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First published online May 28, 2005
doi: 10.1242/10.1242/jcs.02370

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Differential regulation of Cdc2 and Aurora-A in Xenopus oocytes: a crucial role of phosphatase 2A

¹ Laboratoire de Biologie du Développement, UMR-CNRS 7622, Université Pierre et Marie Curie, boîte 24, 4 place Jussieu, 75252 Paris, CEDEX 5, France
² Centre de Recherche de Biochimie Macromoléculaire, CNRS FRE 2593, 1919 Route de Mende, 34293 Montpellier, CEDEX 5, France
³ Signal Transduction and Plasticity in the Nervous System, INSERM U-536, Université Pierre et Marie Curie, Institut du Fer-à-Moulin, 17 rue du Fer-à-Moulin, 75005 Paris, France

View larger version (29K): [in a new window]   Fig. 1. Cdc2 and Aurora-A are activated by okadaic acid and microcystin. Prophase oocyte extracts were incubated at 30°C in the presence of an ATP-regenerating system and either 10–6 M or 10–7 M okadaic acid (OA), or 10–6 M or 10–7 M microcystin (MC). Samples were collected at various times either for western blotting (A) or for histone H1 and H3 kinase assays (B). (A) Western blots were analysed with the anti-Cyclin B2 antibody, the anti-Aurora-A antibody and the anti-P-Aurora-A antibody, as indicated. (B) Samples were pulled down on p13 beads and assayed for Cdc2 kinase activity using histone H1 as substrate, or immunoprecipitated with the anti-Aurora-A antibody and assayed for Aurora-A kinase activity using histone H3-peptide as substrate. Cdc2 and Aurora-A kinase activities are expressed in cpm incorporated in the substrate.

View larger version (63K): [in a new window]   Fig. 2. Oocyte extracts are depleted in PP2A but retain PP1 after incubation with microcystin beads. Prophase oocytes (Pro) or Metaphase II-arrested oocytes (MII) were homogenized in the presence (+OA) or in the absence of 10–6 M okadaic acid. These extracts were incubated once (1), twice (2) or three times (3) for 1 hour at 4°C, either with microcystin-agarose beads (A) or with control beads (B). The supernatants (left panels) and the pellets (right panels) were analysed by western blotting with the anti-Cyclin B2 antibody, the anti-Aurora-A antibody, the anti-PP1 antibody and the anti-PP2A antibodies as indicated.

View larger version (50K): [in a new window]   Fig. 3. Removal of PP2A is sufficient to lead to Cdc2 and Aurora-A activation. Prophase oocyte extracts were incubated in the absence or in the presence of microcystin-agarose beads. The extracts were then incubated at 30°C in the presence or in the absence (–) of an ATP-regenerating system (ATP) with or without okadaic acid (OA). Samples were collected at various times either for histone H1 and H3 kinase assays (A) or for western blotting (B). (A) Samples were pulled down on p13 beads and assayed for Cdc2 kinase activity using histone H1 as substrate, or immunoprecipitated with the anti-Aurora-A antibody and assayed for Aurora-A kinase activity using histone H3 peptide as substrate. Cdc2 and Aurora-A kinase activities are expressed in cpm incorporated in the substrate. (B) Western blots were analysed with the anti-Cyclin B2 antibody, the anti-Aurora-A antibody, the anti-P-Aurora-A antibody, the anti-PP1 antibody and the anti-PP2A antibodies as indicated.

View larger version (55K): [in a new window]   Fig. 4. Aurora-A activation induced by PP2A removal is dependent on Cdc2. Prophase extracts were incubated in the presence of 40 µg/ml of p21Cip1 and then incubated (C) or not (A) with microcystin-agarose beads at 4°C. Control extracts were obtained by incubation with control beads at 4°C (B). After centrifugation, the supernatants were then incubated in the absence or in the presence of an ATP-regenerating system (ATP) with or without okadaic acid (OA) for 2 hours at 30°C. Samples were collected at indicated times either for histone H1 and H3 kinase assays or for western blotting. A pull-down assay on p13 beads was used to measure Cdc2 kinase activity using histone H1 as substrate. Immunoprecipitates were performed with the anti-Aurora-A antibody and assayed for Aurora-A kinase activity using histone H3-peptide as substrate. Cdc2 and Aurora-A kinase activities are expressed in cpm incorporated in the substrate. Western blots were probed with the anti-Cyclin B2 antibody, the anti-Aurora-A antibody, the anti-PP1 antibody, and the anti-PP2A antibodies as indicated.

View larger version (23K): [in a new window]   Fig. 5. Cdc2 and Aurora-A kinase activation is independent of PP1 inhibition. Prophase extracts were depleted (right panels) or not (left panels) in PP2A by incubation at 4°C with microcystin-agarose beads. The extracts were then incubated in the presence or absence of 500 nM P-DARPP-32 at 30°C with an ATP-regenerating system (ATP) plus or minus okadaic acid (OA). Samples were collected at indicated times for histone H1 (A) and histone H3 kinase assays (B). (A) Samples were pulled down on p13 beads and assayed for Cdc2 kinase activity using histone H1 as substrate. (B) Samples were immunoprecipitated with anti-Aurora-A antibody and assayed for Aurora-A kinase activity using histone H3-peptide as substrate. Cdc2 and Aurora-A kinase activities are expressed in cpm incorporated in the substrate.

View larger version (45K): [in a new window]   Fig. 6. Cyclin B2 and Aurora-A phosphorylation is independent of PP1 inhibition. The same extracts as in Fig. 5 were used. Prophase extracts were depleted (C and D) or not (A and B) in PP2A by incubation with microcystin-agarose beads. After centrifugation, the extracts were incubated in the presence (B and D) or absence (A and C) of 500 nM P-DARPP-32 at 30°C in the absence (–) or in the presence of an ATP-regenerating system (ATP) plus or minus okadaic acid (OA). Samples were collected at indicated times and analysed by western blotting with the anti-Cyclin B2 antibody, the anti-Aurora-A antibody, the anti-P-Aurora-A antibody, the DARPP-32 antibody, the anti-P-DARPP-32 antibody, the PP1 antibody and the PP2A antibodies, as indicated.

View larger version (36K): [in a new window]   Fig. 7. PP1 inhibition is not sufficient to trigger Aurora-A phosphorylation and activation. Prophase extracts (Pro) were depleted in PP2A by incubation with microcystin-agarose beads in the presence or in the absence of p21Cip1. After centrifugation, the extracts were incubated for 2 hours in the presence or in the absence of 500 nM P-DARPP-32 at 30°C plus or minus an ATP-regenerating system (ATP). Samples were collected for kinase assays and western blots. A pull-down assay on p13 beads was used to measure Cdc2 kinase activity using histone H1 as substrate. Immunoprecipitates were performed with anti-Aurora-A antibody and assayed for Aurora-A kinase activity using histone H3-peptide as substrate. Cdc2 and Aurora-A kinase activities are expressed in cpm incorporated in the substrate. Western blots were probed with the anti-Cyclin B2 antibody, the anti-Aurora-A antibody, the anti-P-Aurora-A antibody, the DARPP-32 antibody, the anti-P-DARPP-32 antibody, the PP1 antibody and the PP2A antibodies, as indicated.

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