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• W2036851282 abstract "Xenopus oocytes are arrested in meiotic prophase I and resume meiotic divisions in response to progesterone. Progesterone triggers activation of M-phase promoting factor (MPF) or Cdc2-cyclin B complex and neosynthesis of Mos kinase, responsible for MAPK activation. Both Cdc2 and MAPK activities are required for the success of meiotic maturation. However, the signaling pathway induced by progesterone and leading to MPF activation is poorly understood, and most of the targets of both Cdc2 and MAPK in the oocyte remain to be determined. Aurora-A is a Ser/Thr kinase involved in separation of centrosomes and in spindle assembly during mitosis. It has been proposed that in Xenopus oocytes Aurora-A could be an early component of the progesterone-transduction pathway, acting through the regulation of Mos synthesis upstream Cdc2 activation. We addressed here the question of Aurora-A regulation during meiotic maturation by using new in vitro and in vivo experimental approaches. We demonstrate that Cdc2 kinase activity is necessary and sufficient to trigger both Aurora-A phosphorylation and kinase activation in Xenopus oocyte. In contrast, these events are independent of the Mos/MAPK pathway. Aurora-A is phosphorylated in vivo at least on three residues that regulate differentially its kinase activity. Therefore, Aurora-A is under the control of Cdc2 in the Xenopus oocyte and could be involved in meiotic spindle establishment. Xenopus oocytes are arrested in meiotic prophase I and resume meiotic divisions in response to progesterone. Progesterone triggers activation of M-phase promoting factor (MPF) or Cdc2-cyclin B complex and neosynthesis of Mos kinase, responsible for MAPK activation. Both Cdc2 and MAPK activities are required for the success of meiotic maturation. However, the signaling pathway induced by progesterone and leading to MPF activation is poorly understood, and most of the targets of both Cdc2 and MAPK in the oocyte remain to be determined. Aurora-A is a Ser/Thr kinase involved in separation of centrosomes and in spindle assembly during mitosis. It has been proposed that in Xenopus oocytes Aurora-A could be an early component of the progesterone-transduction pathway, acting through the regulation of Mos synthesis upstream Cdc2 activation. We addressed here the question of Aurora-A regulation during meiotic maturation by using new in vitro and in vivo experimental approaches. We demonstrate that Cdc2 kinase activity is necessary and sufficient to trigger both Aurora-A phosphorylation and kinase activation in Xenopus oocyte. In contrast, these events are independent of the Mos/MAPK pathway. Aurora-A is phosphorylated in vivo at least on three residues that regulate differentially its kinase activity. Therefore, Aurora-A is under the control of Cdc2 in the Xenopus oocyte and could be involved in meiotic spindle establishment. Amphibian oocyte meiotic maturation is a pioneer model system to study an unconventional post-transcriptional pathway induced by a steroid hormone, progesterone, and the regulation of M-phase promoting factor (MPF) 1The abbreviations used are: MPF, M-phase promoting factor; Cdc, cell division cycle; GVBD, germinal vesicle breakdown; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; PP, protein phosphatase; GST, glutathione S-transferase; CHX, cycloheximide; IBMX, 3-isobutyl-1-methylxanthine; OA, okadaic acid; WT, wild type. or Cdc2-cyclin B complex, the universal inducer of mitotic and meiotic divisions (1Karaiskou A. Dupre A. Haccard O. Jessus C. Biol. Cell. 2001; 93: 35-46Crossref PubMed Scopus (39) Google Scholar). Xenopus oocytes are arrested in prophase of the first meiotic division, assimilated to the G2 arrest of the cell cycle. When stimulated by progesterone, they resume meiotic division through the activation of MPF. The signaling pathway induced by progesterone and leading to MPF activation is not well understood (1Karaiskou A. Dupre A. Haccard O. Jessus C. Biol. Cell. 2001; 93: 35-46Crossref PubMed Scopus (39) Google Scholar). It involves a drop in cAMP, induced within minutes after progesterone addition, leading to the inhibition of the cAMP-dependent protein kinase, protein kinase A (2Maller J.L. Krebs E.G. J. Biol. Chem. 1977; 252: 1712-1718Abstract Full Text PDF PubMed Google Scholar). Protein kinase A down-regulation could stimulate the synthesis of new proteins, a process required to activate Cdc2-cyclin B complex (3Jessus C. Ozon R. Comp. Biochem. Physiol. 1993; 106A: 431-448Crossref Scopus (24) Google Scholar). The identification of the one or more proteins whose synthesis is needed for Cdc2 activation represents a major challenge. Mos kinase is synthesized and activated simultaneously with that of MPF activation and indirectly induces MAPK activation (4Sagata N. Oskarsson M. Copeland T. Brumbaugh J. Vande Woude G.F. Nature. 1988; 335: 519-525Crossref PubMed Scopus (463) Google Scholar). It has been recently proposed that Mos synthesis and MAPK activation are not required to induce Cdc2-cyclin B activation in Xenopus oocyte; however, they most probably facilitate this process (5Dupre A. Jessus C. Ozon R. Haccard O. EMBO J. 2002; 21: 4026-4036Crossref PubMed Scopus (91) Google Scholar, 6Fisher D.L. Brassac T. Galas S. Doree M. Development. 1999; 126: 4537-4546PubMed Google Scholar, 7Gross S.D. Schwab M.S. Taieb F.E. Lewellyn A.L. Qian Y.W. Maller J.L. Curr. Biol. 2000; 10: 430-438Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Thus, one or more other proteins, not identified yet, must be synthesized in response to progesterone to allow Cdc2-cyclin B activation. Recently, the Aurora kinases emerged as a new family of mitotic Ser/Thr kinases conserved from yeast to humans (8Chan C.S. Botstein D. Genetics. 1993; 135: 677-691Crossref PubMed Google Scholar, 9Glover D.M. Leibowitz M.H. McLean D.A. Parry H. Cell. 1995; 81: 95-105Abstract Full Text PDF PubMed Scopus (700) Google Scholar, 10Schumacher J.M. Ashcroft N. Donovan P.J. Golden A. Development. 1998; 125: 4391-4402Crossref PubMed Google Scholar, 11Roghi C. Giet R. Uzbekov R. Morin N. Chartrain I. LeGuellec R. Couturier A. Doree M. Philippe M. Prigent C. J. Cell Sci. 1998; 111: 557-572Crossref PubMed Google Scholar, 12Gopalan G. Chan C.S. Donovan P.J. J. Cell Biol. 1997; 138: 643-656Crossref PubMed Scopus (117) Google Scholar, 13Kimura M. Kotani S. Hattori T. Sumi N. Yoshioka T. Todokoro K. Okano Y. J. Biol. Chem. 1997; 272: 13766-13771Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). The vertebrate Aurora family is composed of three members, termed Aurora-A, -B, and -C (14Nigg E.A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 21-32Crossref PubMed Scopus (1259) Google Scholar). Aurora-A localizes to the centrosomes and the spindle poles during mitosis (11Roghi C. Giet R. Uzbekov R. Morin N. Chartrain I. LeGuellec R. Couturier A. Doree M. Philippe M. Prigent C. J. Cell Sci. 1998; 111: 557-572Crossref PubMed Google Scholar). Ablation or inactivation of Aurora-A in Caenorhabditis elegans embryos and mammalian culture cells prevents centrosome maturation and spindle assembly, showing a role of Aurora-A in centrosome separation and bipolar establishment of the spindle (9Glover D.M. Leibowitz M.H. McLean D.A. Parry H. Cell. 1995; 81: 95-105Abstract Full Text PDF PubMed Scopus (700) Google Scholar, 15Dutertre S. Descamps S. Prigent C. Oncogene. 2002; 21: 6175-6183Crossref PubMed Scopus (220) Google Scholar, 16Hannak E. Kirkham M. Hyman A.A. Oegema K. J. Cell Biol. 2001; 155: 1109-1116Crossref PubMed Scopus (369) Google Scholar). It has also been demonstrated that, in Xenopus, one substrate of Aurora-A could be the kinesin-related motor Eg5, involved in centrosome separation (17Giet R. Uzbekov R. Cubizolles F. Le Guellec K. Prigent C. J. Biol. Chem. 1999; 274: 15005-15013Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Given the regulation of spindle function by Aurora-A (11Roghi C. Giet R. Uzbekov R. Morin N. Chartrain I. LeGuellec R. Couturier A. Doree M. Philippe M. Prigent C. J. Cell Sci. 1998; 111: 557-572Crossref PubMed Google Scholar, 18Giet R. Prigent C. J. Cell Sci. 1999; 112: 3591-3601Crossref PubMed Google Scholar), it could be proposed that this kinase plays a role downstream MPF activation in the oocyte, as described in human mitotic cell lines (19Marumoto T. Hirota T. Morisaki T. Kunitoku N. Zhang D. Ichikawa Y. Sasayama T. Kuninaka S. Mimori T. Tamaki N. Kimura M. Okano Y. Saya H. Genes Cells. 2002; 7: 1173-1182Crossref PubMed Scopus (189) Google Scholar). Xenopus Aurora-A has been cloned and originally termed “Eg2” in a screen designed to identify mRNAs that are polyadenylated during meiotic maturation and dead-enylated at fertilization (11Roghi C. Giet R. Uzbekov R. Morin N. Chartrain I. LeGuellec R. Couturier A. Doree M. Philippe M. Prigent C. J. Cell Sci. 1998; 111: 557-572Crossref PubMed Google Scholar, 20Paris J. Le Guellec R. Couturier A. Le Guellec K. Omilli F. Camonis J. Macneill S. Philippe M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1039-1043Crossref PubMed Scopus (141) Google Scholar, 21Bouvet P. Omilli F. Arlot-Bonnemains Y. Legagneux V. Roghi C. Bassez T. Osborne H.B. Mol. Cell. Biol. 1994; 14: 1893-1900Crossref PubMed Scopus (59) Google Scholar). Aurora-A protein accumulates during meiotic maturation, in response to progesterone independently of MPF activation, but its kinase activity would be under the control of MPF (22Frank-Vaillant M. Haccard O. Thibier C. Ozon R. Arlot-Bonnemains Y. Prigent C. Jessus C. J. Cell Sci. 2000; 113: 1127-1138Crossref PubMed Google Scholar). The inhibition of Aurora-A by microinjection of a specific antibody in Xenopus oocytes neither affects nuclear envelope breakdown (GVBD, for germinal vesicle breakdown) nor Cdc2 activation. However, oocytes arrest in metaphase I without extruding the first polar body (23Castro A. Mandart E. Lorca T. Galas S. J. Biol. Chem. 2003; 278: 2236-2241Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). This suggests again that Aurora-A functions downstream of MPF activation and controls the meiotic spindle. However, Andrésson and Ruderman (24Andresson T. Ruderman J.V. EMBO J. 1998; 17: 5627-5637Crossref PubMed Scopus (122) Google Scholar) cloned Aurora-A cDNA using a screen designed to identify oocyte proteins whose electrophoretic migration is retarded within 30 min after progesterone stimulation. Moreover, they reported that Aurora-A overexpression accelerates GVBD in response to progesterone (24Andresson T. Ruderman J.V. EMBO J. 1998; 17: 5627-5637Crossref PubMed Scopus (122) Google Scholar). It was then shown in Xenopus oocyte that Aurora-A phosphorylates and regulates CPEB, a protein that binds the CPE sequence present in the 3′ non-coding region of several mRNAs, including Mos mRNA, and allows their transduction (25Mendez R. Hake L.E. Andresson T. Littlepage L.E. Ruderman J.V. Richter J.D. Nature. 2000; 404: 302-307Crossref PubMed Scopus (294) Google Scholar). Therefore, Aurora-A could be an early component of the progesterone pathway stimulating Mos synthesis upstream of Cdc2 activation. Because the Mos/MAPK pathway appears to be dispensable for Cdc2 activation (5Dupre A. Jessus C. Ozon R. Haccard O. EMBO J. 2002; 21: 4026-4036Crossref PubMed Scopus (91) Google Scholar, 6Fisher D.L. Brassac T. Galas S. Doree M. Development. 1999; 126: 4537-4546PubMed Google Scholar, 7Gross S.D. Schwab M.S. Taieb F.E. Lewellyn A.L. Qian Y.W. Maller J.L. Curr. Biol. 2000; 10: 430-438Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), it is difficult to assume that the physiological function of Aurora-A would be restricted to Cdc2 activation. Whether Aurora-A functions upstream or downstream of Cdc2 is important to further understand the regulation of the meiotic process. In this study, we addressed two main questions. First, is Aurora-A kinase activation physiologically controlled by MPF, or by the Mos/MAPK pathway, or by an upstream regulator of MPF? Second, what is the molecular basis of this activation? Several reports have shown that Aurora-A activation correlates with the phosphorylation of several of its residues (26Littlepage L.E. Wu H. Andresson T. Deanehan J.K. Amundadottir L.T. Ruderman J.V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15440-15445Crossref PubMed Scopus (183) Google Scholar). However, the exact role of these phosphorylations remains unclear. Some of them could target the protein to the degradation pathway, others could play a docking function, while a third category could function as activators (19Marumoto T. Hirota T. Morisaki T. Kunitoku N. Zhang D. Ichikawa Y. Sasayama T. Kuninaka S. Mimori T. Tamaki N. Kimura M. Okano Y. Saya H. Genes Cells. 2002; 7: 1173-1182Crossref PubMed Scopus (189) Google Scholar, 26Littlepage L.E. Wu H. Andresson T. Deanehan J.K. Amundadottir L.T. Ruderman J.V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15440-15445Crossref PubMed Scopus (183) Google Scholar, 27Littlepage L.E. Ruderman J.V. Genes Dev. 2002; 16: 2274-2285Crossref PubMed Scopus (237) Google Scholar). To answer these questions, we took advantage of a new in vitro substrate, histone H3, that allowed us to assay Aurora-A kinase activity in a reliable manner. We demonstrate that, in vivo during oocyte maturation as well as in vitro using prophase cytosolic extracts, active MPF is necessary and sufficient to induce Aurora-A phosphorylation and activation, independently of the Mos/MAPK pathway. Material—Xenopus laevis adult females (Horst Kähler, Germany) were bred and maintained under laboratory conditions. Reagents, unless otherwise specified, were from Sigma. Purification of Recombinant Proteins—The 21 N-terminal amino acids of either wild type histone H3 (WT H3) or histone H3 mutant where Ser10 was replaced by Ala (S10A H3) fused with GST were cloned into pGEX vector (28Scrittori L. Hans F. Angelov D. Charra M. Prigent C. Dimitrov S. J. Biol. Chem. 2001; 276: 30002-30010Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). WT H3 and S10A H3 fusion proteins were expressed in bacteria and purified using glutathione-agarose beads (Amersham Biosciences). Recombinant GST-p21cip1, sea urchin cyclin B, and GSTCdc25A were expressed in bacteria and purified as described, respectively, in Refs. 29Frank-Vaillant M. Jessus C. Ozon R. Maller J.L. Haccard O. Mol. Biol. Cell. 1999; 10: 3279-3288Crossref PubMed Scopus (77) Google Scholar, 30Lorca T. Devault A. Colas P. Van L.A. Fesquet D. Lazaro J.B. Doree M. FEBS Lett. 1992; 306: 90-93Crossref PubMed Scopus (38) Google Scholar, 31Karaiskou A. Cayla X. Haccard O. Jessus C. Ozon R. Exp. Cell Res. 1998; 244: 491-500Crossref PubMed Scopus (53) Google Scholar. Xenopus Oocyte Treatments and Homogenization—Fully grown Xenopus oocytes were isolated and prepared as described before (32Jessus C. Thibier C. Ozon R. J. Cell Sci. 1987; 87: 705-712PubMed Google Scholar). Oocytes were injected with various proteins: recombinant GST-p21cip1 at 40 ng per oocyte, recombinant Cdc25A at 25 ng per oocyte, sea urchin cyclin B at 5 ng per oocyte, okadaic acid at 5 × 10–6m intracellular concentration, and morpholino antisense oligonucleotides (Gene Tools LLC) raised against c-Mos mRNA at 100 ng per oocyte (5Dupre A. Jessus C. Ozon R. Haccard O. EMBO J. 2002; 21: 4026-4036Crossref PubMed Scopus (91) Google Scholar). Oocytes were also incubated in the presence of various reagents: 1 μm progesterone, 100 μg/ml cycloheximide (CHX), 1 mm 3-isobutyl-1-methylxanthine (IBMX), or 50 μm U0126 (Promega). GVBD was monitored by the appearance of a white spot at the animal pole. Oocytes were referred as to “metaphase II-arrested oocytes” when they were collected at least 2 h after GVBD. Activation of metaphase II-arrested oocytes was induced by electric shock (33Karsenti E. Newport J. Hubble R. Kirschner M. J. Cell Biol. 1984; 98: 1730-1745Crossref PubMed Scopus (149) Google Scholar). Oocytes were collected by groups of 10 or 20, and lysed at 4 °C in 4 volumes of EB (80 mm β-glycerophosphate, pH 7.3, 20 mm EGTA, 15 mm MgCl2, 1 mm dithiothreitol), supplemented with protease inhibitor mixture (Sigma, P8340) and 1 μm okadaic acid (ICN). Lysates were centrifuged at 15,000 × g at 4 °C for 15 min and frozen at –80 °C. Western blot analysis and kinase activity assays were then performed by using the same lysate. In Vitro Extracts—Xenopus prophase oocyte lysates prepared in the absence of okadaic acid were incubated at 30 °C in the presence of an ATP-regenerating system (10 mm creatine phosphate, 80 μg/ml creatine phosphokinase, 1 mm ATP, 1 mm MgCl2) and either 1 μm okadaic acid (ICN) or 4 μg/ml recombinant Cdc25A. Samples were collected at indicated times for Western blot analysis and kinase assays. Western Blotting—Samples equivalent to two oocytes were electrophoresed on 12.5% SDS-PAGE Anderson system (34Anderson C.W. Baum P.R. Gesteland R.F. J. Virol. 1973; 12: 241-252Crossref PubMed Google Scholar) and transferred to nitrocellulose filters (Schleicher and Schuell) using a semi-dry blotting system (Millipore). The rabbit anti-Xenopus Aurora-A polyclonal antibody and the polyclonal antibody raised against Xenopus cyclin B2 were described previously in Refs. 35Castro A. Arlot-Bonnemains Y. Vigneron S. Labbe J.C. Prigent C. Lorca T. EMBO Rep. 2002; 3: 457-462Crossref PubMed Scopus (128) Google Scholar and 36De Smedt V. Poulhe R. Cayla X. Dessauge F. Karaiskou A. Jessus C. Ozon R. J. Biol. Chem. 2002; 277: 28592-28600Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, respectively. The mouse monoclonal antibody directed against the active phosphorylated form of MAPK, P-MAPK, was purchased from New England BioLabs. The rabbit polyclonal antibodies raised against ERK1 and Xenopus Mos protein were purchased from Santa Cruz Biotechnologies. The primary antibodies were detected with appropriated horseradish peroxidaseconjugated second antibodies (Jackson ImmunoResearch Laboratories) and the Western blot Chemiluminescence Renaissance kit from PerkinElmer Life Sciences. Kinase Assays and Phosphatase Treatment—To measure Cdc2 kinase activity, histone H1 assays were performed on p13suc1 Sepharose pull-down extracts (three oocytes equivalent) in the presence of 1 μCi of [γ-32P]ATP (ICN), 100 μm ATP and 0.2 mg/ml histone H1 (Roche Applied Science) in kinase buffer (50 mm Tris-HCl, pH 7.2, 15 mm MgCl2, 5 mm EGTA, 1 mm dithiothreitol). Histone H3 kinase activity of Aurora-A was assayed in immunoprecipitates, incubated for 30 min at 30 °C in the presence of kinase buffer containing 3 μCi of [γ-32P]ATP (ICN), 50 μm ATP, and 0.5 mg/ml of either WT H3 or S10A H3. Kinase reactions were stopped by adding Laemmli buffer (37Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207472) Google Scholar) and boiling. After electrophoresis and autoradiography, the bands corresponding to histone H1 and histone H3 were excised, and the associated radioactivity was measured in a Wallac 1409 scintillation counter. Phosphatase treatments were carried out in Aurora-A immunoprecipitates, incubated for 1 h at 30 °C in the presence of 2000 units of λ phosphatase (New England BioLabs). Biochemical Characterization of Aurora-A Kinase Activity— To study the regulation of Aurora-A kinase activity, a reproducible kinase assay was developed. MBP and casein are often used to assay Aurora-A kinase activity in vitro (12Gopalan G. Chan C.S. Donovan P.J. J. Cell Biol. 1997; 138: 643-656Crossref PubMed Scopus (117) Google Scholar, 22Frank-Vaillant M. Haccard O. Thibier C. Ozon R. Arlot-Bonnemains Y. Prigent C. Jessus C. J. Cell Sci. 2000; 113: 1127-1138Crossref PubMed Google Scholar, 24Andresson T. Ruderman J.V. EMBO J. 1998; 17: 5627-5637Crossref PubMed Scopus (122) Google Scholar, 26Littlepage L.E. Wu H. Andresson T. Deanehan J.K. Amundadottir L.T. Ruderman J.V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15440-15445Crossref PubMed Scopus (183) Google Scholar, 27Littlepage L.E. Ruderman J.V. Genes Dev. 2002; 16: 2274-2285Crossref PubMed Scopus (237) Google Scholar, 38Walter A.O. Seghezzi W. Korver W. Sheung J. Lees E. Oncogene. 2000; 19: 4906-4916Crossref PubMed Scopus (232) Google Scholar, 39Meraldi P. Honda R. Nigg E.A. EMBO J. 2002; 21: 483-492Crossref PubMed Scopus (576) Google Scholar). However, the activity of Xenopus oocyte Aurora-A kinase toward both substrates is low, rendering the quantification of the kinase activity difficult. It is established that Aurora-B phosphorylates in vivo histone H3 on a single residue, Ser10 (40Hsu J.Y. Sun Z.W. Li X. Reuben M. Tatchell K. Bishop D.K. Grushcow J.M. Brame C.J. Caldwell J.A. Hunt D.F. Lin R. Smith M.M. Allis C.D. Cell. 2000; 102: 279-291Abstract Full Text Full Text PDF PubMed Scopus (719) Google Scholar). Given the high homology between the kinase domains of Aurora-A and Aurora-B, we checked whether histone H3 could be an in vitro substrate of Xenopus Aurora-A. Aurora-A was immunoprecipitated from metaphase II-arrested oocytes that have been reported to contain active Aurora-A kinase (22Frank-Vaillant M. Haccard O. Thibier C. Ozon R. Arlot-Bonnemains Y. Prigent C. Jessus C. J. Cell Sci. 2000; 113: 1127-1138Crossref PubMed Google Scholar, 24Andresson T. Ruderman J.V. EMBO J. 1998; 17: 5627-5637Crossref PubMed Scopus (122) Google Scholar). The kinase activity was measured in immunoprecipitates using the 21 N-terminal amino acids of histone H3 (ARTKQTARKS10TGGKAPRKQLC) fused with a GST tag, as substrate. As a control, we used a mutated histone H3, where Ser10, the unique residue in H3 phosphorylated by Aurora-B, was replaced by Ala (S10A H3). This S10A H3-peptide was never phosphorylated under our conditions. To determine the optimal conditions for the kinase assay, H3-peptide phosphorylation was measured in the presence of various ATP concentrations (from 10 μm to 1 mm) or various histone H3-peptide concentrations (from 2.5 to 500 μg/ml) at 30 °C for 30 min (Fig. 1). The Km for ATP and histone H3-peptide were, respectively, 80 μm and 35 μg/ml (Fig. 1). These values are comparable to those reported for the Cdc2-cyclin B kinase (75 μm for ATP and 40 μg/ml for histone H1 (41Erikson E. Maller J.L. J. Biol. Chem. 1989; 264: 19577-19582Abstract Full Text PDF PubMed Google Scholar)). Aurora-A Is Activated at GVBD Time during Meiotic Maturation—To investigate how Aurora-A is regulated during meiotic maturation, the activity of the protein was estimated, in parallel with its electrophoretic migration. Oocytes were collected at different times following progesterone stimulation (Fig. 2). As expected, MPF activity, measured by histone H1 kinase assay, increased just before GVBD, in correlation with cyclin B2 electrophoretic mobility retardation. Its activity decreased at the metaphase I-metaphase II transition and increased again, inducing entry into metaphase II. MAPK activity was estimated by Western blot, using an antibody that specifically recognizes the phosphorylated active form of the protein. As already reported, MAPK was stably activated at time of GVBD, due to Mos synthesis, until the end of the maturation process (Fig. 2A). Aurora-A kinase activity was similarly measured during meiotic maturation using histone H3-peptide as substrate (Fig. 2B). In prophase-arrested oocytes and until GVBD, Aurora-A was inactive and migrated as a single band (Fig. 2). At GVBD time, Aurora-A accumulated, as previously reported (22Frank-Vaillant M. Haccard O. Thibier C. Ozon R. Arlot-Bonnemains Y. Prigent C. Jessus C. J. Cell Sci. 2000; 113: 1127-1138Crossref PubMed Google Scholar) and underwent an electrophoretic shift, leading to a doublet, in correlation with its kinase activation (Fig. 2). In this experiment, the level of Aurora-A activity increased continuously from GVBD until the arrest in metaphase II. In some cases, Aurora-A kinase activity is maximal at GVBD time and is maintained at a high level until metaphase II (see Figs. 4, 8, and 10 below).Fig. 4Aurora-A kinase activation depends on cAMP and on protein synthesis during Xenopus oocyte meiotic maturation. Prophase oocytes were incubated in the presence (+) or in the absence (–) of cycloheximide (CHX)or 3-isobutyl-1-methylxanthine (IBMX) for 1 h and then stimulated by progesterone (Pg). Oocytes were collected at the indicated times after progesterone addition and homogenized. In control oocytes, GVBD started 4 h after progesterone addition. Lysates were subjected to Western blot with anti-cyclin B2 antibody, anti-Aurora-A antibody, or anti-phospho-MAPK (P-MAPK) antibody, as indicated. Immunoprecipitates were performed in the same lysates with anti-Aurora-A antibody and assayed for Aurora-A kinase activity using histone H3-peptide as substrate. A pull-down assay on p13 beads was used to measure Cdc2 kinase activity using histone H1 as substrate. Aurora-A and Cdc2 kinase activities are expressed in cpm incorporated in the substrate. +, presence of GVBD; –, absence of GVBD.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 8Cdc2-cyclin B activation is necessary for Aurora-A kinase activation during Xenopus oocyte meiotic maturation. Prophase oocytes were injected (+) or not (–) with p21cip1. One hour after, oocytes were incubated in the presence of progesterone (Pg). Oocytes were collected at the indicated times after progesterone addition. In control oocytes, GVBD started 4 h after progesterone addition. Lysates were subjected to Western blot with anti-cyclin B2 antibody, anti-Aurora-A antibody, or anti-MAPK antibody, as indicated, or were immunoprecipitated with anti-Aurora-A antibody and assayed for Aurora-A kinase activity using histone H3-peptide as substrate. A pull-down on p13 beads was assayed for Cdc2 kinase activity using histone H1 as substrate. Aurora-A and Cdc2 kinase activities are expressed in cpm incorporated in the substrate. +, presence of GVBD; –, absence of GVBD.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 10Aurora-A kinase activity is stabilized independently of Cdc2-cyclin B and protein synthesis after GVBD. Prophase oocytes were incubated in the presence of progesterone, and oocytes were collected either in prophase (Pro), at GVBD, or 2 h after GVBD (GVBD+2h). A, GVBD oocytes were either immediately homogenized, incubated in the presence of cycloheximide (CHX), or injected with p21cip1 and collected 2 h later. Lysates were subjected to Western blot with anti-cyclin B2 antibody, anti-Aurora-A antibody, or anti-phospho-MAPK (P-MAPK) antibody, as indicated, or immunoprecipitated with anti-Aurora-A antibody and assayed for Aurora-A kinase activity using histone H3-peptide as substrate. A pull-down on p13 beads was assayed for Cdc2 kinase activity using histone H1 as substrate. Aurora-A and Cdc2 kinase activities are expressed in cpm incorporated in the substrate. B, GVBD oocytes were either immediately homogenized or incubated for 2 h in the presence (+) or in the absence (–) of cycloheximide (CHX). Lysates were immunoprecipitated with anti-Aurora-A antibody and incubated (+) or not (–) in the presence of λ phosphatase (PPλ) for 1 h at 30 °C. Immunoprecipitates were then assayed for Aurora-A kinase activity using histone H3-peptide as substrate.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Metaphase II oocytes were parthenogenetically activated by electric shock. This treatment inactivated MPF within 10 min, due to cyclin B2 degradation (Fig. 2). Mos was degraded 30 min after activation, and consequently, MAPK was inactivated at the same time (Fig. 2A). When oocytes re-entered interphase in response to activation, the upper band of Aurora-A partially disappeared, in correlation with a drop in its kinase activity (Fig. 2). The H3 Kinase Activity of Aurora-A Depends on Its Phosphorylation—We next addressed the question whether Aurora-A activation results from its phosphorylation, as suggested by the electrophoretic retardation that correlates with the active form of the H3 kinase activity. Active Aurora-A kinase was immunoprecipitated from metaphase II-arrested oocytes and incubated in the presence of phosphatase λ (PPλ), that exhibits a broad range of specificity. PPλ treatment abolished Aurora-A activity, whereas heat-inactivated PPλ had no effect (Fig. 3A). The electrophoretic migration of Aurora-A was further analyzed in immunoprecipitates that allow a better resolution than crude extracts. Active Aurora-A migrated as a doublet when metaphase II oocyte extracts were loaded on a gel (see Fig. 2A). The active kinase isolated by immunoprecipitation migrated under three bands (Fig. 3B). The lower band migrated at the same level as the inactive prophase form (Fig. 3B). PPλ treatment induced the disappearance of the upper band, a decrease in the middle band, and the enrichment of the lower band (Fig. 3B). These observations indicate that Aurora-A is activated by phosphorylation and that at least two residues of Aurora-A are phosphorylated in metaphase II-arrested oocytes, resulting in the electrophoretic retardation of the protein. Aurora-A Phosphorylation and Activation Are under the Control of cAMP and Protein Synthesis—Aurora-A phosphorylation and H3 kinase activation take place in response to progesterone at GVBD time. It is well established that progesterone induces an early drop in cAMP and the synthesis of new proteins, both events being required" @default.
• W2036851282 created "2016-06-24" @default.
• W2036851282 creator A5003398310 @default.
• W2036851282 creator A5028094699 @default.
• W2036851282 creator A5033301617 @default.
• W2036851282 creator A5040633585 @default.
• W2036851282 creator A5083127536 @default.
• W2036851282 date "2003-06-01" @default.
• W2036851282 modified "2023-10-01" @default.
• W2036851282 title "Cdc2-Cyclin B Triggers H3 Kinase Activation of Aurora-A in Xenopus Oocytes" @default.
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