The Polo-like Kinase Plx1 Is Required for Activation of the Phosphatase Cdc25C and Cyclin B-Cdc2 in Xenopus Oocytes

In the Xenopus oocyte system mitogen treatment triggers the G(2)/M transition by transiently inhibiting the cAMP-dependent protein kinase (PKA); subsequently, other signal transduction pathways are activated, including the mitogen-activated protein kinase (MAPK) and polo-like kinase pathways. To study the interactions between these pathways, we have utilized a cell-free oocyte extract that carries out the signaling events of oocyte maturation after addition of the heat-stable inhibitor of PKA, PKI. PKI stimulated the synthesis of Mos and activation of both the MAPK pathway and the Plx1/Cdc25C/cyclin B-Cdc2 pathway. Activation of the MAPK pathway alone by glutathione S-transferase (GST)-Mos did not lead to activation of Plx1 or cyclin B-Cdc2. Inhibition of the MAPK pathway in the extract by the MEK1 inhibitor U0126 delayed, but did not prevent, activation of the Plx1 pathway, and inhibition of Mos synthesis by cycloheximide had a similar effect, suggesting that MAPK activation is the only relevant function of Mos. Immunodepletion of Plx1 completely inhibited activation of Cdc25C and cyclin B-Cdc2 by PKI, indicating that Plx1 is necessary for Cdc25C activation. In extracts containing fully activated Plx1 and Cdc25C, inhibition of cyclin B-Cdc2 by p21(Cip1) had no significant effect on either the phosphorylation of Cdc25C or the activity of Plx1. These results demonstrate that maintenance of Plx1 and Cdc25C activity during mitosis does not require cyclin B-Cdc2 activity.     

Dynamic regulation of mitotic ubiquitin ligase APC/C by coordinated Plx1 kinase and PP2A phosphatase action on a flexible Apc1 loop

The anaphase‐promoting complex/cyclosome (APC/C), a multi‐subunit ubiquitin ligase essential for cell cycle control, is regulated by reversible phosphorylation. APC/C phosphorylation by cyclin‐dependent kinase 1 (Cdk1) promotes Cdc20 co‐activator loading in mitosis to form active APC/C‐Cdc20. However, detailed phospho‐regulation of APC/C dynamics through other kinases and phosphatases is still poorly understood. Here, we show that an interplay between polo‐like kinase (Plx1) and PP2A‐B56 phosphatase on a flexible loop domain of the subunit Apc1 (Apc1‐loop⁵⁰⁰) controls APC/C activity and mitotic progression. Plx1 directly binds to the Apc1‐loop⁵⁰⁰ in a phosphorylation‐dependent manner and promotes the formation of APC/C‐Cdc20 via Apc3 phosphorylation. Upon phosphorylation of loop residue T532, PP2A‐B56 is recruited to the Apc1‐loop⁵⁰⁰ and differentially promotes dissociation of Plx1 and PP2A‐B56 through dephosphorylation of Plx1‐binding sites. Stable Plx1 binding, which prevents PP2A‐B56 recruitment, prematurely activates the APC/C and delays APC/C dephosphorylation during mitotic exit. Furthermore, the phosphorylation status of the Apc1‐loop⁵⁰⁰ is controlled by distant Apc3‐loop phosphorylation. Our study suggests that phosphorylation‐dependent feedback regulation through flexible loop domains within a macromolecular complex coordinates the activity and dynamics of the APC/C during the cell cycle.     

A critical balance between Cyclin B synthesis and Myt1 activity controls meiosis entry in Xenopus oocytes

In fully grown oocytes, meiosis is arrested at first prophase until species-specific initiation signals trigger maturation. Meiotic resumption universally involves early activation of M phase-promoting factor (Cdc2 kinase-Cyclin B complex, MPF) by dephosphorylation of the inhibitory Thr14/Tyr15 sites of Cdc2. However, underlying mechanisms vary. In Xenopus oocytes, deciphering the intervening chain of events has been hampered by a sensitive amplification loop involving Cdc2-Cyclin B, the inhibitory kinase Myt1 and the activating phosphatase Cdc25. In this study we provide evidence that the critical event in meiotic resumption is a change in the balance between inhibitory Myt1 activity and Cyclin B neosynthesis. First, we show that in fully grown oocytes Myt1 is essential for maintaining prophase I arrest. Second, we demonstrate that, upon upregulation of Cyclin B synthesis in response to progesterone, rapid inactivating phosphorylation of Myt1 occurs, mediated by Cdc2 and without any significant contribution of Mos/MAPK or Plx1. We propose a model in which the appearance of active MPF complexes following increased Cyclin B synthesis causes Myt1 inhibition, upstream of the MPF/Cdc25 amplification loop.     

Mitotic effects of a constitutively active mutant of the Xenopus polo-like kinase Plx1

During mitosis the Xenopus polo-like kinase 1 (Plx1) plays key roles in the activation of Cdc25C, in spindle assembly, and in cyclin B degradation. Previous work has shown that the activation of Plx1 requires phosphorylation on serine and threonine residues. In the present work, we demonstrate that replacement of Ser-128 or Thr-201 with a negatively charged aspartic acid residue (S128D or T201D) elevates Plx1 activity severalfold and that replacement of both Ser-128 and Thr-201 with Asp residues (S128D/T201D) increases Plx1 activity approximately 40-fold. Microinjection of mRNA encoding S128D/T201D Plx1 into Xenopus oocytes induced directly the activation of both Cdc25C and cyclin B-Cdc2. In egg extracts T201D Plx1 delayed the timing of deactivation of Cdc25C during exit from M phase and accelerated Cdc25C activation during entry into M phase. This supports the concept that Plx1 is a "trigger" kinase for the activation of Cdc25C during the G(2)/M transition. In addition, during anaphase T201D Plx1 reduced preferentially the degradation of cyclin B2 and delayed the reduction in Cdc2 histone H1 kinase activity. In early embryos S128D/T201D Plx1 resulted in arrest of cleavage and formation of multiple interphase nuclei. Consistent with these results, Plx1 was found to be localized on centrosomes at prophase, on spindles at metaphase, and at the midbody during cytokinesis. These results demonstrate that in Xenopus laevis activation of Plx1 is sufficient for the activation of Cdc25C at the initiation of mitosis and that inactivation of Plx1 is required for complete degradation of cyclin B2 after anaphase and completion of cytokinesis.     

Activated Polo-Like Kinase Plx1 Is Required at Multiple Points during Mitosis in Xenopus laevis

Entry into mitosis depends upon activation of the dual-specificity phosphatase Cdc25C, which dephosphorylates and activates the cyclin B-Cdc2 complex. Previous work has shown that the Xenopus polo-like kinase Plx1 can phosphorylate and activate Cdc25C in vitro. In the work presented here, we demonstrate that Plx1 is activated in vivo during oocyte maturation with the same kinetics as Cdc25C. Microinjection of wild-type Plx1 into Xenopus oocytes accelerated the rate of activation of Cdc25C and cyclin B-Cdc2. Conversely, microinjection of either an antibody against Plx1 or kinase-dead Plx1 significantly inhibited the activation of Cdc25C and cyclin B-Cdc2. This effect could be reversed by injection of active Cdc25C, indicating that Plx1 is upstream of Cdc25C. However, injection of Cdc25C, which directly activates cyclin B-Cdc2, also caused activation of Plx1, suggesting that a positive feedback loop exists in the Plx1 activation pathway. Other experiments show that injection of Plx1 antibody into early embryos, which do not require Cdc25C for the activation of cyclin B-Cdc2, resulted in an arrest of cleavage that was associated with monopolar spindles. These results demonstrate that in Xenopus laevis, Plx1 plays important roles both in the activation of Cdc25C at the initiation of mitosis and in spindle assembly at late stages of mitosis.     

Greatwall maintains mitosis through regulation of PP2A

Greatwall (GW) is a new kinase that has an important function in the activation and the maintenance of cyclin B–Cdc2 activity. Although the mechanism by which it induces this effect is unknown, it has been suggested that GW could maintain cyclin B–Cdc2 activity by regulating its activation loop. Using Xenopus egg extracts, we show that GW depletion promotes mitotic exit, even in the presence of a high cyclin B–Cdc2 activity by inducing dephosphorylation of mitotic substrates. These results indicate that GW does not maintain the mitotic state by regulating the cyclin B–Cdc2 activation loop but by regulating a phosphatase. This phosphatase is PP2A; we show that (1) PP2A binds GW, (2) the inhibition or the specific depletion of this phosphatase from mitotic extracts rescues the phenotype induced by GW inactivation and (3) the PP2A‐dependent dephosphorylation of cyclin B–Cdc2 substrates is increased in GW‐depleted Xenopus egg extracts. These results suggest that mitotic entry and maintenance is not only mediated by the activation of cyclin B–Cdc2 but also by the regulation of PP2A by GW.     

Roles of Greatwall kinase in the regulation of cdc25 phosphatase

We previously reported that immunodepletion of Greatwall kinase prevents Xenopus egg extracts from entering or maintaining M phase due to the accumulation of inhibitory phosphorylations on Thr14 and Tyr15 of Cdc2. M phase-promoting factor (MPF) in turn activates Greatwall, implying that Greatwall participates in an MPF autoregulatory loop. We show here that activated Greatwall both accelerates the mitotic G2/M transition in cycling egg extracts and induces meiotic maturation in G2-arrested Xenopus oocytes in the absence of progesterone. Activated Greatwall can induce phosphorylations of Cdc25 in the absence of the activity of Cdc2, Plx1 (Xenopus Polo-like kinase) or mitogen-activated protein kinase, or in the presence of an activator of protein kinase A that normally blocks mitotic entry. The effects of active Greatwall mimic in many respects those associated with addition of the phosphatase inhibitor okadaic acid (OA); moreover, OA allows cycling extracts to enter M phase in the absence of Greatwall. Taken together, these findings support a model in which Greatwall negatively regulates a crucial phosphatase that inhibits Cdc25 activation and M phase induction.     

Human Cdc14A phosphatase modulates the G2/M transition through Cdc25A and Cdc25B

The Cdc14 family of serine-threonine phosphatases antagonizes CDK activity by reversing CDK-dependent phosphorylation events. It is well established that the yeast members of this family bring about the M/G1 transition. Budding yeast Cdc14 is essential for CDK inactivation at the end of mitosis and fission yeast Cdc14 homologue Flp1/Clp1 down-regulates Cdc25 to ensure the inactivation of mitotic CDK complexes to trigger cell division. However, the functions of human Cdc14 homologues remain poorly understood. Here we have tested the hypothesis that Cdc14A might regulate Cdc25 mitotic inducers in human cells. We found that increasing levels of Cdc14A delay entry into mitosis by inhibiting Cdk1-cyclin B1 activity. By contrast, lowering the levels of Cdc14A accelerates mitotic entry. Biochemical analyses revealed that Cdc14A acts through key Cdk1-cyclin B1 regulators. We observed that Cdc14A directly bound to and dephosphorylated Cdc25B, inhibiting its catalytic activity. Cdc14A also regulated the activity of Cdc25A at the G2/M transition. Our results indicate that Cdc14A phosphatase prevents premature activation of Cdk1 regulating Cdc25A and Cdc25B at the entry into mitosis.     

Polo-like kinase confers MPF autoamplification competence to growing Xenopus oocytes

During oogenesis, the Xenopus oocyte is blocked in prophase of meiosis I. It becomes competent to resume meiosis in response to progesterone at the end of its growing period (stage VI of oogenesis). Stage IV oocytes contain a store of inactive pre-MPF (Tyr15-phosphorylated Cdc2 bound to cyclin B2); the Cdc25 phosphatase that catalyzes Tyr15 dephosphorylation of Cdc2 is also present. However, the positive feedback loop that allows MPF autoamplification is not functional at this stage of oocyte growth. We report that when cyclin B is overexpressed in stage IV oocytes, MPF autoamplification does not occur and the newly formed cyclin B-Cdc2 complexes are inactivated by Tyr15 phosphorylation, indicating that Myt1 kinase remains active and that Cdc25 is prevented to be activated. Plx1 kinase (or polo-like kinase), which is required for Cdc25 activation and MPF autoamplification in full grown oocytes is not expressed at the protein level in small stage IV oocytes. In order to determine if Plx1 could be the missing regulator that prevents MPF autoamplification, polo kinase was overexpressed in stage IV oocytes. Under these conditions, the MPF-positive feedback loop was restored. Moreover, we show that acquisition of autoamplification competence does not require the Mos/MAPK pathway.     

Cdc25 Phosphatases Are Required for Timely Assembly of CDK1-Cyclin B at the G2/M Transition

Progression through mitosis requires the coordinated regulation of Cdk1 kinase activity. Activation of Cdk1 is a multistep process comprising binding of Cdk1 to cyclin B, relocation of cyclin-kinase complexes to the nucleus, activating phosphorylation of Cdk1 on Thr¹⁶¹ by the Cdk-activating kinase (CAK; Cdk7 in metazoans), and removal of inhibitory Thr¹⁴ and Tyr¹⁵ phosphorylations. This dephosphorylation is catalyzed by the dual specific Cdc25 phosphatases, which occur in three isoforms in mammalian cells, Cdc25A, -B, and -C. We find that expression of Cdc25A leads to an accelerated G₂/M phase transition. In Cdc25A-overexpressing cells, Cdk1 exhibits high kinase activity despite being phosphorylated on Tyr¹⁵. In addition, Tyr¹⁵-phosphorylated Cdk1 binds more cyclin B in Cdc25A-overexpressing cells compared with control cells. Consistent with this observation, we demonstrate that in human transformed cells, Cdc25A and Cdc25B, but not Cdc25C phosphatases have an effect on timing and efficiency of cyclin-kinase complex formation. Overexpression of Cdc25A or Cdc25B promotes earlier assembly and activation of Cdk1-cyclin B complexes, whereas repression of these phosphatases by short hairpin RNA has a reverse effect, leading to a substantial decrease in amounts of cyclin B-bound Cdk1 in G₂ and mitosis. Importantly, we find that Cdc25A overexpression leads to an activation of Cdk7 and increase in Thr¹⁶¹ phosphorylation of Cdk1. In conclusion, our data suggest that complex assembly and dephosphorylation of Cdk1 at G₂/M is tightly coupled and regulated by Cdc25 phosphatases.     

Cytokinin controls the cell cycle at mitosis by stimulating the tyrosine dephosphorylation and activation of p34cdc2-like H1 histone kinase

In excised pith parenchyma from Nicotiana tabacum L. cv. Wisconsin Havana 38, auxin (naphthalene-1-acetic acid) together with cytokinin (6-benzylaminopurine) induced a greater than 40-fold increase in a p34^cdc2-like protein, recoverable in the p13^suc1-binding fraction, that had high H1 histone kinase activity, but enzyme induced without cytokinin was inactive. In suspension-cultured N. plumbaginifolia Viv., cytokinin (kinetin) was stringently required only in late G2 phase of the cell division cycle (cdc) and cells lacking kinetin arrested in G2 phase with inactive p34^cdc2-like H1 histone kinase. Control of the Cdc2 kinase by inhibitory tyrosine phosphorylation was indicated by high phosphotyrosine in the inactive enzyme of arrested pith and suspension cells. Yeast cdc25 phosphatase, which is specific for removal of phosphate from tyrosine at the active site of p34^cdc2 enzyme, was expressed in bacteria and caused extensive in-vitro activation of p13^suc1-purified enzyme from pith and suspension cells cultured without cytokinin. Cytokinin stimulated the removal of phosphate, activation of the enzyme and rapid synchronous entry into mitosis. Therefore, plants can control cell division by tyrosine phosphorylation of Cdc2 but differ from somatic animal cells in coupling this mitotic control to hormonal signals.Abbreviations BAP 6-benzylaminopurine - BrdUrd 5-bromo-2-deoxyuridine - cdc cell division cycle - Cdc25 cdc phospho-protein phosphatase - CKI cyclin dependent kinase inhibitor - 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 4,6 diamidino-2-phenylindole - GST-cdc25 glutathione sulfur transferase-truncated cdc25 fusion - MS Murashige and Skoog (1962) - NAA naphthalene-1-acetic acid - p34^cdc2 34-kDa product of the cdc2 gene     

A Cdc25A antagonizing K vitamin inhibits hepatocyte DNA synthesis in vitro and in vivo

Thioalkyl containing K vitamin analogs have been shown to be potent inhibitors of hepatoma cell growth and antagonizers of protein tyrosine phosphatase activity. We now show that they inhibit the activity of specific protein tyrosine phosphatases (PTP) in cell-free conditions in vitro, particularly the dual specificity phosphatase Cdc25A. Using primary cultures of adult rat hepatocytes that are in G0/G1 phase until stimulated into DNA synthesis by epidermal growth factor, we found that 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or Compound 5 (Cpd 5) inhibited hepatocyte DNA synthesis and PTP activity in cell culture and in vivo after a two-thirds partial hepatectomy. We found a selective inhibition of Cdc25A activity in vitro, using both synthetic substrates and authentic cellular substrate, immunoprecipitated phospho-Cdk4. Intact Cpd 5-treated cells had decreased cellular Cdc25A activity and increased tyrosine phosphorylation of Cdk4, resulting in decreased phosphorylation of retinoblastoma (Rb). Loss of Cdk4 activity was confirmed using Cdk4 immunoprecipitates from either Cpd 5-treated or untreated cells and measuring its kinase activity using GST-Rb as target. We found a similar order of activity for inhibition of growth and Cdc25A activity using several thiol-containing analogs. Cdc25A inhibitors may thus be useful for defining biochemical pathways involving protein tyrosine phosphorylation that mediate cell growth inhibition.     

Quinone-induced Cdc25A inhibition causes ERK-dependent connexin phosphorylation

Gap junctional intercellular communication (GJC) varies during progression of the cell cycle. We propose here that Cdc25A, a dual specificity phosphatase crucial for cell cycle progression, is linked to connexin (Cx) phosphorylation and the modulation of GJC. Inhibition of Cdc25 phosphatases in rat liver epithelial cells employing a 1,4-naphthoquinone-based inhibitor, NSC95397, induced cell cycle arrest, tyrosine phosphorylation of the epidermal growth factor receptor (EGFR), and activation of extracellular signal-regulated kinases ERK-1 and -2. ERK activation was blocked by specific inhibitors of MAPK/ERK kinases 1/2 or of the EGFR tyrosine kinase. An EGFR-dephosphorylation assay suggested that Cdc25A interacts with the EGFR, with inhibition by NSC95397 resulting in activation of the receptor. As a consequence of ERK activation, Cx43 was phosphorylated, resulting in a downregulation of GJC. Loss of GJC was prevented by inhibition of ERK activation. In summary, cell cycle and GJC are connected via Cdc25A and the EGFR-ERK pathway.     

Arrest of cell cycle by Amida which is phosphorylated by Cdc2 kinase

Amida was first isolated from a rat hippocampal cDNA library as an Arc-associated protein. Although previous studies have shown that Amida mRNA is predominantly expressed and developmentally regulated in rat testis and overexpression induces apoptosis, the function of Amida remains unclear. In this study, we found that overexpression of Amida inhibited cell growth. Flow cytometry analysis showed that Amida caused cell cycle inhibition in the S-phase and blocked cell cycle from entry into mitosis. Attempting to elucidate Amida effect on the cell cycle, we found that Amida was interacted with Cdc2 in mitosis and Amida's overexpression resulted in a decrease in Cdc2 kinase activity. In addition, Amida showed DNA-binding ability with DNA-affinity column chromatography. A region (aa, 76–189) between the two nuclear localization signals was found to be responsible for cell growth inhibition and DNA-binding activity, implying that DNA-binding activity may be necessary for Amida to repress cell cycle. Moreover, Amida was phosphorylated by Cdc2 kinase in vitro and Ser-180 of Amida was identified as the phosphorylation site. Furthermore, AmidaS180G (eliminate phosphorylation of Ser-180) showed stronger DNA-binding activity. Taken together, the data suggest that Amida may play an important role in cell cycle and may be partly regulated by Cdc2 kinase.     

A role for cyclin A1 in the activation of MPF and G2-M transition during meiosis of male germ cells in mice

Cell-cycle transition at G2-M is controlled by MPF (M-phase-promoting factor), a complex consisting of the Cdc2 kinase and a B-type cyclin. We have shown that in mice, targeted disruption of an A-type cyclin gene, cyclin A1, results in a block of spermatogenesis prior to the entry into metaphase I. The meiotic arrest is accompanied by a defect in Cdc2 kinase activation at the G2--M transition, raising the possibility that a cyclin A1-dependent process dictates the activation of MPF. Here we show that like Cdc2, the expression of B-type cyclins is retained in cyclin A1-deficient spermatocytes, while their associated kinases are kept at inactive states. Treatment of arrested germ cells with the protein phosphatase type-1 and -2A inhibitor okadaic acid restores the MPF activity and induces entry into M phase and the formation of normally condensed chromosome bivalents, concomitant with hyperphosphorylation of Cdc25 proteins. Conversely, inhibition of tyrosine phosphatases, including Cdc25s, by vanadate suppresses the okadaic acid-induced metaphase induction. The highest levels of Cdc25A and Cdc25C expression and their subcellular localization during meiotic prophase coincide with that of cyclin A1, and when overexpressed in HeLa cells, cyclin A1 coimmunoprecipitates with Cdc25A. Furthermore, the protein kinase complexes consisting of cyclin A1 and either Cdc2 or Cdk2 phosphorylate both Cdc25A and Cdc25C in vitro. These results suggest that in normal meiotic male germ cells, cyclin A1 participates in the regulation of other protein kinases or phosphatases critical for the G2-M transition. In particular, it may be directly involved in the initial amplification of MPF through the activating phosphorylation on Cdc25 phosphatases.     

Regulation of Cdc25C by ERK-MAP kinases during the G2/M transition

Induction of G(2)/M phase transition in mitotic and meiotic cell cycles requires activation by phosphorylation of the protein phosphatase Cdc25. Although Cdc2/cyclin B and polo-like kinase (PLK) can phosphorylate and activate Cdc25 in vitro, phosphorylation by these two kinases is insufficient to account for Cdc25 activation during M phase induction. Here we demonstrate that p42 MAP kinase (MAPK), the Xenopus ortholog of ERK2, is a major Cdc25 phosphorylating kinase in extracts of M phase-arrested Xenopus eggs. In Xenopus oocytes, p42 MAPK interacts with hypophosphorylated Cdc25 before meiotic induction. During meiotic induction, p42 MAPK phosphorylates Cdc25 at T48, T138, and S205, increasing Cdc25's phosphatase activity. In a mammalian cell line, ERK1/2 interacts with Cdc25C in interphase and phosphorylates Cdc25C at T48 in mitosis. Inhibition of ERK activation partially inhibits T48 phosphorylation, Cdc25C activation, and mitotic induction. These findings demonstrate that ERK-MAP kinases are directly involved in activating Cdc25 during the G(2)/M transition.     

The checkpoint protein Chfr is a ligase that ubiquitinates Plk1 and inhibits Cdc2 at the G2 to M transition.

The checkpoint protein Chfr delays entry into mitosis, in the presence of mitotic stress (Scolnick, D.M., and T.D. Halazonetis. 2000. Nature. 406:430-435). We show here that Chfr is a ubiquitin ligase, both in vitro and in vivo. When transfected into HEK293T cells, Myc-Chfr promotes the formation of high molecular weight ubiquitin conjugates. The ring finger domain in Chfr is required for the ligase activity; this domain auto-ubiquitinates, and mutations of conserved residues in this domain abolish the ligase activity. Using Xenopus cell-free extracts, we demonstrated that Chfr delays the entry into mitosis by negatively regulating the activation of the Cdc2 kinase at the G2-M transition. Specifically, the Chfr pathway prolongs the phosphorylated state of tyrosine 15 in Cdc2. The Chfr-mediated cell cycle delay requires ubiquitin-dependent protein degradation, because inactivating mutations in Chfr, interference with poly-ubiquitination, and inhibition of proteasomes all abolish this delay in mitotic entry. The direct target of the Chfr pathway is Polo-like kinase 1 (Plk1). Ubiquitination of Plk1 by Chfr delays the activation of the Cdc25C phosphatase and the inactivation of the Wee1 kinase, leading to a delay in Cdc2 activation. Thus, the Chfr pathway represents a novel checkpoint pathway that regulates the entry into mitosis by ubiquitin-dependent proteolysis.     

The polo-like kinase Plx1 is required for M phase exit and destruction of mitotic regulators in Xenopus egg extracts.

Polo-like kinases (Plks), named after the Drosophila gene product polo, have been implicated in the regulation of multiple aspects of mitotic progression, including the activation of the Cdc25 phosphatase, bipolar spindle formation and cytokinesis. Genetic analyses performed in yeast and Drosophila suggest a function for Plks at late stages of mitosis, but biochemical data to support such a function in vertebrate organisms are lacking. Here we have taken advantage of Xenopus egg extracts for exploring the function of Plx1, a Xenopus Plk, during the cell cycle transition from M phase to interphase (I phase). We found that the addition of a catalytically inactive Plx1 mutant to M phase-arrested egg extracts blocked their Ca2+-induced release into interphase. Concomitantly, the proteolytic destruction of several targets of the anaphase-promoting complex and the inactivation of the Cdc2 protein kinase (Cdk1) were prevented. Moreover, the M to I phase transition could be abolished by immunodepletion of Plx1, but was restored upon the addition of recombinant Plx1. These results demonstrate that the exit of egg extracts from M phase arrest requires active Plx1, and they strongly suggest an important role for Plx1 in the activation of the proteolytic machinery that controls the exit from mitosis.     

Cdc25B phosphatase participates in maintaining metaphase II arrest in mouse oocytes

Cdc25B is an essential regulator for meiotic resumption in mouse oocytes. However, the role of this phosphatase during the later stage of the meiotic cell cycle is not known. In this study, we investigated the role of Cdc25B during metaphase II (MII) arrest in mouse oocytes. Cdc25B was extensively phosphorylated during MII arrest with an increase in the phosphatase activity toward Cdk1. Downregulation of Cdc25B by antibody injection induced the formation of a pronucleus-like structure. Conversely, overexpression of Cdc25B inhibited Ca²⁺-mediated release from MII arrest. Moreover, Cdc25B was immediately dephosphorylated and hence inactivated during MII exit, suggesting that Cdk1 phosphorylation is required to exit from MII arrest. Interestingly, this inactivation occurred prior to cyclin B degradation. Taken together, our data demonstrate that MII arrest in mouse oocytes is tightly regulated not only by the proteolytic degradation of cyclin B but also by dynamic phosphorylation of Cdk1.     

A p90rsk Mutant Constitutively Interacting with MAP Kinase Uncouples MAP Kinase from p34cdc2/Cyclin B Activation in Xenopus Oocytes

The efficient activation of p90^rsk by MAP kinase requires their interaction through a docking site located at the C-terminal end of p90^rsk. The MAP kinase p42^mpk1 can associate with p90^rsk in G₂-arrested but not in mature Xenopus oocytes. In contrast, an N-terminally truncated p90^rsk mutant named D2 constitutively interacts with p42^mpk1. In this report we show that expression of D2 inhibits Xenopus oocyte maturation. The inhibition requires the p42^mpk1 docking site. D2 expression uncouples the activation of p42^mpk1 and p34^cdc2/cyclin B in response to progesterone but does not prevent signaling through p90^rsk. Instead, D2 interferes with a p42^mpk1-triggered pathway, which regulates the phosphorylation and activation of Plx1, a potential activator of the Cdc25 phosphatase. This new pathway that links the activation of p42^mpk1 and Plx1 during oocyte maturation is independent of p34^cdc2/cyclin B activity but requires protein synthesis. Using D2, we also provide evidence that the sustained activation of p42^mpk1 can trigger nuclear migration in oocytes. Our results indicate that D2 is a useful tool to study MAP kinase function(s) during oocyte maturation. Truncated substrates such as D2, which constitutively interact with MAP kinases, may also be helpful to study signal transduction by MAP kinases in other cellular processes.