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J. Biol. Chem., Vol. 275, Issue 44, 34236-34244, November 3, 2000

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Regulation of the Meiosis-inhibited Protein Kinase, a p38^MAPK Isoform, During Meiosis and Following Fertilization of Seastar Oocytes^*

Donna L. Morrison^§, Arthur Yee^¶, Harry B. Paddon, Dino Vilimek, Ruedi Aebersold, and Steven L. Pelech^**

From the  Department of Medicine, Koerner Pavilion, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada, ^¶ Kinetek Pharmaceuticals, Inc., 1779 W. 75th Avenue, Vancouver, British Columbia V6P 6P2, Canada, and the  Department of Biotechnology, University of Washington, Seattle, Washington 98105

Received for publication, May 30, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A p38^MAPK homolog Mipk (meiosis-inhibited protein kinase) was cloned from seastar oocytes. This 40-kDa protein shares approximately 65% amino acid identity with mammalian p38- isoforms. Mipk was one of the major tyrosine-phosphorylated proteins in immature oocytes arrested at the G₂/M transition of meiosis I. The tyrosine phosphorylation of Mipk was increased in response to anisomycin, heat, and osmotic shock of oocytes. During 1-methyladenine-induced oocyte maturation, Mipk underwent tyrosine dephosphorylation and remained dephosphorylated in mature oocytes and during the early mitotic cell divisions until approximately 12 h after fertilization. At the time of differentiation and acquisition of G phases in the developing embryos, Mipk was rephosphorylated on tyrosine. In oocytes that were microinjected with Mipk antisense oligonucleotides and subsequently were allowed to mature and become fertilized, differentiation was blocked. Because MipK antisense oligonucleotides and a dominant-negative (K62R)Mipk when microinjected into immature oocytes failed to induce germinal vesicle breakdown, inhibition of Mipk function was not sufficient by itself to cause oocyte maturation. These findings point to a putative role for Mipk in cell cycle control as a G-phase-promoting factor.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The meiotic maturation of an immature oocyte into a fertilizable egg is commonly the first developmental step in an organism. Oocytes in many animal species are arrested naturally in the first meiotic prophase or late G₂ phase (1, 2). Hormonal stimulation releases the oocytes from this block, promoting transition of the cells through meiosis I to a second developmental arrest point before or after meiosis II in preparation for fertilization (3). Early events in meiosis I include disintegration of the nuclear envelope leading to germinal vesicle breakdown (GVBD),¹ chromosome condensation, and a dramatic reorganization of many cellular structures. Many of these changes are mediated through the activation of protein kinases (reviewed in Ref. 4).

In seastar oocytes, whose maturation is induced with the hormone 1-methyladenine, several protein kinases appear to contribute to the global protein phosphorylation that precedes GVBD (5). Remarkably, some of these kinases, such as the phosphatidylinositol 3-kinase (6), ribosomal S6 kinase (7-9), and casein kinase 2 (10) appear to mediate the re-entry of G₀-arrested somatic cells into the G₁ phase of the cell cycle, despite the profound differences in the end points. Activation of both somatic cells and oocytes rapidly leads to changes in phospholipid metabolism, transient elevation of free intracellular calcium, a rise in intracellular pH, alteration of the cytoskeleton, and dramatic switches in the patterns of gene transcription and mRNA translation (11). The 34-kDa cyclin-dependent kinase Cdk1, also known as maturation-promoting factor (MPF), is a histone H1 kinase that undergoes cyclic regulation during sea oocyte maturation. Following fertilization, the kinase becomes periodically inhibited just prior to each meiotic and mitotic cell cleavage (8, 9, 12-16). Activation of Cdk1 has been correlated with production of cyclin B, phosphorylation of the Thr-167 site by Cdk7, and dephosphorylation of the Tyr-15 site by fission yeast Cdc25-related phosphatases. Seastar Cdk7 has been shown to be associated with cyclin H and a third stabilizing partner, Mat1 (17).

One of the first members of the mitogen-activated protein (MAPK) family to be identified was the seastar 44-kDa maturation-activated protein kinase (Mapk), which strongly phosphorylates myelin basic protein in vitro (14, 18, 19). p44^mapk is the major tyrosine-phosphorylated protein in maturing seastar oocytes. The precise function of Mapk in oocyte maturation remains equivocal and seems to differ in part from the activity of its counterpart p42^erk2 in the maturing frog oocyte. In Xenopus laevis Erk2 appears to play a role in the formation of the active MPF complex, whereas in the seastar, maximal Mapk stimulation occurs after the initiation of GVBD and later than MPF activation (20). It has been proposed that MAP kinases may act to prevent DNA synthesis during seastar and frog oocyte maturation. Following fertilization of frog oocytes, Erk2 becomes tyrosine dephosphorylated and remains inactive during the early mitotic division (21). We have observed similar results for Mapk in fertilized seastar oocytes (22). Like Erk1 and Erk2, Mapk possesses a Thr-Glu-Tyr motif, which must be phosphorylated on the tyrosine residue by a Mek-like kinase for maximal activation.² While Mek can be activated upon phosphorylation of the two serine residues in a Ser-Met-Ala-Asn-Ser motif by p37^mos (23) and possibly p94^rafB (24, 25) in the frog oocyte, there is no evidence for the presence of these MAP kinase kinase kinases in the seastar. The occurrence of other MAP kinases besides Mapk has not been reported in the seastar.

The initial objective of the present study was to detect and characterize other proteins that undergo marked changes in tyrosine phosphorylation during seastar oocyte maturation. Herein, we describe the major 40-kDa protein that becomes tyrosine dephosphorylated during seastar oocyte maturation and rephosphorylated on tyrosine within 12 h after fertilization. Purification and sequencing permitted its identification as a member of the p38^hog subfamily of MAP kinases. Our findings indicate a possible unrecognized role for stress-activated protein kinases in cell cycle control as G-phase-promoting factors.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- The mouse monoclonal anti-phosphotyrosine antibody 4G10 and the following primary rabbit polyclonal antibodies generated against synthetic peptides were obtained from Upstate Biotechnology Inc. (Lake Placid, NY): Erk1-CT (CGGPFTFDMELDDLPKERLKELIFQETARFQPGAPEAP), Pstaire (EGVPSTAIREISLLKEGGC), Cdk5-CT (CNPVQRISAEEALQHP), and p38 Hog-CT (CDEVISFVPPPLDQEEMES). The phosphorylation site-specific polyclonal antibody for p38^MAPK was bought from New England BioLabs. The various mouse monoclonal antibodies generated against recombinant human p38^hog- were from AbProbes (Portland, Maine). The secondary alkaline phosphatase-conjugated goat anti-rabbit and goat anti-mouse IgG were purchased from Bio-Rad. Purified seastar Mapk, expressed as a recombinant protein in Escherichia coli, was provided by Dr. David Charest (Dept. of Medicine, University of British Columbia). The cDNA for human p38^hog- was generously provided by Dr. Roger Davis (Howard Hughes Medical Institute, Worchester, MA). [-³²P]ATP was purchased from Amersham Pharmacia Biotech Canada (Oakville, ON) and ICN (Aurora, OH). All other reagents were of the highest grade available commercially.

Seastar Oocyte Treatments-- Pisaster ochraceus seastars were collected from beaches in the greater Vancouver area. For the preparation of immature and mature oocytes, the cells were isolated from ovaries and treated with 10 µM 1-methyladenine in natural sea water (NSW) as described (18, 26). Maturation was marked by GVBD with the disappearance of the nucleus, and this typically occurred within 90 min after 1-methyladenine addition for at least 80% of the oocyte population.

For the preparation of P. ochraceus embryos, mature oocytes were collected from seastars that had been injected with 140 µM 1-methyladenine in filtered NSW (26). Equilibrated mature oocytes were fertilized by addition of seastar sperm at a final effective dilution of 1:20,000 (v/v).

For the heat and osmotic shock experiments, oocytes were isolated from seastars that had been kept in captivity for at least 4 months under constant light. These oocytes matured in response to 10 µM 1-methyladenine, essentially as observed for oocytes obtained from seastars harvested during the spawning season (May to mid-July). To study the effect of heat shock, oocytes from individual seastars were resuspended in a measured volume of NSW preheated to 25 or 35 °C. To investigate the effect of osmotic shock, oocytes from individual seastars were resuspended at 12 °C in NSW containing 1.0 M NaCl (based on the initial concentration of NaCl in NSW as 0.5 M). For the zero time points, aliquots of the oocyte suspensions were immediately removed and pelleted, the sea water was aspirated, and the oocytes were quick frozen in a dry ice/ethanol bath.

Preparation of Cytosolic Extracts from Oocytes and Embryos-- Fresh or frozen pellets of oocytes or embryos were resuspended in 2× volume of chilled homogenizing buffer (50 mM -glycerophosphate, 20 mM MOPS, pH 7.2, 5 mM EGTA, 2 mM EDTA, 1 mM Na₃VO₄, 0.25 µM dithiothreitol, 1.0 mM phenylmethylsulfonyl fluoride, and 1.0 mM benzamidine). Large batches of oocytes were homogenized in a Waring blender in 2 × 15 s bursts, whereas small batches were sonicated with 2 × 30 s bursts. The homogenates were centrifuged at 9000 rpm for 15 min in a Beckman J2-HS centrifuge to remove particulate matter and organelles. The postmitochondrial supernatant was centrifuged in a Sorval Combi ultracentrifuge (Dupont, Canada) at 250,000 × g for 30 min, and the supernatants were immediately aliquoted and frozen at 70 °C until required. For the osmotic shock time course experiments, because of the ~5-fold increase in cell volume with medium containing 1 M NaCl, it was necessary to homogenize each cell pellet in a fixed final volume. Therefore, to each cell pellet of ~2.1 × 10⁵ packed cells was added 900 µl of 5× concentrated homogenization buffer, and the volumes were adjusted to 6 ml with water prior to cell lysis with 2 × 30 s bursts with a sonicator.

For the fertilized oocytes and embryos, cells were pelleted at 4 °C in a Beckman J2-HS centrifuge (1500 rpm for 5 min). A 33% (v/v) suspension was prepared in chilled homogenization buffer, and the embryos were disrupted with 2 × 30 s bursts at 19,000 rpm of a Polytron (PT3000, Brinkman). Homogenates were immediately centrifuged in a Sorval Combi ultracentrifuge at 10,000 × g for 10 min, followed by ultracentrifugation of the supernatants at 250,000 × g for 30 min. The resultant cytosols were stored in aliquots at -70 °C.

Protein Analytical Procedures-- Proteins were quantitated by the method of Bradford (27) with bovine serum albumin as the protein standard. For samples containing Brij, the absorbance at 280 nm was measured for each sample, and the protein concentration calculated as follows: protein concentration (mg/ml) = A₂₈₀ × 1.55.

Fast protein liquid chromatography (FPLC) of the cytosolic cell extracts was performed on a 1-ml Resource Q column (Amersham Pharmacia Biotech). Up to 5 mg of cytosolic protein was diluted to 2.1 ml with Buffer A (10 mM MOPS, pH 7.2, 25 mM -glycerophosphate, 2 mM EDTA, 5 mM EGTA, 2 mM Na₃VO₄). Two ml of sample were applied to the column, and the column was developed with a 10-ml 0-0.8 M NaCl linear gradient with collection of 0.25-ml fractions. In some experiments, select Resource Q fractions were subjected to FPLC on a 20-ml Superose 12 column equilibrated and developed with Buffer B (5 mM MOPS, pH 7.2, 5 mM EGTA, 5 mM NaF, 1 mM Na₂VO₃, and 0.25 mM dithiothreitol) and 0.2 M NaCl.

SDS-PAGE was performed as described by Laemmli (28) with 1.5-mm polyacrylamide gels (4% stacking gels and 10% separating gels). Silver staining of SDS-PAGE gels was performed by the method of Merril et al. (29).

Electrophoretic transfer of proteins from SDS-PAGE gels to nitrocellulose membranes (BDH) was carried out at 4 °C for 3 h at 300 mA with a Hoeffer Transfer Cell in the presence of transfer buffer (20 mM Tris, 120 mM glycine, 20% methanol (v/v), pH 8.6). Transferred proteins were visualized with Ponceau S or 0.1% Amido Black in 45% methanol, 10% acetic acid (w/v/v) for 15-30 min at room temperature with shaking. Destaining of Amido Black was carried out in a 45% methanol, 10% acetic acid (v/v) solution.

Immunological Procedures-- Western blotting was performed with primary antibodies in accordance with the supplier's instruction using nitrocellulose membranes that were blocked with 5% skim milk (w/v) in TBS (50 mM Tris base, 150 mM NaCl, pH 7.5) for 2 h. Following incubation for 0.5-2 h at room temperature with alkaline phosphatase-conjugated secondary antibody, Western blots were developed with a mixture of 1 ml of 1.5% 5-bromo-4-chloro-3-indolyl phosphate (ICN) in 100% dimethylformamide, 1 ml of 3% nitro blue tetrazolium (BDH) in 1 ml of 70% dimethylformamide, and 100 ml of 0.1 M Tris, pH 9.5, 0.1 M NaCl, 5 mM MgCl₂. Development was terminated by soaking in deionized H₂O.

For 4G10 blots, blocking of the membrane was achieved using 3% bovine serum albumin in 20 mM Tris, pH 7.5, 50 mM NaCl. Primary and secondary antibodies were diluted in NTBS (0.5% Nonidet-P40 in 20 mM Tris, pH 7.5 and 50 mM NaCl) with incubations of 4 and 2 h, respectively.

For immunoprecipitation of proteins from cell extracts and column fractions, samples were adjusted to 1% SDS (w/v) with addition of 20% SDS, and then diluted with an equal volume of 6% NETF (6% Nonidet P-40 (v/v) in NETF buffer (100 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl, pH 7.4, 50 mM NaF)). The samples were first precleared by incubation at 4 °C for 15 min with rotation with 10 µl of a protein A-Sepharose CL4B (Amersham Pharmacia Biotech) slurry in 3% Nonidet P-40 in NETF buffer, followed by removal of the beads by centrifugation for 1 min at 15,000 rpm. 10 µg of antibody were incubated with the supernatants for 1 h at 4 °C with rotation, followed by the addition of 20 µl of protein A-Sepharose slurry and further incubation for 45 min at 4 °C. The beads were washed twice with 6% NETF and once with NETF. Immunoprecipitates were boiled for 5 min in 4× SDS sample buffer and subjected to SDS-PAGE and Western blotting as described above.

Enrichment of Mipk-- Approximately 3.4 g of cytosolic extract were thawed, diluted to 1:10 with Buffer C (5 mM MOPS, pH 7.2, 5 mM EGTA, 5 mM NaF, 1 mM Na₂VO₃, and 0.25 mM dithiothreitol) and applied to six 10-g hydroxylapatite columns (Bio-Rad). Each column was eluted with a 350-ml 0-0.14 M potassium phosphate (pH 7.2) linear gradient; Mipk eluted in 0.034-0.05 M phosphate. Peak Mipk fractions were diluted 6-fold with Buffer B and applied to two 25-ml Q-Sepharose columns. Proteins were eluted with a 280-ml 0-0.8 M NaCl linear gradient in Buffer B; Mipk eluted between 0.25-0.30 M NaCl. Peak Mipk fractions were diluted 7-fold in Buffer B and applied to a 25-ml heparin-Sepharose column. Mipk did not bind to the heparin-Sepharose, and the flow-through was loaded onto a 25-ml polylysine-agarose column. This column was developed with a 280-ml linear gradient of 0-0.8 M NaCl; Mipk eluted between 0.29-0.33 M NaCl. The peak Mipk fractions were then adjusted to a final concentration of 1 M NaCl and applied to a 5-ml phenyl-Sepharose column and eluted with a 75-ml NaCl/Brij linear gradient, from 1 M NaCl, 0% Brij to 0 M NaCl, 1.5% Brij. Mipk was released between 0.57 M NaCl, 0.65% Brij and 0.53 M NaCl, 0.7% Brij.

For protein microsequencing of enriched Mipk, the purification was scaled up by 5-fold to start with 16 g of immature seastar oocyte cytosolic protein and 24 hydroxylapatite, six Q-Sepharose, three polylysine-agarose, three phenyl-Sepharose, and one Resource Q column were used. When the heparin-agarose column was omitted, the order of the polylysine-agarose and phenyl-Sepharose steps were reversed, and a Resource Q column was added as an extra purification step. The polylysine fractions were diluted 1:1 with Buffer B prior to application on to a 1-ml Resource Q column. The Resource Q column was developed with a 10-ml linear gradient of 0-0.8 M NaCl and 250-µl fractions were collected. Mipk eluted with 0.42-0.44 M NaCl. The 2-peak Mipk fractions from the Resource Q column were loaded into one lane of an 11% SDS-PAGE gel. Following SDS-PAGE, the resolved proteins were transferred onto a nitrocellulose membrane and subjected to Western blotting with Cdk5-CT antibody and Amido Black staining. The Mipk band was excised and placed in deionized H₂O to prevent drying out. This band was subject to trypsinolysis and protein microsequencing as described (30).

Cloning, Sequencing, Site-directed Mutagenesis, and Expression of Mipk-- RNA isolation from 300 µl of packed immature P. ochraceus oocytes was performed using the RNeasy isolation kit from Qiagen. mRNA preparations were made using the Qiagen Oligotex mRNA kit. From approximately 440 µg of total seastar RNA, 4-22 µg of mRNA in 60 µl were isolated. The PerkinElmer Life Sciences GeneAmp RNA PCR kit was used in the synthesis of P. ochraceus cDNA from 10 ng of mRNA.

For degenerate PCR, Mipk peptide sequences were aligned with the human p38 protein sequence. From this information, degenerate oligonucleotides were designed in orientations that permitted amplifications of a ~576-base pair fragment: (S2 sense, 5'-GA(T/C)GA(A/G)CA(T/C)GTICA(A/G)TTC-3'; S5 antisense, 5'-TTIGCIAC(A/G)TAIGGATG-3'); and a ~360-base pair fragment (S4 sense, 5'-CCIGTICA(A/G)TA(T/C)CA(A/G)AAA-3'; S2 antisense, 5'-AA(T/C)TGIAC(A/G)TG(T/C)TC(A/G)TC-3'). The PCR reaction conditions with Taq polymerase were an initial 1 min at 94 °C, 45 cycles of 94 °C for 30 s, 35 °C for 60 s, 72 °C for 90 s, followed by a 10-min incubation at 72 °C to fill all ends. The amplified fragments were cloned into the vector pBluescript SK(-) and the cDNA sequence determined. Cloning into T-tailed pBluescript SK(-) vector was based on the procedure of Holton and Graham (31). Transformations using the pBluescript vector into DH5a competent E. coli cells plated on Luria Broth, ampicillin, 5-bromo-4-chloro-3-indolyl -D-galactopyranoside (X-gal) agar were performed as described (32). Small quantities of plasmids were prepared using the Qiagen QIAprep Plasmid kit, whereas large quantity DNA preparations were performed using the Qiagen Plasmid Midi kit.

5'- and 3'-rapid amplification of cDNA ends (RACE) reactions were performed using the CLONTECH Marathon cDNA Amplification kit (33). PCR amplifications of the 3'- and 5'-ends of the Mipk cDNA were undertaken with the CLONTECH Advantage KlenTaq polymerase mix. To 5 µl of diluted Ad-cDNA were added 5 µl of 10× KlenTaq buffer, 20 µM dNTPs, 0.2 µM gene-specific primer (sense primers for 3'-RACE: 5'-AAACTCTCCGCAGTGGGAGC-3' or antisense primers for 5'-RACE: 5'-GATGACTCAGTGCCTCTTCAG-3'), 0.2 µM adaptor primer 1. A touchdown PCR method of amplification was used with the following conditions: 94 °C for 1 min, 5 cycles of 94 °C for 30 s; 72 °C for 3 min, 5 cycles of 94 °C for 30 s; 70 °C for 3 min, 25 cycles of 94 °C for 30 s; 68 °C for 3 min and final elongation at 72 °C for 10 min. The reaction products were purified on a low melting point agarose gel, and the appropriate product was used for ligation using the method of Kalvakolanu and Livingston (34).

Clones were sequenced by automated fluorescent DNA sequencing using the dideoxy chain termination method (35) and ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase (PerkinElmer Life Sciences). The construction of kinase-inactive (K62R)Mipk using PCR site-directed mutagenesis was based on the method of Ali and Steinkasserer (36) and used the primers: antisense KR (5'-GCGGATAGCAATCTTTATGCCAG-3'), STOPS, sense KK (5'-AAGCTTTCTCGACCATTTCAG-3') and ATGE.

Gluthionine S-transferase (GST) Mipk fusion proteins were produced in 100 µM isopropyl-1-thio--D-galactopyranoside (IPTG)-treated DH5a or UT5600 E. coli cells according to manufacturer's instructions (32). Cells were pelleted and resuspended in 1-5 ml of lysis buffer (1 ml of STE-500 (500 mM NaCl in TE buffer), 1 mg of lysozyme, 1 µg of soybean trypsin inhibitor, 1 µg of phenylmethylsulfonyl fluoride). The suspension was incubated on ice for 15 min, sonicated 3 × 15 s, and centrifuged in an Eppendorf tube at 15,000 rpm for 5 min. 100 µl of GST-agarose slurry was added to the supernatant and allowed to incubate for 1-2 h with rotation at 4 °C. The beads were washed with STE-500 buffer and phosphate-buffered saline before being resuspended in an equal volume of phosphate-buffered saline. These beads were then either stored at 70 °C or used for thrombin cleavage. For thrombin cleavage, 1 unit of thrombin was added to 100 µl of GST fusion protein-agarose slurry in phosphate-buffered saline and incubation continued at room temperature for 1 h. The reaction was stopped with 10 µl of benzamidine-Sepharose slurry (in phosphate-buffered saline) and incubated for 1 h at 4 °C with rotation. The beads were pelleted, and the supernatant with cleaved Mipk was aliquoted and stored at 70 °C until required.

Oocyte Microinjection Studies-- Seastar oocytes in filtered NSW at 6 °C were sorted visually with a dissecting microscope (WILD-M3B) at × 16 power. At this magnification, oocytes (150 µm in diameter, ~2500 picoliters in volume) were inspected, and healthy immature oocytes were individually picked and transferred to the microinjection slide. Usually about 20 oocytes were placed in 100 µl of NSW on each slide. The NSW was localized within a ring of rubber cement 1 cm in diameter made with a PAP pen (The Binding Site, Institute of Research and Development, Birmingham, United Kingdom). The glass slides were kept at a temperature of 12-13 °C on a three-eighths of an inch thick aluminum plate on a thermoelectric cold plate (Thermoelectrics Unlimited Inc.). Evaporation was minimized by using a blue plastic cap from a 15-ml centrifuge tube (Falcon) to cover the seawater ring.

Microinjection of the oocytes was performed using a Leitz Labovert FS inverted microscope with differential interference contrast optics. The microscope was flanked by right and left Leitz micromanipulators, which held the injection needles and holding pipettes, respectively. During the microinjection, the slide was kept at 10-12 °C using a refrigerated microscope stage based on a Peltier element, receiving variable direct current 12 V, 0-2 A. Injections of 50-150 picoliters of samples were performed as judged by the initial diameter of the injected materials.

For the oligonucleotide injections, Mipk sense-1 (5'-ATGAACAACCCAGTAACAGGA-3'), Mipk antisense-1 (5'-TCCTGTTACTGGGTTGTTCAT-3'), and Mipk antisense-2 (5'-CTGAACCGGCACCTCCCA-3') were diluted to 100 µM in sterile water and filtered through 0.45-µm filters prior to microinjection of approximately 65 µg of each oligonucleotide. For the (K62R)Mipk, Mapk, and SB203580 p38^hog inhibitor studies, oocytes were injected with approximately 10 pg of the recombinant (K62R)Mipk or 2 µM final concentration of SB203580 and monitored for ~24 h for spontaneous maturation events. In all microinjection experiments, the injected oocytes were allowed to incubate at 10 °C for about 24 h to allow protein turnover to occur. Spontaneous maturations were quantitated prior to maturation of the oocytes with 10 µM 1-methyladenine. The oocytes were allowed to mature for 4 h and then fertilized as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Detection of a Tyrosine-dephosphorylated Protein in Maturing Seastar Oocytes-- Relatively few proteins undergo alterations in their state of tyrosine phosphorylation (as visualized with the 4G10 anti-phosphotyrosine monoclonal antibody) when seastar oocytes are released from their block at the G₂/M border of the cell cycle by the natural hormone 1-methyladenine to resume meiotic maturation (Fig. 1, A and E). By the onset of GVBD prior to the first meiotic cell division, the tyrosine phosphorylation of the MAP kinase p44^mapk (Fig. 1, B and F) and the dephosphorylation of p34^cdk1 (Fig. 1, C and G) were evident as described previously (8, 9, 12-16). Even more striking than the reduced phosphorylation of p34^cdk1 was the dephosphorylation of a 40-kDa protein (Fig. 1, A and E, open arrow). We observed that this cytosolic, tyrosine-phosphorylated protein cofractionated on Resource Q with immunoreactivity toward an antibody developed to recognize the C terminus of the 31-kDa protein cyclin-dependent kinase 5 (Cdk5-CT) (Fig. 1, D and H). The size of the 40-kDa tyrosine-phosphorylated protein and its detection with a protein kinase antibody compelled us to characterize it further. The characterization described below, revealed the 40-kDa protein to be a meiosis-inhibited protein kinase, which we have called p40^mipk.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Immunological detection of tyrosine-phosphorylated protein kinases in seastar oocyte extracts fractionated by Resource Q chromatography. Cytosolic extracts prepared from either immature oocytes (A-D) or oocytes treated with 10 µM 1-methyladenine for 90 min (E-H) were fractionated over a Resource Q anion exchange column, and proteins were separated by SDS-PAGE. Western blots were probed with the 4G10 anti-phosphotyrosine monoclonal antibody (A and E) and polyclonal antibodies raised against C-terminal sequences in p44erk1 (Erk1-CT, B and F) and p31cdk5 (Cdk5-CT, D and H), and a catalytic subdomain III sequence from p34cdk1 (Pstaire, C and G). The p44mapk is identified by the filled arrowhead, p34cdk1 by the arrow, and p40mipk by the open arrowhead. Migration of molecular size standards is shown to the left.

To verify that the Cdk5-CT cross-reactive protein was tyrosine phosphorylated in immature oocytes and less so in maturing oocytes, we exploited the ability of the Cdk5-CT antibody to immunoprecipitate the 40-kDa protein under denaturing conditions in the presence of 1% SDS (Fig. 2A, lane 3). Although Cdk5-CT failed to detect p44^mapk by Western blotting analysis, this antibody was also able to immunoprecipitate this MAP kinase (Fig. 2A, lane 1), which could be immunoblotted and immunoprecipitated with Erk1-CT antibody directed against the C terminus of rat MAP kinase Erk1 (Fig. 2A, lane 2). There was a progressive tyrosine dephosphorylation of the Cdk5-CT-immunoprecipitated 40-kDa protein following 1-methyladenine treatment of the oocytes that preceded the tyrosine phosphorylation of p44^mapk (Fig. 2B). The level of tyrosine phosphorylation of the 40-kDa protein continued to decline after GVBD (at 80 min), but it was restored 12 h after fertilization of the oocytes prior to gastrulation (Fig. 2C). The amount of the 40-kDa protein was unchanged during maturation and up to 24-h postfertilization (Fig. 2D). These results demonstrate that the tyrosine phosphorylation states of p44^mapk and the 40-kDa protein were reciprocally regulated.

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Coordinate regulation of p44mapk and p40mipk tyrosine phosphorylation during seastar oocyte maturation and following fertilization. Cytosolic extracts were prepared from seastar oocytes that incubated with 10 µM 1-methyladenine for various times up to 3 h and then for up to 48 h following fertilization. A, Western blots of lysates from either immature oocytes (lanes 1 and 3) or GVBD+ oocytes following treatment with 1-methyladenine for 90 min (lanes 2 and 4) probed with Erk1-CT (lanes 1 and 2) and Cdk5-CT (lanes 3 and 4) following immunoprecipitation with Erk1-CT (lanes 2 and 4) and Cdk5-CT (lanes 1 and 3). B, quantitation of the p44mapk and p40mipk bands in immunoblots of Cdk5-CT immunoprecipitates probed with 4G10 anti-phosphotyrosine antibody of lysates from oocytes treated for 0-2 h with 1-methyladenine. One arbitrary unit corresponded to the phosphotyrosine signals generated by p40mipk in extracts from immature oocytes and by p44mapk in extracts from oocytes treated with 1-methyladenine for 2 h. Values are the mean ± S.E. of three independent experiments. C and D, Western blots probed with 4G10 and Cdk5-CT antibodies show Cdk5-CT immunoprecipitates from immature oocytes (lane 1), oocytes treated with 1-methyladenine for 90 min (lane 2), and mature oocytes following fertilization after 3 h (lane 3), 6 h (lane 4), 12 h (lane 5), 24 h (lane 6), and 48 h (lane 7).

Purification and Identification of p40^mipk-- To enrich the 40-kDa phosphoprotein sufficiently for its identification, the Cdk5-CT and 4G10 antibodies were used to monitor purification by conventional column chromatography (Fig. 3). Starting with cytosol from 20 ml of packed immature oocytes (~4 × 10⁶ oocytes), the 40-kDa Cdk5-CT immunoreactive protein was observed to copurify with the major 4G10 immunoreactivity over 5 consecutive column steps (Fig. 3, G-P). Insufficient amounts of the 40-kDa protein were purified by this method, making it necessary to increase the amount of starting protein by 5-fold and add a Resource Q step at the end of the purification. As shown in the silver-stained gel of selected Resource Q fractions in Fig. 3F, the final preparation was still impure. Nevertheless, the Cdk5-CT immunoreactive protein was unequivocally identified on the silver-stained gel by alignment with the Ponceau S-stained Western blot in Fig. 3Q. The 40-kDa protein represented less than 0.5% of the protein at this stage. However, the SDS-PAGE step permitted sufficient resolution of the other proteins so that protein microsequencing was feasible. Based on the amount of cytosolic protein used at the start of the purification (i.e. 16 g), this amounted to a purification of greater than 16,000,000-fold to obtain less than 1 µg of the SDS-PAGE purified 40-kDa protein. This indicated that the 40-kDa protein was expressed at relatively low levels in the order of 7.5 × 10⁵ molecules/cell, especially when the large size of the oocyte is considered. By comparison, approximately 5 × 10⁶ molecules of p44^mapk are found per oocyte (18).

View larger version (56K): [in this window] [in a new window]   Fig. 3.   Purification of p40mipk from immature seastar oocytes. Cytosol from immature oocytes was fractionated as described under "Experimental Procedures" by sequential column chromatography on hydroxylapatite (A, G, and H), Q-Sepharose (B, I, and J), heparin-agarose (C, K, and L), polylysine-agarose (D, M, and N), phenyl-Sepharose (E, O, and P) and Resource Q (F and Q). Immunoblots are shown for the fractions that encompass the positions of peak immunoreactivity of the 40-kDa protein with the Cdk5-CT (G, I, M, O, and Q) and 4G10 anti-phosphotyrosine antibody (H, J, N, and P). The 40-kDa immunoreactive protein did not bind to heparin-agarose, but this resin did retain other proteins (C). The fractions from each column step that were pooled for subsequent application to the next column are indicated by the stippled regions (A, B, D, and E). A nearly full-length silver-stained SDS-PAGE gel of the most purified p40mipk preparation following MonoQ chromatography is shown in F. The CDK5-CT Western blot of these Resource Q fractions shown in Q was also Ponceau S-stained to facilitate comparison with F. It is evident that p40mipk was highly enriched by this stage, but still represents a minor constituent.

The purified 40-kDa protein was subjected to trypsin digestion followed by peptide separation by reverse phase-high performance liquid chromatography. Mass spectrometry (MS/MS) sequence data was obtained for six discreet tryptic peptides from the 40-kDa protein (Fig. 4A, boxed peptides S1-S6). Protein database searches indicate that the 40-kDa protein was related to the MAP kinase superfamily of protein kinases, with the S2, S3, and S4 tryptic peptides displaying closest homology to the p38^hog subfamily of stress-activated kinases. Because p38^hog isoforms are tyrosine phosphorylated in their active state, by analogy it would appear that the 40-kDa protein was inactivated during oocyte maturation. Consequently, this p38^hog-related kinase was subsequently referred to as Mipk for meiosis or maturation-inhibited protein kinase.

View larger version (55K): [in this window] [in a new window]   Fig. 4.   Primary structure of p40mipk and comparison with p38hog isoforms. A, predicted amino acid sequence of Mipk and p38hog- isoform from rat, mouse, carp, and X. laevis are compared with human p38hog-. Only amino acid residues that differed from the human sequence are shown; identical residues are indicated by dashes. Amino acid sequences for tryptic peptides generated from the purified seastar p40mipk are boxed below the full-length cDNA-derived amino acid sequence of this kinase. Roman numerals indicate the positions of the conserved protein kinase subdomains. B shows the percent amino acid identities and homologies between seastar p40mipk and the human -, 2-, -, and -p38 isoforms as well as fission yeast StyI and budding yeast Hog1. Alignments and percentages of identity and homology were generated using the AGALIGN program developed by Dr. Allen Delaney (Kinetek Pharmaceuticals, Inc.).

Cloning and Sequencing of p40^mipk-- Using the peptide sequence data for Mipk and the likely orientation of these peptides by alignment with the primary structures of p38^hog isoforms, a PCR strategy was used to clone Mipk from seastar oocyte cDNA. The open reading frame of Mipk was 1089 base pairs, which predicted a 363 amino acid protein-serine/threonine kinase of the p38^hog family (Fig. 4). The Thr-189 and Tyr-191 residues in the activation loop between conserved kinase subdomains VII and VIII were in an equivalent position to the Thr-Gly-Tyr dual phosphorylation site in known p38^hog isoforms. All of the tryptic peptides sequenced from the purified Mipk protein matched perfectly with the predicted amino acid sequence, except for peptide S1. The first 5 residues of S1 corresponded to amino acids 230-234 of Mipk, but Ser-235 was identified as a Tyr in the peptide; perhaps because of inaccuracies in peptide sequencing.

A search of various nucleotide and protein databases revealed that the deduced amino acid sequence of Mipk is more closely related to human p38^hog-, than the , , or  isoforms (Fig. 4B). In mammals, Xenopus and carp, p38^hog- cognates typically share 84-99% amino acid identity, but Mipk exhibited only 65% overall identity with p38^hog- from these species, and there are several other significant differences (Fig. 4A). The N terminus of Mipk contains an extra 8 amino acids that were missing from other p38^hog isoforms. There is a stretch of 10 amino acids between subdomains IV and V that is 80% different from other p38^hog subfamily members. The largest region of sequence variation between Mipk and other p38^hog isoforms begins in subdomain X and continues to the C terminus of the protein. In this 124-amino acid region, there is only 46% identity between Mipk and human p38^hog-. In the final 36 amino acids, the identity drops to 28%, with the last 6 residues in p38^hog- missing from Mipk.

Immunological Characterization of p40^mipk and p38^hog--- To confirm that the Cdk5-CT antibody cross-reacted with Mipk on Western blots, seastar Mipk was expressed as a 66-kDa GST fusion protein in DH5a (standard) and UT5600 (protease-deficient) strains of E. coli. The UT5600 cells permitted the highest levels of Mipk expression, with approximately 30% of the protein present in the soluble fraction (data not shown). The Cdk5-CT antibody immunoblotted recombinant GST·Mipk (data not shown). Site-directed mutagenesis was used to create a kinase-inactive mutant of Mipk (i.e. GST·(K62R)Mipk) in which the conserved lysine residue in subdomain II required for catalytic activity was converted to arginine. This mutant exhibited a similar expression pattern in bacteria to GST·Mipk. Thrombin cleavage permitted the generation of 40-kDa soluble forms of wild-type and kinase-inactive Mipk (data not shown).

Characterization of the Catalytic Activity of p40^mipk-- The conservation of the Thr-Gly-Tyr dual phosphorylation site in Mipk with p38^hog isoforms strongly indicated that tyrosine-phosphorylated Mipk represented the active form of the enzyme. To confirm this, Mipk purified to the Resource Q step (Fig. 3) was tested for catalytic activity toward a panel of substrates including ATF-2, myelin basic protein, histone H1, core histones, phosvitin, protamine, casein, and a peptide based on the C terminus of ribsomal protein S6. The Resource Q fractions containing the Mipk peak failed to demonstrate phosphotransferase activity toward these substrates (data not shown). When GST·Mipk was tested for phosphotransferase activity with the aforementioned proteins as well as ATF-2 and c-Jun, the kinase was apparently inactive, as might be expected in view of the absence of tyrosine phosphorylation of GST·Mipk (data not shown).

Without a suitable substrate with which to correlate the catalytic activity of Mipk with its tyrosine phosphorylation state, the autophosphorylating activity of Mipk was assessed. Extracts from immature (Fig. 5, lane 1), GVBD+ (Fig. 5, lane 2), 3-h postfertilization (Fig. 5, lane 3), and 18-h postfertilization (Fig. 5, lane 4) were resolved on a Resource Q column, and the peak Mipk fractions were identified. These fractions were incubated with 50 µM [-³²P]ATP for 1 h followed by immunoprecipitation with Cdk5-CT antibody and Western blotting with 4G10 anti-phosphotyrosine antibody (Fig. 5B). Autoradiography of this Western blot revealed that a 40-kDa protein underwent enhanced ³²P labeling in concert with the state of tyrosine phosphorylation of Mipk (Fig. 5A). However, it still remains possible that the phosphorylation of Mipk was catalyzed by another kinase that copurified with Mipk rather than by autophosphorylation.

View larger version (44K): [in this window] [in a new window]   Fig. 5.   Autophosphorylation of partially purified p40mipk. Cytosolic extracts were prepared from immature (lane 1), mature oocytes following exposure to 10 µM 1-methyladenine for 90 min (lane 2), 3 h (lane 3), and 18 h (lane 4) after fertilization. The extracts were further fractionated by Resource Q chromatography, and then the Mipk peak fractions were incubated with 50 µM [-32P]ATP and subsequently immunoprecipitated with Cdk5-CT antibody. A shows an autoradiogram of an SDS-PAGE gel of the Cdk5-CT immunoprecipitates. B shows the p40mipk region of a Western blot of a similar SDS-PAGE gel that was probed with 4G10 anti-phosphotyrosine antibody.

Regulation of p40^mipk by Stress Stimuli-- Although Mipk was controlled in a cell cycle-dependent fashion in the oocyte and was distinct from p38^hog- in this system, it was of interest to evaluate whether p38^hog Mipk was also regulated by stress stimuli. We observed that seastars that were kept under constant light retained their oocytes for at least 4 months after the conclusion of the spawning season. Interestingly, the level of tyrosine phosphorylation of Mipk was substantially reduced in these oocytes, but they underwent GVBD normally in response to 1-methyladenine. Therefore, these oocytes were used to investigate whether heat shock and osmotic shock stimulated the tyrosine phosphorylation of Mipk and presumably its phosphotransferase activity.

P. ochraceus normally encounter temperatures of around 10-14 °C in the ocean shores around southwestern British Columbia, but they are able to endure higher temperatures on the surface of rocks at low tide. The effects of incubation of the oocytes at temperatures of 25, 35, and 45 °C were tested. At 45 °C, the oocytes immediately underwent gross changes in morphology and appeared to melt (data not shown). At 25 °C, an increase in tyrosine phosphorylation of Mipk became evident after 30 min of incubation (Fig. 6, A-C). At 35 °C, Mipk became strongly tyrosine phosphorylated within 30 min. After 30 min, while the level of tyrosine phosphorylation was maintained, there was a reduction in the total amount of Mipk protein. It appeared that the remaining Mipk was tyrosine phosphorylated to a higher stoichiometry that compensated for degradation of the kinase.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Post-translational regulation of p40mipk by stress stimuli. Immature oocytes were obtained from seastars maintained in a constant light tank for 3 months after their sisters spawned in the wild. These oocytes exhibited lower levels of tyrosine phosphorylation of p40mipk than detected in oocytes obtained during the normal spawning season for P. ochraceus. A-C, after continuous incubation at 14 °C, oocyte pellets were resuspended in sea water at either 25 or 35 °C for up to 1 h prior to harvesting. D-E, oocyte pellets were resuspended in sea water with a final NaCl concentration of 1 M for up to 1 h prior to harvesting. Lysates were prepared from the oocytes and subject to immunoprecipitation with the Cdk5-CT antibody and Western blotting. The immunoblots were probed with 4G10 anti-phosphotyrosine antibody (A and D) and Cdk5-CT antibody (B and E). Alkaline phosphatase signals for the p40mipk band on the Western blots were quantitated by densitometric analysis and phosphotyrosine levels corrected for the amount of p40mipk protein detected (C and F). Data are the mean ± S.E. of three independent experiments. G, oocyte lysates from seastars held captive for 3 months (lanes 3-5) and freshly captured (lanes 1 and 2) were probed in immunoblots with a phosphorylation site-specific polyclonal antibody for p38MAPK. The immature oocytes were untreated (lanes 1 and 3), or exposed to 1-methyladenine for 90 min (lanes 2 and 4) or 60 µM anisomycin for 3 h.

To investigate the effect of osmotic shock on Mipk in the seastar oocytes, concentrations of NaCl that exceeded 0.5 M were tested as natural sea water has a salt concentration of approximately 0.5 M. When oocytes were placed in sea water at 14 °C with 1, 1.5, 2, and 3 M NaCl, 1 M NaCl was the highest concentration that permitted survival of the oocytes over a 1-h period. Within 30 s of placement of the oocytes in sea water with 1 M NaCl, there was a marked increase in the tyrosine phosphorylation of Mipk (Fig. 6D). After this period, there was a drastic reduction in the amount of Cdk5-CT-immunoreactive Mipk despite the maintenance of the level of tyrosine phosphorylation of the remaining kinase for up to 60 min (Fig. 6E).

We further explored the effect of the protein synthesis inhibitor anisomycin on Mipk phosphorylation using a polyclonal antibody that recognizes only p38^MAPK that are phosphorylated at the activating phosphorylation sites in the Thr-Gly-Tyr motif. As shown in Fig. 6G, treatment of immature oocytes from seastars kept captive for 3 months with 60 µM anisomycin for 3 h produced a marked increase in Mipk phosphorylation. This was comparable with the level of Mipk phosphorylation in immature oocytes from freshly captured seastars. Collectively, these findings demonstrate that Mipk is subject to regulation by diverse stress stimuli as well as during normal meiosis and early mitotic cell divisions in the seastar.

Oocyte Microinjection Studies-- The fact that the tyrosine phosphorylation of Mipk was markedly decreased in immature oocytes from seastar held in captivity for several months indicated that the inactivation of this kinase was insufficient to permit their release from the arrest at prophase of meiosis I. This was further substantiated by experiments in which we attempted to deplete the level of Mipk in immature oocyte with two antisense oligonucleotides based on the seastar Mipk cDNA sequence. Antisense-1 was created to block the start codon of the Mipk transcript whereas antisense-2 was designed to bind downstream of the start codon. When either antisense oligonucleotide was injected into immature oocytes for up to 48 h, there was no induction of maturation, and neither was there an inhibition of the ability of 1-methyladenine to induce GVBD in these oocytes (data not shown). The 24 h antisense microinjected oocytes maintained their immature morphology.

To examine a role for Mipk in early development, oocytes that had been previously microinjected with the Mipk antisense oligonucleotides for 24 h, were incubated with 1-methyladenine for 4 h and then fertilized with seastar sperm. In control oocytes injected with water, the oocytes proceeded to undergo reductive, mitotic divisions, and eventually differentiation, with 4 cells typically evident at 5-h postfertilization (Fig. 7D), with approximately 64 undifferentiated cells by 10-h postfertilization (Fig. 7G), and blastulas with gastrulation by 24-h postfertilization (Fig. 7I). Gastrulation was also evident within 24 h after fertilization of oocytes that had been microinjected with the Mipk sense oligonucleotide counterpart to Mipk antisense-1 (Fig. 7K). In the oocytes that were injected with Mipk antisense-1 (Fig. 7, E and H) or Mipk antisense-2 (data not shown), initial cell divisions following maturation and fertilization proceeded as observed in the H₂O and Mipk sense controls. However, even 24 h after fertilization, the Mipk antisense-1 and antisense-2-injected cells did not undergo differentiation (Fig. 7J and data not shown), which was evident within 16 h of fertilization in control embryos.

View larger version (117K): [in this window] [in a new window]   Fig. 7.   Effect of Mipk antisense microinjection into immature seastar oocytes on meiosis and early embryonic development. Immature oocytes were microinjected with water (A, D, G, and I), Mipk sense-1 oligonucleotide (K), Mipk antisense-1 oligonucleotide (B, E, H, and J), and recombinant (K62R)Mipk (C and F) and incubated for ~24 h at 14 °C as described under "Experimental Procedures." Cells were photographed either 10 min after microinjection (A) or after 24 h (B and C). The oocytes were then stimulated to undergo meiotic maturation with 10 µM 1-methyladenine for 4 h followed by fertilization. The fertilized cells were photographed 5 h (D-F), 10 h (G and H), and 20 h (I-K) after fertilization.

The inability of either antisense oligonucleotide to induce oocyte maturation may have reflected technical difficulties (such as low turnover of Mipk and inadequate time to deplete the level of Mipk with the antisense oligonucleotides) or that inactivation of Mipk was necessary but not sufficient for resumption of cell cycle progression and GVBD. In an alternative strategy, immature oocytes were microinjected with a kinase-inactive version of Mipk (i.e. (K62R)Mipk) that was expressed as a GST fusion protein and thrombin-cleaved to remove the GST portion. However, this dominant-negative protein also failed to induce GVBD in the immature oocytes, and it did not affect the rate of 1-methyladenine-induced GVBD (Fig. 7C). However, the kinase-inactive Mipk mutant completely blocked cell division following 1-methyladenine treatment and fertilization of the microinjected oocytes. Within 5 h of fertilization, (K62R)Mipk-injected eggs appeared to undergo cell death (Fig. 7F). This lethal effect with the Mipk mutants did not appear to be nonspecific, because fertilized eggs that had been microinjected with recombinant seastar p44^mapk were viable (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The identification of one of the major proteins that undergoes tyrosine dephosphorylation during oocyte maturation as a novel isoform of the p38^hog subfamily of MAP kinases was unexpected. To our knowledge, this represents the first time that a stress-activated protein kinase has been implicated in normal cell cycle control. Recently, Takenaka et al. (37) demonstrated that p38^hog- is activated in nocodazole-treated NIH-3T3 and HeLa cells arrested in M-phase. However, this appeared to be a stress-related response because p38^hog- was not activated during normal cell cycle progression through M-phase. After our study was completed, Suzanne et al. (38) reported that a p38 MAP kinase kinase (licorne) in Drosophila is required for correct anterior-posterior and dorsal-ventral patterning during oogenesis. These effects were mediated in part through licorne regulation of the activity of transforming growth factor-, encoded by the gurken gene in Drosophila (38).

A plethora of stress stimuli has been shown to activate p38^hog isoforms in diverse species from yeast to man. Four distinct p38^hog isoforms (, 2,  (alias Sapk3 or Erk6), and  (alias Sapk4)) have been characterized in mammalian cells, and they exhibit very similar properties (39, 40). All four were activated in response to treatments of cells with interleukin-1, tumor necrosis factor-, sorbitol, and UV light, but were unaffected by insulin-like growth factor-1 or phorbol esters. They were all capable of phosphorylating myelin basic protein, Mapkapk-2, Mapkapk-3, ATF-2, Elk-1, and Sap-1, although there were some differences in substrate preferences. The mammalian p38^hog isoforms differed in their tissue distribution, with  and 2 broadly expressed,  found primarily in muscle and brain, and  expressed predominantly in salivary gland, pituitary gland, adrenal gland, and placenta (41).

Seastar p40^mipk featured the characteristic Thr-Gly-Tyr dual phosphorylation site motif as well as most of the other residues located between kinase subdomains VII and VIII found in other p38^hog isoforms. Overall it shared 71% identity within its kinase domain with human p38^hog-, which appeared to be its closest homolog, and 65% identity over the entire protein. Mipk was tyrosine phosphorylated in response to anisomycin, heat, and osmotic shock (Fig. 6) as has been observed for p38^hog- (42, 43). However, we were unable to confirm that Mipk phosphorylated any of the known substrates of p38^hog- such as myelin basic protein or ATF-2.

The high level of tyrosine phosphorylation of Mipk in immature oocytes and its subsequent dephosphorylation early in the maturation process pointed to a potential role for this kinase in the cell cycle block at the beginning of prophase in meiosis I. Presumably, a Mipk-dependent signaling pathway participated in the maintenance of the oocytes in an arrested state and inhibition of this kinase is necessary for transition through M phase. We were unable to obtain direct evidence to support this hypothesis. It would appear that inactivation of Mipk was insufficient by itself to induce meiotic maturation in the oocytes as revealed by the absence of germinal vesicle breakdown. Because of the limitations of microinjection experiments, it remains unclear whether inactivation of Mipk promoted stimulation of Mapk, Cdk1, S6 kinase, casein kinase 2, or any of the other protein kinases that are activated in maturing oocytes. Stimulation of cAMP-dependent protein kinase or protein kinase C inhibits the maturation of seastar oocytes by 1-methyladenine and the activation of the aforementioned protein kinases (8, 44, 45). It will be interesting to evaluate whether treatment of oocytes with cAMP analogs and phorbol esters prevents the dephosphorylation of Mipk in response to 1-methyladenine.

More definitive evidence was obtained for a role for Mipk in early embryonic development. For approximately 12 h immediately following fertilization at 12 °C, P. ochraceus embryos undergo a rapid increase in cell number to the 256-cell stage through 8 cycles of synchronous cleavage without discernable G₁ or G₂ phases. Subsequently, the individual cells within the embryo assume independent division rates, and there is a flattening of the developmental curve (26). Mipk underwent a marked increase in tyrosine phosphorylation apparently coincident with the transition from synchronous cell cleavages to differential cleavages (Fig. 2C). Because Mipk was also highly tyrosine phosphorylated in prophase-arrested immature oocyte, it would seem that the kinase may act as a G₂ promotion factor and a cytostatic factor. A reduction in the rate of cell division would require activation of enzymes which promote entry into G₂ in preparation for growth and for differentiation. Mipk activation may prevent the transit of cells that have complete DNA synthesis directly into M phase. In this regard, it is worthwhile considering that while Cdk1 acts as an MPF, it must be inactivated at the end of M phase before cell division will occur.

The concept of Mipk as a G₂ promotion factor was supported by the microinjection experiments with two Mipk antisense oligonucleotides (Fig. 7), but not with microinjected (K62R)Mipk. The absence of cell division in the oocytes that were microinjected with the (K62R)Mipk mutant following fertilization might have arisen because Mipk also serves a stress protective function. Not only did these cells not divide, but they appeared to undergo cell death. It is possible that the microinjection experiments with (K62R)Mipk evoked a level of stress for which endogenous Mipk can normally compensate. In the absence of Mipk function, the fertilized oocytes were triggered to apoptose. The results of the anisomycin, heat, and osmotic shock experiments demonstrated that Mipk can participate in stress signaling (Fig. 6). Future studies should provide a tighter correlation between the growth of cells (with the introduction of G₁ and G₂ phases) and Mipk activation during the transition from reductive cell division to differentiation in the developing seastar embryo.

	ACKNOWLEDGEMENTS

We thank Dr. Allen Delaney (Kinetek Pharmaceuticals, Inc.) for his aid in the protein database searches and sequence alignments. Drs. J. Sanghera, C. Palaty, D. Charest, D. Lefebvre, and L. Charlton in our laboratory are also acknowledged for their helpful advice and materials. We are also grateful to Dr. Roger Davis (Howard Hughes Medical Institute) for the human p38^hog cDNA clone used in this study.

	FOOTNOTES

^* This research was supported in part by a grant-in-aid from the Medical Research Council of Canada (MRCC) (to S. L. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF084574.

^§ Recipient of an MRCC Studentship Award.

^** Recipient of an MRCC Industrial Scientist Award.

To whom correspondence should be addressed: Dept. of Medicine, 2nd Floor, Koerner Pavilion, 2211 Wesbrook Mall, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada. Tel.: 604-822-8086; Fax: 604-822-8693; E-mail: spelech@home.com.

Published, JBC Papers in Press, July 20, 2000, DOI 10.1074/jbc.M004656200

² D. L. Charest, A. Yee, and S. L. Pelech, unpublished data.

	ABBREVIATIONS

The abbreviations used are: GVBD, germinal vesicle breakdown; Mipk, meiosis-inhibited protein kinase; MPF, maturation promoting factor; NSW, natural sea water; MAPK, mitogen-activated protein kinase; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Mapk, maturation-activated protein kinase; MOPS, 4-morpholinepropanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES