Inhibition of Cdc7/Dbf4 kinase activity affects specific phosphorylation sites on MCM2 in cancer cells

The Cdc7/Dbf4 kinase is required for initiation of DNA replication and also plays a role in checkpoint function in response to replication stress. Exactly how Cdc7/Dbf4 mediates those activities remains to be elucidated. Cdc7/Dbf4 physically interacts with and phosphorylates the minichromosome maintenance complex (MCM), such as MCM2, MCM4 and MCM6. Cdc7/Dbf4 activity is required for association of Cdc45 followed by recruitment of DNA polymerase on the chromatin. Using high resolution mass spectrometry, we identified six phosphorylation sites on MCM2, two of them have not been described before. We provide evidence that Cdc7/Dbf4 mediates phosphorylation on serine 108 and serine 40 on human MCM2 in vitro and in vivo in cancer cells in the absence of DNA damage. Antibodies specific to pS108 or pS40 confirmed the sites and established useful read-outs for inhibition of Cdc7/Dbf4. This report demonstrates the utility of an in vitro to in vivo workflow utilizing immunoprecipitation and mass spectrometry to map phosphorylation sites on endogenous kinase substrates. The approach can be readily generalized to identify target modulation read-outs for other potential kinase cancer targets.     

Aurora Kinase A Is Not Involved in CPEB1 Phosphorylation and cyclin B1 mRNA Polyadenylation during Meiotic Maturation of Porcine Oocytes

Regulation of mRNA translation by cytoplasmic polyadenylation is known to be important for oocyte maturation and further development. This process is generally controlled by phosphorylation of cytoplasmic polyadenylation element binding protein 1 (CPEB1). The aim of this study is to determine the role of Aurora kinase A in CPEB1 phosphorylation and the consequent CPEB1-dependent polyadenylation of maternal mRNAs during mammalian oocyte meiosis. For this purpose, we specifically inhibited Aurora kinase A with MLN8237 during meiotic maturation of porcine oocytes. Using poly(A)-test PCR method, we monitored the effect of Aurora kinase A inhibition on poly(A)-tail extension of long and short cyclin B1 encoding mRNAs as markers of CPEB1-dependent cytoplasmic polyadenylation. Our results show that inhibition of Aurora kinase A activity impairs neither cyclin B1 mRNA polyadenylation nor its translation and that Aurora kinase A is unlikely to be involved in CPEB1 activating phosphorylation.     

Essential role of phosphorylation of MCM2 by Cdc7/Dbf4 in the initiation of DNA replication in mammalian cells

We report the identification of Cdc7/Dbf4 phosphorylation sites in human MCM2 and the determination of the role of Cdc7/Dbf4 phosphorylation of MCM2 in the initiation of DNA replication. Using immunoblotting, immunofluorescence, and high-speed automated cell-imaging analyses with antibodies specific against MCM2 and Cdc7/Dbf4 phosphorylated MCM2, we show that the chromatin recruitment and phosphorylation of MCM2 are regulated during the cell cycle in HeLa cells. Chromatin-bound MCM2 is phosphorylated by Cdc7/Dbf4 during G1/S, which coincides with the initiation of DNA replication. Moreover, we show that baculovirus-expressed purified MCM2-7 complex and its phosphomimetic MCM2E-7 complex display higher ATPase activity when compared with the nonphosphorylatable MCM2A-7 complex in vitro. Furthermore, suppression of MCM2 expression in HeLa cells by siRNA results in the inhibition of DNA replication. The inhibition can be rescued by the coexpression of wild type MCM2 or MCM2E but not MCM2A. Taken together, these results indicate that Cdc7/Dbf4 phosphorylation of MCM2 is essential for the initiation of DNA replication in mammalian cells.     

Growth hormone and gene expression of in vitro‐matured rhesus macaque oocytes

Growth hormone (GH) in rhesus macaque in vitro oocyte maturation (IVM) has been shown to increase cumulus expansion and development of embryos to the 9–16 cell stage in response to 100 ng/ml recombinant human GH (r‐hGH) supplementation during IVM. Although developmental endpoints for metaphase II (MII) oocytes and embryos are limited in the macaque, gene expression analysis can provide a mechanism to explore GH action on IVM. In addition, gene expression analysis may allow molecular events associated with improved cytoplasmic maturation to be detected. In this study, gene expression of specific mRNAs in MII oocytes and cumulus cells that have or have not been exposed to r‐hGH during IVM was compared. In addition, mRNA expression was compared between in vitro and in vivo‐matured metaphase II (MII) oocytes and germinal vesicle (GV)‐stage oocytes. Only 2 of 17 genes, insulin‐like growth factor 2 (IGF2) and steroidogenic acute regulator (STAR), showed increased mRNA expression in MII oocytes from the 100 ng/ml r‐hGH treatment group compared with other IVM treatment groups, implicating insulin‐like growth factor (IGF) and steroidogenesis pathways in the oocyte response to GH. The importance of IGF2 is notable, as expression of IGF1 was not detected in macaque GV‐stage or MII oocytes or cumulus cells. Mol. Reprod. Dev. 77: 353–362, 2010. © 2009 Wiley‐Liss, Inc.     

The existence of two distinct Wee1 isoforms in Xenopus: implications for the developmental regulation of the cell cycle

In eukaryotic cells, the Wee1 protein kinase phosphorylates and inhibits Cdc2, thereby creating an interphase of the cell cycle. In Xenopus, the conventional Wee1 homolog (termed Xe-Wee1A, or Wee1A for short) is maternally expressed and functions in pregastrula embryos with rapid cell cycles. Here, we have isolated a second, zygotic isoform of Xenopus Wee1, termed Xe-Wee1B (or Wee1B for short), that is expressed in postgastrula embryos and various adult tissues. When ectopically expressed in immature oocytes, Wee1B inhibits Cdc2 activity and oocyte maturation (or entry into M phase) much more strongly than Wee1A, due to its short C-terminal regulatory domain. Moreover, ectopic Wee1B, unlike Wee1A, is very labile during meiosis II and cannot accumulate in mature oocytes due to the presence of PEST-like sequences in its N-terminal regulatory domain. Finally, when expressed in fertilized eggs, ectopic Wee1B but not Wee1A does affect cell division and impair cell viability in early embryos, due primarily to its very strong kinase activity. These results suggest strongly that the differential expression of Wee1A and Wee1B is crucial for the developmental regulation of the cell cycle in XENOPUS:     

NEK5 regulates cell cycle progression during mouse oocyte maturation and preimplantation embryonic development

NEK5, a member of never in mitosis‐gene A‐related protein kinase, is involved in the regulation of centrosome integrity and centrosome cohesion at mitosis in somatic cells. In this study, we investigated the expression and function of NEK5 during mouse oocyte maturation and preimplantation embryonic development. The results showed that NEK5 was expressed from germinal vesicle (GV) to metaphase II (MII) stages during oocyte maturation with the highest level of expression at the GV stage. It was shown that NEK5 localized in the cytoplasm of oocytes at GV stage, concentrated around chromosomes at germinal vesicle breakdown (GVBD) stage, and localized to the entire spindle at prometaphase I, MI and MII stages. The small interfering RNA‐mediated depletion of Nek5 significantly increased the phosphorylation level of cyclin‐dependent kinase 1 in oocytes, resulting in a decrease of maturation‐promoting factor activity, and severely impaired GVBD. The failure of meiotic resumption caused by Nek5 depletion could be rescued by the depletion of Wee1B. We found that Nek5 depletion did not affect CDC25B translocation into the GV. We also found that NEK5 was expressed from 1‐cell to blastocyst stages with the highest expression at the blastocyst stage, and Nek5 depletion severely impaired preimplantation embryonic development. This study demonstrated for the first time that NEK5 plays important roles during meiotic G2/M transition and preimplantation embryonic development.     

Role of Greatwall kinase in release of mouse oocytes from diplotene arrest

In eukaryotes, mitosis entry is induced by activation of maturation‐promoting factor (MPF), which is regulated by a network of kinases and phosphatases. It has been suggested that Greatwall (GWL) kinase was crucial for the M‐phase entry and could maintain cyclin B–Cdc2 activity through regulation of protein phosphatase 2A (PP2A), a counteracting phosphatase of MPF. Here, the role of GWL was assessed during release of mouse oocytes from prophase I arrest. GWL was crucial for meiotic maturation in mouse oocytes. As a positive regulator for meiosis resumption, GWL was continually expressed in germinal vesicle (GV) and MII stage oocytes and two‐cell stage embryos. Additionally, GWL localized to the nucleus and dispersed into cytoplasm during GV breakdown (GVBD). Furthermore, downregulation of GWL or overexpression of catalytically‐inactive GWL inhibited partial meiotic maturation. This prophase I arrest induced by GWL depletion could be rescued by the PP2A inhibition. However, both GWL‐depleted and rescued oocytes had severe spindle defects that hardly reached MII. In contrast, oocytes overexpressing wild‐type GWL resumed meiosis and progressed to MII stage. Thus, our data demonstrate that GWL acts in a pathway with PP2A which is essential for prophase I exit and metaphase I microtubule assembly in mouse oocytes.     

Oligomers of the Cdc7/Dbf4 protein kinase exist in the yeast cell

Cdc7/Dbf4 protein kinase is required for the initiation of DNA replication in Saccharomyces cerevisiae. Cdc7/Dbf4 protein kinase is not a cyclin-dependent kinase (CDK), but is regulated in a similar fashion in that the Cdc7 kinase subunit is inactive in the absence of the regulatory subunit Dbf4. In contrast to what is known about CDKs, Cdc7/Dbf4 protein kinase is shown to be an oligomer in the cell in this report. Genetic data that support this claim include interallelic complementation between several cdc7ts alleles and the cdc7T281A allele and also the results of experiments using the two-hybrid system with Cdc7 in both DNA-binding and transactivation domain plasmids. A molecular interaction between two different Cdc7 molecules was shown by using a HA-tagged Cdc7 protein that differs in size from the wild-type Cdc7 protein: an anti-HA antibody immunoprecipitates both proteins in appproximately equal stoichiometry. Analysis of the native molecular weight of Cdc7/Dbf4 protein kinase is consistent with oligomerization of the Cdc7 protein in that complexes of about 180 and 300 kDa were found. Oligomers of Cdc7 protein may exist for the purpose of allosteric regulation or to allow phosphorylation of multiple substrate protein molecules. Received: 4 January 1998 / Accepted: 16 June 1998     

The temporal control of Wee1 mRNA translation during Xenopus oocyte maturation is regulated by cytoplasmic polyadenylation elements within the 3'-untranslated region

The Wee1 protein tyrosine kinase is a key regulator of cell cycle progression. Wee1 activity is necessary for the control of the first embryonic cell cycle following the fertilization of meiotically mature Xenopus oocytes. Wee1 mRNA is present in immature oocytes, but Wee1 protein does not accumulate in immature oocytes or during the early stages of progesterone-stimulated maturation. This delay in Wee1 translation is critical since premature Wee1 protein accumulation has been shown to inhibit oocyte maturation. In this study we provide evidence that Wee1 protein accumulation is regulated at the level of mRNA translation. This translational control is directed by sequences within the Wee1 mRNA 3'-untranslated region (3' UTR). Specifically, cytoplasmic polyadenylation element (CPE) sequences within the Wee1 3' UTR are necessary for full translational repression in immature oocytes. Our data further indicate that while CPE-independent mechanisms may regulate the levels of Wee1 protein accumulation during progesterone-stimulated oocyte maturation, the timing of Wee1 mRNA translational induction is directed through a CPE-dependent mechanism.     

A novel mRNA 3' untranslated region translational control sequence regulates Xenopus Wee1 mRNA translation

Cell cycle progression during oocyte maturation requires the strict temporal regulation of maternal mRNA translation. The intrinsic basis of this temporal control has not been fully elucidated but appears to involve distinct mRNA 3′ UTR regulatory elements. In this study, we identify a novel translational control sequence (TCS) that exerts repression of target mRNAs in immature oocytes of the frog, Xenopus laevis, and can direct early cytoplasmic polyadenylation and translational activation during oocyte maturation. The TCS is functionally distinct from the previously characterized Musashi/polyadenylation response element (PRE) and the cytoplasmic polyadenylation element (CPE). We report that TCS elements exert translational repression in both the Wee1 mRNA 3′ UTR and the pericentriolar material-1 (Pcm-1) mRNA 3′ UTR in immature oocytes. During oocyte maturation, TCS function directs the early translational activation of the Pcm-1 mRNA. By contrast, we demonstrate that CPE sequences flanking the TCS elements in the Wee1 3′ UTR suppress the ability of the TCS to direct early translational activation. Our results indicate that a functional hierarchy exists between these distinct 3′ UTR regulatory elements to control the timing of maternal mRNA translational activation during oocyte maturation.     

Non-dependence of cyclin E/Cdk2 kinase activity on the initiation of oocyte maturation in goldfish

Cdk2 kinase activity increases during oocyte maturation but neither cyclin A nor B is associated with Cdk2 in mature oocytes in goldfish. As a potential Cdk2 partner in meiosis, a cyclin E homolog was isolated from a goldfish oocyte cDNA library. A monoclonal antibody was raised against bacterially produced full-length goldfish cyclin E. Both cyclin E and Cdk2 were already present in immature oocytes and their protein levels did not change remarkably during oocyte maturation. Cyclin E formed a complex mainly with Cdk2 just at the time of germinal vesicle breakdown (GVBD) in association with the increase in Cdk2 kinase activity, although a fraction of cyclin E bound to Cdk(s) other than Cdk2 and Cdc2. Ectopic activation of cyclin E/Cdk2 by the injection of cyclin E messenger RNA (mRNA) into immature oocytes did not induce maturation-promoting factor (MPF) activation and GVBD. Furthermore, inhibition of cyclin E/Cdk2 kinase activity by the injection of p21SDI1 into the oocytes treated with 17alpha,20beta-dihydroxy-4-pregnen-3-one had no effect on MPF activation and GVBD. These results indicate that cyclin E/Cdk2 kinase activity is insufficient and unnecessary for initiating goldfish oocyte maturation.     

Developmental Competence of Vitrified-Warmed Bovine Oocytes at the Germinal-Vesicle Stage is Improved by Cyclic Adenosine Monophosphate Modulators during In Vitro Maturation

Cryopreservation of mature oocytes and embryos has provided numerous benefits in reproductive medicine. Although successful cryopreservation of germinal-vesicle stage (GV) oocytes holds promise for further advances in reproductive biology and clinical embryology fields, reports regarding cryopreservation of immature oocytes are limited. Oocyte survival and maturation rates have improved since vitrification is being performed at the GV stage, but the subsequent developmental competence of GV oocytes is still low. The purpose of this study was to evaluate the effects of supplementation of the maturation medium with cyclic adenosine monophosphate (cAMP) modulators on the developmental competence of vitrified-warmed GV bovine oocytes. GV oocytes were vitrified-warmed and cultured to allow for oocyte maturation, and then parthenogenetically activated or fertilized in vitro. Our results indicate that addition of a cAMP modulator forskolin (FSK) or 3-isobutyl-1-methylxanthine (IBMX) to the maturation medium significantly improved the developmental competence of vitrified-warmed GV oocytes. We also demonstrated that vitrification of GV oocytes led to a decline in cAMP levels and maturation-promoting factor (MPF) activity in the oocytes during the initial and final phases of maturation, respectively. Nevertheless, the addition of FSK or IBMX to the maturation medium significantly elevated cAMP levels and MPF activity during IVM. Taken together, our results suggest that the cryopreservation-associated meiotic and developmental abnormalities observed in GV oocytes may be ameliorated by an artificial increase in cAMP levels during maturation culture after warming.     

Ion current activity and molecules modulating maturation and growth stages of ascidian (Ciona intestinalis) oocytes

Electrophysiological techniques were used to study ion currents in the ascidian Ciona intestinalis oocyte plasma membranes during different stages of growth and meiosis. Three stages (A, B, C) of immature oocytes were discriminated in the ovary, with the germinal vesicle (GV) showing specific different features of growth and maturation. Stage A (pre‐vitellogenic) oocytes exhibited the highest L‐type Ca²⁺current activity, and were incompetent for meiosis resumption. Stage B (vitellogenic) oocytes showed Na⁺ currents that remained high during the maturation, up to the post‐vitellogenic stage C oocytes. The latter had acquired meiotic competence, undergoing spontaneous maturation and interacting with the spermatozoon. However, fertilized oocytes did not produce normal larvae, suggesting that cytoplasmic maturation plays a specific role in embryo development. Spontaneous maturation was inhibited at low pH whereas trypsin was able to trigger germinal vesicle breakdown (GVBD) regardless of pH; in addition spontaneous maturation was not affected by removal of follicle cells or by inhibiting junctional communication between oocyte and follicle cells. Taken together these results imply: (i) Ca²⁺ and Na⁺ currents are involved in meiotic progression, growth, and acquisition of meiotic competence; (ii) trypsin‐like molecules may have a role as candidates for providing the physiological stimulus to resume meiosis. Finally, we provide evidence that follicle cells in Ciona are not involved in triggering GVBD as it occurs in other ascidians. Mol. Reprod. Dev. 76: 1084–1093, 2009. © 2009 Wiley‐Liss, Inc.     

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     

Effect of roscovitine on nuclear maturation, MPF and MAP kinase activity and embryo development of prepubertal goat oocytes

The low number of embryos obtained from IVM-IVF-IVC of prepubertal goat oocytes could be due to an incomplete cytoplasmic maturation. Roscovitine (ROS) inhibits MPF and MAP kinase activity and maintains the oocyte at Germinal Vesicle (GV) stage. The aim of this study was to determine if meiotic activity is arrested in prepubertal goat oocytes cultured with 0, 12.5, 25, 50 and 100 microM of ROS for 24 h. A group of oocytes from adult goats was cultured with 25 microM of ROS to compare the effect of ROS on prepubertal and adult goat oocytes. A sample of oocytes was stained to evaluate the nuclear stage at oocyte collection time and after ROS incubation. IVM-oocytes not exposed to ROS formed the control group. Prepubertal goat IVM-oocytes were inseminated and cultured for 8 days. The percentage of oocytes at GV stage, after exposition to ROS was significantly higher in adult goat oocytes (64.5%) than in prepubertal goat oocytes. No differences were found among 25, 50 and 100 microM ROS concentrations (29, 23 and 26%, oocytes at GV stage, respectively). After 8 days of culture, no differences in total embryos were observed between control oocytes and oocytes treated with 12.5 and 25 microM (45.2, 36.1 and 39.4%, respectively), however the percentage of blastocysts was higher in the control group. Western blot for the MAPK and p34(cdc2) showed that both enzymes were active in prepubertal goat oocytes after 24h of ROS exposition. In conclusion, a low percentage of prepubertal goat oocytes reached GV stage after ROS incubation; possibly because most of them had reinitiated the meiosis inside the follicle. ROS did not affect fertilization or total embryos but ROS showed a negative effect on blastocyst development.