Effect of Kisspeptin on the Developmental Competence and Early Transcript Expression in Porcine Oocytes Parthenogenetically Activated with Different Methods.

Recent studies showed the modulatory effect of kisspeptin (KP) on calcium waves through the cell membrane and inside the cell. Spermatozoon can induce similar ooplasmic calcium oscillations at fertilization to trigger meiosis II. Here, we evaluated the effect of KP supplementation with 6-dimethylaminopurine (6-DMAP) for 4 h on embryonic development after oocyte activation with single electric pulse, 5 µM ionomycin, or 8% ethanol. Compared to control nonsupplemented groups, KP significantly improved embryo developmental competence electric- and ethanol-activated oocytes in terms of cleavage (75.3% and 58.6% versus 64% and 48%, respectively, p < 0.05) and blastocyst development (31.3% and 10% versus 19.3% and 4%, respectively, p < 0.05). MOS expression was increased in electrically activated oocytes in presence of KP while it significantly reduced CCNB1 expression. In ionomycin treated group, both MOS and CCNB1 showed significant increase with no difference between KP and control groups. In ethanol-treated group, KP significantly reduced CCNB1 but no effect was observed on MOS expression. The early alterations in MOS and CCNB1 mRNA transcripts caused by KP may explain the significant differences in the developmental competence between the experimental groups. Kisspeptin supplementation may be adopted in protocols for porcine oocyte activation through electric current and ethanol to improve embryonic developmental competence.

The dynamics of MAPK inactivation at fertilization in mouse eggs.

Egg activation at fertilization in mammals is initiated by prolonged Ca(2+) oscillations that trigger the completion of meiosis and formation of pronuclei. A fall in mitogen-activated protein kinase (MAPK) activity is essential for pronuclear formation, but the precise timing and mechanism of decline are unknown. Here, we have measured the dynamics of MAPK pathway inactivation during fertilization of mouse eggs using novel chemiluminescent MAPK activity reporters. This reveals that the MAPK activity decrease begins during the Ca(2+) oscillations, but MAPK does not completely inactivate until after pronuclear formation. The MAPKs present in eggs are Mos, MAP2K1 and MAP2K2 (MEK1 and MEK2, respectively) and MAPK3 and MAPK1 (ERK1 and ERK2, respectively). Notably, the MAPK activity decline at fertilization is not explained by upstream destruction of Mos, because a decrease in the signal from a Mos-luciferase reporter is not associated with egg activation. Furthermore, Mos overexpression does not affect the timing of MAPK inactivation or pronuclear formation. However, the late decrease in MAPK could be rapidly reversed by the protein phosphatase inhibitor, okadaic acid. These data suggest that the completion of meiosis in mouse zygotes is driven by an increased phosphatase activity and not by a decline in Mos levels or MEK activity.

A soft cortex is essential for asymmetric spindle positioning in mouse oocytes.

At mitosis onset, cortical tension increases and cells round up, ensuring correct spindle morphogenesis and orientation. Thus, cortical tension sets up the geometric requirements of cell division. On the contrary, cortical tension decreases during meiotic divisions in mouse oocytes, a puzzling observation because oocytes are round cells, stable in shape, that actively position their spindles. We investigated the pathway leading to reduction in cortical tension and its significance for spindle positioning. We document a previously uncharacterized Arp2/3-dependent thickening of the cortical F-actin essential for first meiotic spindle migration to the cortex. Using micropipette aspiration, we show that cortical tension decreases during meiosis I, resulting from myosin-II exclusion from the cortex, and that cortical F-actin thickening promotes cortical plasticity. These events soften and relax the cortex. They are triggered by the Mos-MAPK pathway and coordinated temporally. Artificial cortex stiffening and theoretical modelling demonstrate that a soft cortex is essential for meiotic spindle positioning.

Transforming growth factor-ß-induced protein (TGFBI) suppresses mesothelioma progression through the Akt/mTOR pathway.

As an uncommon cancer, mesothelioma is very hard to treat with a low average survival rate owing to its usual late detection and being highly invasive. The link between asbestos exposure and the development of mesothelioma in humans is unequivocal. TGFBI, a secreted protein that is induced by transforming growth factor-ß in various human cell types, has been shown to be associated with tumorigenesis in various types of tumors. It has been demonstrated that TGFBI expression is markedly suppressed in asbestos-induced tumorigenic cells, while an ectopic expression of TGFBI significantly suppresses tumorigenicity and progression in human bronchial epithelial cells. In order to delineate a potential role of TGFBI in mediating the molecular events that occur in mesothelioma tumorigenesis, we generated stable TGFBI knockdown mutants from the mesothelium cell line Met-5A by using an shRNA approach, and secondly created ectopic TGFBI overexpression mutants from the mesothelioma cell line H28 in which TGFBI is absent. We observed that in the absence of TGFBI, the knockdown mesothelial and mesothelioma cell lines exhibited an elevated proliferation rate, enhanced plating efficiency, increased anchorage-independent growth, as well as an increased cellular protein synthesis rate as compared with their respective controls. Furthermore, cell cycle regulatory proteins c-myc/cyclin D1/phosphor-Rb were upregulated; a more active PI3K/Akt/mTOR signaling pathway was also detected in TGFBI-depleted cell lines. These findings suggest that TGFBI may repress mesothelioma tumorigenesis and progression via the PI3K/Akt signaling pathway.

Nanobiology for the pharmacology of cellular ion channels.

Writing this editorial is especially pleasing. First, it provides us an opportunity to introduce new directives to the field of Neuroimmune Pharmacology and to explain why the field of nanomedicine is likely an important part of its future growth and development. Second, it is an opportunity to showcase research in this area currently operative in Russia that may not be readily accessible to the readership. Third, it is a platform to better explain why the Journal Editorial leadership was enthusiastic about the science and its relationship to the Society on NeuroImmune Pharmacology strategic goals. All are brought to bear in this issue of the Journal of Neuroimmune Pharmacology. The issue includes articles presented at a recent joint US-Russian workshop entitled, "Health in the 21st Century: Nanomedicine and Self-Organization of Biological Systems" held at M.V. Lomonosov Moscow State University (MSU), Moscow, Russia, December 10-11, 2007. The conjoint meeting was organized through the Departments of Biology, Chemistry, and Physics, MSU and by the Center for Drug Delivery and Nanomedicine and Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center (Omaha, NE). The speakers included established internationally regarded scientists from these institutions as well as graduate students and faculties at MSU. In addition to selected papers by workshop contributors, we have included several papers closely aligned to the theme of nanomedicine and nanopharmacology of the central nervous system in order to provide a biological anchor for this research. We understand that such works are new to many but hope that its organization and interdisciplinary approaches will appeal to this audience. All together, it is our hope that, by gathering basic and clinical scientists with the common interest of using nanotechnology in the delivery of therapeutic agents with a focus on nanopharmacology and complex supramolecular biological assembly, the papers included will provide a platform for thought, discussion, and future translational research.

Activation of both Mos and Cdc25 is required for G2-M transition in perch oocyte.

Resumption of meiosis from diplotene arrest during the first meiotic prophase in vertebrate oocytes is universally controlled by MPF, a heterodimer of Cdk1 and cyclin B. Activation of MPF depends on the withdrawal of Cdk1 inhibition by Wee1/Myt1 kinase on the one hand and the activation of Cdk1 by Cdc25 phosphatase on the other. It is relevant to know whether both these pathways are necessary to rescue diplotene arrest or if either one of them is sufficient. In MIH (17alpha, 20beta dihydroxy-4-pregnen-3-one) incubated perch (Anabas testudineus) oocytes we have examined these possibilities. Perch oocyte extract following MIH incubation showed a significant increase in Myt1 phosphorylation from 12 to 16 hr indicating its progressive deactivation. MIH induced Mos expression markedly increased at 16 hr effecting 95% GVBD. Cycloheximide inhibited MIH induced Mos expression and its phosphorylation, which in turn reduced Myt1 phosphorylation and GVBD. Myt1 phosphorylation was blocked in Mos immunodepleted oocytes. All these suggest the involvement of Mos in Myt1 phosphorylation. Oocytes incubated in MIH for 16 hr activated Cdc25, but such activation could not rescue the inhibition of GVBD due to Myt1 in Mos immunodepleted oocytes. Blocking Cdc25 with an antisense oligo significantly inhibited GVBD even though Myt1 remained deactivated during this period. Taken together, our findings indicate that MIH requires both pathways for perch oocyte maturation: the expression and activation of Mos, which is linked to Myt1 deactivation on the one hand, and the activation of Cdc25 on the other, as blocking either pathway compromised G2-M transition in perch oocytes.

Versatile retargeting of SH3 domain binding by modification of non-conserved loop residues.

Src-homology (SH3) domain belongs to a class of ubiquitous modular protein domains found in nature. SH3 domains have a conserved surface that recognises proline-rich peptides in ligand proteins, but additional contacts also contribute to binding. Using the SH3 domain of hematopoietic cell kinase as a test case, we show that SH3 binding properties can be profoundly altered by modifications within a hexapeptide sequence in the RT-loop region that is not involved in recognition of currently known consensus SH3 target peptides. These results highlight the role of non-conserved regions in SH3 target selection, and introduce a strategy that may be generally feasible for generating artificial SH3 domains with desired ligand binding properties.

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.

[Establishment of a stable and regulable 293 cell line expressing mycoplasma hyorhinis protein P37].

OBJECTIVE: To establish a stable cell line, which can express P37 protein of mycoplasma hyorhinis and be regulated by tetracycline, for investigating the effect of p37 on phenotype of cells and its mechanism. METHODS: Recombinant plasmid PcDNA5/FRT/TO-p37 was constructed and cotransfected with pOG44 into Flp-In-T-REx-293 cells by lipofectamine. Positive clones were screened with Hygromycin and Blasticidin. RT-PCR and Western blot were used to exam the mRNA and protein expression in selected clones. The expression level at different inducing times and concentrations of tetracycline were examined. MTT assay was used to observe the effect of P37 on proliferation of 293 cells. RESULTS: P37 protein, which is 43.5x10(3), was expressed in the selected clone as well as secreted from cells. Tetracycline showed a good regulation on the expression of P37 protein, which was not detectable without tetracycline induction. When induced with 2 mg/L tetracycline for 60 hours, the P37 protein expression reached maximum level. Cell growth was promoted after being transfected with p37. CONCLUSION: A stable cell line expressing P37 regularly was established, which provides a good cell model for studying p37 function and its molecular mechanism.

Phosphoinositide3-kinase regulates actin polymerization during delayed phagocytosis of Helicobacter pylori.

We have shown previously that ulcerogenic (type I) strains of Helicobacter pylori (Hp) retard their entry into macrophages. However, the signaling pathways that regulate Hp phagocytosis are largely undefined. We show here that Hp strongly activated class IA phosphoinositide3-kinases (PI3Ks) in macrophages, coincident with phagocytosis, and endogenous p85 and active protein kinase Balpha accumulated on forming phagosomes. PI3K inhibitors, wortmannin and LY294002, inhibited phagocytosis of Hp in a dose-dependent manner, and blockade of engulfment correlated directly with loss of 3'-phosphoinositides in the membrane subjacent to attached bacteria. During uptake of large immunoglobulin G (IgG)-coated particles, PI3Ks regulate pseudopod extension and phagosome closure. In marked contrast, we show here that 3'-phosphoinositides regulated actin polymerization at sites of Hp uptake. Moreover, Hp and IgG beads activated distinct PI3K isoforms. Phagosomes containing IgG-coated particles accumulated 3'-phosphatase and tensin homologue deleted on chromosome 10 and Src homology 2 domain-containing inositol 5'-phosphatase, yet Hp phagosomes did not. Finally, rapid uptake of IgG-opsonized Hp or a less-virulent type II Hp was PI3K-independent. We conclude that Hp and IgG beads are ingested by distinct mechanisms and that PI3Ks regulate the actin cytoskeleton during slow phagocytosis of ulcerogenic Hp.

Mitogen-activated protein kinase dynamics during the meiotic G2/MI transition of mouse spermatocytes.

Cellular and genetic approaches were used to investigate the requirements for activation during spermatogenesis of the extracellular signal-regulated protein kinases (ERKs), more commonly known as the mitogen-activated protein kinases (MAPKs). The MAPKS and their activating kinases, the MEKs, are expressed in specific developmental patterns. The MAPKs and MEK2 are expressed in all premeiotic germ cells and spermatocytes, while MEK1 is not expressed abundantly in pachytene spermatocytes. Phosphorylated (active) variants of these kinases are diminished in pachytene spermatocytes. Treatment of pachytene spermatocytes with okadaic acid (OA), to induce transition from meiotic prophase to metaphase I (G2/MI), resulted in phosphorylation and enzymatic activation of ERK1/2. However, U0126, an inhibitor of the ERK-activating kinases, MEK1/2, did not inhibit OA-induced MAPK activation or chromosome condensation. Analysis of spermatocytes lacking MOS, a mitogen-activated protein kinase kinase kinase responsible for MEK and MAPK activation, revealed that MOS is not required for OA-induced activation of the MAPKs. OA-induced MAPK activation was inhibited by butyrolactone I, an inhibitor of cyclin-dependent kinases 1 and 2 (CDK1, CDK2); thus, these kinases may regulate MAPK activity. Additionally, spermatocytes lacking CDC25C condensed bivalent chromosomes and activated both MPF and MAPKs in response to OA treatment; therefore, there is a CDC25C-independent pathway for MPF and MAPK activation. These studies reveal that spermatocytes do not require either MOS or CDC25C for onset of the meiotic division phase or for activation of MPF and the MAPKs, thus implicating a novel pathway for activation of the ERK1/2 MAPKs in spermatocytes.

Ca(2+)(cyt) negatively regulates the initiation of oocyte maturation.

Ca(2+) is a ubiquitous intracellular messenger that is important for cell cycle progression. Genetic and biochemical evidence support a role for Ca(2+) in mitosis. In contrast, there has been a long-standing debate as to whether Ca(2+) signals are required for oocyte meiosis. Here, we show that cytoplasmic Ca(2+) (Ca(2+)(cyt)) plays a dual role during Xenopus oocyte maturation. Ca(2+) signals are dispensable for meiosis entry (germinal vesicle breakdown and chromosome condensation), but are required for the completion of meiosis I. Interestingly, in the absence of Ca(2+)(cyt) signals oocytes enter meiosis more rapidly due to faster activation of the MAPK-maturation promoting factor (MPF) kinase cascade. This Ca(2+)-dependent negative regulation of the cell cycle machinery (MAPK-MPF cascade) is due to Ca(2+)(cyt) acting downstream of protein kinase A but upstream of Mos (a MAPK kinase kinase). Therefore, high Ca(2+)(cyt) delays meiosis entry by negatively regulating the initiation of the MAPK-MPF cascade. These results show that Ca(2+) modulates both the cell cycle machinery and nuclear maturation during meiosis.

Spindle checkpoint proteins Mad1 and Mad2 are required for cytostatic factor-mediated metaphase arrest.

In cells containing disrupted spindles, the spindle assembly checkpoint arrests the cell cycle in metaphase. The budding uninhibited by benzimidazole (Bub) 1, mitotic arrest-deficient (Mad) 1, and Mad2 proteins promote this checkpoint through sustained inhibition of the anaphase-promoting complex/cyclosome. Vertebrate oocytes undergoing meiotic maturation arrest in metaphase of meiosis II due to a cytoplasmic activity termed cytostatic factor (CSF), which appears not to be regulated by spindle dynamics. Here, we show that microinjection of Mad1 or Mad2 protein into early Xenopus laevis embryos causes metaphase arrest like that caused by Mos. Microinjection of antibodies to either Mad1 or Mad2 into maturing oocytes blocks the establishment of CSF arrest in meiosis II, and immunodepletion of either protein blocked the establishment of CSF arrest by Mos in egg extracts. A Mad2 mutant unable to oligomerize (Mad2 R133A) did not cause cell cycle arrest in blastomeres or in egg extracts. Once CSF arrest has been established, maintenance of metaphase arrest requires Mad1, but not Mad2 or Bub1. These results suggest a model in which CSF arrest by Mos is mediated by the Mad1 and Mad2 proteins in a manner distinct from the spindle checkpoint.

Regulation of the G2/M transition in oocytes of xenopus tropicalis.

The molecular events regulating hormone-induced oocyte activation and meiotic maturation are probably best understood in Xenopus laevis. In X. laevis, progesterone activates the G2-arrested oocyte, induces entry into M phase of meiosis I (MI) and resumption of the meiotic cell cycles, and leads to the formation of a mature, fertilizable egg. Oocytes of Xenopus tropicalis offer several practical advantages over those of X. laevis, including faster and more synchronous meiotic cell cycle progression, less seasonal variability, and the availability of transgenic approaches. Previous work found several similarities in the pathways regulating oocyte maturation in the two species. Here, we report several additional ones that are conserved in X. tropicalis. (1). Injection of Mos mRNA into G2-arrested oocytes activates the MAP kinase cascade and induces the G2/MI transition. (2). Injection of the beta subunit of the kinase CK2 (a negative regulator of Mos and oocyte activation) delays the G2/MI transition. (3). Elevating PKA activity blocks progesterone-induced maturation; repressing PKA activity induces entry into MI in the absence of progesterone. (4). LF (anthrax lethal factor), which cleaves certain MAP kinase kinases, strongly reduces both the rate and extent of entry into MI. In contrast to the one previously reported major difference between oocytes of the two species, we find that injection of egg cytoplasm ("MPF activity") into G2-arrested X. tropicalis oocytes induces entry into meiosis I even when protein synthesis is blocked, just as it does in oocytes of X. laevis. These results indicate that much of what we have learned from studies of X. laevis oocytes holds for those of X. tropicalis, and suggest that X. tropicalis oocytes offer a good experimental system for investigating certain questions that require a rapid, synchronous progression through the G2/meiosis I transition.

Interactive effects of insulin-like growth factor-1 and beta-estradiol on endothelial nitric oxide synthase activity in rat aortic endothelial cells.

Insulin-like growth factor-1 (IGF-1) and beta-estradiol (E2) have vasodilatory effects, in part, through stimulation of vascular nitric oxide (NO) production. However, their interactive effects on endothelial nitric oxide synthase (eNOS) and NO production have not been previously studied in endothelial cells (EC). Employing rat aortic EC (RAEC), the effects of acute (20 and 30 minutes) and prolonged (4 hours) stimulation with 100 nmol/L IGF-1 and 1 nmol/L E2 (alone or in combination) were assessed with respect to protein levels and enzymatic activities for phosphatidyl inositol 3-kinase (PI3K) and serine/threonine kinase Akt (Akt), enzymes involved in eNOS activation. Exposure to IGF-1 for 30 minutes or E2 for 20 minutes increased insulin receptor substrate-1 (IRS-1) association with the regulatory (p85) subunit of PI3K, enhanced tyrosine phosphorylation of p85, and increased PI3K activity. Combined treatment had a greater effect on p85 phosphorylation and PI3K activity then either agonist alone. Moreover, IGF-1 and E2 enhanced Akt Ser(473) phosphorylation, with the effect of IGF-1 being much greater. Acute expose to both E2 (20 minutes) and IGF-1 (30 minutes) were associated with an increase in eNOS activity. Prolonged exposure (4 hours) to either IGF-1 or E2 increased expression of the p85 subunit as well as eNOS activity. Pretreatment with PI3K antagonist wortmannin (WT) prevented this increase in eNOS activity. The results suggest that IGF-1 and E2 may interact through PI3K/Akt-related pathways to increase eNOS activity.

Maturation-promoting factor governs mitogen-activated protein kinase activation and interphase suppression during meiosis of rat oocytes.

Meiosis is a particular example of a cell cycle, characterized by two successive divisions without an intervening interphase. Resumption of meiosis in oocytes is associated with activation of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK). The activity of MPF declines during the transition between the two meiotic divisions, whereas the activity of MAPK is sustained. Attempts to disclose the interplay between these key regulators of meiosis in both amphibian and mammalian oocytes generated contradictory results. Furthermore, the enzyme that governs the suppression of interphase in mammals is still unidentified. To our knowledge, we provide herein the first demonstration in a mammalian system that inhibition of MPF at reinitiation of meiosis abrogated Mos expression and MAPK activation. We also show that oocytes, in which reactivation of MPF at completion of the first telophase was prevented, exhibited an interphase nucleus with decondensed chromosomes. Inhibition of MAPK did not interfere with the progression to the second meiotic metaphase but, rather, resulted in parthenogenic activation. We conclude that in rat oocytes, MPF regulates MAPK activation and its timely reactivation prevents the oocytes from entering interphase.

Expression of mos in astrocytic tumors and its potential role in neoplastic progression.

The c-mos gene and its protein product mos, components of the mitogen-activated protein kinase transduction pathway, are known to be involved in the control of meiosis and mitosis. Apart from a study on lung carcinomas, there is little information about its role in human neoplasia. The aim of this study was to investigate expression of mos in astrocytic tumors and to correlate it with accumulation of p53. We studied expression of mos in 62 cases of supratentorial astrocytic tumor. Intracytoplasmic immunostaining for mos was found in 28 (45%) cases: 3 of 20 (15%) grade 2 astrocytomas, 9 of 20 (45%) grade 3 anaplastic astrocytomas, and 16 of 22 (73%) glioblastomas. Immunopositivity for mos correlated significantly (P < 0.01) with tumor grade but not with p53 expression. In contrast to the findings in relation to lung tumors, immunopositivity for mos in astrocytic tumors did not predict recurrence-free or overall survival time. Cytoplasmic immunostaining was observed in scattered large cortical neurons adjacent to tumors, possibly due to stress-induced abortive entry into the cell cycle. The correlation of mos immunopositivity with tumor grade may reflect the expansion of more malignant mos-positive clones. This study provides evidence that mos may be involved in the neoplastic progression of a proportion of astrocytic tumors.

MyoD binds to Mos and inhibits the Mos/MAP kinase pathway.

When ectopically expressed, the serine/threonine kinase Mos can induce oncogenic transformation of somatic cells by direct phosphorylation of MAP kinase/ERK kinase (MEK1), activating the mitogen-activated protein kinases ERK1 and ERK2. On the other hand, overexpression of Mos in C2C12 myoblasts is not transforming. Mos activates myogenic differentiation by promoting heterodimerization of the MyoD/E12 proteins, increasing the expression of myogenic markers and the positive autoregulatory loop of MyoD. In this study, we show that in myogenic cells, the mitogenic and oncogenic signalling from the Mos/MEK/ERK pathway is suppressed by MyoD through the formation of a heterotrimeric complex.

Evidence of a functional interaction between serine 3 and serine 25 Mos phosphorylation sites. A dominant inhibitory role of serine 25 phosphorylation on Mos protein kinase.

Recently, we identified the major in vivo phosphorylation site on v-Mos as Ser-56, which is phosphorylated by cyclic AMP dependent protein kinase (PKA). Others have shown that c-Mos phosphorylation at Ser-3 (equivalent to Ser-34 in v-Mos) is important for the interaction of c-Mos with its substrate MEK and for its stability and cytostatic factor activity in eggs. To investigate the role of Ser-56 phosphorylation, we generated site-directed mutants of v-Mos that would mimic phosphorylation in terms of charge at positions 56 and 34. After mutating serine (S) residues with alanine (A) or glutamic acid (E) in different combinations, various v-Mos mutants were expressed in a rabbit reticulocyte lysate in vitro translation system and in COS-1 or NIH/3T3 cells. The effect of mutations on Mos function was evaluated by in vitro protein kinase assays and by the ability of Mos to cause neoplastic transformation of NIH/3T3 cells. The S56E but not the S56A mutation inhibited v-Mos kinase activity suggesting that Ser-56 phosphorylation has an inhibitory role. As predicted from Xenopus c-Mos studies, S34A but not S34E mutation inhibited v-Mos activity. Studies with the double mutants showed that the S56E mutation but not S56A mutation inhibited v-Mos kinase activity of both S34A and S34E mutants. Interestingly, the S56A mutation blocked the inhibitory effect of the S34A mutation on v-Mos kinase suggesting that in c-Mos the corresponding serine (Ser-25) can influence the regulation of c-Mos by Ser-3. Results showing inhibition of v-Mos kinase activity of the S34E mutant by the S56E mutation is significant as it suggests that doubly phosphorylated Mos at these residues would be inactive. Because residues corresponding to both v-Mos Ser-34 and Ser-56 are evolutionarily conserved in c-Mos, the kinase activity of c-Mos during meiosis may also be regulated in the same manner as v-Mos kinase activity.

Transcription factor E2F and cyclin E-Cdk2 complex cooperate to induce chromosomal DNA replication in Xenopus oocytes.

Although no chromosomal DNA replication actually occurs during Xenopus oocyte maturation, the capability develops during the late meiosis I (MI) phase in response to progesterone. This ability, however, is suppressed by Mos proteins and maturation/mitosis promoting factor during the second meiosis phase (meiosis II; MII) until fertilization. Inhibition of RNA synthesis by actinomycin D during early MI prevented induction of the replication ability, but did not interfere with initiation of the meiotic cell cycle progression characterized by oscillation of the maturation/mitosis promoting factor activity and germinal vesicle breakdown. Microinjection of recombinant proteins such as dominant-negative E2F or universal Cdk inhibitors, p21 and p27, but not wild type human E2F-1 or Cdk4-specific inhibitor, p19, into maturing oocytes during MI abolished induction of the DNA replication ability. Co-injection of human E2F-1 and cyclin E proteins into immature oocytes allowed them to initiate DNA replication even in the absence of progesterone treatment. Injection of cyclin E alone, which was sufficient to activate endogenous Cdk2 kinase, failed to induce DNA replication. Moreover, the activation of Cdk2 was not affected under the conditions where DNA replication was blocked by actinomycin D. Thus, like somatic cells, both activities of E2F and cyclin E-Cdk2 complex are required for induction of the DNA replication ability in maturing Xenopus oocytes, and enhancement of both activities enables oocytes to override DNA-replication inhibitory mechanisms that specifically lie in maturing oocytes.