Cloning of Ribosomal Protein S6 Kinase cDNA and ...

Mol. Cells, Vol. 14, No. 1, pp. 16-23

M olecules and Cells KSMCB 2002

Cloning of Ribosomal Protein S6 Kinase cDNA and Its Involvement in Meiotic Maturation in Rana dybowskii Oocytes Hyang Min Byun, Sung Goo Kang1, and Hae Mook Kang* Department of Genetic Engineering, Chongju University, Chongju 360-764, Korea; 1 School of Biology, Inje University, Kimhae 621-749, Korea. (Received January 23, 2002; Accepted April 18, 2002)

Several protein kinases are involved in the meiotic maturation of frog oocytes in order to activate the maturation-promoting factor (MPF). Among these kinases, the 90 kDa ribosomal protein S6 kinase (p90Rsk or Rsk) is directly phosphorylated and activated by the mitogen-activated protein kinase (MAPK). During Xenopus oocyte maturation, the activation of Rsk closely parallels that of MAPK. Both enzymes are dephosphorylated when the cytostatic factor (CSF) disappears after fertilization. Therefore, Rsk seems to play an essential role in the activation of MPF. To evaluate it in other frog oocytes, we cloned and characterized Rsk cDNA in Rana dybowskii oocytes. The cloned Rana Rsk cDNA had 2,961 bp of nucleotides, which contained a complete single open-reading frame with ATG codon and polyadenylation signal. The deduced amino acid sequence of Rana Rsk is 733 amino acids with 83 kDa. Rana Rsk shows a high homology (about 88%) with Xenopus Rsk. It also had two wellconserved kinase domains with specific phosphorylation sites, which are known to be essential for the activation of Rsk. A Northern analysis showed that Rana Rsk mRNA was strongly expressed in ovary tissue, but weakly in other tissue. Rana Rsk protein is expressed with the pTYB1 vector and purified with the IMPACT-CN system. The purified Rana Rsk crossreacted with Xenopus, a p90Rsk2 antiserum. Therefore, we examined the phosphorylation of Rana Rsk during Rana oocyte maturation. In P4-treated oocytes, Rana Rsk was phosphorylated about 6−9 h, which correlated well with the germinal vesicle breakdown of Rana oocytes. Therefore, it is likely that Rana Rsk plays an important role in the meiotic maturation of seasonal breeding animals.

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* To whom correspondence should be addressed. Tel: 82-43-229-8565; Fax: 82-43-229-8432 E-mail: [email protected]

Keywords: cDNA; Frog; Maturation-promoting Factor (MPF); Oocyte Maturation; Rana dybowskii; Ribosomal Protein S6 Kinase (Rsk).

Introduction Full-grown oocytes of amphibian are arrested at the diplotene stage of the first meiotic prophase, the same as most vertebrates’ oocytes. Stimulation with progesterone, insulin, or the insulin-like growth factor-1 releases this arrest by activating a number of protein kinases, including the maturation-promoting factor (MPF). It initiates the germinal vesicle breakdown (GVBD) and meiotic events that lead to the production of an unfertilized egg, which is also arrested at the metaphase of meiosis II. Upon fertilization, an increase of free-calcium inactivates the cytostatic factor (CSF), which is responsible for the maintenance of mature oocytes in their normal metaphase arrest state (Masui and Markert, 1971). Over the past decade, studies have identified the MAPK pathway that is required for meiotic maturation. The most important event in this process may be the activation of MPF for the resumption of the meiotic maturation, then the appearance of CSF activity for the metaphase arrest. MPF ultimately proved to be a complex of the universal M-phase regulator that is composed of the Cdc2 kinase and cyclin B. (reviewed by Yamashita, 1998). CSF has not been characterized, but the MAPK pathway is required for the generation of CSF activity (Sagata, 1997). In Xenopus oocytes and other systems, the 90-kDa ribosomal protein S6 kinase (Rsk) is directly phosphorylated and activated by MAPK (Grove et al., 1993; Sturgill et al., Abbreviations: CSF, cytostatic factor; GVBD, germinal vesicle breakdown; MAPK, mitogen-activated protein kinase; MPF, maturation promoting-factor; PCR, polymerase chain reaction; Rsk, ribosomal protein S6 kinase.

Hyang Min Byun et al.

1988). During meiotic maturation, Rsk activation is closely related to that of MAPK, and Rsk activity disappeared after fertilization (Hartley et al., 1996; Roy et al., 1996). Cloning of the Rsk cDNA in vertebrate revealed three types of Rsk isoforms, all of them have two distinct domains of kinase activity (Alcorta et al., 1989; Jones et al., 1988; Moller et al., 1994). Rsk activation requires phosphorylation at two specific sites by MAPK, as well as autophosphorylation at a specific site by the C-terminal kinase domain (Bjørbœk et al., 1995; Dalby et al., 1998). Recently, in Xenopus oocytes, Rsk appeared to be a mediator of CSF activity. Immunodepletion of CSF activity in MII-arrested oocyte extracts lost their capacity to undergo mitotic arrest in response to the activation of the MAPK cascade (Bhatt and Ferrell, 1999). Constitutively, the Rsk expression in early Xenopus blastomere by a microinjection of Rsk mRNA resulted in cleavage arrest in the M-phase with a prominent spindle that is characteristic of the meiotic metaphase (Gross et al., 1999). Evidence demonstrates that oocyte maturation is associated with the translation of specific maternal mRNAs, which includes proto-oncogene c-mos and mitotic cyclins (Maller, 1990; Nebreda et al., 1995; Sagata et al., 1988). It is also characterized by major changes in intracellular protein phosphorylation (Erickson and Maller, 1989; Maller, 1990). Phosphorylation of the ribosomal protein S6 is an important regulatory step for translation mRNA in eukaryotes, including amphibian oocytes (Dufner and Thomas, 1999). In frog oocyte, the progesterone-induced Rsk activity is closely associated with the induction of protein synthesis and activation of MPF during oocyte maturation. In contrast to Xenopus oocyte in the Rana dybowskii oocyte, the inhibition of Rsk activity by the treatment of rapamycin showed the failure of GVBD and protein synthesis (Bandyopadhyay et al., 1999). This indicates the existence of different signaling systems between Xenopus and Rana oocytes in relation to the initiation of meiotic maturation. In spite of extensive studies on Rsk in Xenopus oocyte, there is little available information on Rsk in meiotic resumption of oocytes from seasonally breeding amphibian, such as R. dybowskii, which has been used as a model for oocyte maturation over the last decade. Therefore, we cloned and characterized Rsk cDNA and examined its phosphorylation during P4-induced meiotic maturation in R. dybowskii oocytes.

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surgically removed and immediately placed in an amphibian Ringer’s medium (AR) (Kwon and Schuetz, 1985). Full-grown oocytes were manually defolliculated as described previously (Kwon and Lee, 1991). Denuded oocytes were incubated with progesterone (3 µM) for 24 h in a shaking incubator at 22−24°C. At the indicated time of culture, the oocytes were fixed with 5% trichloroacetic acid (TCA) and examined for GVBD. Preparation of Rana Rsk probe Degenerate primers for the isolation of a Rana Rsk cDNA fragment were designed, based on the conserved nucleotide sequences from the known vertebrate Rsk genes. The forward and backward primers are 5′GCMCAYCAGCTNTTYCGNGG -3′ and 5′-GCTNGGYTTSARRTCYCKRTG-3′, respectively. These were commercially synthesized (Bioneer, Korea). Total RNA from ovary tissues was isolated using Tri-reagent (Sigma). First-strand DNA was synthesized with AMV reverse transcriptase (TaKaRa). A polymerase chain reaction (PCR) was performed with Taq polymerase (TaKaRa) for 35 cycles (94°C, 30 s; 45°C, 30 s; and 72°C, 30 s) with degenerate primers in a thermal cycler (PerkinElmer). It produced a single band of 511 bp, which was inserted into a pGEM-T Easy vector (Promega). The nucleotide sequences of a 511 bp PCR product showed a partial clone of putative Rsk cDNA. This fragment was further used for screening a full-length Rsk cDNA from the R. dybowskii ovary cDNA library, following the plaque hybridization procedure (Bandyopadhyay et al., 2000).

Materials and Methods

Screening of cDNA library The [32P]-labeled probe was prepared with [α-32P]-dCTP (NEN) by the random-primed DNA labeling kit (Roche Molecular Biochemical). Briefly, about 3 × 105 plaques were plated and transferred to nylon membranes (Amersham Pharmacia Biotech). The membranes were prehybridized at 42°C for 2 h in a hybridization mixture that contained 0.25 M Na2HPO4 (pH 7.2), 0.25 M NaCl, 1 mM EDTA, 7% SDS, 50% formamide, 5% dextran sulfate, and 100 µg/ml denatured salmon sperm DNA at 42°C. Then, [32P]-labeled 283 bp of PCR fragment was added to the hybridization mixture (1 × 106 cpm/ml) and incubated at 42°C overnight. The membranes were washed twice with 2× SSC, 0.1% SDS at RT for 5 m, followed by a final washing with 0.2× SSC, 0.1% SDS at 65°C for 1 h, then exposed to X-ray film (Kodak). Finally, four positive clones were obtained through secondary and tertiary screenings. The cDNA inserts were subcloned into a pBluescript SK vector by the in vivo excision method using the ExAssist/SOLR system (Stratagene). Sequences of the cloned fragments were determined with the gene-specific synthetic oligonucleotides, as well as T7 or T3 primers, in an Applied Biosystems DNA sequencer (Prism 877 model).

Animal collection and oocyte culture Hibernating female frogs (Rana dybowskii) were purchased from a local commercial store. They were collected during the winter season (December to January) in the Kwangwon province of Korea. The frogs were kept at 4°C with moisture. Ovaries of the female frogs were

Analysis of nucleotide and amino acid sequence All of the nucleotide sequence analyses (including the open-reading frame, PCR primers, and Blast searching) were performed with a MacVector 7.0 sequence analysis package (Oxford Molecular Ltd.). Nucleotide and amino acid sequences were compared

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Rana Ribosomal Protein S6 Kinase

after retrieving the sequence data from Entrez using ClustalW alignment program of MacVector. Northern blot hybridization From adult frogs of R. dybowskii, the total RNA of various tissues were isolated and fractionated on a 1% formaldehyde-agarose gel as described preciously (Bandyopadhyay et al., 2000). RNA was transferred onto a nylon membrane that was followed by UV cross-linking. The [32P]-labeled PCR product, 511 bp fragment, of the Rana Rsk cDNA was used for a probe. The membrane was probed with a formamide-containing hybridization buffer at 42°C for 24 h as described previously. The membrane was washed, then exposed to X-ray film for 72 h. In vitro translation of Rana Rsk Rana Rsk was expressed and purified with the IMPACT-CN system (New England Biolabs). All of the procedures were carried out according to the manufacture’s protocol. Briefly, the cloned Rana Rsk cDNA was amplified with EV1 primer (5′-GGTGGTCATATGCCGCTCGCACAATTGGC-3′) and the EV2 primer (5′-GGTGGTTGCT-

CTTCCGCAGAGAGTGGTGGATG-3′) with ExTaq polymerase (TaKaRa) for 25 cycles at 94°C, 30 s; 58°C, 30 s; and 72°C, 120 s. The amplified Rana Rsk coding region was inserted into the SapI and NdeI sites of the pTYB1 vector. The constructed vector was transformed into the ER2566 competent E. coli strain. The cells that contained the Rsk expression plasmid were cultured into 200 ml of a LB media at 37°C until OD600 = 0.5. After adding 0.3 mM IPTG, the cells were further cultured overnight at 20°C. The harvested cells were homogenated by sonication in 6 ml of a lysis buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 20 µM PMSF). The Rana Rsk protein was purified using a column with chitin bead. Immunoblotting The in vitro expressed Rana Rsk protein was verified by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech) in a transfer buffer (25 mM Tris, 193 mM glycine and 20% methanol) at 50 V for 1 h. In a separate experiment, P4-treated oocytes were collected at the designated time. Ten oocytes were homogenated in 400 µl of

Fig. 1. Nucleotide and deduced amino acid sequences of the cloned Rana Rsk cDNA. A complete nucleotide sequence of cDNA clones was determined. It generates a single open-reading frame of 733 amino acids from nucleotide number of 100 to 2298. The conserved polyadenylation signal sequence, ATTAAA, is underlined. The sequence was deposited in GenBank accession No. AF363966.


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Fig. 2. Comparison of the deduced amino acid sequence of Rana Rsk with those of several vertebrata. The aligned sequences and their GenBank accession numbers are as follows: Xenopus (P10665), chickens (P18652), mice (NP-035429), and humans (NP-004577). The alignment was performed with the Clustal W program. Asterisks indicate identical amino acid residues. Amino acid residues that have chemically similar characteristics (negatively-charged, positively-charged, or aliphatic amino acids) are shown by dots. The gray boxes indicate the most conserved residues in the catalytic domains of protein kinases (Dalby et al., 1998). The degree of identity is expressed as a percentage at the end.

an oocyte extraction buffer (100 mM β-glycerophosphate, 20 mM HEPES, pH 7.5, 15 mM MgCl2, 5 mM EGTA, 1 mM DTT, 100 µM PMSF, 3 µg/ml leupeptin) and centrifuged at 10,000 × g for 10 m. Thirty microliter of extracts were separated in 12% SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was incubated with mouse anti-Xenopus p90Rsk2 monoclonal IgG (Santa Cruz) in a blocking buffer (10% non-fat dried milk in a Tris-buffered solution that contained 20 mM Tris, pH 7.6, 137 mM NaCl, and 0.1% Tween 20), and subsequently with HRP-labeled goat anti-mouse IgG (Santa Cruz). Immunoreactive bands were visualized by an ECL Western blotting detection kit (Amersham Pharmacia Biotech) and exposed to Xray film for 15 s.

Results Four independent clones of the Rana Rsk cDNA were

isolated through the screening of colony-lift hybridization with a PCR-amplified Rsk cDNA fragment as a probe. The nucleotide and deduced amino acid sequences of the largest insert (approximately 3.0 kb) are shown in Fig. 1. This Rana Rsk cDNA consists of 2961 nucleotides. It has a single open-reading frame, encoding Rsk of 733 amino acids, with the Met (ATG) codon located at position 100 nucleotide and the terminating stop (TAG) codon located at position 2299 nucleotide. The surrounding nucleotide sequence of the first Met codon has a good consensus sequence for translation initiation (Kozak, 1986). The cloned Rana cDNA has a short 5′ untranslated region and a relatively long (about 650 bp) 3′-untranslated region. The polyadenylation signal, ATTAAA sequence, was located at 620 nucleotides downstream from the stop codon that is underlined in Fig. 1, and the poly(A+) tail followed. Three other clones also had a part in the same nucleotide sequence.

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A. SDS-PAGE

B. Western blotting

Fig. 3. Northern blot analysis of the Rana Rsk gene expression in frog tissue. The total RNAs were isolated from the ovary, testis, kidney, liver, and muscle tissues. Thirty micrograms of total RNA from each tissue were separated on a 1% agaroseformaldehyde gel and transferred to the membrane. The blot was probed with [32P]-labeled Rana Rsk cDNA. The arrows indicate the positions of 28S and 18S RNA. A transcript of 4.0 kb was strongly expressed in ovary tissues, but weakly in other somatic tissues.

Fig. 4. In vitro expression and purification of Rana Rsk. The cloned Rana Rsk is inserted into the pTYB1 vector with a correct open-reading frame. The Rsk protein was expressed as described in Materials and Methods. The expressed Rsk protein was purified with the IMPACT-CN system, and immunoblotted with Xenopus p90Rsk2 antiserum. A. Purification of Rana Rsk protein expressed in vitro by IMPACT-CN system. B. Detection of Rana Rsk protein cross-reacted with Xenopus p90Rsk2 antiserum. Arrows indicate the Rsk position in SDSPAGE. Molecular weight (MW) is marked as kDa.

Figure 2 shows the amino acid sequence alignment of Rana Rsk with Xenopus, chickens, humans, and mice. The deduced amino acid sequence of Rana Rsk was 88% identical to that of Xenopus and 76% to humans. It also showed a relatively high homology (70−80%) with other known vertebrata Rsks (data not shown). The cloned Rana Rsk especially seems to be type 2, because it has a higher homology to type 2 Rsk of Xenopus than the other types. Rana Rsk also shows the conserved phosphorylation sites of Ser363 and Tyr574 by MAPK and autophosphorylation sites of Ser221 and Ser380 by two kinase domains. These sites are observed in Xenopus Rsk2 (Dalby et al., 1998) and marked in a box in Fig. 2. The high homology of Rsk amino acids in both species is specially exhibited in the two kinase domains. The Rsk mRNA expression levels were analyzed by a Northern blot in several frog tissues (Fig. 3). Total RNAs (30 µg per tissue) were separated, transferred onto a membrane, then hybridized with the [32P]-labeled PCRamplified 511 fragment of Rana Rsk cDNA. Using stringent washing conditions, a 4.0 kb of the mRNA transcript was expressed in most of the tissues that were examined. The size of the Rana Rsk transcript was bigger than about 3.0 kb of the cloned Rsk cDNA, indicating that the actual size of the Rsk mRNA is about 4.0 kb. The Rana Rsk mRNA was strongly expressed in ovary tissues, but faint signals were also observed in the testis, kidney, lung, brain,

spleen, and liver. As expected, the lower expression of the Rsk gene in other tissues, except the ovary, may be due to the hibernation period, in which the gene expression maintains a very low level. A high expression of Rsk in the ovary plays a functional role that is related to meiotic maturation in the winter season. To examine the synthesis of the Rana Rsk protein from the cloned cDNA, we expressed it in vitro by using the Impact-CN system. A PCR-amplified coding region of Rana Rsk was inserted into the pTYB1 vector. Then, the Rsk protein that was induced by the IPTG treatment was purified with a chitin-bead column. The expression product was analyzed by SDS-PAGE (Fig. 4A). The expressed Rana Rsk had about a 83 kDa molecular mass, which matched well with the estimated molecular weight from the deduced amino acid of the cloned cDNA sequence. Western blot data showed that the expressed Rsk protein cross-reacted with the Xenopus p90Rsk2 antibody (Fig. 5B). This reflected the high degree of conservation with Xenopus Rsk at the amino acid sequence level (Fig. 2). To determine whether or not Rsk is directly involved in meiotic maturation, the Rsk level was examined with time-course by immunoblotting in P4-treated R. dybowskii oocytes. The GVBD of P4-treated oocytes started at 9 h and reached 90% at 12 h (data not shown). The control oocytes without the P4 treatment did not change in their amount of Rsk (Fig. 5). But, the band of Rsk in the P 4 -


Fig. 5. Modification by Rsk phosphorylation during oocyte maturation in R. dybowskii. Immature oocytes were cultured in the absence (control) or presence of progesterone (P4-treated). The GVBD of oocytes was observed at the indicated times. At the same time, oocytes collected at the indicated times were subjected to SDS-PAGE and immunoblotted with Xenopus p90Rsk2 antibody. Phosphorylation of Rana Rsk was detected by shifts in their electrophoretic mobility on an immunoblot as indicated by an arrow.

treated oocytes began to disappear and shifted to a new upper band at 9 h. It is clear that the lower band of Rsk almost disappeared at 12 h. Only the upper band of Rsk reminded afterwards. Modification of Rsk correlated well with the GVBD of P4-treated oocytes. This indicates, therefore, that the Rsk protein is modified by phosphorylation, and its phosphorylation may be involved in the activation of MPF in the R. dybowskii oocyte.

Discussion The MAPK cascade may regulate progesterone-induced meiotic maturation in amphibian oocytes. Phosphorylation of the S6 ribosomal protein by the S6 kinase constitutes an important phosphorylation event that is associated with MPF activation. Studies that use full-grown oocytes of a seasonally breeding frog, R. dybowskii, have suggested that Rsk plays a necessary role in mediating meiotic maturation (Bandyopadhyay et al., 1999). Therefore, we isolated Rsk cDNA and examined its expression in the R. dybowskii oocyte. A 3.0 kb of Rana Rsk cDNA was successfully cloned from a Rana ovary cDNA library and encoded about 84 kDa of the Rsk protein that consisted of 733 amino acid residues. The cloned Rsk cDNA also expressed in vitro about 90 kDa protein, which cross-reacted with the Xenopus p90Rsk-2 antibody. A comparison of the deduced amino acid sequence of Rana Rsk with type 2 Rsk of humans and Xenopus showed a higher conserved nature with over a 70% homology in amino acid identity level than that with other types of Rsk (Fig. 2). It indicates that the cloned Rana Rsk is type 2. Among three different isoforms of Rsk1, Rsk2, and Rsk3 that are known to be present in the vertebrate system, Rsk1 and Rsk2 are involved in the MAPK pathway during meiotic maturation (Hsiao et al., 1994; Palmer et al., 1998). These proteins have two kinase domains, as

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well as two kinds of phosphorylation sites by MAPK, and are autophosphorylated by the COOH terminal kinase, respectively (Bjørbœk et al., 1995: Dalby et al., 1998). Rana Rsk also has conserved phosphorylation sites (marked by box in Fig. 2) and shows a relatively higher amino acid homology in its two kinase domains with Xenopus Rsk2. In the screening of Rsk cDNA in the R. dybowskii ovary cDNA library, we isolated four independent clones. All of the clones encoded Rsk2, but not other types. Interestingly, Rsk2 mRNA is expressed higher in the ovary than other tissues (Fig. 3). It seems that Rsk2 plays a major role in the oocyte maturation event in R. dybowskii. In a previous study on the R. dybowskii oocyte, Bandyopadhyay et al. (1999) showed that specific Rsk components play a necessary role in mediating oocyte maturation. This is based on the result that rapamycin, a potent p70Rsk inhibitor, blocked progesterone-induced Rsk activity, protein synthesis, MPF activity, and oocyte maturation. Interestingly, this result was not observed in the rapamycin-treated Xenopus oocytes (Morley and Pain, 1992). This strongly suggests that protein synthesis is under the control of Rsk in the R. dybowskii oocytes, but not in the Xenopus oocytes. The role difference of Rsk isoforms during meiotic maturation in both species of frogs may affect the appearance of MPF activity. In the R. dybowskii oocytes, Rsk2 is phosphorylated parallel to GVBD during P4-induced meiotic maturation (Fig. 5). This suggests that p70Rsk plays a role in the translational control that is related to the appearance of MPF activity, whereas Rsk2 is involved in the signaling pathway of MPF activation in the R. dybowskii oocytes. It is well known that MPF, which consists of the Cdc2 kinase and cyclin, is directly involved in the meiotic oocyte maturation in vertebrates, including frogs. MPF activity appears at the same time as GVBD, decreases between the two meiotic divisions, and increases again at the second meiotic metaphase. The Cdc2 kinase activity should be tightly regulated through association of cyclin B in a diverse manner, according to the reproductive cycle of the animal (reviewed by Yamashita, 1998). Most of the amphibians and fish oocytes required de novo protein synthesis in order to undergo GVBD, which needed the prerequisite activation of MPF. The cyclin protein must be synthesized by triggering the hormonal signal to associate with Cdc2 that already exists (Nebreda et al., 1995; Tanaka and Yamashita, 1995; Yamashita et al., 1995). Ihara et al. (1998) also showed that the synthesis of cyclin needs MPF activation during oocyte maturation in Rana japonica. This situation is comparable to Xenopus oocytes since these prophase-blocked oocytes already contain cyclin B1 (Izumi and Maller, 1995; Kobayashi et al., 1991). In this process during the meiotic maturation in the R. dybowskii oocytes, Rana Rsk may be involved in the translational control of cyclin synthesis.

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The R. dybowskii oocytes also seem to require protein synthesis for MPF activation during maturation, because a blockade of protein synthesis in progesterone-treated GV oocytes inhibits the meiotic maturation (Bandyopadhyay et al., 1999; Kwon and Lee, 1991). However, the reproductive behavior of this frog species is distinctly different from that of other frogs, including Xenopus. We cloned the cDNAs of Cdc2 (Bandyopadhyay et al., 2000), as well as cyclin B1 and B2 (unpublished) as two components of MPF in the R. dybowskii oocytes. An examination of the functional change of Rana Rsk isoforms will be helpful to understand the subtle difference in the regulation of MPF activation during the oocyte maturation of the wild frog. Recently, it was reported that Rsk is essential for, and a mediator of, MAPK-dependent CSF arrest, which is the metaphase arrest of meiosis II in Xenopus oocytes before fertilization (Bhatt and Ferrell, 1999; Gross et al., 1999). These results indicate that the meiotically-activated MOS/MOPK pathway induces CSF arrest through the MAPK-dependent activation of Rsk, and that the activation of Rsk is necessary for the CSF appearance. This strongly suggests that Rana Rsk is also involved in CSF arrest in the metaphase II-arrested oocytes of frogs. Therefore, the cloned Rana Rsk cDNA will serve as an important tool for future work in characterizing CSF in the oocytes of seasonal-breeding frogs.

Acknowledgments We thank H. B. Kwon, Ph.D. and S. Y. Sung, Ph.D. (Hormone Research Center, Chonnam National University, Korea) for their helpful discussions, as well as their technical assistance on the manuscript. A Korea Research Foundation Grant (KRF- 2000-015-DP0369) supported this work.

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