis, we first studied the expression
of the three mammalian p90Rsk isoforms
(Frodin and Gammentiolft, 1999). Specific
cDNA probes for northern blot analysis of
p90Rsk1, p90Rsk2 and p90Rsk3 expression
were obtained by PCR amplification as
described in the Materials and Methods.
Specificity of the probes was assessed by
analysis of mRNA obtained from COS cells
transfected with the three different isoforms (data not shown).
p90Rsk1 transcript is absent in both pre-meiotic and postmeiotic spermatogenic cells (Fig. 2A). The mRNA transcript
for p90Rsk2 was mainly detected in spermatogonia and
in pachytene spermatocytes, whereas p90Rsk3 was mainly
expressed in haploid round spermatids (Fig. 2A). To confirm
the results obtained with mRNA analysis at the protein
level, we performed immunoprecipitation experiments using
antibodies specific to p90Rsk1, p90Rsk2 or p90Rsk3, and
cell extracts from different spermatogenic cells.
Immunoprecipitated proteins were analyzed by western blot
with the same specific antibodies and we used cell extracts
from COS cells transfected with p90Rsk1, p90Rsk2 or
p90Rsk3 as positive controls. We were unable to detect
p90Rsk1 in any of the germ cells analyzed, confirming that this
isoform is not expressed in the testis (Fig. 2B). A specific band
of 82 kDa that corresponds to p90Rsk2 was detected in cell
extracts from spermatogonia and spermatocytes (Fig. 2B). A
faint band was also detected in round spermatids, and it could
either reflect the small amount of mRNA detected in these cells
(Fig. 2A) or derive from contaminating spermatocytes and/or
spermatogonia in the spermatid cell population. Confirming the
mRNA analysis, p90Rsk3 was mainly expressed in round
spermatids (Fig. 2B), and to a smaller extent in spermatogonia.
To validate the observations obtained using isolated germ
cells and to avoid problems caused by small levels of cross
contamination in the cell populations used, we investigated
the expression of p90Rsk isoforms directly in situ by
Fig. 2. Specific expression of p90Rsk
isoforms during mouse spermatogenesis.
(A) Northern blot analysis of total RNA
(20 µg) isolated from total testis or
purified cell populations (see Materials
and Methods) with specific cDNA probes
for p90Rsk1, p90Rsk2 and p90Rsk3.
(B) Western blot analysis of p90Rsk
isoforms as in A confirms the results
obtained with mRNAs analysis at the
protein level. Immunoprecipitation of soluble protein extracts (500 µg) obtained from
same cell populations as in A was performed using antibodies specific to p90Rsk1,
p90Rsk2 or p90Rsk3, and immunoprecipitated proteins were analyzed by western blot
using the same specific antibodies. As positive control, immunoprecipitated proteins
obtained from COS cells transfected with recombinant p90Rsk1, p90Rsk2 or p90Rsk3
were used as control for antibody specificity. Molecular weights were calculated using
the Gibco Benchmark protein ladder as standard.
1720 S. Di Agostino and others
immunofluorescence analysis of testis histological sections. As
shown in Fig. 3, p90Rsk2 was specifically expressed in
spermatogonia and in spermatocytes, which are positioned,
respectively, inside and just above the basal compartment of
the tubule. As suggested by northern and western analyses,
p90Rsk3 expression was restricted to mitotic spermatogonia
and to haploid spermatids, but it was absent in meiotic
spermatocytes. p90Rsk1 was absent in all testicular germ cells
(Fig. 3).
p90Rsk2 is activated during the OAinduced meiotic G2/M transition
The stage-specific expression of p90Rsk2
during spermatogenesis suggests that it
could play a role in meiotic cells. To study
whether p90Rsk2 is activated during
the OA-induced G2/M progression of
mouse spermatocytes, the kinase was
immunoprecipitated from extracts of
either control or OA-treated cells, and the
activity was assayed using S6 peptide and
32P-γ-ATP as substrates. We observed a
10-fold increase of p90Rsk2 activity in
spermatocytes incubated with OA for 4-6
hours (Fig. 4). As a control for specificity
of immunoprecipitation and p90Rsk2
activation, we also used antibodies
directed against the other two p90Rsk
isoforms. The anti-p90Rsk1 did not
precipitate any S6 activity from cell
extracts (Fig. 4), although it could
immunoprecipitate recombinant p90Rsk1
from transfected COS cell extracts
(Fig. 2B). The anti-p90Rsk3 antibody
precipitated a small amount of S6 kinase
activity from both control and OA-treated
spermatocyte extracts (Fig. 4), which
could be due to small amounts of
p90Rsk3 derived from contaminating
round spermatids in the spermatocyte cell
population (see Fig. 1).
p90Rsk kinases can be activated by at
least two pathways in several cell systems:
the Erk1/2 pathway and the PDK1 pathway
(Frodin and Gammentolft, 1999; Gross et
al., 1999). We tested whether the Erk1/2
pathway plays a role in p90Rsk2 activation
during
OA-induced
G2/M
male
meiotic progression. Preincubation of
spermatocytes with the MEK1/2 inhibitor
U0126 (10 µM) completely suppresses
Erks activation by OA (Fig. 1E). Under this
condition, activation of p90Rsk2 by OAtreatment was also completely suppressed,
as measured by both S6 kinase activity
after immunoprecipitation (Fig. 5A) and
electrophoretic mobility in SDS-PAGE,
where phosphorylated and activated
p90Rsk2 shows an upward mobility
shift (Fig. 5B). These results suggest
that p90Rsk2 activation during G2/M
progression of spermatocytes is entirely mediated by the
MAPK pathway.
Immunolocalization of p90Rsk2 in mouse
spermatocytes
We have previously shown that activation of the MAPK
pathway is required for efficient chromosome condensation
during the OA-induced G2/M progression and that Erk1
Fig. 3. Differential expression of p90Rsk2 and p90Rsk3 in mouse spermatocytes and
spermatids. Histological sections obtained from adult testis were stained with antibodies
directed against the three p90Rsk isoforms. Immunostaining was carried out using the antip90Rsk1, anti-p90Rsk2 or anti-p90Rsk3 antibody and a rhodamine-conjugated secondary
antibody as described in the Materials and Methods. Sections were also stained with
Hoechst 3332 to detect chromatin (right panels). In all panels, L stands for lumen of the
tubule and B for basal lamina. In the anti-p90Rsk2 panels, the arrowheads indicate a
representative spermatocyte, whereas in the anti-p90Rsk3 panels, the arrowheads point to a
representative elongated spermatid (central tubule) and a representative round spermatid
(lower tubule). The layer of spermatogonia just above the basal lamina is stained by both the
anti-p90Rsk2 and the anti-p90Rsk3 antibody.
Nek2 activation 1721
Control
2000
S6 kinase activity (CPM/mg protein)
Okadaic acid
1500
1000
500
0
α-p90Rsk1
α-p90Rsk2
α-p90Rsk3
Fig. 4. p90Rsk2 is activated during meiotic G2/M progression
induced by okadaic acid. Soluble extracts (500 µg) obtained from
either control pachytene spermatocytes or spermatocytes treated for 6
hours with 0.5 µM OA were immunoprecipitated using antibodies
specific for the three p90Rsk isoforms. The immunoprecipitated
kinase activity was assayed using 10 µM S6 peptide and 10 µM 32Pγ-ATP as substrates. p90Rsk2 activity was increased ~10-fold by
incubation of spermatocytes with okadaic acid. Data represent the
mean±s.d. of triplicate determinations from three separate
experiments.
colocalizes with the meiotic spindle in close proximity with the
condensed chromosomes (Sette et al., 1999). As p90Rsks are
downstream effectors of Erks, it is possible that p90Rsk2 plays
a direct role in chromatin condensation. As a first step, we
investigated the subcellular localization of p90Rsk2 in mouse
spermatocytes. In control cells, the distribution of p90Rsk2
was mainly nuclear (Fig. 6), and after OA treatment the
staining was more punctuate than in control cells and it seemed
to localize with the condensed chromosomes (Fig. 6). To
investigate whether p90Rsk2 was indeed associated with
meiotic chromosomes, cells were lysed in hypotonic solution
to disrupt cytoplasmic and nuclear membranes, and chromatin
was dispersed directly on a microscope slide for
immunofluorescence analysis. Metaphase chromosomes were
strongly stained by the p90Rsk2 antibody, demonstrating a
tight association of the kinase with condensed chromatin (Fig.
7A). Staining with the anti-p90Rsk2 antibody was specific,
because neither the anti-p90Rsk1, which is absent in these
cells, nor secondary antibody alone stained metaphase
chromosomes (data not shown).
Since p90Rsk2 is activated by Erks (see Fig. 5), we tested
whether phosphorylated and activated Erks were also
associated with meiotic chromosomes. To this end,
immunofluorescence analysis of dispersed chromatin was
carried out using an antibody specific for the phosphorylatedactivated forms of Erk1/2. We found that, similar to p90Rsk2,
active Erks also colocalized with metaphase chromosomes
(Fig. 7B), suggesting that an active Erk-p90Rsk complex forms
on meiotic chromatin and plays a role during condensation into
metaphase chromosomes.
Fig. 5. Activation of p90Rsk2 in mouse spermatocytes is mediated
by the MAPK pathway. (A) Extracts from control or OA-treated cells
preincubated in the absence or presence of 10 µM U0126 to inhibit
signaling through the MAPK pathway were immunoprecipitated
using the anti-p90Rsk2 antibody. Kinase activity present in the
immunoprecipitates was assayed as described in the legend to Fig. 3.
Data represent the mean±s.d. of triplicate determinations from three
separate experiments. (B) Western blot analysis of p90Rsk2 in
representative samples of the immunoprecipitates. The upward shift
in electrophoretic mobility in SDS-PAGE of the sample obtained
from cells treated with OA indicate that p90Rsk2 was
hyperphosphorylated and activated during G2/M progression of
mouse spermatocytes.
Histone H3 phosphorylation is not mediated by the
MAPK pathway in mouse spermatocytes
During the G2/M progression of mitotic and meiotic cells,
histone H3 is phosphorylated at Ser10 (Wei et al., 2000; Cobb
et al., 1999b), and phosphorylation is thought to mediate
interaction of nucleosomes with factors that regulate both gene
transcription and chromatin condensation (reviewed by
Cheung et al., 2000). It has been shown that p90Rsk2 is able
to phosphorylate H3 at Ser10 in vitro, and that p90Rsk2mediated phosphorylation of H3 is necessary for gene
transcription in mitotic cells (Sassone-Corsi et al., 1999).
To study the role of the MAPK pathway in H3
phosphorylation during male meiosis, we used two approaches.
First, p90Rsk2 was immunoprecipitated from control or OAtreated spermatocytes and purified H3 was used as substrate in
an in vitro immunokinase assay. Phosphorylated H3 was then
detected by western blot analysis using an antibody raised
against phosphorylated Ser10 of H3. As shown in Fig. 8A,
p90Rsk2 was able to phosphorylate H3 in vitro, and
phosphorylation was induced by activation of the enzyme after
treatment of cells with OA. Inhibition of the MAPK pathway
by U0126, which prevents activation of p90Rsk2 by OA,
blocks phosphorylation of H3 in this in vitro kinase assay,
1722 S. Di Agostino and others
demonstrating that the activity is due to p90Rsk2 (Fig. 8A).
Second, we studied the impact of activation of MAPKs on H3
phosphorylation in vivo by western blot analysis of extracts of
cells after different treatments. In control spermatocytes, H3 is
not phosphorylated at Ser10, but H3 phosphorylation was
strongly induced after 6 hours treatment with OA (Fig. 8B).
However, preincubation of spermatocytes with the MAPK
inhibitor U0126 did not prevent OA-induced H3
phosphorylation, indicating that Erks and p90Rsk2 are not
required for this event in vivo (Fig. 8B). As activation of
Erks and p90Rsk2 are necessary for efficient chromatin
condensation in these cells, these data suggest that H3
phosphorylation correlates with appearance of metaphase
chromosome but it is not sufficient to trigger this event in male
meiotic cells.
incubation, Nek2 was immunoprecipitated from the cytosolic
extracts and its activity tested using MBP as substrate. We
found that Nek2 was activated after incubation with active
p90Rsk2, when compared with Nek2 incubated with the
inactive form, indicating that p90Rsk2 is able to stimulate the
activity of Nek2 in vitro (Fig. 9B). Similar results were also
obtained using casein as substrate for Nek2 activity (data not
shown). When cytosolic extracts were immunodepleted of
Nek2 before incubation with p90Rsk2, no MBP activity
could be immunoprecipitated with anti-Nek2 antibodies,
demonstrating that the MBP kinase activity was not due to
nonspecific binding of a kinase to the beads (data not shown).
Finally, we tested whether p90Rsk2 was able to
phosphorylate purified Nek2 in vitro. We were unable to
express and purify full length GST-Nek2 from E. coli.
Therefore, we produced two fusion proteins, one containing the
N-terminal kinase domain of Nek2 (amino acids 1-272) and
another containing the C-terminal domain (amino acids 273444) (Fig. 9C), which is supposed to play a regulatory role
(Rhee and Wogelmuth, 1997). p90Rsk2 was immunopurified
using a specific antibody and in vitro kinase assays were
performed using GST-Nek21-272 or GST-Nek2273-444 as
substrates in the presence of 32P-γ-ATP. We found that GSTNek21-272 was phosphorylated by activated p90Rsk2 (Fig. 9D),
whereas GST-Nek2273-444 (Fig. 9D) or GST alone (data not
shown) was not. The highly phosphorylated band at 30-35 kDa
(indicated by an arrowhead in Fig. 9D) is due to
Activation of Nek2 in mouse spermatocytes
The Ser/Thr protein kinase NIMA was first isolated in
Aspergillus nidulans and shown to play a role in chromosome
condensation during mitosis (Fry et al., 1995). The NIMA
murine homolog Nek2 is expressed in pachytene and diplotene
spermatocytes where it is found associated with meiotic
chromosomes (Tanaka et al., 1997; Rhee and Wolgemuth,
1997). During the G2/M progression triggered by OA, Nek2 is
activated with a time course similar to that of MPF and Erk1
(Rhee and Wogelmuth, 1997); however, the roles played
by Nek2 and the pathway leading to its activation in
spermatocytes are unknown.
To test whether the MAPK pathway
is required for Nek2 activation,
spermatocytes were preincubated in the
presence or absence of 10 µM U0126 for
12 hours and then treated for 6 hours with
or without 0.5 µM OA. Nek2 activity was
assayed using either MBP or casein as
substrate. Activation of Nek2 by OA
correlated with chromosome condensation
and G2/M progression as previously
reported (Rhee and Wogelmuth, 1997)
(Fig. 9A). Interestingly, inhibition of the
MAPK pathway by preincubation with
U0126 completely blocked OA-induced
activation of Nek2, whereas the basal
activity of Nek2 was not affected in control
cells (Fig. 9A). This result suggests that
activation of ERKs and p90Rsk2 is
required for activation of Nek2
during G2/M progression of mouse
spermatocytes.
To investigate whether p90Rsk2 was
able to induce activation of Nek2, we used
an in vitro approach. As a source of active
and inactive p90Rsk2, COS cells were
transfected with a p90Rsk2 cDNA and
incubated respectively with or without
OA for 3 hours to induce activation of
p90Rsk2 (~10-fold, data not shown). Fig. 6. Subcellular localization of p90Rsk2 in mouse spermatocytes. The panels on the left
Immunopurified active or inactive show the immunofluorescence analysis of control (top) or OA-treated (6 hours with 0.5 µM,
p90Rsk2 was then incubated for 30 bottom) cells. Immunostaining was carried out using the anti-p90Rsk2 antibody and a
minutes at 30°C with cytosolic extracts rhodamine-conjugated secondary antibody as described in the Materials and Methods. Cells
from control spermatocytes. After the were also stained with Hoechst 3332 to detect chromatin (right panels).
Nek2 activation 1723
phosphorylation of proteolytic fragments of GST-Nek21-272
that were routinely purified from E. coli together with the fulllength protein (arrowheads in Fig. 9C). As these fragments are
much better substrates for p90Rsk2 in vitro than is the entire
kinase domain (Fig. 9D), it is possible that full-length Nek2
may need a particular conformation to be phosphorylated by
p90Rsk2.
The data presented demonstrate that activation of
the MAPK pathway during the G2/M progression of
mouse spermatocytes leads to phosphorylation and
activation of Nek2 by p90Rsk2 (Fig. 10).
only for the maintenance of chromatin condensed between the
two meiotic divisions and for the prevention of DNA synthesis
during interphase. The role of MAPK at this earlier meiotic
stage could represent another level of the dimorphism observed
between male and female meiosis in higher eukaryotes (Handel
and Eppig, 1998).
In agreement with the role of Erk1 activation during
DISCUSSION
At the end of the pachytene stage of meiotic
prophase, the synaptonemal complex dissolves and
chromatin condenses into metaphase chromosomes,
which align on the equatorial plate of the spindle.
We have previously demonstrated that activation of
the MAPK Erk1 is required for efficient chromatin
condensation during the G2/M progression triggered
by OA in mouse pachytene spermatocytes (Sette et
al., 1999). We have extended these studies and
identified p90Rsk2 as a downstream effector of the
MAPK pathway in these cells. Furthermore, we
show that Nek2, a serine-threonine kinase involved
in chromatin condensation and centrosome
duplication, is activated by the MAPK pathway and
is a substrate of p90Rsk2 in vitro.
Three p90Rsk isoforms, p90Rsk1, p90Rsk2 and
p90Rsk3, are expressed in mouse tissues (Frodin and
Gammeltoft, 1999). Our northern blot, western blot
and immunofluorescence analyses demonstrate that
these isoforms are specifically expressed during
spermatogenesis. p90Rsk2 is expressed during the
mitotic and meiotic stages of spermatogenesis, from
spermatogonia to pachytene spermatocytes, and its
expression decreases or ceases in post-meiotic
spermatids. p90Rsk3 is present at low levels in
spermatogonia, is absent (or expressed at very low
levels) in meiotic spermatocytes and is abundant in
post-meiotic cells, suggesting that it could play a
role during differentiation of round spermatids into
mature sperm. p90Rsk1 is not expressed in germ
cells at any stage examined.
In mitotic cells and in maturing Xenopus oocytes,
p90Rsk2 was found to be the main effector of Erks.
Indeed, activation of p90Rsk2 is required for all the
known effects exerted by the MAPK pathway in
meiotic oocytes, such as formation of a meiotic
spindle during the first division, suppression of
DNA replication between the two metaphases, and
inhibition of the cycle at metaphase of the second
division after progesterone-induced maturation
(Bhatt and Ferrell, 1999; Gross et al., 1999; Gross
et al., 2000). We found that p90Rsk2 was also
activated by Erks during mouse male meiosis.
However, our data suggest that in spermatocytes,
activation of the MAPK pathway is also important
for triggering chromosome condensation, and not
Fig. 7. Activated Erk1/2 and p90Rsk2 associate with meiotic chromosomes.
Chromatin and/or chromosomes were obtained by hypotonic treatment of
spermatocytes and were attached directly on a microscope slide for
immunofluorescence analysis as described in the legend to Fig. 5. (A) Chromatin
was stained with anti-p90Rsk2 antibody (right panels) or Hoechst dye (left
panels). (B) Chromatin was stained with anti-phosphoErk1/2 antibody (right
panels) or Hoechst (left panels). Both p90Rsk2 and activated Erks colocalize with
meiotic chromosomes.
1724 S. Di Agostino and others
Fig. 8. Histone H3 phosphorylation is not mediated by the MAPK
pathway in mouse spermatocytes. (A) Immunokinase assay of
p90Rsk2 activity using H3 as substrate. p90Rsk2 was
immunoprecipitated as described in the legend to Fig. 4 from cells
treated as indicated in the figure and in the text, and activity was
assayed using 0.1 mg/ml H3 and 10 mM ATP as substrates. At the
end of the incubation, proteins were separated on a 10% SDS-PAGE
and analyzed by western blot using either anti-phospho-H3 to detect
phosphorylation of Ser10 in H3, or anti-p90Rsk2 to verify that equal
amounts of enzyme were used in the assay. (B) Western blot analysis
of extracts (20 µg) of cells treated as indicated in the figure and in
the text using either the anti-phosphoH3 antibody to detect
phosphorylation of Ser10 in H3 in intact cells, or the anti-H3
antibody to quantify the amount of H3 in the samples.
chromatin condensation (Sette et al., 1999), we show that
activated Erks localize in close association with condensed
chromosomes at metaphase in mouse spermatocyte.
Furthermore, their effector p90Rsk2 is always nuclear, and it
also associates with condensed chromosomes at metaphase.
Therefore, both Erks and p90Rsk2 are able to directly or
indirectly interact with chromatin and regulate its state of
condensation, probably by phosphorylating proteins that
modulate nucleosomal assembly (Fig. 10). Whereas interaction
of activated Erks with chromosomes had been previously
reported in mitotic cells (Zecevic et al., 1998), where they
associate with the kinetochores and the motor protein CENPE, a similar localization for p90Rsk2 has never been observed
and it could suggest the direction for searching new p90Rsk
substrates. Recently, it has been shown that in Xenopus eggs
p90Rsk phosphorylates and activates Bub1, an upstream
Fig. 9. Activation of the Ser/Thr protein
kinase Nek2 in mouse spermatocytes is
MAPK dependent. (A) Immunokinase
assay of Nek2 activity. Nek2 was
immunoprecipitated from control or OAtreated cells preincubated or not with
U0126 (10 µM for 12 hours) and incubated
for 20 minutes at 30°C in the presence of 1
µg full-length MBP and 10 µM 32P-γ-ATP
as substrates. Reactions were terminated by
adding SDS-sample buffer, samples were
boiled for 5 minutes and protein were
separated on a 13% SDS-PAGE. The dried
gel was then autoradiographed. (B)
Reconstitution of Nek2 activation in vitro.
Active and inactive p90Rsk2 were
immunopurified as described in the
Materials and Methods, and incubated with
cytosolic extracts from control
spermatocytes for 30 minutes at 30°C in the
presence of 10 µM ATP. At the end of the
incubation, cytosolic extracts were
separated by centrifugation and Nek2 was
immunopurified using 1 mg anti-Nek2
antibody and proteinA-sepharose beads.
The activity of Nek2 was assayed as
described in A and the dried gel was
autoradiographed. (C) Coomassie Blue
staining of GST-Nek21-272 and GSTNek2273-444 purified from E. coli that were
used in D as substrates for p90Rsk2.
Arrowheads on the left side point to
degradation products routinely observed in purified GST-Nek21-272. (D) In vitro assay for phosphorylation of GST-Nek21-272 and GSTNek2273-444 by p90Rsk2. Active or inactive immunopurified p90Rsk2 was incubated with GST-Nek2 proteins for 30 minutes at 30°C in the
presence of 10 µM ATP. The reaction was terminated adding SDS-PAGE sample buffer and proteins were separated on a 10% SDS-PAGE. The
gel was dried and autoradiographed. Arrowhead on the left shows the position of GST-Nek21-272 degradation products.
Nek2 activation 1725
Erk1
extracellular signal?
p90Rsk2
Nek2
Erk1
p90Rsk2
Erk1-p
Nek2
p90Rsk2-p
Nek2-p
chromosome condensation
Fig. 10. Hypothetical model of the activation of Nek2 and induction
of chromatin condensation by Erk1 and p90Rsk2. Erk1 may be
activated by local extracellular signals when spermatocytes are
mature to enter the meiotic divisions. Activation of Erk1 allows its
translocation into the nucleus where it phosphorylates and activates
p90Rsk2. Activated Erk1 and p90Rsk2 associates with chromatin,
and p90Rsk2 phosphorylates and activates the chromatin-bound
Nek2. Activation of Nek2 triggers chromatin condensation into
metaphase chromosomes.
component of the kinetochore attachment checkpoint,
suggesting that APC inhibition and the cytostatic activity exerted
by the MAPK pathway might be mediated by this checkpoint
protein (Schwab et al., 2001). Our data suggest that in
mammalian cells, in addition to regulation of the exit from
metaphase after the attachment of the kinetochores to the
spindle, the MAPK pathway also regulates earlier steps involved
in assembly of chromatin into metaphase chromosomes.
A candidate for regulation of chromatin assembly is histone
3 (H3). It has been reported that phosphorylation of H3 at
Ser10 temporally correlates with entry into metaphase and
chromatin condensation during both mitosis and meiosis (Hsu
et al., 2000; Chadee et al., 1999; Cobb et al., 1999b). Several
protein kinases, including MAPKs, p90Rsks, Aurora A and B
kinases have been shown to phosphorylate H3 in vitro and/or
in vivo (Sassone-Corsi et al., 1999; Murnion et al., 2001;
Scrittori et al., 2001). However, the role played by p90Rsk in
H3 phosphorylation is not completely understood. SassoneCorsi et al. (Sassone-Corsi et al., 1999) have reported that
p90Rsk2 phosphorylates H3 in mitotic cells and that this
phosphorylation is required for induction of early genes in G1.
However, p90Rsk2 does not seem to be required for
phosphorylation of H3 later in the cell cycle, as inhibition of
the MAPK pathway does not block this event during the G2/M
progression in Xenopus egg cycling extracts (Murnion et al.,
2001). Our data extend these results to mammalian meiotic
cells, because we have demonstrated that inhibition of MAPKs
and p90Rsk2 activation does not affect phosphorylation of H3
during meiotic G2/M transition. Because inhibition of the
MAPK pathway strongly affects chromatin condensation, our
data also indicate that H3 phosphorylation is not sufficient to
trigger chromosome assembly. Indeed, a similar observation
has also been reported by Murnion et al. (Murnion et al., 2001)
in mitotic cycling extracts, where stimulation of H3
phosphorylation in interphase cytosols was not sufficient to
drive chromosome condensation or targeting of condensins to
chromatin. Therefore, proteins other than H3 must be targets
of p90Rsk2 in vivo, and play a role in proper condensation of
chromatin during both mitosis and meiosis.
In this study, we have identified Nek2 as one of the
possible effectors of p90Rsk2 during meiotic chromosome
condensation. Nek2 is a member of the NIMA kinases, for
which a role in chromosome condensation and centrosome
duplication has been suggested in organisms as diverse as yeast
and vertebrates (Fry and Nigg, 1995; Fry et al., 1998a; Fry et
al., 1998b; Uto and Sagata, 2000). In Aspergillus nidulans,
mutations in NIMA arrest cells in late G2 (Morris, 1976), even
when Cdc2 is activated, indicating that NIMA is part of a
Cdc2-independent mitotic pathway (Osmani et al., 1991).
However, although not required for its basal activity, CyclinBCdc2 phosphorylates NIMA and stimulates its kinase activity,
suggesting that a positive crosstalk between the two pathways
exists and could play a role in coordinating mitotic events (Ye
et al., 1995). In metazoan cells, the expression and activity of
the NIMA homologues (Nek1, Nek2 and Nek3) are cell cycle
regulated during mitosis (Fry and Nigg, 1995; Fry and Nigg,
1997). Nek2 expression levels and kinase activity are high in
late S-phase and G2, decrease at metaphase, and are absent in
G1 (Fry et al., 1995). Furthermore, inappropriate activation of
Nek2 triggers premature condensation of chromatin in a Cdc2independent fashion (Lu and Hunter, 1995; O’Connell et al.,
1994), suggesting that its physiological role is to control the
timing of chromatin condensation at the end of S-phase. It was
previously observed that Nek2 is specifically expressed in
mouse pachytene spermatocytes, and that its activity increases
during the G2/M progression triggered by OA in these cells
(Tanaka et al., 1997; Rhee and Wogelmuth, 1997). We
demonstrate that Nek2 activity is modulated by the MAPK
pathway in vivo, and that it can be stimulated by p90Rsk2 in
an in vitro reconstitution experiment. Furthermore, we show
that a recombinant GST-Nek2 protein, which contains the Nterminal half of the kinase (including the kinase domain), is a
substrate for p90Rsk2 in vitro. These results indicate a
connection between the MAPK pathway and the pathway that
leads to chromosome condensation at the end of the first
meiotic prophase (Fig. 10).
Little is known about upstream regulators of Nek2 or
pathways that lead to its activation during meiosis or mitosis.
Therefore, activation of Nek2 by Erks-p90Rsk2 represents the
first connection between a NIMA kinase and pathways
regulated by extracellular signals. Although we have used OA
to trigger MAPK activation and meiotic progression, several
observations suggest that the events described here have a
physiological relevance. First, chromosomes condense as
normal meiotic bivalents (Wiltshire et al., 1995), showing that
this progression occurs following the physiological route, and
inhibition of the MAPK pathway by U0126 interferes with this
event. Therefore, we hypothesize that MAPK activation is not
a mere epiphenomenon caused by treatment of cells with a
serine-threonine phosphatase inhibitor, but it may also play a
role during the natural meiotic progression. Second, MAPK is
necessary for the activation of Nek2, a protein known
physiologically to induce chromatin condensation and
centrosome duplication. Third, we were able to isolate
1726 S. Di Agostino and others
spermatocytes at late pachytene or diplotene stage and have
observed an increase in MAPK activity in these cells (S. D.,
P. R., R. G. and C. S., unpublished), reinforcing the idea that
this pathway plays a role in male meiosis.
It is not currently known what stimuli physiologically lead to
MAPK activation during the prophase of male meiosis;
however, it is conceivable to hypothesize the involvement of
paracrine factors temporally expressed in the seminiferous
tubule. Indeed, although meiotic events such as crossing over
and DNA repair after recombination have already occurred at
mid-pachytene (Wiltshire et al., 1995; Cobb et al., 1999a),
spermatocytes wait for several days at this cell-cycle stage
before entering metaphase. This time is used to accumulate
RNAs and proteins that will allow two subsequent cell divisions
without intervening growth. Further meiotic progression might
depend on appropriate signals from the neighboring germ cells
or the nursing Sertoli cell when the spermatocyte has reached
its mature size. The observation that activation of extracellularregulated enzymes such as Erk1 and p90Rsk2 is required for
activation of Nek2 and proper chromosome condensation in
pachytene spermatocytes supports such hypothesis. Future
studies will be aimed at identifying physiological factors
involved in regulation of the MAPK pathway and meiotic
progression in mouse spermatocytes.
We thank Drs M. Frödin and Christian Bjørbaek for generous gift
of p90Rsk2 and 3 cDNAs, Dr J. Avruch for p90Rsk1 cDNA, Dr S.
Dolci for help with preparation of histological sections, and Drs S.
Dolci and P. Grimaldi for useful comments on the manuscript. This
work was supported by a grant from the Agenzia Spaziale Italiana
(ASI) and by the projects Molecular Mechanisms of Germ Cell
Development and Differentiation and Molecular Triggering of the
Embryo Developmental Program in the Mouse by MURST 2000.
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