Metaphase arrest by cyclin E-Cdk2 requires the spindle-checkpoint kinase Mps1

Cytostatic factor (CSF) arrests vertebrate eggs in metaphase of meiosis II through several pathways that inhibit activation of the anaphase-promoting complex/cyclosome (APC/C). In Xenopus, the Mos-MEK1-MAPK-p90(Rsk) cascade utilizes spindle-assembly-checkpoint components to effect metaphase arrest. Another pathway involves cyclin E-Cdk2, and sustained cyclin E-Cdk2 activity in egg extracts causes metaphase arrest in the absence of Mos; this latter finding suggests that an independent pathway contributes to CSF arrest. Here, we demonstrate that metaphase arrest with cyclin E-Cdk2, but not with Mos, requires the spindle-checkpoint kinase monopolar spindles 1 (Mps1), a cyclin E-Cdk2 target that is also implicated in centrosome duplication. xMps1 is synthesized and activated during oocyte maturation and inactivated upon CSF release. In egg extracts, CSF release by calcium was inhibited by constitutively active cyclin E-Cdk2 and delayed by wild-type xMps1. Ablation of cyclin E by antisense oligonucleotides blocked accumulation of xMps1, suggesting that cyclin E-Cdk2 controls Mps1 levels. During meiosis II, activated cyclin E-Cdk2 significantly inhibited the APC/C even in the absence of the Mos-MAPK pathway, but this inhibition was not sufficient to suppress S phase between meiosis I and II. These results uniquely place xMps1 downstream of cyclin E-Cdk2 in mediating a pathway of APC/C inhibition and metaphase arrest.     

Bub1 is activated by the protein kinase p90(Rsk) during Xenopus oocyte maturation

BACKGROUND: The kinetochore attachment (spindle assembly) checkpoint arrests cells in metaphase to prevent exit from mitosis until all the chromosomes are aligned properly at the metaphase plate. The checkpoint operates by preventing activation of the anaphase-promoting complex (APC), which triggers anaphase by degrading mitotic cyclins and other proteins. This checkpoint is active during normal mitosis and upon experimental disruption of the mitotic spindle. In yeast, the serine/threonine protein kinase Bub1 and the WD-repeat protein Bub3 are elements of a signal transduction cascade that regulates the kinetochore attachment checkpoint. In mammalian cells, activated MAPK is present on kinetochores during mitosis and activity is upregulated by the spindle assembly checkpoint. In vertebrate unfertilized eggs, a special form of meiotic metaphase arrest by cytostatic factor (CSF) is mediated by MAPK activation of the protein kinase p90(Rsk), which leads to inhibition of the APC. However, it is not known whether CSF-dependent metaphase arrest caused by p90(Rsk) involves components of the spindle assembly checkpoint. RESULTS: xBub1 is present in resting oocytes and its protein level increases slightly during oocyte maturation and early embryogenesis. In Xenopus oocytes, Bub1 is localized to kinetochores during both meiosis I and meiosis II, and the electrophoretic mobility of Bub1 upon SDS-PAGE decreases during meiosis I, reflecting phosphorylation and activation of the enzyme. The activation of Bub1 can be induced in interphase egg extracts by selective stimulation of the MAPK pathway by c-Mos, a MAPKKK. In oocytes treated with the MEK1 inhibitor U0126, the MAPK pathway does not become activated, and Bub1 remains in its low-activity, unshifted form. Injection of a constitutively active target of MAPK, the protein kinase p90(Rsk), restores the activation of Bub1 in the presence of U0126. Moreover, purified p90(Rsk) phosphorylates Bub1 in vitro and increases its protein kinase activity. CONCLUSIONS: Bub1, an upstream component of the kinetochore attachment checkpoint, is activated during meiosis in Xenopus in a MAPK-dependent manner. Moreover, a single substrate of MAPK, p90(Rsk), is sufficient to activate Bub1 in vitro and in vivo. These results indicate that in vertebrate eggs, kinetochore attachment/spindle assembly checkpoint proteins, including Bub1, are downstream of p90(Rsk) and may be effectors of APC inhibition and CSF-dependent metaphase arrest by p90(Rsk).     

p90Rsk is not involved in cytostatic factor arrest in mouse oocytes

Vertebrate oocytes arrest in metaphase of the second meiotic division (MII), where they maintain a high cdc2/cyclin B activity and a stable, bipolar spindle because of cytostatic factor (CSF) activity. The Mos-MAPK pathway is essential for establishing CSF. Indeed, oocytes from the mos-/- strain do not arrest in MII and activate without fertilization, as do Xenopus laevis oocytes injected with morpholino oligonucleotides directed against Mos. In Xenopus oocytes, p90Rsk (ribosomal S6 kinase), a MAPK substrate, is the main mediator of CSF activity. We show here that this is not the case in mouse oocytes. The injection of constitutively active mutant forms of Rsk1 and Rsk2 does not induce a cell cycle arrest in two-cell mouse embryos. Moreover, these two mutant forms do not restore MII arrest after their injection into mos-/- oocytes. Eventually, oocytes from the triple Rsk (1, 2, 3) knockout present a normal CSF arrest. We demonstrate that p90Rsk is not involved in the MII arrest of mouse oocytes.     

More than G1 or G2 arrest: useful starfish oocyte system for investigating skillful MAP kinase

During the meiotic cycles in starfish oocytes, MAP kinase (MAPK) is involved in the meta-I arrest, the meiosis I to II transition, and the arrest at pronucleus stage (G1 and/or G2 phase). The eventual fate, either development or death, of mature eggs is also under the control of MAPK. Starfish oocytes are thus a useful model system to study multiple functions of skillful MAPK.     

Mos is not required for the initiation of meiotic maturation in Xenopus oocytes

In Xenopus oocytes, the c-mos proto-oncogene product has been proposed to act downstream of progesterone to control the entry into meiosis I, the transition from meiosis I to meiosis II, which is characterized by the absence of S phase, and the metaphase II arrest seen prior to fertilization. Here, we report that inhibition of Mos synthesis by morpholino antisense oligonucleotides does not prevent the progesterone-induced initiation of Xenopus oocyte meiotic maturation, as previously thought. Mos-depleted oocytes complete meiosis I but fail to arrest at metaphase II, entering a series of embryonic-like cell cycles accompanied by oscillations of Cdc2 activity and DNA replication. We propose that the unique and conserved role of Mos is to prevent mitotic cell cycles of the female gamete until the fertilization in Xenopus, starfish and mouse oocytes.     

Postmeiotic unfertilized starfish eggs die by apoptosis

Fertilization of starfish eggs during meiosis results in rapid progression to embryogenesis as soon as meiosis II is completed. Unfertilized eggs complete meiosis and arrest in postmeiotic interphase for an, until now, indeterminate time. If they remain unfertilized, the mature postmeiotic eggs ultimately die. The aim of this study is to characterize the mechanism of death in postmeiotic unfertilized starfish eggs. We report that, in two species of starfish, in the absence of fertilization, postmeiotic interphase arrest persists for 16-20 h, after which time the cells synchronously and rapidly die. Dying eggs extrude membrane blebs, undergo cytoplasmic contraction and darkening, and fragment into vesicles in a manner reminiscent of apoptotic cells. The DNA of dying eggs is condensed, fragmented, and labeled by the TUNEL assay. Taken together, these data suggest that the default fate of postmeiotic starfish eggs, like their mammalian counterparts, is death by apoptosis. We further report that the onset and execution of apoptosis in this system is dependent on ongoing protein synthesis and is inhibited by a rise in intracellular Ca(2+), an essential component of the fertilization signaling pathway. We propose starfish eggs as a useful model to study developmentally regulated apoptosis.     

Phosphorylation of Cdc25C by pp90Rsk Contributes to a G2 Cell Cycle Arrest in Xenopus Cycling Egg Extracts

In unfertilized Xenopus eggs, the p42 mitogen activated protein kinase (p42MAPK) pathway isknown to maintain cell cycle arrest at metaphase of meiosis II. However, constitutive activation ofp42MAPK in post-meiotic, cycling Xenopus egg extracts can lead to either a G2 or M-phase arrestof the cell cycle, depending on the timing of p42MAPK activation. Here, we examined themolecular mechanism by which activation of the p42MAPK pathway during interphase leads to cellcycle arrest in G2. When either a recombinant wild type Cdc25C(WT) or a mutated form ofCdc25C, in which serine 287 was replaced by an alanine (S287A), was added to cycling eggextracts, S287A accelerated entry into M-phase. Furthermore, the addition of S287A overcame theG2 arrest caused by p42MAPK, driving the extract into M-phase. p90Rsk, a kinase that is the targetof p42MAPK, was phosphorylated and activated (pp90Rsk) in the G2-arrested egg extracts, and wasable to phosphorylate WT but not S287A in vitro. 14-3-3 proteins were associated with endogenousCdc25C in G2-arrested extracts. Cdc25C(WT) that had been phosphorylated by pp90Rsk bound 14-3-3?, whereas S287A could not. These data suggest that the link between the p42MAPK signalingpathway and Cdc25C involves the activation of pp90Rsk and its phosphorylation of Cdc25C at S287,causing the binding of 14-3-3 proteins. We propose that the binding of 14-3-3 proteins to pp90Rskphosphorylated-Cdc25C results in a G2 arrest in a manner similar to the cell cycle delays inducedby differentiation signals that occur later in embryonic development.     

Regulation of Intracellular pH by p90Rsk-dependent Activation of an Na+/H+ Exchanger in Starfish Oocytes

Starfish oocytes arrest at metaphase of the first meiotic division (MI arrest) in the ovary and resume meiosis after spawning into seawater. MI arrest is maintained by lower intracellular pH (pH_i) and release from arrest by cellular alkalization. To elucidate pH_i regulation in oocytes, we cloned the starfish (Asterina pectinifera) Na⁺/H⁺ exchanger 3 (ApNHE3) expressed in the plasma membrane of oocytes. The cytoplasmic domain of ApNHE3 contains p90 ribosomal S6 kinase (p90Rsk) phosphorylation sites, and injection of a constitutively active p90Rsk and the upstream regulator Mos to immature oocytes, stimulated an increase in pH_i. This increase was blocked by 5-(N-ethyl-N-isopropyl)-amiloride, a NHE inhibitor, and SL0101, a specific Rsk inhibitor. The MAPK kinase (MEK) inhibitor U0126 blocked the Mos-induced, but not the p90Rsk-induced, pH_i increase, suggesting that the Mos-MEK-MAPK-p90Rsk pathway promotes ApNHE3 activation. In a cell-free extract, the Mos-MEK-MAPK-p90Rsk pathway phosphorylates ApNHE3 at Ser-590, -606, and -673. When p90Rsk-dependent ApNHE3 phosphorylation was blocked by a dominant-negative C-terminal fragment, or neutralizing antibody, the p90Rsk-induced pH_i increase was suppressed in immature oocytes. However, ApNHE3 is up-regulated via the upstream phosphatidylinositol 3-kinase pathway before MAPK activation and the active state is maintained until spawning, suggesting that the p90Rsk-dependent ApNHE3 phosphorylation is unlikely to be the primary regulatory mechanism involved in MI arrest exit. After meiosis is completed, unfertilized eggs maintain their elevated pH_i (∼7.4) until the onset of apoptosis. We suggest that the p90Rsk/ApNHE3-dependent elevation of pH_i increases fertilization success by delaying apoptosis initiation.     

Functional interaction between p90Rsk2 and Emi1 contributes to the metaphase arrest of mouse oocytes

Vertebrate eggs arrest at metaphase of the second meiotic division before fertilization under the effect of a cytostatic factor (CSF). This arrest is established during oocyte maturation by the MAPK kinase module, comprised of Mos, MEK, MAPKs and p90Rsk. Maintenance of CSF arrest at metaphase requires inhibitors of the anaphase-promoting complex (APC) like Emi1, which sequesters the APC activator Cdc20. Although it was proposed that the Mos pathway and Emi1 act independently, neither one alone is sufficient to entirely reproduce CSF arrest. Herein we demonstrate that p90Rsk2 associates with and phosphorylates Emi1 upstream of the binding region for Cdc20, thus stabilizing their interaction. Experiments in transfected cells and two-cell embryos indicate that Emi1 and p90Rsk2 cooperate to induce the metaphase arrest. Moreover, oocyte maturation was impaired by interfering with the interaction between p90Rsk2 and Emi1 or by RNA interference of Emi1. Our results indicate that p90Rsk2 and Emi1 functionally interact during oocyte maturation and that the Mos pathway establishes CSF activity through stabilization of an APC-inhibitory complex composed by Emi1 and Cdc20 before fertilization.     

A constitutively active form of the protein kinase p90Rsk1 is sufficient to trigger the G2/M transition in Xenopus oocytes

The protein kinase p90(Rsk) has previously been implicated as a key target of the MAPK pathway during M phase of meiosis II in Xenopus oocytes. To determine whether Rsk is a mediator of MAPK for stimulation of the G(2)/M transition early in meiosis I, we sought to generate a form of Rsk that would be constitutively active in resting, G(2) phase oocytes. Initial studies revealed that an N-terminal truncation of 43 amino acids conferred enhanced specific activity on the enzyme in G(2) phase, and stability was highest if the C terminus was not truncated. The full-length enzyme is known to be activated by phosphorylation at five sites. Two of these sites and flanking residues were replaced with either aspartic or glutamic acid, and Tyr(699) was mutated to alanine. The resulting construct, termed fully activated (FA) Rsk, had constitutive activity in G(2) phase, with a specific activity equivalent to that of wild type Rsk in M phase. In eight independent experiments approximately 45% of oocytes expressing FA-Rsk underwent germinal vesicle breakdown (GVBD, the G(2)/M transition) in the absence of progesterone, and this effect could be observed even in the presence of the MAPK kinase inhibitor U0126. Moreover, the specific activity of FA-Rsk in vivo was unaffected by U0126. In oocytes that did not undergo GVBD with FA-Rsk expression, subsequent treatment with progesterone resulted in a very rapid rate of GVBD even in the presence of U0126 to inhibit the endogenous MAPK/Rsk pathway. These results indicate that Rsk is the mediator of MAPK effects for the G(2)/M transition in meiosis I and in a subpopulation of oocytes Rsk is sufficient to trigger the G(2)/M transition.     

Emi1 Class of Proteins Regulate Entry into Meiosis and the Meiosis I to Meiosis II Transition in Xenopus Oocytes

Xenopus oocytes are arrested at the G2/prophase boundary of meiosis I and enter meiosis in response to progesterone. A hallmark of meiosis is the absence of DNA replication between the successive cell division phases meiosis I (MI) and meiosis II (MII). After the MI-MII transition, Xenopus eggs are locked in metaphase II by the cytostatic factor (CSF) arrest to prevent parthenogenesis. Early Mitotic Inhibitor 1 (Emi1) maintains CSF arrest by inhibiting the ability of the Anaphase Promoting Complex (APC) to direct the destruction of cyclin B. To investigate whether Emi1 has an earlier role in meiosis, we injected Xenopus oocytes with neutralizing antibodies against Emi1 at G2/prophase and during the MI-MII transition. Progesterone-treated G2/prophase oocytes injected with anti-Emi1 antibody fail to activate Maturation Promoting Factor (MPF), a complex of cdc2/cyclin B, and the MAPK pathway, and do not undergo germinal vesicle breakdown (GVBD). Injection of purified ?90 cyclin B protein or blocking anti-Emi1 antibody with purified Emi1 protein rescues these meiotic processes in Emi1-neutralized oocytes. Acute inhibition of Emi1 in progesterone treated oocytes immediately after GVBD causes rapid loss of cdc2 activity with simultaneous loss of cyclin B levels and inactivation of the MAPK pathway. These oocytes decondense their chromosomes and enter a DNA replication phase instead of progressing to MII. Prior ablation of Cdc20, addition of methyl-ubiquitin, or addition of indestructible ?90 cyclin B rescues the MI-MII transition in Emi1 inhibited oocytes.     

蛋白激酶在卵母细胞减数分裂和受精中的作用

脊椎动物卵母细胞的减数分裂和受精过程受到多种蛋白激酶的调节。近年来对于卵母细胞成熟、活化和受精的分子机制研究取得了长足进步 ,发现促成熟因子 (MPF)和促分裂原活化蛋白激酶 (MAPK)是调节卵母细胞细胞周期的关键分子 ,二者的激活和失活导致了减数分裂的恢复、阻滞和完成。许多蛋白激酶通过调节MPF和MAPK活性来影响减数分裂。Polo like激酶活化MPF ,Mos激活MAPK而启动成熟分裂并维持中期阻滞。CaMKII通过泛素途径灭活MPF使卵突破MII期阻滞。另外 ,p90 rsk作为MAPK的下游分子参与减数分裂调节 ,蛋白激酶C(PKC)诱导皮质颗粒排放并抑制MAPK激活 ,酪氨酸蛋白激酶家族成员介导受精诱发的Ca2 释放。这些蛋白激酶的协同作用推动了卵母细胞正常的成熟与受精     

APC/C-Cdc20-mediated degradation of cyclin B participates in CSF arrest in unfertilized Xenopus eggs

In vertebrates, unfertilized eggs are arrested at meiotic metaphase II (meta-II) by cytostatic factor (CSF), with Cdc2 activity maintained at a constant, high level. CSF is thought to suppress cyclin B degradation through the inhibition of the anaphase-promoting complex/cyclosome (APC/C)-Cdc20 while cyclin B synthesis continues in unfertilized eggs. Thus, it is a mystery how Cdc2 activity is kept constant during CSF arrest. Here, we show that the APC/C-Cdc20 can mediate cyclin B degradation in CSF-arrested Xenopus eggs and extracts, in such a way that when Cdc2 activity is elevated beyond a critical level, APC/C-Cdc20-dependent cyclin B degradation is activated and Cdc2 activity consequently declines to the critical level. This feedback control of Cdc2 activity is shown to be required for keeping Cdc2 activity constant during meta-II arrest. We have also shown that Mos/MAPK pathway is essential for preventing the cyclin B degradation from inactivating Cdc2 below the critical level required to sustain meta-II arrest. Our results indicate that under CSF arrest, Mos/MAPK activity suppresses cyclin B degradation, preventing Cdc2 activity from falling below normal meta-II levels, whereas activation of APC/C-Cdc20-mediated cyclin B degradation at elevated levels of Cdc2 activity prevents Cdc2 activity from reaching excessively high levels.     

MAP kinase links the fertilization signal transduction pathway to the G1/S-phase transition in starfish eggs.

The mechanism by which fertilization initiates S-phase in the zygote is examined by manipulating the activity of MAP kinase in mature starfish eggs. These unfertilized eggs, which are arrested at G1-phase after the completion of meiosis, have high MAP kinase activity but undetectable cdc2 kinase activity. Either fertilization or inhibition of protein synthesis causes a decrease in MAP kinase activity, which is followed by DNA synthesis. Inactivation of MAP kinase with its specific phosphatase, CL100, initiates DNA synthesis in the absence of fertilization, while constitutive activation of MAP kinase with MEK represses the initiation of DNA synthesis following fertilization. Thus, in unfertilized mature starfish eggs, a capacity for DNA replication is already acquired, but entry into S-phase is negatively regulated by MAP kinase activity that is supported by a continuously synthesized protein(s) but not by cdc2 kinase. Upon fertilization, downregulation of MAP kinase activity is necessary and sufficient for triggering the G1/S-phase transition.     

Metaphase I arrest of starfish oocytes induced via the MAP kinase pathway is released by an increase of intracellular pH

Reinitiation of meiosis in oocytes usually occurs as a two-step process during which release from the prophase block is followed by an arrest in metaphase of the first or second meiotic division [metaphase I (MI) or metaphase II (MII)]. The mechanism of MI arrest in meiosis is poorly understood, although it is a widely observed phenomenon in invertebrates. The blockage of fully grown starfish oocytes in prophase of meiosis I is released by the hormone 1-methyladenine. It has been believed that meiosis of starfish oocytes proceeds completely without MI or MII arrest, even when fertilization does not occur. Here we show that MI arrest of starfish oocytes occurs in the ovary after germinal vesicle breakdown. This arrest is maintained both by the Mos/MEK/MAP kinase pathway and the blockage of an increase of intracellular pH in the ovary before spawning. Immediately after spawning into seawater, activation of Na+/H+ antiporters via a heterotrimeric G protein coupling to a 1-methyladenine receptor in the oocyte leads to an intracellular pH increase that can overcome the MI arrest even in the presence of active MAP kinase.     

DOC1R: a MAP kinase substrate that control microtubule organization of metaphase II mouse oocytes

For the success of fertilization, spindles of vertebrate oocytes must remain stable and correctly organized during the arrest in metaphase II of meiosis. Using a two-hybrid screen with MAPK as a bait, we have recently identified MISS (MAPK interacting and spindle stabilizing) which controls mouse oocyte metaphase II spindle stability. Using the same screen, we identify another MAPK partner, DOC1R (Deleted in oral cancer one related), a murine homologue of a potential human tumor suppressor gene. We characterize DOC1R during mouse oocyte meiosis resumption. DOC1R is regulated by phosphorylation during meiotic maturation by MPF (M-phase promoting factor) and by the MOS/./MAPK pathway. DOC1R and a DOC1R-GFP fusion localize to microtubules during meiotic maturation. Consistent with this microtubular localization, we show, by antisense and double-stranded RNA injection, that depletion of DOC1R induces microtubule defects in metaphase II oocytes. These defects are rescued by overexpressing a Xenopus DOC1R, showing that they are specific to DOC1R. Thus, the discovery of DOC1R, a substrate of MAPK that regulates microtubule organization of metaphase II mouse oocytes, reinforces the importance of this pathway in the control of spindle stability during the metaphase II arrest.     

CK2 beta, which inhibits Mos function, binds to a discrete domain in the N-terminus of Mos

Progesterone stimulates G2-arrested Xenopus oocytes to synthesize Mos, a MAPK kinase kinase required for the coordinated activation of cdc2 and the G2/Meiosis I (MI) transition. Mos leads to activation of MAPK, Rsk, and the inhibition of the cdc2 inhibitor Myt1. Previous work identified CK2 beta as a Mos-interacting protein, and suggested that CK2 beta acts as a negative regulator by setting a threshold above which newly made Mos must accumulate to activate MAPK. However, it had not been demonstrated that CK2 beta directly inhibits Mos. We report here that Mos (52-115) is required for CK2 beta binding and can serve as a portable binding domain. To test whether CK2 beta acts at the level of Mos or on a downstream component, we took advantage of previous work that showed injection of Mos arrests rapidly dividing embryonic cells. We find that coinjection of CK2 beta and Mos into embryonic cells inhibits the ability of Mos to arrest cell division. In contrast, CK2 beta does not inhibit the mitotic arrest induced by injection of active Rsk. These results argue that CK2 beta directly binds and inhibits Mos rather than a downstream component, and support that CK2 beta functions as a molecular buffer that prevents premature MAPK activation and oocyte maturation.     

The spindle checkpoint kinase bub1 and cyclin e/cdk2 both contribute to the establishment of meiotic metaphase arrest by cytostatic factor

In vertebrate unfertilized eggs, metaphase arrest in Meiosis II is mediated by an activity known as cytostatic factor (CSF). CSF arrest is dependent upon Mos-dependent activation of the MAPK/Rsk pathway, and Rsk activates the spindle checkpoint kinase Bub1, leading to inhibition of the anaphase-promoting complex (APC), an E3 ubiquitin ligase required for the metaphase/anaphase transition. However, it is not known whether Bub1 is required for the establishment of CSF arrest or whether other pathways also contribute. Here, we show that immunodepletion of Bub1 from egg extracts blocks the ability of Mos to establish CSF arrest, and arrest can be restored by the addition of wild-type, but not kinase-dead, Bub1. The appearance of CSF arrest at Meiosis II may result from coexpression of cyclin E/Cdk2 with the MAPK/Bub1 pathway. Cyclin E/Cdk2 was able to cause metaphase arrest in egg extracts even in the absence of Mos and could also inhibit cyclin B degradation in oocytes when expressed at anaphase of Meiosis I. Once it has been established, metaphase arrest can be maintained in the absence of MAPK, Bub1, or cyclin E/Cdk2 activity. Both pathways are independent of each other, but each appears to block activation of the APC, which is required for cyclin B degradation and the metaphase/anaphase transition.     

The MAPK pathway triggers activation of Nek2 during chromosome condensation in mouse spermatocytes

Chromosome condensation during the G2/M progression of mouse pachytene spermatocytes induced by the phosphatase inhibitor okadaic acid (OA) requires the activation of the MAPK Erk1. In many cell systems, p90Rsks are the main effectors of Erk1/2 function. We have identified p90Rsk2 as the isoform that is specifically expressed in mouse spermatocytes and have shown that it is activated during the OA-triggered meiotic G2/M progression. By using the MEK inhibitor U0126, we have demonstrated that activation of p90Rsk2 during meiotic progression requires activation of the MAPK pathway. Immunofluorescence analysis indicates that activated Erks and p90Rsk2 are tightly associated with condensed chromosomes during the G2/M transition in meiotic cells. We also found that active p90Rsk2 was able to phosphorylate histone H3 at Ser10 in vitro, but that the activation of the Erk1/p90Rsk2 pathway was not necessary for phosphorylation of H3 in vivo. Furthermore, phosphorylation of H3 was not sufficient to cause condensation of meiotic chromosomes in mouse spermatocytes. Other proteins known to associate with chromatin may represent effectors of Erk1 and p90Rsk2 during chromosome condensation. Nek2 (NIMA-related kinase 2), which associates with chromosomes, plays an active role in chromatin condensation and is stimulated by treatment of pachytene spermatocytes with okadaic acid. We show that inhibition of the MAPK pathway by preincubation of spermatocytes with U0126 suppresses Nek2 activation, and that incubation of spermatocyte cell extracts with activated p90Rsk2 causes stimulation of Nek2 kinase activity. Furthermore, we show that the Nek2 kinase domain is a substrate for p90Rsk2 phosphorylation in vitro. These data establish a connection between the Erk1/p90Rsk2 pathway, Nek2 activation and chromosome condensation during the G2/M transition of the first meiotic prophase.     

Inhibition of MEK or cdc2 kinase parthenogenetically activates mouse eggs and yields the same phenotypes as Mos(-/-) parthenogenotes

Mammalian eggs are arrested in metaphase II of meiosis until fertilization. Arrest is maintained by cytostatic factor (CSF) activity, which is dependent on the MOS-MEK-MAPK pathway. Inhibition of MEK1/2 with a specific inhibitor, U0126, parthenogenetically activated mouse eggs, producing phenotypes similar to Mos(-/-) parthenogenotes (premature, unequal cleavages and large polar bodies). U0126 inactivated MAPK in eggs within 1 h, in contrast to the 5 h required after fertilization, while the time course of MPF inactivation was similar in U0126-activated and fertilized eggs. We also found that inactivation of MPF by the cdc2 kinase inhibitor roscovitine induced parthenogenetic activation. Inactivation of MPF by roscovitine resulted in the subsequent inactivation of MAPK with a time course similar to that following fertilization. Notably, roscovitine also produced some Mos(-/-)-like phenotypes, indistinguishable from U0126 parthenogenotes. Simultaneous inhibition of both MPF and MAPK in eggs treated with roscovitine and U0126 produced a very high proportion of eggs with the more severe phenotype. These findings confirm that MEK is a required component of CSF in mammalian eggs and imply that the sequential inactivation of MPF followed by MAPK inactivation is required for normal spindle function and polar body emission.