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.     

Emi2 at the Crossroads: Where CSF Meets MPF

Vertebrate eggs arrest at metaphase of meiosis II due to an activity known as cytostatic factor (CSF). CSF antagonizes the ubiquitin ligase activity of the anaphase-promoting complex/cyclosome (APC/C), preventing cyclin B destruction and meiotic exit until fertilization occurs. A puzzling feature of CSF arrest is that APC/C inhibition is leaky. Ongoing cyclin B synthesis is counterbalanced by a limited amount of APC/C-mediated cyclin B destruction; thus, cyclin B/Cdc2 activity remains at steady state. How the APC/C can be slightly active toward cyclin B, and yet restrained from ubiquitinating cyclin B altogether, is unknown. Emi2/XErp1 is the critical CSF component directly responsible for APC/C inhibition during CSF arrest. Fertilization triggers the Ca2+-dependent destruction of Emi2, releasing the APC/C to ubiquitinate the full pool of cyclin B and initiate completion of meiosis. Previously, we showed that a phosphatase maintains Emi2’s APC/C-inhibitory activity in CSF-arrested Xenopus egg extracts. Here, we demonstrate that phosphatase inhibition permits Emi2 phosphorylation at thr-545 and -551, which inactivates Emi2. Furthermore, we provide evidence that adding excess cyclin B to CSF extracts stimulates Cdc2 phosphorylation of these same residues, antagonizing Emi2-APC/C association. Our findings suggest a model wherein the pool of Emi2 acts analogously to a rheostat by integrating Cdc2 and phosphatase activities to prevent cyclin B overaccumulation and Cdc2 hyperactivity during the indefinite period of time between arrival at metaphase II and eventual fertilization. Finally, we propose that inactivation of Emi2 by Cdc2 permits mitotic progression during early embryonic cleavage cycles.     

The HECT E3 ligase Smurf2 is required for Mad2-dependent spindle assembly checkpoint

Activation of the anaphase-promoting complex/cyclosome (APC/C) by Cdc20 is critical for the metaphase–anaphase transition. APC/C-Cdc20 is required for polyubiquitination and degradation of securin and cyclin B at anaphase onset. The spindle assembly checkpoint delays APC/C-Cdc20 activation until all kinetochores attach to mitotic spindles. In this study, we demonstrate that a HECT (homologous to the E6-AP carboxyl terminus) ubiquitin ligase, Smurf2, is required for the spindle checkpoint. Smurf2 localizes to the centrosome, mitotic midbody, and centromeres. Smurf2 depletion or the expression of a catalytically inactive Smurf2 results in misaligned and lagging chromosomes, premature anaphase onset, and defective cytokinesis. Smurf2 inactivation prevents nocodazole-treated cells from accumulating cyclin B and securin and prometaphase arrest. The silencing of Cdc20 in Smurf2-depleted cells restores mitotic accumulation of cyclin B and securin. Smurf2 depletion results in enhanced polyubiquitination and degradation of Mad2, a critical checkpoint effector. Mad2 is mislocalized in Smurf2-depleted cells, suggesting that Smurf2 regulates the localization and stability of Mad2. These data indicate that Smurf2 is a novel mitotic regulator.     

Loss of Cdc20 causes a securin-dependent metaphase arrest in two-cell mouse embryos

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase mediating targeted proteolysis through ubiquitination of protein substrates to control the progression of mitosis. The APC/C recognizes its substrates through two adapter proteins, Cdc20 and Cdh1, which contain similar C-terminal domains composed of seven WD-40 repeats believed to be involved in interacting with their substrates. During the transition from metaphase to anaphase, APC/C-Cdc20 mediates the ubiquitination of securin and cyclin B1, allowing the activation of separase and the onset of anaphase and mitotic exit. APC/C-Cdc20 and APC/C-Cdh1 have overlapping substrates. It is unclear whether they are redundant for mitosis. Using a gene-trapping approach, we have obtained mice which lack Cdc20 function. These mice show failed embryogenesis. The embryos were arrested in metaphase at the two-cell stage with high levels of cyclin B1, indicating an essential role of Cdc20 in mitosis that is not redundant with that of Cdh1. Interestingly, Cdc20 and securin double mutant embryos could not maintain the metaphase arrest, suggesting a role of securin in preventing mitotic exit.     

Coupling of Cdc20 inhibition and activation by BubR1

Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by cyclin B1–Cdk1 independently inhibits APC/C–Cdc20 activation. This creates a conundrum for how Cdc20 is activated before cyclin B1 degradation. Here, we show that the MCC component BubR1 harbors both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically, BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest, arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively, our work reveals how Cdc20 can be dephosphorylated in the presence of cyclin B1-Cdk1 activity without causing premature anaphase onset.     

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.     

XCdh1 is involved in progesterone-induced oocyte maturation

The molecular events triggered during progesterone-induced oocyte maturation in Xenopus are not well understood. One of the first events is the activation of the MAPK cascade and the maturation-promoting factor (MPF). The latter triggers meiosis I resumption and meiosis II progression until the metaphase II arrest. The release of the metaphase II is mediated by the anaphase-promoting complex (APC)-dependent degradation of cyclin B. This degradation activity requires the APC activator Cdc20 that activates ubiquitination reactions by recruiting substrates to the APC. However, recent reports in different organisms involve other APC regulators during different phases of the meiotic cycle. Therefore, we investigated the role of another APC regulator, XCdh1 during the G2/M transition in meiosis I in the Xenopus oocyte. Here, we report that XCdh1 protein is expressed in oocytes. Besides, injection of specific XCdh1 antisense inhibits progesterone-induced G2/M transition that can be rescued by adding back the purified human Cdh1 protein. On the other hand, ectopic expression of low levels of XCdh1 protein has a positive effect on the G2/M transition by facilitating this process. Moreover, the sole injection of XCdh1 mRNA triggers Mos protein synthesis and biphosphorylation of MAPK in absence of progesterone. Altogether, these data show that XCdh1 has a positive role during the G2/M transition in the oocyte. According to our results, its role could be independent of the APC.     

The pathway of MAP kinase mediation of CSF arrest in Xenopus oocytes.

A cytoplasmic activity in mature oocytes responsible for second meiotic metaphase arrest was identified over 30 years ago in amphibian oocytes. In Xenopus oocytes CSF activity is initiated by the progesterone-dependent synthesis of Mos, a MAPK kinase kinase, which activates the MAPK pathway. CSF arrest is mediated by a sole MAPK target, the protein kinase p90Rsk which leads to inhibition of cyclin B degradation by the anaphase-promoting complex. Rsk phosphorylates and activates the Bub1 protein kinase, which may cause metaphase arrest due to inhibition of the anaphase-promoting complex (APC) by a conserved mechanism defined genetically in yeast and mammalian cells. CSF arrest in vertebrate oocytes by p90Rsk provides a potential link between the MAPK pathway and the spindle assembly checkpoint in the cell cycle.     

A role for Cdc2- and PP2A-mediated regulation of Emi2 in the maintenance of CSF arrest

BACKGROUND: Vertebrate oocytes are arrested in metaphase II of meiosis prior to fertilization by cytostatic factor (CSF). CSF enforces a cell-cycle arrest by inhibiting the anaphase-promoting complex (APC), an E3 ubiquitin ligase that targets Cyclin B for degradation. Although Cyclin B synthesis is ongoing during CSF arrest, constant Cyclin B levels are maintained. To achieve this, oocytes allow continuous slow Cyclin B degradation, without eliminating the bulk of Cyclin B, which would induce release from CSF arrest. However, the mechanism that controls this continuous degradation is not understood. RESULTS: We report here the molecular details of a negative feedback loop wherein Cyclin B promotes its own destruction through Cdc2/Cyclin B-mediated phosphorylation and inhibition of the APC inhibitor Emi2. Emi2 bound to the core APC, and this binding was disrupted by Cdc2/Cyclin B, without affecting Emi2 protein stability. Cdc2-mediated phosphorylation of Emi2 was antagonized by PP2A, which could bind to Emi2 and promote Emi2-APC interactions. CONCLUSIONS: Constant Cyclin B levels are maintained during a CSF arrest through the regulation of Emi2 activity. A balance between Cdc2 and PP2A controls Emi2 phosphorylation, which in turn controls the ability of Emi2 to bind to and inhibit the APC. This balance allows proper maintenance of Cyclin B levels and Cdc2 kinase activity during CSF arrest.     

Interplay between Cdc2 kinase and the c-Mos/MAPK pathway between metaphase I and metaphase II in Xenopus oocytes

Xenopus oocytes arrested in prophase I resume meiotic division in response to progesterone and arrest at metaphase II. Entry into meiosis I depends on the activation of Cdc2 kinase [M-phase promoting factor (MPF)]. To better understand the role of Cdc2, MPF activity was specifically inhibited by injection of the CDK inhibitor, Cip1. When Cip1 is injected at germinal vesicle breakdown (GVBD) time, Cdc25 and Plx1 are both dephosphorylated and Cdc2 is rephosphorylated on tyrosine. The autoamplification loop characterizing MPF is therefore not only required for MPF generation before GVBD, but also for its stability during the GVBD period. The ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), responsible for cyclin degradation, is also under the control of Cdc2; therefore, Cdc2 activity itself induces its own inactivation through cyclin degradation, allowing the exit from the first meiotic division. In contrast, cyclin accumulation, responsible for Cdc2 activity increase allowing entry into metaphase II, is independent of Cdc2. The c-Mos/mitogen-activated protein kinase (MAPK) pathway remains active when Cdc2 activity is inhibited at GVBD time. This pathway could be responsible for the sustained cyclin neosynthesis. In contrast, during the metaphase II block, the c-Mos/MAPK pathway depends on Cdc2. Therefore, the metaphase II block depends on a dynamic interplay between MPF and CSF, the c-Mos/MAPK pathway stabilizing cyclin B, whereas in turn, MPF prevents c-Mos degradation.     

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.     

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.     

Involvement of Ca2+-dependent proteasome in the degradation of both cyclin B1 and Mos during spontaneous activation of matured rat oocytes

In matured rat oocytes, spontaneous activation from the metaphase-II (MII) stage occurred after collection from the oviducts. It is well known that the mitogen-activated protein kinase (MAPK) pathway and p34(cdc2) kinase play an important role in the arrest at MII in other species. However, there is no information about the difference in these factors among strains of rats. In the present study, in spontaneously activated oocytes from the Wistar rat, the Mos protein level and the activity of MAPK kinase (MEK)/MAPK were decreased at 120 min (13.8, 25.7, and 19.3, respectively, P<0.05), whereas Sprague-Dawley (SD) oocytes, which were not spontaneously activated, had a high level of Mos protein and MEK/MAPK activity (75.9, 76.2, and 87.9, respectively, P<0.05). Phosphorylation of MAPK in the SD oocytes was significantly suppressed by MEK inhibitor, U0126 at 60 min; this treatment decreased p34(cdc2) kinase activity via cyclin B1 degradation in a time-dependent manner. The treatment with proteasome inhibitor, MG132 or Ca2+-chelator, BAPTA-AM, overcame the spontaneous degradation of both Mos and cyclin B1 in a dose-dependent manner in Wistar oocytes. More than 90% of Wistar oocytes treated with BAPTA-AM were arrested at MII until 120 min. In conclusion, SD oocytes carrying Mos/MEK/MAPK, maintained a high activity of p34(cdc2) kinase by stabilizing cyclin B1, thus involved in their meiotic arrest. In contrast, Wistar oocytes had a relatively low cytostatic factor activity; rapid decrease of Mos/MEK/MAPK failed to stabilize both cyclin B1 and Mos, and these oocytes were likely to spontaneously activate.     

p90Rsk is required for G1 phase arrest in unfertilized starfish eggs

The cell cycle in oocytes generally arrests at a particular meiotic stage to await fertilization. This arrest occurs at metaphase of meiosis II (meta-II) in frog and mouse, and at G1 phase after completion of meiosis II in starfish. Despite this difference in the arrest phase, both arrests depend on the same Mos-MAPK (mitogen-activated protein kinase) pathway, indicating that the difference relies on particular downstream effectors. Immediately downstream of MAPK, Rsk (p90 ribosomal S6 kinase, p90(Rsk)) is required for the frog meta-II arrest. However, the mouse meta-II arrest challenges this requirement, and no downstream effector has been identified in the starfish G1 arrest. To investigate the downstream effector of MAPK in the starfish G1 arrest, we used a neutralizing antibody against Rsk and a constitutively active form of Rsk. Rsk was activated downstream of the Mos-MAPK pathway during meiosis. In G1 eggs, inhibition of Rsk activity released the arrest and initiated DNA replication without fertilization. Conversely, maintenance of Rsk activity prevented DNA replication following fertilization. In early embryos, injection of Mos activated the MAPK-Rsk pathway, resulting in G1 arrest. Moreover, inhibition of Rsk activity during meiosis I led to parthenogenetic activation without meiosis II. We conclude that immediately downstream of MAPK, Rsk is necessary and sufficient for the starfish G1 arrest. Although CSF (cytostatic factor) was originally defined for meta-II arrest in frog eggs, we propose to distinguish ;G1-CSF' for starfish from ;meta-II-CSF' for frog and mouse. The present study thus reveals a novel role of Rsk for G1-CSF.     

Cdc2 and Mos regulate Emi2 stability to promote the meiosis I-meiosis II transition

The transition of oocytes from meiosis I (MI) to meiosis II (MII) requires partial cyclin B degradation to allow MI exit without S phase entry. Rapid reaccumulation of cyclin B allows direct progression into MII, producing a cytostatic factor (CSF)-arrested egg. It has been reported that dampened translation of the anaphase-promoting complex (APC) inhibitor Emi2 at MI allows partial APC activation and MI exit. We have detected active Emi2 translation at MI and show that Emi2 levels in MI are mainly controlled by regulated degradation. Emi2 degradation in MI depends not on Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), but on Cdc2-mediated phosphorylation of multiple sites within Emi2. As in MII, this phosphorylation is antagonized by Mos-mediated recruitment of PP2A to Emi2. Higher Cdc2 kinase activity in MI than MII allows sufficient Emi2 phosphorylation to destabilize Emi2 in MI. At MI anaphase, APC-mediated degradation of cyclin B decreases Cdc2 activity, enabling Cdc2-mediated Emi2 phosphorylation to be successfully antagonized by Mos-mediated PP2A recruitment. These data suggest a model of APC autoinhibition mediated by stabilization of Emi2; Emi2 proteins accumulate at MI exit and inhibit APC activity sufficiently to prevent complete degradation of cyclin B, allowing MI exit while preventing interphase before MII entry.     

Ca(2+) oscillations promote APC/C-dependent cyclin B1 degradation during metaphase arrest and completion of meiosis in fertilizing mouse eggs

Cyclin B1, the regulatory component of M phase-promoting factor (MPF), is degraded during the metaphase-anaphase transition in an anaphase-promoting complex/cyclosome (APC/C)-dependent process. MPF activity is stable in eggs, and a sperm-triggered Ca(2+) signal is needed to promote cyclin degradation. In frogs, a single Ca(2+) spike promotes cell cycle resumption, but, in mammals, the Ca(2+) signal is more complex, consisting of a series of spikes that stop several hours after sperm fusion. Using dual imaging in mouse eggs, we have examined how the Ca(2+) signal generates cyclin B1 destruction using destructible and nondestructible GFP-tagged constructs. APC/C activity was present in unfertilized eggs, giving cyclin B1 a half-life of 1.15 +/- 0.28 hr. However, APC/C-dependent cyclin degradation was elevated 6-fold when sperm raised cytosolic Ca(2+) levels above 600 nM. This activation was transitory since cyclin B1 levels recovered between Ca(2+) spikes. For continued cyclin degradation at basal Ca(2+) levels, multiple spikes were needed. APC/C-mediated degradation was observed until eggs had completed meiosis with the formation of pronuclei, and, at this time, Ca(2+) spikes stopped. Therefore, the physiological need for a repetitive Ca(2+) signal in mammals is to ensure long-term cyclin destruction during a protracted exit from meiosis.     

Activation of the anaphase-promoting complex and degradation of cyclin B is not required for progression from Meiosis I to II in Xenopus oocytes.

Sister chromatid separation and cyclin degradation in mitosis depend on the association of the anaphase-promoting complex (APC) with the Fizzy protein (Cdc20), leading to the metaphase/anaphase transition and exit from mitosis [1--3]. In Xenopus, after metaphase of the first meiotic division, only partial cyclin degradation occurs, and chromosome segregation during anaphase I proceeds without sister chromatid separation [4--7]. We investigated the role of xFizzy during meiosis using an antisense depletion approach. xFizzy accumulates to high levels in Meiosis I, and injection of antisense oligonucleotides to xFizzy blocks nearly all APC-mediated cyclin B degradation and Cdc2/cyclin B (MPF) inactivation between Meiosis I and II. However, even without APC activation, xFizzy-ablated oocytes progress to Meiosis II as shown by cyclin E synthesis, further accumulation of cyclin B, and evolution of the metaphase I spindle to a metaphase II spindle via a disc-shaped aggregate of microtubules known to follow anaphase I [8]. Inhibition of the MAPK pathway by U0126 in antisense-injected oocytes prevents cyclin B accumulation beyond the level that is present at metaphase I. Full synthesis and accumulation can be restored in the presence of U0126 by the expression of a constitutively active form of the MAPK target, p90(Rsk). Thus, p90(Rsk) is sufficient not only to partially inhibit APC activity [7], but also to stimulate cyclin B synthesis in Meiosis II.     

Activation of Wee1 by p42 MAPK in vitro and in cycling xenopus egg extracts

Xenopus oocytes and eggs provide a dramatic example of how the consequences of p42 mitogen-activated protein kinase (p42 MAPK) activation depend on the particular context in which the activation occurs. In oocytes, the activation of Mos, MEK, and p42 MAPK is required for progesterone-induced Cdc2 activation, and activated forms of any of these proteins can bring about Cdc2 activation in the absence of progesterone. However, in fertilized eggs, activation of the Mos/MEK/p42 MAPK pathway has the opposite effect, inhibiting Cdc2 activation and causing a G2 phase delay or arrest. In the present study, we have investigated the mechanism and physiological significance of the p42 MAPK-induced G2 phase arrest, using Xenopus egg extracts as a model system. We found that Wee1-depleted extracts were unable to arrest in G2 phase in response to Mos, and adding back Wee1 to the extracts restored their ability to arrest. This finding formally places Wee1 downstream of Mos/MEK/p42 MAPK. Purified recombinant p42 MAPK was found to phosphorylate recombinant Wee1 in vitro at sites that are phosphorylated in extracts. Phosphorylation by p42 MAPK resulted in a modest ( approximately 2-fold) increase in the kinase activity of Wee1 toward Cdc2. Titration experiments in extracts demonstrated that a twofold increase in Wee1 activity is sufficient to cause the delay in mitotic entry seen in Mos-treated extracts. Finally, we present evidence that the negative regulation of Cdc2 by Mos/MEK/p42 MAPK contributes to the presence of an unusually long G2 phase in the first mitotic cell cycle. Prematurely inactivating p42 MAPK in egg extracts resulted in a corresponding hastening of the first mitosis. The negative effect of p42 MAPK on Cdc2 activation may help ensure that the first mitotic cell cycle is long enough to allow karyogamy to be accomplished successfully.     

Cdc20 proteolysis requires p38 MAPK signaling and Cdh1‐independent APC/C ubiquitination during spindle assembly checkpoint activation by cadmium

Cdc20, an activator of the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase, initiates the destruction of key mitotic regulators to facilitate mitosis, while it is negatively regulated by the spindle assembly checkpoint (SAC) to prevent premature anaphase entry. Activation of the p38 mitogen‐activated protein kinase could contribute to mitotic arrest, but the underlying mechanism is unknown. Here we report a novel pathway in which the p38 signaling triggers Cdc20 destruction under SAC elicited by cadmium, a human carcinogen. We found that the cadmium‐induced prometaphase arrest was linked to decreased Cdc20 and accumulated cyclin A protein levels in human cells, whereas the activity of cyclin B1–Cdk1 was unaffected. The Cdc20 half‐life was markedly shortened along with its ubiquitination and degradation via 26S proteasome in cadmium‐treated asynchronous or G₂‐enriched cells. Depletion of APC3 markedly suppressed the cadmium‐induced Cdc20 ubiquitination and proteolysis, while depletion of Cdh1, another activator of APC/C, did not. Intriguingly, blockage of p38 activity restored the Cdc20 levels for continuing mitosis under cadmium, while inhibition of JNK activity had no effect. The cadmium‐induced Cdc20 proteolysis was also suppressed during transient depletion of p38α or stable expression a dominant negative form of p38. Inhibition of p38 abolished the induction of Mad2–Cdc20–APC3 complex by cadmium. Moreover, forced expression of MKK6–p38 signaling could promote Cdc20 degradation in a Cdh1‐independent APC/C pathway. In summary, accelerated ubiquitination and proteolysis of Cdc20 is essential for prometaphase arrest that is mediated via the p38 signaling during SAC activation. J. Cell. Physiol. 223: 327–334, 2010. © 2010 Wiley‐Liss, Inc.     

Activation of the p42 Mitogen-activated Protein Kinase Pathway Inhibits Cdc2 Activation and Entry into M-Phase in Cycling Xenopus Egg Extracts

We have added constitutively active MAP kinase/ERK kinase (MEK), an activator of the mitogen-activated protein kinase (MAPK) signaling pathway, to cycling Xenopus egg extracts at various times during the cell cycle. p42MAPK activation during entry into M-phase arrested the cell cycle in metaphase, as has been shown previously. Unexpectedly, p42MAPK activation during interphase inhibited entry into M-phase. In these interphase-arrested extracts, H1 kinase activity remained low, Cdc2 was tyrosine phosphorylated, and nuclei continued to enlarge. The interphase arrest was overcome by recombinant cyclin B. In other experiments, p42MAPK activation by MEK or by Mos inhibited Cdc2 activation by cyclin B. PD098059, a specific inhibitor of MEK, blocked the effects of MEK(QP) and Mos. Mos-induced activation of p42MAPK did not inhibit DNA replication. These results indicate that, in addition to the established role of p42MAPK activation in M-phase arrest, the inappropriate activation of p42MAPK during interphase prevents normal entry into M-phase.