The mitogen-activated protein kinase signaling pathway stimulates mos mRNA cytoplasmic polyadenylation during Xenopus oocyte maturation.
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The Mos protein kinase is a key regulator of vertebrate oocyte maturation. Oocyte-specific Mos protein expression is subject to translational control. In the frog Xenopus, the translation of Mos protein requires the progesterone-induced polyadenylation of the maternal Mos mRNA, which is present in the oocyte cytoplasm. Both the Xenopus p42 mitogen-activated protein kinase (MAPK) and maturation-promoting factor (MPF) signaling pathways have been proposed to mediate progesterone-stimulated oocyte maturation. In this study, we have determined the relative contributions of the MAPK and MPF signaling pathways to Mos mRNA polyadenylation. We report that progesterone-induced Mos mRNA polyadenylation was attenuated in oocytes expressing the MAPK phosphatase rVH6. Moreover, inhibition of MAPK signaling blocked progesterone-induced Mos protein accumulation. Activation of the MAPK pathway by injection of RNA encoding Mos was sufficient to induce both the polyadenylation of synthetic Mos mRNA substrates and the accumulation of endogenous Mos protein in the absence of MPF signaling. Activation of MPF, by injection of cyclin B1 RNA or purified cyclin B1 protein, also induced both Mos protein accumulation and Mos mRNA polyadenylation. However, this action of MPF required MAPK activity. By contrast, the cytoplasmic polyadenylation of maternal cyclin B1 mRNA was stimulated by MPF in a MAPK-independent manner, thus revealing a differential regulation of maternal mRNA polyadenylation by the MAPK and MPF signaling pathways. We propose that MAPK-stimulated Mos mRNA cytoplasmic polyadenylation is a key component of the positive-feedback loop, which contributes to the all-or-none process of oocyte maturation. (+info)
Biochemical and biological effects of KN-93, an inhibitor of calmodulin-dependent protein kinase II, on the initial events of mouse egg activation induced by ethanol.
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Calmodulin-dependent protein kinase II (CaMKII) is transiently activated in mouse eggs by the increase in calcium that occurs upon activation with ethanol. This study investigated the biological and biochemical effects of KN-93, a reported selective inhibitor of CaMKII, to explore the potential role of this kinase in the initial events of egg activation. Mouse eggs were incubated for 30 min in the presence of different concentrations of KN-93 and induced to activate by 7% ethanol. KN-93 elicited a dose-dependent inhibition of polar body emission that resulted from the failure of the eggs to undergo meiosis resumption and inactivation of maturation-promoting factor (MPF). Furthermore, 15 mumol KN-93 l-1 produced a marked reduction in ethanol-induced loss of cortical granules. In vivo biochemical analysis revealed that 15 mumol KN-93 l-1 was responsible for significant inhibition of ethanol-stimulated CaMKII. The activity of the enzyme remained at a resting value, in spite of the presence of a calcium signal similar to that measured in control activated eggs. The inhibitory effects of KN-93 on the parameters tested in this study could not be mimicked by the inactive analogue KN-92. These results show that in mouse eggs, when ethanol-induced CaMKII activation was prevented, cortical granule exocytosis and meiosis resumption were inhibited. This suggests that CaMKII acts as a switch in the transduction of the calcium signal triggering mammalian egg activation. (+info)
Instabilities in phosphorylation-dephosphorylation cascades and cell cycle checkpoints.
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The G2-M checkpoint in the cell cycle is identified with a set of phosphorylation-dephosphorylation (PD) cycles involving Cdc25 and the maturation-promoting factor (MPF); these PD cycles are coupled in a way that generates an instability. This instability arises out of a transcritical bifurcation which could be exploited by the G2 DNA damage checkpoint pathway in order to arrest or delay entry into mitosis. The coupling between PD cycles involving Wee1 and MPF does not lead to an instability and therefore Wee1 may not be a crucial target of the checkpoint pathway. A set of PD cycles exhibiting transcritical bifurcation also possesses the integrative ability of a checkpoint for 'checking' that prerequisites are satisfied prior to the next cell cycle event. Such a set of coupled PD cycles is suggested to be a core mechanism of cell cycle checkpoints. (+info)
Interplay of maturation-promoting factor and mitogen-activated protein kinase inactivation during metaphase-to-interphase transition of activated bovine oocytes.
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The objective of the present study was to examine the activity changes in histone H1 kinase (also known as maturation-promoting factor [MPF]) and mitogen-activated protein kinase (MAPK) and their constituent proteins in in vitro-matured bovine oocytes after in vitro fertilization (IVF) or after parthenogenetic activation induced by calcium ionophore A23187 alone or by the ionophore followed by either 6-dimethylaminopurine (6-DMAP) or cycloheximide (CHX). Inactivation of both H1 kinase and MAPK occurred after both A23187+6-DMAP treatment and IVF; inactivation of H1 kinase preceded inactivation of MAPK. However, MAPK was inactivated much earlier in 6-DMAP-treated oocytes. Further analysis of constituent cell cycle proteins of these kinases by Western blot showed that A23187 alone could not induce changes in cdc2, cdc25, or ERK2 but induced reduction of cyclin B1. IVF and A23187+CHX induced similar changes: cyclin B1 was destroyed shortly after activation followed by accumulation of cyclin B1, phosphorylation of cdc2, and dephosphorylation of ERK2 at pronuclear formation 15 h after activation. No change in cdc25 was observed at this time. In contrast, A23187+6-DMAP treatment resulted in earlier phosphorylation of cdc2 and dephosphorylation of ERK2 at 4 h after treatment when the pronucleus formed. Moreover, accumulation of both cdc25 and cyclin B1 was detected at 15 h. Microinjection of ERK2 antibody into A23187-treated oocytes resulted in pronuclear formation. In conclusion, activation of bovine oocytes with 6-DMAP led to earlier inactivation of MAPK, while CHX induced inactivation of MAPK parallel to that following sperm-induced oocyte activation. Destruction of cyclin B is responsible for inactivation of MPF, while phosphorylation of cdc2 is likely responsible for maintaining its low activity. Inactivation of MAPK is closely associated with pronuclear development regardless of the activation protocol used. (+info)
Comparative investigation on spindle behavior and MPF activity changes during oocyte maturation between gynogenetic and amphimictic crucian carp.
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The spindle behavior and MPF activity changes in the progression of oocyte maturation were investigated and compared with cytological observation and kinase assay between gynogenetic silver crucian carp and amphimictic colored crucian carp. MPF activity was measured by using histone H1 as phosphorylation substrate. There were two similar oscillatory MPF kinase activity changes during oocyte maturation in two kinds of fishes with different reproductive modes, but there existed some subtle difference between them. The subtle difference was that the first peak of MPF kinase activity was kept to a longer-lasting time in the gynogenetic silver crucian carp than in the amphimictic colored crucian carp. It was suggested that the difference may be related to the spindle behavior changes, such as tripolar spindle formation and spindle rearrangement in the gynogenetic crucian carp. (+info)
Plo1 kinase recruitment to the spindle pole body and its role in cell division in Schizosaccharomyces pombe.
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Polo kinases execute multiple roles during cell division. The fission yeast polo related kinase Plo1 is required to assemble the mitotic spindle, the prophase actin ring that predicts the site for cytokinesis and for septation after the completion of mitosis (Ohkura et al., 1995; Bahler et al., 1998). We show that Plo1 associates with the mitotic but not interphase spindle pole body (SPB). SPB association of Plo1 is the earliest fission yeast mitotic event recorded to date. SPB association is strong from mitotic commitment to early anaphase B, after which the Plo1 signal becomes very weak and finally disappears upon spindle breakdown. SPB association of Plo1 requires mitosis-promoting factor (MPF) activity, whereas its disassociation requires the activity of the anaphase-promoting complex. The stf1.1 mutation bypasses the usual requirement for the MPF activator Cdc25 (Hudson et al., 1990). Significantly, Plo1 associates inappropriately with the interphase SPB of stf1.1 cells. These data are consistent with the emerging theme from many systems that polo kinases participate in the regulation of MPF to determine the timing of commitment to mitosis and may indicate that pole association is a key aspect of Plo1 function. Plo1 does not associate with the SPB when septation is inappropriately driven by deregulation of the Spg1 pathway and remains SPB associated if septation occurs in the presence of a spindle. Thus, neither Plo1 recruitment to nor its departure from the SPB are required for septation; however, overexpression of plo1+ activates the Spg1 pathway and causes transient Cdc7 recruitment to the SPB and multiple rounds of septation. (+info)
Regulation of APC activity by phosphorylation and regulatory factors.
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Ubiquitin-dependent proteolysis of Cut2/Pds1 and Cyclin B is required for sister chromatid separation and exit from mitosis, respectively. Anaphase-promoting complex/cyclosome (APC) specifically ubiquitinates Cut2/Pds1 at metaphase-anaphase transition, and ubiquitinates Cyclin B in late mitosis and G1 phase. However, the exact regulatory mechanism of substrate-specific activation of mammalian APC with the right timing remains to be elucidated. We found that not only the binding of the activators Cdc20 and Cdh1 and the inhibitor Mad2 to APC, but also the phosphorylation of Cdc20 and Cdh1 by Cdc2-Cyclin B and that of APC by Polo-like kinase and cAMP-dependent protein kinase, regulate APC activity. The cooperation of the phosphorylation/dephosphorylation and the regulatory factors in regulation of APC activity may thus control the precise progression of mitosis. (+info)
A novel p34(cdc2)-binding and activating protein that is necessary and sufficient to trigger G(2)/M progression in Xenopus oocytes.
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The activation of maturation-promoting factor (MPF) is required for G(2)/M progression in eukaryotic cells. Xenopus oocytes are arrested in G(2) and are induced to enter M phase of meiosis by progesterone stimulation. This process is known as meiotic maturation and requires the translation of specific maternal mRNAs stored in the oocytes. We have used an expression cloning strategy to functionally identify proteins involved in G(2)/M progression in Xenopus oocytes. Here we report the cloning of two novel cDNAs that when expressed in oocytes induce meiotic maturation efficiently. The two cDNAs encode proteins of 33 kD that are 88% identical and have no significant homologies to other sequences in databases. These proteins, which we refer to as p33(ringo) (rapid inducer of G(2)/M progression in oocytes), induce very rapid MPF activation in cycloheximide-treated oocytes. Conversely, ablation of endogenous p33(ringo) mRNAs using antisense oligonucleotides inhibits progesterone-induced maturation, suggesting that synthesis of p33(ringo) is required for this process. We also show that p33(ringo) binds to and activates the kinase activity of p34(cdc2) but does not associate with p34(cdc2)/cyclin B complexes. Our results identify a novel p34(cdc2) binding and activating protein that regulates the G(2)/M transition during oocyte maturation. (+info)