RESUMEN
In most tumor cells a chromosomal instability leads to an abnormal chromosome number (aneuploidy). The mitotic checkpoint is essential for ensuring accurate chromosome segregation by allowing mitotic delay in response to a spindle defect. This checkpoint delays the onset of anaphase until all the chromosomes are correctly aligned on the mitotic spindle. When unattached kinetochores are present, the metaphase/anaphase transition is not allowed and the time available for chromosome-microtubule capture increases. Genes required for this delay were first identified in Saccharomyces cerevisiae (the MAD, BUB and MPS1 genes) and subsequently, homologs have been identified in higher eucaryotes showing that the spindle checkpoint pathway is highly conserved. The checkpoint functions by preventing an ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC) from ubiquitinylating proteins whose destruction is required for anaphase onset.
Asunto(s)
División Celular , Cromosomas/fisiología , Aneuploidia , Animales , Segregación Cromosómica , Humanos , Cinetocoros , Mitosis , Neoplasias/genética , Saccharomyces cerevisiae/genética , Huso AcromáticoRESUMEN
Members of the polo-like family of protein kinases have been involved in the control of APC (anaphase-promoting complex) during the cell cycle, yet how they activate APC is not understood in any detail. In Xenopus oocytes, Ca2+-dependent degradation of cyclin B associated with release from arrest at second meiotic metaphase was demonstrated to require the polo-like kinase Plx1. The aim of the present study was to examine, beyond Ca2+-dependent resumption of meiosis, the possible role of Plx1 in the control of cyclin degradation during the early mitotic cell cycle. Plx1 was found to be dispensable for MPF to turn on the cyclin degradation machinery. However, it is required to prevent premature inactivation of the APC-dependent proteolytic pathway. Microcystin suppresses the requirement for Plx1 in both Ca2+-dependent exit from meiosis, associated with degradation of both cyclin B and A downstream of CaMK2 activation, and prevention of premature APC(Fizzy) inactivation in the early mitotic cell cycle. These results are consistent with the view that Plx1 antagonizes an unidentified microcystin-sensitive phosphatase that inactivates APC(Fizzy).
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ligasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Complejos de Ubiquitina-Proteína Ligasa , Proteínas de Xenopus , Ciclosoma-Complejo Promotor de la Anafase , Animales , Proteína Quinasa CDC2/metabolismo , Proteínas Cdc20 , Ciclo Celular , Ciclina B/metabolismo , Cisteína Endopeptidasas/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Microcistinas , Complejos Multienzimáticos/metabolismo , Péptidos Cíclicos/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/metabolismo , Complejo de la Endopetidasa Proteasomal , Proteínas Serina-Treonina Quinasas/genética , Estrellas de Mar , Factores de Tiempo , Ubiquitina-Proteína Ligasas , XenopusRESUMEN
The auto-catalytic activation of the cyclin-dependent kinase Cdc2 or MPF (M-phase promoting factor) is an irreversible process responsible for the entry into M phase. In Xenopus oocyte, a positive feed-back loop between Cdc2 kinase and its activating phosphatase Cdc25 allows the abrupt activation of MPF and the entry into the first meiotic division. We have studied the Cdc2/Cdc25 feed-back loop using cell-free systems derived from Xenopus prophase-arrested oocyte. Our findings support the following two-step model for MPF amplification: during the first step, Cdc25 acquires a basal catalytic activity resulting in a linear activation of Cdc2 kinase. In turn Cdc2 partially phosphorylates Cdc25 but no amplification takes place; under this condition Plx1 kinase and its activating kinase, Plkk1 are activated. However, their activity is not required for the partial phosphorylation of Cdc25. This first step occurs independently of PP2A or Suc1/Cks-dependent Cdc25/Cdc2 association. On the contrary, the second step involves the full phosphorylation and activation of Cdc25 and the initiation of the amplification loop. It depends both on PP2A inhibition and Plx1 kinase activity. Suc1-dependent Cdc25/Cdc2 interaction is required for this process.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Meiosis/fisiología , Oocitos/enzimología , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Schizosaccharomyces pombe , Tirosina 3-Monooxigenasa , Proteínas de Xenopus , Xenopus/fisiología , Fosfatasas cdc25/antagonistas & inhibidores , Fosfatasas cdc25/metabolismo , Proteínas 14-3-3 , Animales , Sistema Libre de Células , Niño , Proteínas Fúngicas/metabolismo , Humanos , Proteína Fosfatasa 2 , Proteínas/metabolismoRESUMEN
MAP kinase activation occurs during meiotic maturation of oocytes from all animals, but the requirement for MAP kinase activation in reinitiation of meiosis appears to vary between different classes. In particular, it has become accepted that MAP kinase activation is necessary for progesterone-stimulated meiotic maturation of Xenopus oocytes, while this is clearly not the case in other systems. In this paper, we demonstrate that MAP kinase activation in Xenopus oocytes is an early response to progesterone and can be temporally dissociated from MPF activation. We show that MAP kinase activation can be suppressed by treatment with geldanamycin or by overexpression of the MAP kinase phosphatase Pyst1. A transient and low-level early activation of MAP kinase increases the efficiency of cell cycle activation later on, when MAP kinase activity is no longer essential. Many oocytes can still undergo reinitiation of meiosis in the absence of active MAP kinase. Suppression of MAP kinase activation does not affect the formation or activation of Cdc2-cyclin B complexes, but reduces the level of active Cdc2 kinase. We discuss these findings in the context of a universal mechanism for meiotic maturation in oocytes throughout the animal kingdom.
Asunto(s)
Factor Promotor de Maduración/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Xenopus/crecimiento & desarrollo , Xenopus/metabolismo , Animales , Benzoquinonas , Proteína Quinasa CDC2/metabolismo , Ciclina B/metabolismo , Fosfatasa 6 de Especificidad Dual , Activación Enzimática/efectos de los fármacos , Femenino , Insulina/farmacología , Lactamas Macrocíclicas , Meiosis/efectos de los fármacos , Meiosis/fisiología , Oocitos/efectos de los fármacos , Progesterona/farmacología , Proteínas Tirosina Fosfatasas/metabolismo , Quinonas/farmacologíaRESUMEN
X-PAKs are involved in negative control of the process of oocyte maturation in Xenopus (). In the present study, we define more precisely the events targetted by the kinase in the inhibition of the G2/M transition. We show that microinjection of recombinant X-PAK1-Cter active kinase into progesterone-treated oocytes prevents c-Mos accumulation and activation of both MAPK and maturation-promoting factor (MPF). In conditions permissive for MAPK activation, MPF activation still fails. We demonstrate that a constitutive truncated version of X-PAK1 (X-PAK1-Cter) does not prevent the association of cyclin B with p34(cdc2) but rather prevents the activation of the inactive complexes present in the oocyte. Proteins participating in the MPF amplification loop, including the Cdc25-activating Polo-like kinase are all blocked. Indeed, using active MPF, the amplification loop is not turned on in the presence of X-PAK1. Our results indicate that X-PAK and protein kinase A targets in the control of oocyte maturation are similar and furthermore that this negative regulation is not restricted to meiosis, because we demonstrate that G2/M progression is also prevented in Xenopus cycling extracts in the presence of active X-PAK1.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fase G2 , Mitosis , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Animales , Catálisis , Proteínas de Ciclo Celular/antagonistas & inhibidores , Activación Enzimática , Femenino , Oocitos/efectos de los fármacos , Oocitos/enzimología , Progesterona/farmacología , Proteínas Recombinantes/metabolismo , Xenopus , Quinasas p21 ActivadasRESUMEN
We have investigated whether Plx1, a kinase recently shown to phosphorylate cdc25c in vitro, is required for activation of cdc25c at the G2/M-phase transition of the cell cycle in Xenopus. Using immunodepletion or the mere addition of an antibody against the C terminus of Plx1, which suppressed its activation (not its activity) at G2/M, we show that Plx1 activity is required for activation of cyclin B-cdc2 kinase in both interphase egg extracts receiving recombinant cyclin B, and cycling extracts that spontaneously oscillate between interphase and mitosis. Furthermore, a positive feedback loop allows cyclin B-cdc2 kinase to activate Plx1 at the G2/M-phase transition. In contrast, activation of cyclin A-cdc2 kinase does not require Plx1 activity, and cyclin A-cdc2 kinase fails to activate Plx1 and its consequence, cdc25c activation in cycling extracts.