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• W2114435526 abstract "Fully grown Xenopus oocyte is arrested at prophase I of meiosis. Re-entry into meiosis depends on the activation of MPF (M-phase promoting factor or cyclin B·Cdc2 complex), triggered by progesterone. The prophase-arrested oocyte contains a store of Cdc2. Most of the protein is present as a monomer whereas a minor fraction, called pre-MPF, is found to be associated with cyclin B. Activation of Cdc2 depends on two key events: cyclin binding and an activating phosphorylation on Thr-161 residue located in the T-loop. To get new insights into the regulation of Thr-161 phosphorylation of Cdc2, monomeric Cdc2 was isolated from prophase oocytes. Based on its activation upon cyclin addition and detection by an antibody directed specifically against Cdc2 phosphorylated on Thr-161, we show for the first time that the prophase oocyte contains a significant amount of monomeric Cdc2 phosphorylated on Thr-161. PP2C, a Mg2+-dependent phosphatase, negatively controls Thr-161 phosphorylation of Cdc2. The unexpected presence of a population of free Cdc2 already phosphorylated on Thr-161 could contribute to the generation of the Cdc2 kinase activity threshold required to initiate MPF amplification. Fully grown Xenopus oocyte is arrested at prophase I of meiosis. Re-entry into meiosis depends on the activation of MPF (M-phase promoting factor or cyclin B·Cdc2 complex), triggered by progesterone. The prophase-arrested oocyte contains a store of Cdc2. Most of the protein is present as a monomer whereas a minor fraction, called pre-MPF, is found to be associated with cyclin B. Activation of Cdc2 depends on two key events: cyclin binding and an activating phosphorylation on Thr-161 residue located in the T-loop. To get new insights into the regulation of Thr-161 phosphorylation of Cdc2, monomeric Cdc2 was isolated from prophase oocytes. Based on its activation upon cyclin addition and detection by an antibody directed specifically against Cdc2 phosphorylated on Thr-161, we show for the first time that the prophase oocyte contains a significant amount of monomeric Cdc2 phosphorylated on Thr-161. PP2C, a Mg2+-dependent phosphatase, negatively controls Thr-161 phosphorylation of Cdc2. The unexpected presence of a population of free Cdc2 already phosphorylated on Thr-161 could contribute to the generation of the Cdc2 kinase activity threshold required to initiate MPF amplification. M-phase promoting factor cyclin-dependent kinase CDK-activating kinase S. cerevisiae CDK-activating kinase 1 germinal vesicle breakdown protein phosphatase 2C dithiothreitol endoplasmic reticulum glutathioneS-transferase reduced glutathione protein kinase A okadaic acid CDK-associated phosphatase The fully grown Xenopus oocyte is physiologically arrested at the diplotene stage of meiotic prophase; it contains a maternal store of Cdc2 or Cdk1 (cyclin-dependent kinase). The majority of the protein is present as a monomer in the cytoplasmic compartment of the oocyte, whereas a minor fraction (10% as estimated by Western blotting) is found to be associated with B2 and B5 cyclins (1Kobayashi H. Minshull J. Ford C. Golsteyn R. Poon R. Hunt T. J. Cell Biol. 1991; 114: 755-765Crossref PubMed Scopus (223) Google Scholar, 2Hochegger H. Klotzbucher A. Kirk J. Howell M. le Guellec K. Fletcher K. Duncan T. Sohail M. Hunt T. Development. 2001; 128: 3795-3807PubMed Google Scholar). The cyclin B·Cdc2 complex, which accumulates during oogenesis, is maintained inactive by two inhibitory phosphorylations on Thr-14 and Tyr-15 of Cdc2 catalyzed by the membrane-associated Myt1 kinase (3Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (533) Google Scholar, 4Nakajo N. Yoshitome S. Iwashita J. Iida M. Uto K. Ueno S. Okamoto K. Sagata N. Genes Dev. 2000; 14: 328-338PubMed Google Scholar). Another phosphorylation of Cdc2, on the Thr-161 residue located in the T-loop of the protein, is known to be required for Cdc2 kinase activation (5Norbury C. Blow J. Nurse P. EMBO J. 1991; 10: 3321-3329Crossref PubMed Scopus (397) Google Scholar). The inactive cyclin B·Cdc2 complex present in fully grown oocyte, also known as pre-MPF,1 contains a triphosphorylated Cdc2 subunit on Thr-14, Tyr-15, and Thr-161 (6Coleman T.R. Dunphy W.G. Curr. Opin. Cell Biol. 1994; 6: 877-882Crossref PubMed Scopus (325) Google Scholar). Two hypotheses can be envisaged concerning the timing and the location of Cdc2 phosphorylation on Thr-161 and the enzymes responsible on this process during oogenesis: 1) Newly synthesized cyclin B associates with Cdc2 in the cytoplasm. Then the neocomplex is translocated to the nucleus where it becomes a substrate of a CDK-activating kinase (CAK), composed of CDK7, cyclin H, and the assembly factor MAT1, a complex known to be strictly located within the Xenopus oocyte nucleus (7Labbe J.C. Martinez A.M. Fesquet D. Capony J.P. Darbon J.M. Derancourt J. Devault A. Morin N. Cavadore J.C. Doree M. EMBO J. 1994; 13: 5155-5164Crossref PubMed Scopus (67) Google Scholar, 8Fesquet D. Labbe J.C. Derancourt J. Capony J.P. Galas S. Girard F. Lorca T. Shuttleworth J. Doree M. Cavadore J.C. EMBO J. 1993; 12: 3111-3121Crossref PubMed Scopus (325) Google Scholar). CAK exhibits a stronger affinity for cyclin-associated CDKs than for monomeric CDKs (9Kaldis P. Russo A.A. Chou H.S. Pavletich N.P. Solomon M.J. Mol. Biol. Cell. 1998; 9: 2545-2560Crossref PubMed Scopus (91) Google Scholar). To prevent premature activation of Cdc2, the complex needs to be inactivated by phosphorylations on Thr-14 and Tyr-15 of Cdc2 by the ER membrane-associated Myt1 kinase (3Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (533) Google Scholar), to accumulate as pre-MPF in the cytoplasm. 2) Cyclin-free Cdc2 is a substrate of another cytoplasmic CAK. In Saccharomyces cerevisiae, the only known CAK is a cytoplasmic monomeric enzyme called Cak1 or Civ1 (10Thuret J.Y. Valay J.G. Faye G. Mann C. Cell. 1996; 86: 565-576Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 11Kaldis P. Sutton A. Solomon M.J. Cell. 1996; 86: 553-564Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 12Espinoza F.H. Farrell A. Erdjument-Bromage H. Tempst P. Morgan D.O. Science. 1996; 273: 1714-1717Crossref PubMed Scopus (146) Google Scholar). In contrast to the CDK7·cyclin H complex, it preferentially phosphorylates monomeric CDKs rather than cyclin-associated CDKs (9Kaldis P. Russo A.A. Chou H.S. Pavletich N.P. Solomon M.J. Mol. Biol. Cell. 1998; 9: 2545-2560Crossref PubMed Scopus (91) Google Scholar). Recently, a “monomeric CAK” activity has been also detected in human cells (13Nagahara H. Ezhevsky S.A. Vocero-Akbani A.M. Kaldis P. Solomon M.J. Dowdy S.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14961-14966Crossref PubMed Scopus (91) Google Scholar,14Kaldis P. Solomon M.J. Eur. J. Biochem. 2000; 267: 4213-4221Crossref PubMed Scopus (49) Google Scholar). If such an enzyme is expressed, then monomeric Cdc2 would be phosphorylated on Thr-161 in the cytoplasm prior its association with newly synthesized cyclin B, and, in the case of the growing oocyte, prior to its inactivation by the membrane-associated Myt1 kinase, leading to pre-MPF formation.During progesterone-induced meiotic maturation, the abrupt activation of pre-MPF into MPF occurs through an autoamplification process whereby the protein phosphatase Cdc25 removes the inhibitory phosphates on Thr-14 and Tyr-15 of Cdc2 (6Coleman T.R. Dunphy W.G. Curr. Opin. Cell Biol. 1994; 6: 877-882Crossref PubMed Scopus (325) Google Scholar). A two-step mechanism, involving proteins such as protein phosphatase 2A and Plx1 kinase, allows active Cdc2 to positively regulate Cdc25 (15Karaiskou A. Jessus C. Brassac T. Ozon R. J. Cell Sci. 1999; 112: 3747-3756PubMed Google Scholar). A major unanswered question is how the feedback loop between Cdc25 and Cdc2 is initiated. One possibility could be that an unstable or a neosynthetized “Cdc25-like” phosphatase, such as Cdc25B, is activated before Cdc25C and serves as a threshold for the Cdc2-Cdc25C autoamplification loop. Until now, no experimental evidence supports this hypothesis in theXenopus oocytes undergoing meiotic division. Another possibility is that, upon progesterone stimulation, a newly synthesized cyclin, or another Cdc2 partner, would associate with free Cdc2; the neoformed complex would then escape inactivating phosphorylations by Myt1 kinase and would serve as a threshold to initiate MPF autoamplification (15Karaiskou A. Jessus C. Brassac T. Ozon R. J. Cell Sci. 1999; 112: 3747-3756PubMed Google Scholar, 16Nebreda A. Gannon J. Hunt T. EMBO J. 1995; 14: 5597-5607Crossref PubMed Scopus (125) Google Scholar, 17Frank-Vaillant M. Jessus C. Ozon R. Maller J.L. Haccard O. Mol. Biol. Cell. 1999; 10: 3279-3288Crossref PubMed Scopus (76) Google Scholar). In this context, the presence of monomeric Cdc2 already phosphorylated on Thr-161 would favor the formation of this small starter amount of active MPF.The phosphorylation of Cdc2 on Thr-161 represents a potential key regulation step at two essential periods of the oocyte development; first, during late oogenesis when pre-MPF accumulates in prophase-arrested oocyte, and second, at the time preceding GVBD, when pre-MPF is activated. A major insight is to identify the enzymes, kinase and phosphatase, that control this critical event.In a first approach to understand how the Thr-161 phosphorylation of Cdc2 is regulated, we decided to undertake a biochemical purification of cyclin B-free Cdc2 and analyzed its Thr-161 phosphorylation level. The major observation of this study indicates that fully grown resting oocyte contains a significant amount of monomeric Cdc2 phosphorylated on Thr-161, whose phosphorylation level is negatively regulated by a Mg2+-dependent phosphatase, PP2C.EXPERIMENTAL PROCEDURESMaterialsXenopus laevis adult females (CNRS, Rennes, France) were bred and maintained under laboratory conditions. [γ-32P]ATP (6000 Ci/mmol, NEG502Z) was purchased from PerkinElmer Life Sciences. Reagents, unless otherwise specified, were from Sigma Chemical Co.Xenopus Oocyte ExtractsFully grown Xenopus prophase oocytes were obtained as described previously (18Jessus C. Thibier C. Ozon R. J. Cell Sci. 1987; 87: 705-712PubMed Google Scholar). For extract preparations, oocytes were lysed in 4 volumes of EB (80 mm β-glycerophosphate, 20 mm EGTA, 15 mm MgCl2, 1 mm DTT, pH 7.3) or modified EB (80 mmβ-glycerophosphate, 10 mm EDTA, 30 mm NaCl, 1 mm DTT, pH 7.3) supplemented with 10 mm ATP, 50 mm NaF, 100 μm sodium orthovanadate, and protease inhibitor mixture (Sigma P8340). Lysates were centrifuged at 100,000 × g, and the supernatants were recovered and termed “cytosolic extracts” or “S100.” Proteins of cytosolic extracts were precipitated by salting out using ammonium sulfate, successively 40 and 60%, as described in a previous study (15Karaiskou A. Jessus C. Brassac T. Ozon R. J. Cell Sci. 1999; 112: 3747-3756PubMed Google Scholar). Ammonium sulfate pellets, respectively, P40 and P60, were stored at −80 °C for further analysis.Gel FiltrationP40 and P60 precipitates from 200 oocytes were resuspended in 160 μl of column buffer (EB or modified EB, adjusted to 0.1m NaCl) and then chromatographed on a Superose 12 gel filtration column (Amersham Biosciences) at 0.5 ml/min. Ten fractions of 1 ml were collected and subject to Western blot and kinase and phosphatase assays.ImmunoblottingProteins were separated on 12% SDS-PAGE (Amresco) and transferred to nitrocellulose filters (Schleicher and Schuell). Anti-Xenopus cyclin B2 and cyclin B1 antibodies were obtained from goats immunized with inclusion bodies containing bacterially expressed Xenopus cyclins B2 and B1 and affinity-purified. The monoclonal mouse anti-Xenopus Cdc2 antibodies (mixture of A17 and 3E1) were initially described in a previous study (19Kobayashi H. Stewart E. Poon R.Y.C. Hunt T. J. Biol. Chem. 1994; 269: 29153-29160Abstract Full Text PDF PubMed Google Scholar). The anti-MO15 and anti-Cak1 polyclonal rabbit antibodies were described previously (Refs. 8Fesquet D. Labbe J.C. Derancourt J. Capony J.P. Galas S. Girard F. Lorca T. Shuttleworth J. Doree M. Cavadore J.C. EMBO J. 1993; 12: 3111-3121Crossref PubMed Scopus (325) Google Scholar and 10Thuret J.Y. Valay J.G. Faye G. Mann C. Cell. 1996; 86: 565-576Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, respectively). Polyclonal rabbit anti-phospho-Cdc2 (Tyr-15) and anti-phospho-Cdc2 (Thr-161) antibodies were purchased from Cell Signaling Technology, and polyclonal rabbit anti-PSTAIR antibody and polyclonal sheep anti-human PP2Cα were purchased from Upstate Biotechnology. The primary antibodies were detected with appropriated horseradish peroxidase-conjugated second antibodies (Jackson ImmunoResearch laboratories) and the Western blot Chemiluminescence Renaissance kit from PerkinElmer Life Sciences.Cdc2 Activation and Cdk2 PhosphorylationRecombinant GST-Cdc2GSH-Sepharose beads bound to purified and refolded GST-Cdc2 were washed in kinase buffer (50 mmTris-HCl, pH 7.2, 1 mm DTT, 15 mmMgCl2, 5 mm EGTA) and then incubated for 30 min at 30 °C in kinase buffer in the presence of 100 μmATP and various effectors: Cak1 (0.06 μg/μl), GST-cyclin A (0.1–0.2 μg/μl) or His-cyclin B1 (0.1 μg/μl). For histone H1 kinase assay, 0.2 mg/ml histone H1 (Roche Diagnostics) and 1 μCi of [γ-32P]ATP were added for a further 15 or 30 min at 30 °C. The reaction was stopped by adding Laemmli buffer (20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205998) Google Scholar) and by boiling for 3 min.Endogenous Monomeric Cdc2Ammonium sulfate was removed from P40 and P60 by ultrafiltration with an Ultrafree Biomax system (Millipore). The amount of proteins recovered in each fraction was evaluated by Bradford analysis (21Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213288) Google Scholar). One oocyte corresponds to 24, 8, 12, and 1.2 μg of proteins, respectively, in S100, P40, P60, and F9. Activation of endogenous Cdc2 present in P60 or F9 was performed under the same conditions as for recombinant Cdc2, by adding Cak1 (0.06 μg/μl), GST-cyclin A (0.2 μg/μl), or His-cyclin B1 (0.1 μg/μl). In some experiments, GST·cyclin A·Cdc2 complexes were recovered by GST-cyclin A binding for 4 h at 4 °C on GSH-agarose beads. After several washes in EB (modified or not) or in kinase buffer, the bead pellets were, respectively, submitted to Western blot analysis or histone H1 kinase assay.Recombinant GST-Cdk2Phosphorylation of GST-Cdk2 was performed by incubating the protein (0.1 μg/μl) for 1 h at 30 °C in the presence of Cak1 (0.06 μg/μl) in kinase buffer containing 10 μm ATP and 1 μCi of [γ-32P]ATP. GST-Cdk2 was then ultrafiltrated on a Microcon system (Millipore) to eliminate free [γ-32P]ATP or purified on GSH-agarose beads for 4 h at 4 °C. In some experiments, incubation was performed in the presence of various concentrations of P60 and F9. After GST pull-down, pellets were washed, resuspended in sample buffer, and heated at 100 °C for 3 min, and proteins were separated on 12% SDS-PAGE. The radioactivity incorporated in GST-Cdk2 was revealed by autoradiography and counted after excision from the gel in a Wallac counter.Phosphatase AssaySubstrate Preparation for PP2C Isolation AssayCasein (Sigma C4765, 5 mg) was phosphorylated by 250 milliunits of the catalytic subunit of PKA (Sigma P2645) for 2 h at 30 °C in the presence of 100 μm ATP and 250 μCi of [γ-32P]ATP. GST-Cdk2 (1 mg) was phosphorylated by Cak1 (15 μg) for 16 h at 30 °C in the presence of 200 μm ATP and 500 μCi of [γ-32P]ATP. Reactions were stopped by addition of 10 mm EDTA, 30 mm NaF, and 2 mm pyrophosphate. Proteins were then precipitated twice at 0 °C with an equal volume of 90% saturated ammonium sulfate solution. Free nucleotides were removed by chromatography on Sephadex G-25 (Amersham Biosciences).Phosphatase Reaction32P-Phosphorylated GST-Cdk2 or 32P-phosphorylated casein was incubated for 20 min at 30 °C in the presence of either F9 (1 μg of proteins), recombinant Xenopus PP2C, or fractions from the purification procedure in the presence of bovine serum albumin (5 μg) and various amounts of Mg2+ and OA. Reactions were stopped by addition of 10 volumes of 20% trichloroacetic acid, centrifuged for 5 min, and the released 32P label was counted.Xenopus PP2C PurificationPhosphatase activity was determined using casein phosphorylated by PKA and Cdk2 phosphorylated by Cak1 as substrates. The selected fractions contain a phosphatase activity toward both substrates that is dependent on Mg2+ and insensitive to 1 μmokadaic acid. The entire purification procedure was carried out at 4 °C. Ovaries from 30 females were homogenized in 3 volumes of the following buffer: 50 mm Tris-HCl (pH 7.5), 2 mmEGTA, 2 mm EDTA, 0.1% β-mercaptoethanol, 1 mm 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride (Pentapharm), 1 mm benzamidine. The lysate was centrifuged at 10,000 × g for 20 min, and the supernatant was filtered through glass wool and centrifuged again at 21,000 × g for 3 h, leading to a cytosolic extract. P40 and P60 were then prepared (15Karaiskou A. Jessus C. Brassac T. Ozon R. J. Cell Sci. 1999; 112: 3747-3756PubMed Google Scholar). 30–40% of PP2C was recovered in the P40 while 60–70% was recovered in P60, as estimated by Western blot signal. The P60 fraction was resuspended in 1 liter of buffer A (25 mm Tris-HCl (pH 7.5), 1 mm EGTA, 1 mm EDTA, 0.1% β-mercaptoethanol) and mixed with 500 ml of DEAE-Sepharose Fast-Flow resin equilibrated in buffer A. Fractions were eluted by steps in buffer A containing increasing NaCl concentrations (100 mm, 250 mm, 500 mm, 750 mm, and 1 m). The active fraction, eluted in 500 mm NaCl, was concentrated on Centricon Plus-80 (Amicon) and loaded on a Sephacryl S200 column equilibrated in buffer A plus 150 mm NaCl. The active fraction was desalted on Centricon Plus-80 and then loaded on a UnoQ column (Bio-Rad) equilibrated in buffer A. Proteins were eluted with a linear gradient from 0 to 1 m NaCl in buffer A. PP2C activity was eluted around 500 mm NaCl. NaCl was increased up to 3 m in the active fraction, which was then loaded on a phenyl-Superose HR5/5 column (Amersham Biosciences) equilibrated in buffer A plus 3 m NaCl. Proteins were eluted with a linear gradient from 3 to 0 m NaCl in buffer A, and the phosphatase activity was recovered at around 750 mm NaCl. The active fraction was desalted on Centricon Plus-80 with buffer A, then supplemented with 25 mm MgCl2 and loaded on a 5-ml Hi-Trap Blue column (Amersham Biosciences) equilibrated with buffer A plus 25 mm MgCl2. Proteins were eluted with a linear gradient from 25 to 0 mm MgCl2 in buffer A, and the active fraction was recovered at 13 mmMgCl2. After concentration by dialysis against 10 mm Tris-HCl, pH 7.5, 50 mm NaCl, 0.1% β-mercaptoethanol, and 20% polyethylene glycol 40,000, the active fraction was stored at −80 °C.Xenopus PP2Cα CloningBased on the sequences dd98g09.x1 (December 2000) and dc59g09.y1 (September 2000) of two X. laevis expressed sequence tag cDNA clones (IMAGE (National Institutes of Health): 3436624 and 3401440) homologous to the human protein phosphatase 2Cα, two oligoprimers containing BglII sites (underlined) were designed: 5′-GAA GAT CTC ATG GGA GCA TTT TTA GAT AAG CC-3′ (corresponding to the amino-terminal part of the protein and used as upstream primer), and 5′-GAA GAT CTC TTA CCA CAT ATC ATC TGT TGA TGC-3′ (corresponding to the carboxyl-terminal part of the protein and used as downstream primer). PCR was performed with a mix (50/50) of Pfu DNA polymerase and Taq DNA polymerase (Promega, #M7741 and #M2661) using a cDNA library fromX. laevis oocytes (λ ZAP Express phages, kind gift of Dr. J. Maller). The amplified PCR product was subcloned in the pGEM-T easy vector, and the cDNA sequences were determined on automated DNA Sequencer ALF-express (Amersham Biosciences) with a Thermo Sequenase CY5 Dye Terminator kit (Amersham Pharmacia) using T3- and T7-CY5 primers. The entire encoding nucleotide sequence has been deposited at the EMBL nucleotide sequence data base under the accession numberAJ438209.Preparation of Recombinant ProteinsGST- and His-tagged recombinant proteins were expressed and purified as described in a previous study (19Kobayashi H. Stewart E. Poon R.Y.C. Hunt T. J. Biol. Chem. 1994; 269: 29153-29160Abstract Full Text PDF PubMed Google Scholar), using the following plasmids: human GST-cyclin A (kind gift of Dr. C. Bréchot, INSERM, France), Xenopus GST-Cdk2, Xenopus wild type GST-Cdc2, and Xenopus Thr-161 → Ala mutant GST-Cdc2 (kind gifts of Dr. T. Hunt, Imperial Cancer Research Fund, UK).S. cerevisiae His-Cak1 protein and human His-cyclin B1 protein were kindly provided by Dr. C. Mann (Commissariată l'Energie Aromique, Saclay, France) and Dr. B. Ducommun (CNRS, Toulouse, France), respectively.Bacterially produced Xenopus GST-Cdc2 is inactive and requires a refolding step (19Kobayashi H. Stewart E. Poon R.Y.C. Hunt T. J. Biol. Chem. 1994; 269: 29153-29160Abstract Full Text PDF PubMed Google Scholar, 22Poon R.Y.C. Yamashita K. Adamczewski J.P. Hunt T. Shuttleworth J. EMBO J. 1993; 12: 3123-3132Crossref PubMed Scopus (330) Google Scholar). This was performed by incubating 1 μg of wild type or Thr-161 → Ala mutant GST-Cdc2 in 2.5 μl of prophase oocyte extracts, prepared as described previously (23Murray A.W. Methods Cell Biol. 1991; 36: 581-605Crossref PubMed Scopus (800) Google Scholar), for 30 min at room temperature. After refolding, GST-Cdc2 was isolated using GSH-Sepharose beads (Amersham Biosciences).Xenopus PP2Cα cDNA was cloned into the expression vector pThioHisB (Invitrogen). Expression of recombinant ThioHis-PP2C was induced with 0.5 mmisopropylthio-β-d-galactoside. The bacterial pellet was lysed in 20 mm NaH2PO4, 500 mm NaCl, N-octylglucoside (0.5% v/v), 1% (v/v) protease inhibitor mixture (Sigma P8340) and centrifuged (10 min, 14,000 × g, 4 °C). The supernatant was chromatographed on a nickel column (Probond, Invitrogen), and the imidazole eluate was chromatographed on a phenyl arsine oxide-agarose column (Thiobond, Invitrogen). Step-elution was performed with β-mercaptoethanol from 50 mm to 1 m. Fractions of interest were dialyzed and concentrated in 10 mm Tris-HCl, pH 7.5, 50 mm NaCl, and stored at −80 °C before use.DISCUSSIONMPF or Cdc2 protein kinase drives Xenopus oocyte meiotic maturation. It is regulated by the availability of cyclin subunits and phosphorylation/dephosphorylation reactions. The activating phosphorylation on Thr-161 within the T-loop is required for kinase activity (5Norbury C. Blow J. Nurse P. EMBO J. 1991; 10: 3321-3329Crossref PubMed Scopus (397) Google Scholar) and should be regulated at different critical phases of oogenesis: first, during pre-MPF accumulation occurring during the last period of oocyte growth; second at the entry into metaphase I (or GVBD) when MPF is first activated; and third, during the metaphase I-metaphase II transition when cyclin turnover occurs. Does the addition of a phosphate to Thr-161 of Cdc2 precede or follow cyclin binding at each of these steps? Our results show that a significant fraction of monomeric Cdc2 partially purified in a Mg2+-free buffer from prophase resting oocytes is directly activable in vitro by cyclin addition. This result indirectly suggests that Thr-161-phosphorylated free Cdc2 is present in the oocyte. We directly evidenced the presence of this phosphorylated form of Cdc2 by using an antibody specifically directed against Thr-161-phosphorylated Cdc2. This antibody is able to recognize monomeric Cdc2, only when prepared in the absence of Mg2+. To ascertain the specificity of the antibody, recombinantXenopus Cdk2 or partially purified Xenopusmonomeric Cdc2 (F9) were phosphorylated in vitro on Thr-160 or Thr-161, respectively, by recombinant Cak1. Western blots illustrated in Figs. 6 and 7 clearly demonstrate the high detection specificity of the phosphorylated activating Thr of the T-loop of Cdk2 and Cdc2 by this antibody. Therefore, through two distinct experimental approaches, it is possible to conclude that monomeric Thr-161-phosphorylated Cdc2 is present in the oocyte where it represents a latent form of Cdc2 directly activable by cyclin binding. Consequently, one might postulate that Xenopus oocyte contains the enzymes that control the addition and removal of phosphate on residue Thr-161 of monomeric Cdc2. Mg2+ chelation in purification buffers is required for the immunodetection of phosphorylated monomeric Cdc2, which appears to be directly activable by cyclin binding. This prompted us to search for the presence of a Mg2+-dependent protein phosphatase specific of Thr-161-phosphorylated Cdc2 in the Xenopus oocyte. A partial purification led to the isolation of a 45-kDa Mg2+-dependent phosphatase, which is recognized by an antibody raised against human PP2Cα and exhibits the enzymatic properties of PP2C. This purified enzyme was able to dephosphorylate recombinant Thr-160-phosphorylated Cdk2 as well as Thr-161-phosphorylated Xenopus Cdc2. After bacterial expression, the recombinant Xenopus PP2Cα similarly possesses a Mg2+-dependent phosphatase activity able to dephosphorylate the Thr-161 residue of monomeric Cdc2. When cyclins are added to prephosphorylated Cdc2, PP2C is not able to dephosphorylate Thr-161 anymore, indicating that cyclin·Cdc2 complex is not a PP2C substrate.In a previous study, Poon and Hunter (30Poon R.Y. Hunter T. Science. 1995; 270: 90-93Crossref PubMed Scopus (149) Google Scholar) reported that an EDTA-treated extract prepared from Xenopus eggs contains a “KAP”-like activity that dephosphorylates monomeric Thr-160-phosphorylated recombinant Cdk2. This phosphatase activity present in egg extracts has not been further characterized. Under our experimental conditions, no KAP-like phosphatase could be found in extracts from prophase oocytes prepared in the presence of EDTA. An intriguing possibility, which remains to be experimentally explored, could be that a KAP-like phosphatase activity, absent or inactive in prophase oocyte, is neosynthetized or unmasked during meiotic maturation.The copurification of a PP2C-like phosphatase with monomeric Cdc2 explains why Thr-161-phosphorylated Cdc2 had not been previously identified in Xenopus oocytes. Indeed, the standard EB buffer used to isolate pre-MPF or MPF contains a high Mg2+concentration (15 mm) (24Wu M. Gerhart J.C. Dev. Biol. 1980; 79: 465-477Crossref PubMed Scopus (150) Google Scholar), allowing full activity of Mg2+-dependent phosphatases and leading consequently to dephosphorylation of Cdc2.Solomon and co-workers (31Cheng A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2946-2957Crossref PubMed Scopus (127) Google Scholar) identified genetically and biochemically Ptc2p and Ptc3p in S. cerevisiae as the two major type 2C phosphatases that dephosphorylate monomeric CDC28. Therefore, PP2C physically opposes the biological functions of monomeric Cak1 in budding yeast. Human HeLa cells also contain two PP2C isoforms, PP2Cα and β2, that dephosphorylate monomeric human Cdk2/Cdk6 in vitro (32Cheng A. Kaldis P. Solomon M.J. J. Biol. Chem. 2000; 275: 34744-34749Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). These new observations raise, by analogy, the possibility that phosphorylated monomeric Cdc2 isolated fromXenopus oocyte could also be regulated by a monomeric CAK and PP2C. Whereas our results establish that a phosphatase 2C catalyzes the removal of phosphate on Thr-161/Thr-160 of Cdc2/Cdk2, it is at present uncertain whether the Xenopus oocyte contains an enzyme that catalyzes the phosphorylation of Thr-161 in monomeric Cdc2. Identification of monomeric Cdc2 phosphorylated on Thr-161 together with the low affinity of CDK7·cyclin H enzyme for monomeric CDKs (9Kaldis P. Russo A.A. Chou H.S. Pavletich N.P. Solomon M.J. Mol. Biol. Cell. 1998; 9: 2545-2560Crossref PubMed Scopus (91) Google Scholar) favors the view that such an enzyme would be present and functional in the oocyte. A difficulty encountered for the purification of this putative kinase is to inhibit or to remove the Mg2+-dependent phosphatase activity that opposes to this kinase activity.Our results show for the first time that monomeric Thr-161-phosphorylated Cdc2 can be isolated from Xenopusextracts and that it is a substrate of an endogenous PP2C. A specific regulation, implying the Thr-161 kinase and/or PP2C, allows the presence of two monomeric Cdc2 subpopulations in the oocyte, one being phosphorylated on Thr-161 and directly activable by cyclin binding while the other one is not. These results have important physiological implications. Of particular interest is the possible role of phosphorylated monomeric Cdc2 in the initiation of the MPF autoamplification loop. A small increase in cyclin B availability might be sufficient to bind with and to activate Cdc2 already phosphorylated on Thr-161 and then to generate a threshold Cdc2 kinase activity able to trigger MPF autoamplification. A recent study, using an antisense strategy, reported that the synthesis of cyclins B1, B2, B4, and B5 is not required in ovo for the initiation of MPF amplification during oocyte maturation (2Hochegger H. Klotzbucher A. Kirk J. Howell M. le Guellec K. Fletcher K. Duncan T. Sohail M. Hunt T. Development. 2001; 128: 3795-3807PubMed Google Scholar). It cannot be excluded, however, that beyond cyclins B, another cyclin or an unknown partner of Thr-161-phosphorylated monomeric Cdc2 could be involved in the switching on of its kinase activity. A major objective will be to determine the levels of Thr-161-phosphorylated monomeric Cdc2 and to study how the phosphatase 2C and its opposed kinase are subject to regulation during the whole meiotic maturation process. The fully grown Xenopus oocyte is phys" @default.
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