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: settec{at}seneca.uniroma2.it)
Accepted 1 January 2002
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SUMMARY |
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Key words: Meiosis, Spermatocytes, MAPK, Nek2, Chromosome, Mouse
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INTRODUCTION |
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Most of the knowledge on meiotic cell cycle events derives 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 Polo-like-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 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 anaphase-promoting 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 centrosome-associated 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.
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MATERIALS AND METHODS |
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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-ß-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 glutathione-Sepharose 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 manufacturers 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 10x 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, 3xSSC, 10 mM EDTA (pH 7.2), 0.2% SDS and 5xDenhardt solution. Hybridization was carried out overnight under the same conditions with p90Rsk1, p90Rsk2 and p90Rsk3-cDNA clones radiolabeled with [32 P]dATP by random examer labeling.
The membrane was washed once with 1xSSC, 0.1% SDS at room temperature and three times with 0.2xSSC, 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, anti-p90Rsk2 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 5x105 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 anti-goat 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.
Immunoprecipitation experiments
Control or treated spermatocytes (approximately 2x106 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 ß-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 ß-glicerophosphate, 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 ß-glicerophosphate, 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--ATP (0.2 µCi/µl), 1 µg cAMP-dependent protein kinase inhibitor and the appropriate substrate (500 µM MBP-derived 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 SDS-sample 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.
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RESULTS |
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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 p90Rsk2-mediated 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 OA-treated 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, 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.
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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.
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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 273-444) (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-
-ATP. We found that GST-Nek21-272 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 phosphorylation of proteolytic fragments of GST-Nek21-272 that were routinely purified from E. coli together with the full-length 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).
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DISCUSSION |
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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 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 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 CENP-E, 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 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. Sassone-Corsi 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, CyclinB-Cdc2 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 Cdc2-independent fashion (Lu and Hunter, 1995
; OConnell 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 N-terminal 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 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 extracellular-regulated 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.
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ACKNOWLEDGMENTS |
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