Cytostatic Factor Arrest Research Articles

MAPK3/1 (ERK1/2) and Myosin Light Chain Kinase in Mammalian Eggs Affect Myosin-II Function and Regulate the Metaphase II State in a Calcium- and Zinc-Dependent Manner.

Vertebrate eggs are arrested at metaphase of meiosis II, a state classically known as cytostatic factor arrest. Maintenance of this arrest until the time of fertilization and then fertilization-induced exit from metaphase II are crucial for reproductive success. Another key aspect of this meiotic arrest and exit is regulation of the metaphase II spindle, which must be appropriately localized adjacent to the egg cortex during metaphase II and then progress into successful asymmetric cytokinesis to produce the second polar body. This study examined the mitogen-activated protein kinases MAPK3 and MAPK1 (also known as ERK1/2) as regulators of these two related aspects of mammalian egg biology, specifically testing whether this MAPK pathway affected myosin-II function and whether myosin-II perturbation would produce some of the same effects as MAPK pathway perturbation. Inhibition of the MEK1/2-MAPK pathway with U0126 leads to reduced levels of phosphorylated myosin-regulatory light chain (pMRLC) and causes a reduction in cortical tension, effects that are mimicked by treatment with the myosin light chain kinase (MLCK) inhibitor ML-7. These data indicate that one mechanism by which the MAPK pathway acts in eggs is by affecting myosin-II function. We further show that MAPK or MLCK inhibition induces loss of normal cortical spindle localization or parthenogenetic egg activation. This parthenogenesis is dependent on cytosolic and extracellular calcium and can be rescued by hyperloading eggs with zinc, suggesting that these effects of inhibition of MLCK or the MAPK pathway are linked with dysregulation of ion homeostasis.

Involvement of Mos–MEK–MAPK pathway in cytostatic factor (CSF) arrest in eggs of the parthenogenetic insect, Athalia rosae

Extensive survey of meiotic metaphase II arrest during oocyte maturation in vertebrates revealed that the mitogen-activated protein kinase (MAPK) pathway regulated by the c-mos proto-oncogene product, Mos, has an essential role in cytostatic activity, termed cytostatic factor (CSF). In contrast, little is known in invertebrates in which meiotic arrest occurs in most cases at metaphase I (MI arrest). A parthenogenetic insect, the sawfly Athalia rosae, in which artificial egg activation is practicable, has advantages to investigate the mechanisms of MI arrest. Both the MAPK/extracellular signal-regulated protein kinase kinase (MEK) and MAPK were phosphorylated and maintained active in MI-arrested sawfly eggs, whereas they were dephosphorylated soon after egg activation. Treatment of MI-arrested eggs with U0126, an inhibitor of MEK, resulted in dephosphorylation of MAPK and MI arrest was resumed. The sawfly c-mos gene orthologue encoding a serine/threonine kinase was cloned and analyzed. It was expressed in nurse cells in the ovaries. To examine CSF activity of the sawfly Mos, synthesized glutathione S-transferase (GST)-fusion sawfly Mos protein was injected into MI-resumed eggs in which MEK and MAPK were dephosphorylated. Both MEK and MAPK were phosphorylated again upon injection. In these GST-fusion sawfly Mos-injected eggs subsequent mitotic (syncytial) divisions were blocked and embryonic development was ceased. These results demonstrated that the MEK–MAPK pathway was involved in maintaining CSF arrest in sawfly eggs and Mos functioned as its upstream regulatory molecule.

Control of Emi2 activity and stability through Mos-mediated recruitment of PP2A

Before fertilization, vertebrate eggs are arrested in meiosis II by cytostatic factor (CSF), which holds the anaphase-promoting complex (APC) in an inactive state. It was recently reported that Mos, an integral component of CSF, acts in part by promoting the Rsk-mediated phosphorylation of the APC inhibitor Emi2/Erp1. We report here that Rsk phosphorylation of Emi2 promotes its interaction with the protein phosphatase PP2A. Emi2 residues adjacent to the Rsk phosphorylation site were important for PP2A binding. An Emi2 mutant that retained Rsk phosphorylation but lacked PP2A binding could not be modulated by Mos. PP2A bound to Emi2 acted on two distinct clusters of sites phosphorylated by Cdc2, one responsible for modulating its stability during CSF arrest and one that controls binding to the APC. These findings provide a molecular mechanism for Mos action in promoting CSF arrest and also define an unusual mechanism, whereby protein phosphorylation recruits a phosphatase for dephosphorylation of distinct sites phosphorylated by another kinase.

Phosphorylation of Erp1 by p90rsk is required for cytostatic factor arrest in Xenopus laevis eggs

Until fertilization, the meiotic cell cycle of vertebrate eggs is arrested at metaphase of meiosis II by a cytoplasmic activity termed cytostatic factor (CSF), which causes inhibition of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets mitotic cyclins-regulatory proteins of meiosis and mitosis-for degradation. Recent studies indicate that Erp1/Emi2, an inhibitor protein for the APC/C, has an essential role in establishing and maintaining CSF arrest, but its relationship to Mos, a mitogen-activated protein kinase (MAPK) kinase kinase that also has an essential role in establishing CSF arrest through activation of p90 ribosomal S6 kinase (p90rsk), is unclear. Here we report that in Xenopus eggs Erp1 is a substrate of p90rsk, and that Mos-dependent phosphorylation of Erp1 by p90rsk at Thr 336, Ser 342 and Ser 344 is crucial for both stabilizing Erp1 and establishing CSF arrest in meiosis II oocytes. Semi-quantitative analysis with CSF-arrested egg extracts reveals that the Mos-dependent phosphorylation of Erp1 enhances, but does not generate, the activity of Erp1 that maintains metaphase arrest. Our results also suggest that Erp1 inhibits cyclin B degradation by binding the APC/C at its carboxy-terminal destruction box, and this binding is also enhanced by the Mos-dependent phosphorylation. Thus, Mos and Erp1 collaboratively establish and maintain metaphase II arrest in Xenopus eggs. The link between Mos and Erp1 provides a molecular explanation for the integral mechanism of CSF arrest in unfertilized vertebrate eggs.

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.

Translational Unmasking of Emi2 Directs Cytostatic Factor Arrest in Meiosis II

Cytostatic factor (CSF) arrests unfertilized vertebrate eggs in metaphase of meiosis II by inhibiting the anaphase-promoting complex/cyclosome (APC/C) from mediating cyclin destruction. The APC/C inhibitor Emi2/XErp1 satisfies a number of historical criteria for the molecular identification of CSF, but the mechanism by which CSF is activated selectively in meiosis II is the remaining unexplained criterion. Here we provide an explanation by showing that Emi2 is expressed specifically in meiosis II through translational de-repression or “unmasking” of its mRNA. We find that Emi2 protein is undetectable in immature, G2/prophase-arrested Xenopus oocytes and accumulates ~90 minutes after germinal vesicle breakdown. The 3’ untranslated region of Emi2 mRNA contains cytoplasmic polyadenylation elements that directly bind the CPEB protein and confer temporal regulation of Emi2 polyadenylation and translation. Our results demonstrate that cytoplasmic polyadenylation and translational unmasking of Emi2 directs meiosis II-specific CSF arrest.

New insight into metaphase arrest by cytostatic factor: from establishment to release

Since the discovery of cytostatic factor (CSF) 35 years ago, significant progress has been made in identifying molecular components of CSF activity and the mechanism of CSF-induced metaphase II arrest (CSF arrest). This short review focuses on recent discoveries in the field and discusses the implication of these results for a general picture of CSF establishment and release. One recent focus is on the cyclin E/Cdk2 pathway. The discovery of a downstream target for cyclin E/Cdk2, the spindle checkpoint protein Mps1, provides insight into how cyclin E/Cdk2 contributes to CSF arrest. The anaphase promoting complex/cyclosome (APC/C) inhibitor Emi2 is another recent focus of work in the field. It is now clear that not only is degradation of Emi2 critical for CSF release, but its abrupt accumulation during meiosis II (M II) is also required for the establishment of CSF arrest. Thus, by discrete pathways of APC/C inhibition operative during CSF arrest, the stability of cell cycle arrest in the egg appears to be reinforced by multiple mechanisms.

A Role for Cdc2- and PP2A-Mediated Regulation of Emi2 in the Maintenance of CSF Arrest

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. 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. 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.

How eggs arrest at metaphase II: MPF stabilisation plus APC/C inhibition equals Cytostatic Factor

Oocytes from higher chordates, including man and nearly all mammals, arrest at metaphase of the second meiotic division before fertilization. This arrest is due to an activity that has been termed 'Cytostatic Factor'. Cytostatic Factor maintains arrest through preventing loss in Maturation-Promoting Factor (MPF; CDK1/cyclin B). Physiologically, Cytostatic Factor – induced metaphase arrest is only broken by a Ca2+ rise initiated by the fertilizing sperm and results in degradation of cyclin B, the regulatory subunit of MPF through the Anaphase-Promoting Complex/Cyclosome (APC/C). Arrest at metaphase II may therefore be viewed as being maintained by inhibition of the APC/C, and Cytostatic Factor as being one or more pathways, one of which inhibits the APC/C, consorting in the preservation of MPF activity.Many studies over several years have implicated the c-Mos/MEK/MAPK pathway in the metaphase arrest of the two most widely studied vertebrates, frog and mouse. Murine downstream components of this cascade are not known but in frog involve members of the spindle assembly checkpoint, which act to inhibit the APC/C. Interesting these downstream components appear not to be involved in the arrest of mouse eggs, suggesting a lack of conservation with respect to c-Mos targets. However, the recent discovery of Emi2 as an egg specific APC/C inhibitor whose degradation is Ca2+ dependent has greatly increased our understanding of MetII arrest. Emi2 is involved in both the establishment and maintenance of metaphase II arrest in frog and mouse suggesting a conservation of metaphase II arrest. Its identity as the physiologically relevant APC/C inhibitor involved in Cytostatic Factor arrest prompted us to re-evaluate the role of the c-Mos pathway in metaphase II arrest.This review presents a model of Cytostatic Factor arrest, which is primarily induced by Emi2 mediated APC/C inhibition but which also requires the c-Mos pathway to set MPF levels within physiological limits, not too high to induce an arrest that cannot be broken, or too low to induce parthenogenesis.

The Anaphase-promoting Complex/Cyclosome Inhibitor Emi2 Is Essential for Meiotic but Not Mitotic Cell Cycles

Vertebrate oocytes awaiting fertilization are arrested at metaphase of meiosis II by cytostatic factor (CSF). This arrest is due to inhibition of the anaphase-promoting complex/cyclosome, in part by a newly identified protein, Emi2 (xErp1). Emi2 is required for maintenance of CSF arrest in egg extracts, but its function in CSF establishment in oocytes and the normal embryonic cell cycle is unknown. Here we show that during oocyte maturation, Emi2 appears only after metaphase I, and its level peaks at CSF arrest (metaphase II). In M phase, Emi2 undergoes a phosphorylation-dependent electrophoretic shift. Microinjection of antisense oligonucleotides against Emi2 into stage VI oocytes blocks progression through meiosis II and the establishment of CSF arrest. Recombinant Emi2 rescues CSF arrest in these oocytes and also causes CSF arrest in egg extracts and in blastomeres of two-cell embryos. Fertilization triggers rapid, complete degradation of Emi2, but it is resynthesized in the first embryonic cell cycle to reach levels 5-fold lower than during CSF arrest. However, depletion of the protein from cycling egg extracts does not prevent mitotic cell cycle progression. Thus, Emi2 plays an essential role in meiotic but not mitotic cell cycles.

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.

Cytostatic factor: an activity that puts the cell cycle on hold

Fertilization is the fundamental process in which two gametes - sperm and oocyte - fuse to generate a zygote that will form a new multicellular organism. In most vertebrates, oocytes await fertilization while arrested at metaphase of meiosis II. This resting state can be stable for many hours and depends on a cytoplasmic activity termed cytostatic factor (CSF). Recently, members of the novel Emi/Erp family of proteins have been put forward as important components of CSF. These proteins inhibit the anaphase-promoting complex/cyclosome (APC/C), which acts at the very core of the cell cycle regulatory machinery. Initially, Xenopus early mitotic inhibitor 1 (Emi1) was proposed to be a component of CSF, but newer work suggests that a structural relative, Emi-related protein 1 (Erp1/Emi2), is essential for maintenance of CSF arrest in Xenopus. Most importantly, studies on Erp1/Emi2 regulation have led to a detailed molecular understanding of the Ca2+-mediated release from CSF arrest that occurs upon fertilization.

Mammalian Emi2 mediates cytostatic arrest and transduces the signal for meiotic exit via Cdc20

Fertilizable mammalian oocytes are arrested at the second meiotic metaphase (mII) by the cyclinB-Cdc2 heterodimer, maturation promoting factor (MPF). MPF is stabilized via the activity of an unidentified cytostatic factor (CSF), thereby suspending meiotic progression until fertilization. We here present evidence that a conserved 71 kDa mammalian orthologue of Xenopus XErp1/Emi2, which we term endogenous meiotic inhibitor 2 (Emi2) is an essential CSF component. Depletion in situ of Emi2 by RNA interference elicited precocious meiotic exit in maturing mouse oocytes. Reduction of Emi2 released mature mII oocytes from cytostatic arrest, frequently inducing cytodegeneration. Mos levels autonomously declined to undetectable levels in mII oocytes. Recombinant Emi2 reduced the propensity of mII oocytes to exit meiosis in response to activating stimuli. Emi2 and Cdc20 proteins mutually interact and Cdc20 ablation negated the ability of Emi2 removal to induce metaphase release. Consistent with this, Cdc20 removal prevented parthenogenetic or sperm-induced meiotic exit. These studies show in intact oocytes that the interaction of Emi2 with Cdc20 links activating stimuli to meiotic resumption at fertilization and during parthenogenesis in mammals.

CaMKII and Polo-like kinase 1 sequentially phosphorylate the cytostatic factor Emi2/XErp1 to trigger its destruction and meiotic exit

In vertebrate meiosis, unfertilized eggs are arrested in metaphase II by cytostatic factor (CSF), which is required to maintain mitotic cyclin-dependent kinase activity. Fertilization triggers a transient increase in cytosolic free Ca(2+), which leads to CSF inactivation and ubiquitin-dependent cyclin destruction through the anaphase promoting complex or cyclosome (APC/C). The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and the Polo-like kinase Plx1 are essential factors for Ca(2+)-induced meiotic exit, but the critical targets of these kinases were unknown. The APC/C inhibitor Emi2 or XErp1 has recently been characterized as a pivotal CSF component, required to maintain metaphase II arrest and rapidly destroyed in response to Ca(2+) signaling through phosphorylation by Plx1 and ubiquitination by the SCF(betaTrCP) complex. An important question is how the increase in free Ca(2+) targets Plx1 activity toward Emi2. Here, we demonstrate that CaMKII is required for Ca(2+)-induced Emi2 destruction, and that CaMKII functions as a "priming kinase," directly phosphorylating Emi2 at a specific motif to induce a strong interaction with the Polo Box domain of Plx1. We show that the strict requirement for CaMKII to phosphorylate Emi2 is a specific feature of CSF arrest, and we also use phosphatase inhibitors to demonstrate an additional mode of Emi2 inactivation independent of its destruction. We firmly establish the CSF component Emi2 as the first-known critical and direct target of CaMKII in CSF release, providing a detailed molecular mechanism explaining how CaMKII and Plx1 coordinately direct APC/C activation and meiotic exit upon fertilization.

Calcium triggers exit from meiosis II by targeting the APC/C inhibitor XErp1 for degradation

Vertebrate eggs awaiting fertilization are arrested at metaphase of meiosis II by a biochemical activity termed cytostatic factor (CSF). This activity inhibits the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that triggers anaphase onset and mitotic/meiotic exit by targeting securin and M-phase cyclins for destruction. On fertilization a transient rise in free intracellular calcium causes release from CSF arrest and thus APC/C activation. Although it has previously been shown that calcium induces the release of APC/C from CSF inhibition through calmodulin-dependent protein kinase II (CaMKII), the relevant substrates of this kinase have not been identified. Recently, we characterized XErp1 (Emi2), an inhibitor of the APC/C and key component of CSF activity in Xenopus egg extract. Here we show that calcium-activated CaMKII triggers exit from meiosis II by sensitizing the APC/C inhibitor XErp1 for polo-like kinase 1 (Plx1)-dependent degradation. Phosphorylation of XErp1 by CaMKII leads to the recruitment of Plx1 that in turn triggers the destruction of XErp1 by phosphorylating a site known to serve as a phosphorylation-dependent degradation signal. These results provide a molecular explanation for how the fertilization-induced calcium increase triggers exit from meiosis II.

Calcium Elevation at Fertilization Coordinates Phosphorylation of XErp1/Emi2 by Plx1 and CaMK II to Release Metaphase Arrest by Cytostatic Factor

Vertebrate oocytes are arrested at second meiotic metaphase by cytostatic factor (CSF) while awaiting fertilization. Accumulating evidence has suggested that inhibition of the anaphase-promoting complex/cyclosome (APC/C) is responsible for this arrest. Xenopus polo-like kinase 1 (Plx1) is required for activation of the APC/C at the metaphase-anaphase transition, and calcium elevation, upon fertilization/activation of eggs, acting through calmodulin-dependent kinase II (CaMKII) is sufficient to activate the APC/C and terminate CSF arrest. However, connections between the Plx1 pathway and the CaMKII pathway have not been identified. Overexpression of Plx1 causes CSF release in the absence of calcium, and depletion of Plx1 from egg extracts blocks induction of CSF release by calcium and CaMKII. Prior phosphorylation of the APC/C inhibitor XErp1/Emi2 by CaMK II renders it a good substrate for Plx1, and phosphorylation by both kinases together promotes its degradation in egg extracts. The pathway is enhanced by the ability of Plx1 to cause calcium-independent activation of CaMKII. The results identify the targets of CaMKII and Plx1 that promote egg activation and define the first known pathway of CSF release in which an APC/C inhibitor is targeted for degradation only when both CaMKII and Plx1 are active after calcium elevation at fertilization. Plx1 with an intact polo-box domain is necessary for release of CSF arrest and sufficient when overexpressed. It acts at the same level as CaMKII in the pathway of calcium-induced CSF release by cooperating with CaMKII to regulate APC/C regulator(s), such as XErp1/Emi2, rather than by directly activating the APC/C itself.

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.

Inhibition of the anaphase-promoting complex by the Xnf7 ubiquitin ligase

Degradation of specific protein substrates by the anaphase-promoting complex/cyclosome (APC) is critical for mitotic exit. We have identified the protein Xenopus nuclear factor 7 (Xnf7) as a novel APC inhibitor able to regulate the timing of exit from mitosis. Immunodepletion of Xnf7 from Xenopus laevis egg extracts accelerated the degradation of APC substrates cyclin B1, cyclin B2, and securin upon release from cytostatic factor arrest, whereas excess Xnf7 inhibited APC activity. Interestingly, Xnf7 exhibited intrinsic ubiquitin ligase activity, and this activity was required for APC inhibition. Unlike other reported APC inhibitors, Xnf7 did not associate with Cdc20, but rather bound directly to core subunits of the APC. Furthermore, Xnf7 was required for spindle assembly checkpoint function in egg extracts. These data suggest that Xnf7 is an APC inhibitor able to link spindle status to the APC through direct association with APC core components.

Phosphorylation of Maskin by Aurora-A Participates in the Control of Sequential Protein Synthesis during Xenopus laevis Oocyte Maturation

At the end of oogenesis, Xenopus laevis stage VI oocytes are arrested at the G2/M transition (prophase) waiting for progesterone to release the block and begin maturation. Progesterone triggers a cascade of phosphorylation events such as a decrease of pK(a) and an increase of maturating-promoting factor activity. Progression through meiosis was controlled by the sequential synthesis of several proteins. For instance, the MAPK kinase kinase c-Mos is the very first protein to be produced, whereas cyclin B1 appears only after meiosis I. After the meiotic cycles, the oocyte arrests at metaphase of meiosis II with an elevated c-Mos kinase activity (cytostatic factor). By using a two-hybrid screen, we have identified maskin, a protein involved in the control of mRNA sequential translation, as a binding partner of Aurora-A, a protein kinase necessary for oocyte maturation. Here we showed that, in vitro, Aurora-A directly binds to maskin and that both proteins can be co-immunoprecipitated from oocyte extracts, suggesting that they do associate in vivo. We also demonstrated that Aurora-A phosphorylates maskin on a Ser residue conserved in transforming acidic coiled coil proteins from Drosophila to human. When the phosphorylation of this Ser was inhibited in vivo by microinjection of synthetic peptides that mimic the maskin-phosphorylated sequence, we observed a premature maturation. Under these conditions, proteins such as cyclin B1 and Cdc6, which are normally detected only in meiosis II, were massively produced in meiosis I before the occurrence of the nuclear envelope breakdown. This result strongly suggests that phosphorylation of maskin by Aurora-A prevents meiosis II proteins from being produced during meiosis I.

A role for the anaphase-promoting complex inhibitor Emi2/XErp1, a homolog of early mitotic inhibitor 1, in cytostatic factor arrest of Xenopus eggs

Unfertilized vertebrate eggs are arrested in metaphase of meiosis II with high cyclin B/Cdc2 activity to prevent parthenogenesis. Until fertilization, exit from metaphase is blocked by an activity called cytostatic factor (CSF), which stabilizes cyclin B by inhibiting the anaphase-promoting complex (APC) ubiquitin ligase. The APC inhibitor early mitotic inhibitor 1 (Emi1) was recently found to be required for maintenance of CSF arrest. We show here that exogenous Emi1 is unstable in CSF-arrested Xenopus eggs and is destroyed by the SCF(betaTrCP) ubiquitin ligase, suggesting that endogenous Emi1, an apparent 44-kDa protein, requires a stabilizing factor. However, anti-Emi1 antibodies crossreact with native Emi2/Erp1/FBXO43, a homolog of Emi1 and conserved APC inhibitor. Emi2 is stable in CSF-arrested eggs, is sufficient to prevent CSF release, and is rapidly degraded in a Polo-like kinase 1-dependent manner in response to calcium-mediated egg activation. These results identify Emi2 as a candidate CSF maintenance protein.