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.     

Synergy between the Mos/mitogen-activated protein kinase pathway and loss of p53 function in transformation and chromosome instability.

Constitutive activation of mitogen-activated protein kinase (MAPK) is a property common to many oncoproteins, including Mos, Ras, and Raf, and is essential for their transforming activities. We have shown that high levels of expression of the Mos/MAPK pathway in Swiss 3T3 fibroblast cause cells in S phase to undergo apoptosis, while cells in G1 irreversibly growth arrest. Interestingly, cells in G2 and M phases also arrest at a G1-like checkpoint after proceeding through mitosis. These cells fail to undergo cytokinesis and are binucleated. Thus, constitutive overexpression of Mos and MAPK cannot be tolerated, and fibroblasts transformed by Mos express only low levels of the mos oncogene product. Here, we show that p53 plays a key role in preventing oncogene-mediated activation of MAPK. In the absence of p53 (p53-/-), the growth arrest normally observed in wild-type p53 (p53+/+) mouse embryo fibroblasts (MEFs) is markedly reduced. The mos transformation efficiency in p53-/- MEFs is two to three orders of magnitude higher than that in p53+/+ cells, and p53-/- cells tolerate > 10-fold higher levels of both Mos and activated MAPK. Moreover, we show that, like Mos, both v-ras and v-raf oncogene products induce apoptosis in p53+/+ MEFs. These oncogenes also display a high transforming activity in p53-/- MEFs, as does a gain-of-function MAPK kinase mutant (MEK*). Thus, the p53-dependent checkpoint pathway is responsive to oncogene-mediated MAPK activation in inducing irreversible G1 growth arrest and apoptosis. Moreover, we show that the chromosome instability induced by the loss of p53 is greatly enhanced by the constitutive activation of the Mos/MAPK pathway.     

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.     

Meiotic spindle stability depends on MAPK-interacting and spindle-stabilizing protein (MISS), a new MAPK substrate

Vertebrate oocytes arrest in the second metaphase of meiosis (metaphase II [MII]) by an activity called cytostatic factor (CSF), with aligned chromosomes and stable spindles. Segregation of chromosomes occurs after fertilization. The Mos/.../MAPK (mitogen-activated protein kinases) pathway mediates this MII arrest. Using a two-hybrid screen, we identified a new MAPK partner from a mouse oocyte cDNA library. This protein is unstable during the first meiotic division and accumulates only in MII, where it localizes to the spindle. It is a substrate of the Mos/.../MAPK pathway. The depletion of endogenous RNA coding for this protein by three different means (antisense RNA, double-stranded [ds] RNA, or morpholino oligonucleotides) induces severe spindle defects specific to MII oocytes. Overexpressing the protein from an RNA not targeted by the morpholino rescues spindle destabilization. However, dsRNA has no effect on the first two mitotic divisions. We therefore have discovered a new MAPK substrate involved in maintaining spindle integrity during the CSF arrest of mouse oocytes, called MISS (for MAP kinase-interacting and spindle-stabilizing protein).     

The Mos/MAPK pathway is involved in metaphase II arrest as a cytostatic factor but is neither necessary nor sufficient for initiating oocyte maturation in goldfish

Mos plays a crucial role in meiotic cell division in vertebrates. In Xenopus, Mos is involved in the initiation of oocyte maturation as an initiator and in the arrest at the metaphase II stage (MII) as a component of the cytostatic factor (CSF). The function of Mos is mediated by MAP kinase (MAPK). We investigated the function of the Mos/MAPK pathway during goldfish oocyte maturation induced by 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP), a natural maturation-inducing hormone in fishes. Mos was absent in immature goldfish oocytes. It appeared before the onset of germinal vesicle breakdown (GVBD), increased to a maximum in mature oocytes arrested at MII and disappeared after fertilization. MAPK was activated after Mos synthesis but before maturation-promoting factor (MPF) activation, and its activity reached maximum at MII. Injection of either Xenopus or goldfish c-mos mRNA into one blastomere of 2-cell-stage Xenopus and goldfish embryos induced metaphase arrest, suggesting that goldfish Mos has a CSF activity. Injection of constitutively active Xenopus c-mos mRNA into immature goldfish oocytes induced MAPK activation, but neither MPF activation nor GVBD occurred. Conversely, the injection of goldfish c-mos antisense RNA inhibited both Mos synthesis and MAPK activation in the 17α,20β-DP-treated oocytes, but these oocytes underwent GVBD. These results indicate that the Mos/MAPK pathway is not essential for initiating goldfish oocyte maturation despite its general function as a CSF. We discuss the general role of Mos/MAPK during oocyte maturation, with reference to the difference in contents of inactive MPF (pre-MPF) stored in immature oocytes. Received: 10 February 2000 / Accepted: 25 April 2000     

Involvement of mitogen-activated protein kinase (MAPK) pathway in LH- and meiosis-activating sterol (MAS)-induced maturation in rat and mouse oocytes

Gonadotropic stimulation of meiotic resumption in mice is dependent upon mitogen-activated protein kinase (MAPK) activation in the somatic compartment of the follicle. By contrast, spontaneous resumption of meiosis is independent of MAPK activation. In view of the suggested role of meiosis-activating sterol (MAS) in oocyte maturation we have (i) compared MAPK activation in rat preovulatory follicles stimulated by LH or by accumulation of endogenous MAS by using an inhibitor of MAS conversion, AY9944; (ii) examined whether stimulation of meiosis by MAS is dependent upon MAPK activation using denuded oocytes (DO) of Mos- null mice (hereafter Mos(-/-)) with oocytes unable to activate MAPK. Rat preovulatory follicles responded to LH or AY9944 stimulation by MAPK activation. Inhibition of MAPK phosphorylation blocked both LH- and AY9944 triggered resumption of meiosis. In mouse cumulus-enclosed oocytes (CEOs) and DOs AY9944 stimulated GVB in wild-type and Mos(-/-) mouse CEOs cultured with hypoxanthine (Hx). Addition of MAS or AY9944 to mouse DOs cultured with Hx induced resumption of meiosis only in wild-type and Mos(+/-) oocytes, but they were ineffective in Mos(-/-) oocytes. The observed sluggish activation of MAPK induced by AY9944 in rat follicle-enclosed oocytes (FEO) may cause the delay in meiotic resumption in response to MAS and AY9944 stimulation. Further, it is incompatible with the suggested role of MAS as an obligatory mediator of LH in the induction of meiotic maturation. MAPK/MOS activation, whether in the somatic compartment or in denuded oocytes, is required for MAS- like LH-, FSH-, or EGF-induced resumption of meiosis.     

Function of the Mos/MAPK pathway during oocyte maturation in the Japanese brown frog Rana japonica

Fully grown immature oocytes acquire the ability to be fertilized with sperm after meiotic maturation, which is finally accomplished by the formation and activation of the maturation-promoting factor (MPF). MPF is the complex of Cdc2 and cyclin B, and its function in promoting metaphase is common among species. The Mos/mitogen-activated protein kinase (MAPK) pathway is also commonly activated during vertebrate oocyte maturation, but its function seems to be different among species. We investigated the function of the Mos/MAPK pathway during oocyte maturation of the frog Rana japonica. Although MAPK was activated in accordance with MPF activation during oocyte maturation, MPF activation and germinal vesicle breakdown (GVBD) was not initiated when the Mos/MAPK pathway was activated in immature oocytes by the injection of c-mos mRNA. Inhibition of Mos synthesis by c-mos antisense RNA and inactivation of MAPK by CL100 phosphatase did not prevent progesterone-induced MPF activation and GVBD. However, continuous MAPK activation and MAPK inhibition through oocyte maturation accelerated and delayed MPF activation, respectively. Furthermore, Mos induced a low level of cyclin B protein synthesis in immature oocytes without the aid of MAPK. These results suggest that the general function of the Mos/MAPK pathway, which is not essential for MPF activation and GVBD in Rana oocytes, is to enhance cyclin B translation by Mos itself and to stabilize cyclin B protein by MAPK during oocyte maturation.     

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.     

Protein kinase A acts at multiple points to inhibit Xenopus oocyte maturation.

In Xenopus oocytes, initiation of maturation is dependent on reduction of cyclic AMP-dependent protein kinase (PKA) activity and the synthesis of the mos proto-oncogene product. Mos is required during meiosis I for the activation of both maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Here we show that injection of the catalytic subunit of PKA (PKAc) prevented progesterone-induced synthesis of endogenous Mos as well as downstream MPF and MAPK activation. However, PKAc did not prevent injected soluble Mos product from activating MAPK. While MAPK is activated during Mos-PKAc coinjection, attendant MPF activation is blocked. Additionally, PKAc caused a potent block in the electrophoretic mobility shift of cdc25 that is associated with phosphatase activation. This inhibition of cdc25 activity was not reversed by progesterone, Mos, or MPF. We conclude that PKAc acts as a negative regulator at several points in meiotic maturation by preventing both Mos translation and MPF activation.     

Ser-3 is important for regulating Mos interaction with and stimulation of mitogen-activated protein kinase kinase.

Mos is a germ cell-specific serine/threonine protein kinase that activates mitogen-activated protein kinase (MAPK) through MAPK kinase (MKK). In Xenopus oocytes, Mos synthesis is required for progesterone-induced activation of MAPK and maturation promoting factor. Injection of Mos or active MAPK causes mitotic arrest in early embryos, suggesting that Mos also acts via MKK and MAPK to induce the arrest of unfertilized eggs in metaphase of meiosis II. We have investigated whether Mos activity is regulated by phosphorylation. Previous studies have identified Ser-3 as the principal autophosphorylation site. We show that Mos interacts with the catalytic domain of MKK in a Saccharomyces cerevisiae two-hybrid test. Acidic substitutions of the sites phosphorylated by Mos in MKK reduce the interaction, implying that the complex may dissociate after phosphorylation of MKK by Mos. Furthermore, the Mos-MKK interaction requires Mos kinase activity, suggesting that Mos autophosphorylation may be involved in the interaction. Substitution of Ser-3 of Mos with Ala reduces the interaction with MKK and also reduces both the activation of MKK by Mos in vitro and cleavage arrest induced by Mos fusion protein in Xenopus embryos. By contrast, substitution of Ser-3 by Glu, an acidic amino acid that mimics phosphoserine, fosters the Mos interaction with MKK and permits activation of MKK in vitro and Mos-induced cleavage arrest. Moreover, the Glu-3 substitution increases the interaction of a kinase-inactive Mos mutant with MKK. Taken together, these results suggest that an important step in Mos activation involves the phosphorylation at Ser-3, which promotes Mos interaction with and activation of MKK.     

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).     

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.     

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.     

RINGO efficiently triggers meiosis resumption in mouse oocytes and induces cell cycle arrest in embryos.

RINGO was identified as a Cdc2-binding and activating protein which is necessary and sufficient to trigger G2/M progression in Xenopus oocytes. We have investigated whether the function of RINGO is conserved in mouse oocytes. We show that RINGO induces Germinal Vesicle BreakDown (GBVD) in mouse oocytes. Mos is known to induce GVBD in mouse oocytes, and is also involved in the metaphase II arrest, which is due to the CSF (CytoStatic Factor) activity. We found that RINGO also has CSF activity and induces cleavage arrest after injection into one blastomere of a late two-cell mouse embryo, like Mos. However, RINGO also inhibits polar body extrusion of wild type mouse oocytes. The same effect of RINGO on first and second polar body extrusion was observed in Mos -/- mouse oocytes. The injection of RINGO mimics Mos effects: GVBD induction and efficient cleavage arrest. However, our results in mouse oocytes suggest that RINGO may have additional functions in meiosis regulation.     

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.     

Mitogen activated protein kinase plays a significant role in metaphase II arrest, spindle morphology, and maintenance of maturation promoting factor activity in bovine oocytes

Mammalian oocytes are arrested at the G2/M transition of the first meiotic division from which, after reaching full size and subsequent to an LH surge, they undergo final maturation. Oocyte maturation, which involves germinal vesicle breakdown, progression through metaphase I (MI), and arrest at MII, is triggered and regulated by the coordinated action of two kinases, maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK). The importance of the role of MPF in mammalian oocyte maturation is well established, while the role of MAPK, although well understood in mouse oocytes, has not been fully elucidated in oocytes of large domestic species, especially bovine oocytes. Here we show that injection of MKP-1 mRNA, which encodes a dual specificity MAPK phosphatase, into germinal vesicle stage bovine oocytes prevents the activation of MAPK during maturation. Despite the lack of MAPK activity, MKP-1-injected oocytes resume and progress through meiosis, although they are unable to arrest at MII stage and, by 22-26-hour post-maturation, exhibit decondensed pronucleus-like chromatin, a clear sign of parthenogenetic activation. MKP-1-injected bovine oocytes exhibit normal activation of MPF activity; however, by 18-hour post-maturation, MPF activity starts to decline and by 22-26 hr MPF activity is absent. MKP-1-injected oocytes also show disorganized MII spindles with poorly aligned chromosomes. In summary, our results demonstrate that in bovine oocytes MAPK activity is required for MII arrest, maintenance of MPF activity, and spindle organization.     

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 effect of PD98059 on MAPK regulation in cumulus-enclosed and cumulus-free mouse oocytes

The effect of the p42/44 mitogen-activated kinase (MAPK) inhibitor, PD98059, on MAPK activation and meiosis resumption in mouse oocytes was studied. When germinal vesicle (GV)-stage denuded oocytes (DOs) were cultured continuously in 50 microM PD98059, germinal vesicle breakdown (GVBD) was postponed for 2-3 h. MAPK phosphorylation and activation was delayed as well. However, PD98059 did not impair histone H1 kinase activation. After 14 h of culture there was no significant difference in the rate of DOs reaching metaphase II (MII) arrest in either control or experimental conditions. The effect of PD98059 on MAPK inhibition was further tested in epidermal growth factor (EGF)-treated oocytecumulus complexes (OCCs). Exposure of GV-stage OCCs for 5 min to EGF (10 ng/ml) induced a considerable increase in MAPK phosphorylation. After OCCs were further cultured in 50 microM PD98059 a rapid dephosphorylation of MAPK was induced. Already after 1 min of treatment the non-phosphorylated form of MAPK dominated, indicating the high effectivity of PD98059. This result indicates that short EGF/PD98059 treatment of OCCs induced MAPK phosphorylation/dephosphorylation in cumulus cells only. As only a transient delay in MAPK phosphorylation and activation was observed in PD98059-treated DOs we conclude that there is also another PD98059-nonsensitive pathway(s) leading to MAPK activation in mouse oocytes. The data obtained suggest that meiosis resumption in mouse oocytes is somehow influenced by the MEK/MAPK activation pathway.     

Mos induces the in vitro activation of mitogen-activated protein kinases in lysates of frog oocytes and mammalian somatic cells.

Mitogen-activated protein kinases (MAPKs) are rapidly and transiently activated when both quiescent Go-arrested cells and G2-arrested oocytes are stimulated to reenter the cell cycle. We previously developed a cell-free system from lysates of quiescent Xenopus oocytes that responds to oncogenic H-ras protein by activating a MAPK, p42MAPK. Here, we show that the oncogenic protein kinase mos is also a potent activator of p42MAPK in these lysates. Mos also induces p42MAPK activation in lysates of activated eggs taken at a time when neither mos nor p42MAPK is normally active, showing that the mos-responsive MAPK activation pathway persists beyond the stage where mos normally functions. Similarly, lysates of somatic cells (rabbit reticulocytes) also retain a mos-inducible MAPK activation pathway. The mos-induced activation of MAPKs in all three lysates leads to phosphorylation of the pp90rsk proteins, downstream targets of the MAPK signaling pathway in vivo. The in vitro activation of MAPKs by mos in cell-free systems derived from oocytes and somatic cells suggests that mos contributes to oncogenic transformation by inappropriately inducing the activation of MAPKs.     

Changes in the activity of type 2A protein phosphatases during meiotic maturation and the first mitotic cell cycle in mouse oocytes.

We have established an assay to measure protein phosphatase activity in mouse oocytes using [32P]-radiolabeled phosphorylase a as the substrate. Removal of the radiolabel from the substrate in vitro was linear with time and could be inhibited totally by the addition of okadaic acid (inhibitor of type 1 and type 2 protein phosphatases), or partially by protein inhibitor 2 (inhibitor of type 1 protein phosphatases). We performed a detailed study of the activity of type 2A protein phosphatases in mouse oocytes undergoing meiotic maturation and after parthenogenetic activation of mature oocytes arrested in metaphase II. Significant changes in the activity of type 2A protein phosphatases were observed during the first meiotic and the first mitotic cell cycles. These alterations in type 2A protein phosphatase activity occurred in the absence of changes in the quantity of the catalytic sub-unit and can be correlated with changes in the activity of protein kinases and rearrangement of the cellular cytoskeleton. Our observations support a role for type 2A protein phosphatases in cell cycle regulation and demonstrate that, like the protein kinases, the type 2A phosphatases also undergo changes in their activity during early mammalian development.