RESUMO
The M phase promoting factor (MPF) is a dimer composed of a catalytic Cdk1 subunit and a Cyclin B regulatory subunit. We have characterized a cDNA containing the entire coding sequence of an axolotl Cyclin B1 protein that is able to promote MPF activity when added to a fraction from prophase I oocytes that contains monomeric Cdk1. The axolotl cyclin B1 gene is expressed as a maternal mRNA in oocytes and early embryos. Its poly(A) tail length increases in metaphase II oocytes and then decreases regularly during the first embryonic cell cycles. Endogenous Cyclin B1 protein is first expressed during oocyte meiotic maturation. Its level oscillates after fertilization and is coordinated to the phosphorylation level of tyrosine 15 residue of Cdk1 (pTyr15), with both maxima preceding each cell division. As expected, when translated into microinjected oocytes, axolotl Cyclin B1 induces the resumption of meiosis. In electrically activated unfertilized eggs (UFE), Cyclin B1 and pTyr15 cyclic accumulations are observed with kinetics different from those of the early embryonic cycles. The axolotl embryo and UFE provide interesting in vivo comparative models for studying events controlling Cyclin B1 regulation during development.
Assuntos
Ambystoma mexicanum/embriologia , Ambystoma mexicanum/genética , Ciclina B/genética , Oogênese/genética , Ambystoma mexicanum/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Ciclina B1 , DNA Complementar/genética , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Fator Promotor de Maturação/química , Fator Promotor de Maturação/genética , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclina B/metabolismo , Histonas/metabolismo , Proteínas Quinases/metabolismo , Animais , Aurora Quinases , Western Blotting , Proteínas de Ciclo Celular , AMP Cíclico/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Ativação Enzimática , Cinética , Meiose , Mitose , Oócitos/enzimologia , Peptídeos/química , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Progesterona/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fuso Acromático , Fatores de Tempo , Regulação para Cima , Xenopus , Proteínas de Xenopus , Fosfatases cdc25/metabolismoRESUMO
Spatial control is a key issue in cell division. The Ran GTPase regulates several fundamental processes for cell life, largely acting through importin molecules. The best understood of these is protein import through the nuclear envelope in interphase, but roles in mitotic spindle assembly are also established. In mammalian cells, in which centrosomes are major spindle organizers, a link is emerging between the Ran network, centrosomes and spindle poles. Here, we show that, after nuclear envelope breakdown, importin beta is transported to the spindle poles in mammalian cells. This localization is temporally regulated from prometaphase until anaphase, when importin beta dissociates from poles and is recruited back around reforming nuclei. Importin beta sediments with mitotic microtubules in vitro and its accumulation at poles requires microtubule integrity and dynamics in vivo. Furthermore, RNA interference-dependent inactivation of TPX2, the major Ran-dependent spindle organizer, abolishes importin beta accumulation at poles. Importin beta has a functional role in spindle pole organization, because overexpression yields mitotic spindles with abnormal, fragmented poles. Coexpression of TPX2 with importin beta mitigates these abnormalities. Together, these results indicate that the balance between importins and spindle regulators of the TPX2 type is crucial for spindle formation. Targeting of TPX2/importin-beta complexes to poles is a key aspect in Ran-dependent control of the mitotic apparatus in mammalian cells.