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Results for “Maturation-Promoting Factor”

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PubMed1992

Dephosphorylation of cdc2 on threonine 161 is required for cdc2 kinase inactivation and normal anaphase.

Exit from metaphase of the cell cycle requires inactivation of MPF, a stoichiometric complex between the cdc2 catalytic and the cyclin B regulatory subunits, as well as that of cyclin A-cdc2 kinase. Inactivation of both complexes depends on proteolytic degradation of the cyclin subunit, yet cyclin proteolysis is not sufficient to inactivate the H1 kinase activity of cdc2. Genetic evidence strongly suggests that type 1 phosphatase plays a key role in the metaphase-anaphase transition of the cell cycle. Here we report that inhibition of both type 1 and type 2A phosphatases by okadaic acid allows cyclin degradation to occur, but prevents cdc2 kinase inactivation. Complete inhibition of type 2A phosphatase alone is not sufficient to prevent cdc2 kinase inactivation following cyclin proteolysis. We show further that residue 161 of cdc2 is phosphorylated in active cyclin A or cyclin B complexes at metaphase, whilst unassociated cdc2 is not phosphorylated. Proteolysis of cyclin releases a free cdc2 subunit, which subsequently undergoes dephosphorylation and then migrates more slowly than its Thr161 phosphorylated counterpart in Laemmli gels. Removal of phosphothreonine 161 requires cyclin proteolysis. However, it does not occur even after cyclin proteolysis, when both type 1 and type 2A phosphatases are inhibited. We conclude that both cyclin degradation and dephosphorylation of Thr161 on cdc2, catalysed at least in part by type 1 phosphatase, are required to inactivate either cyclin B- or cyclin A-cdc2 kinases and thus for cells to exit from M phase.

Amino Acid Sequence↗
PubMed1992

Regulation of a major microtubule-associated protein by MPF and MAP kinase.

The interphase-M phase transition of microtubule dynamics is thought to be induced by phosphorylation reactions mediated by MPF and by MAP kinase functioning downstream of MPF. We have now identified and purified from Xenopus eggs a major microtubule-associated protein, p220, that may be a target protein for these two M phase-activated kinases. p220, when purified from interphase cells, potently bound to microtubules and stimulated tubulin polymerization, whereas p220 purified from M phase cells showed little or no such activities. Cell staining with a monoclonal anti-p220 antibody revealed that p220 is localized on cytoplasmic microtubule networks during interphase, while it is distributed rather diffusely throughout the cell during M phase. We have further found that p220 is phosphorylated specifically in M phase. Moreover, p220 purified from interphase cells served as a good substrate for MAP kinase and MPF in vitro, and two-dimensional phosphopeptide mapping pattern of the p220 phosphorylated in vitro was very similar to that of p220 phosphorylated at M phase in vivo. These results suggest that the drastic change in p220 activity during the transition from interphase to M phase may be induced by its phosphorylation in M phase probably catalyzed by MAP kinase and MPF.

Animals↗
PubMed1992

The fall of biological maturation promoting factor (MPF) and histone H1 kinase activity during anaphase and telophase in mouse oocytes.

Cell fusions have been used to determine the biological activity of the MPF complex in murine oocytes during their progression through anaphase and telophase to metaphase II. Oocytes (1) at metaphase I, (2) during the anaphase-telophase transition, or (3) at metaphase II were fused to germinal vesicle-staged (immature) oocytes. The hybrids were cultured for 1 h in the presence of db cAMP before fixation and nuclear evaluation. Metaphase I oocytes invariably induced germinal vesicle breakdown (GVBD) in the immature partner. By contrast, anaphase/telophase oocytes never induced GVBD in immature oocytes. The capacity to induce GVBD reappears after the formation of the second metaphase plate. In a second study, histone H1 kinase activity was measured during mouse oocyte maturation in single oocytes. H1 kinase activity was low in GV oocytes, increased sharply at MI, declined during anaphase and telophase and increased again at MII. After egg activation, H1 kinase activity was reduced to basal levels. These results provide direct evidence that a drop in activity of MPF in murine oocytes occurs concomitantly with the exit from metaphase I; MPF activity remains low until the cell re-enters metaphase.

Anaphase↗
PubMed1992

Okadaic acid and p13suc1 modulate the reinitiation of meiosis in mouse oocytes.

Short-term exposure to okadaic acid (OA), a specific inhibitor of protein phosphatases 1 and 2A, induced resumption of meiosis, including metaphase spindle formation, in mouse oocytes treated with a phosphodiesterase inhibitor, while long incubations with OA arrested oocyte maturation at a step prior to spindle formation. To explore the basis for this difference, the overall patterns of protein synthesis and phosphorylation and the production of tissue-type plasminogen activator (tPA), the synthesis of which is induced after germinal vesicle breakdown (GVBD), were analyzed under various OA treatments. Short-term exposure to OA led to tPA production and did not greatly affect the maturation-associated changes in protein phosphorylation. By contrast, a long application of OA did not result in tPA production and induced more marked changes in protein phosphorylation. Microinjection into prophase oocytes of the product of the fission yeast gene p13suc1, known to inhibit p34cdc2 kinase activation and/or activity, prevented meiotic reinitiation. This effect was overcome by microinjection of OA, at concentrations higher than those required for induction of maturation in the absence of p13suc1. These observations suggest that inhibition of phosphatase 1 or 2A or both triggers meiotic resumption by acting at the same site or at a site proximal to the p13suc1-sensitive step of cdc2 kinase activation.

Animals↗
PubMed1992

A deficiency in the mechanism for p34cdc2 protein kinase activation in mouse embryos arrested at 2-cell stage.

Mouse embryos of the ddY strain fertilized in vitro undergo the first cleavage to the 2-cell stage but not the second cleavage even 45 hr after insemination (2-cell block). We examined the phosphorylation state of p34cdc2 and histone H1 kinase activity in mouse 2-cell embryos to investigate the relationship of p34cdc2 with 2-cell block. In the first mitotic cell cycle, the amount of phosphorylated forms of p34cdc2, which were detected as the bands of retarded mobility on SDS-PAGE followed by immunoblotting with anti-p34cdc2 antibody, increased during interphase and abruptly decreased at M phase. Concomitant with this dephosphorylation, histone H1 kinase activity was increased. After the embryos cleaved to the 2-cell stage, the amounts of phosphorylated forms of p34cdc2 increased up to 33 hr after insemination. However, the activation of histone H1 kinase did not occur and the states of phosphorylation of p34cdc2 did not show any significant changes until 45 hr. In contrast, 2-cell embryos of B6C3F1 mice, which do not show a 2-cell block and develop normally to blastocysts in vitro, exhibit the dephosphorylation of p34cdc2 and an increase in histone H1 kinase activity between 31 and 45 hr after insemination. When the ddY mouse embryos arrested at the 2-cell stage were treated with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, the dephosphorylation of p34cdc2 occurred and histone H1 kinase activity increased. The chromosomes of these embryos stained with 4',6'-diamidino-2-phenylindole revealed the initiation of condensation. These results suggest that 2-cell-blocked embryos contain enough p34cdc2 to induce mitotic events but the protein remains in a latent form.

Animals↗
PubMed1992

Isolation and characterization of goldfish cdk2, a cognate variant of the cell cycle regulator cdc2.

This paper reports the nucleotide and predicted amino acid sequences of the goldfish cdk2, a cognate variant of the cell cycle regulator cdc2. The predicted protein sequence shows strong homology to the other known cdk2 (88% for Xenopus and 90% for human). A monoclonal antibody against the C-terminal sequence of goldfish cdk2 recognized a 34-kDa protein in extracts from various goldfish tissues. The protein level was high in such tissues as testis and ovary containing actively dividing cells. Protein cdk2 binds to p13sucl, the fission yeast suc1+ gene product, but not to cyclin B, with which cdc2 forms a complex. The kinase activity of cdk2 increased 30-fold when oocytes matured, although its protein level did not remarkably change. Anti-cdk2 immunoprecipitates from 32P-labeled mature oocyte extracts contained a 47-kDa protein, which was not recognized by either anti-cyclin A or anti-cyclin B antibody, indicating complex formation of cdk2 with a protein other than cyclins A or B.

Amino Acid Sequence↗
PubMed1992

The cell cycle then and now.

In the last few years a general model of cell cycle control has been established for all eukaryotic cells. Experiments from a variety of organisms and from a variety of experimental approaches have identified a protein kinase and its unstable regulatory subunit as the activator of mitosis; related molecules seem to be involved in the activation of chromosome replication. The identification of the biochemical components of these important regulatory pathways is providing several new insights into homeostatic and developmental control mechanisms in higher organisms.

Animals↗
PubMed1992

Meiotic initiation by the mos protein in Xenopus.

When fully grown Xenopus oocytes are stimulated by progesterone, a period of protein synthesis is necessary for maturation. Synthesis of the mos proto-oncogene product, pp39mos, is necessary for the activation of M-phase promoting factor (MPF) in meiosis I. On the basis that mos is translated de novo on hormonal stimulation of Xenopus oocytes and that injecting mos RNA into oocytes induces their maturation, we have proposed that the mos protein is a candidate initiator of oocyte maturation, needed to trigger the conversion of precursor MPF into its active form. To determine whether mos is the only protein required for initiating maturation, we have produced a soluble, active recombinant mos protein and injected it into Xenopus oocytes. We report here that in the absence of protein synthesis that mos protein efficiently induces germinal vesicle breakdown and the activation of MPF. The oocytes, however, do not proceed into meiosis II. Thus, the mos protein fulfills the requirements of an initiator protein, but the synthesis of one or more additional proteins may be necessary to complete oocyte maturation.

Animals↗
PubMed1992

Requirement for p34cdc2 kinase is restricted to mitosis in the mammalian cdc2 mutant FT210.

The mouse FT210 cell line is a temperature-sensitive cdc2 mutant. FT210 cells are found to arrest specifically in G2 phase and unlike many alleles of cdc2 and cdc28 mutants of yeasts, loss of p34cdc2 at the nonpermissive temperature has no apparent effect on cell cycle progression through the G1 and S phases of the division cycle. FT210 cells and the parent wild-type FM3A cell line each possess at least three distinct histone H1 kinases. H1 kinase activities in chromatography fractions were identified using a synthetic peptide substrate containing the consensus phosphorylation site of histone H1 and the kinase subunit compositions were determined immunochemically with antisera prepared against the "PSTAIR" peptide, the COOH-terminus of mammalian p34cdc2 and the human cyclins A and B1. The results show that p34cdc2 forms two separate complexes with cyclin A and with cyclin B1, both of which exhibit thermal lability at the non-permissive temperature in vitro and in vivo. A third H1 kinase with stable activity at the nonpermissive temperature is comprised of cyclin A and a cdc2-like 34-kD subunit, which is immunoreactive with anti-"PSTAIR" antiserum but is not recognized with antiserum specific for the COOH-terminus of p34cdc2. The cyclin A-associated kinases are active during S and G2 phases and earlier in the division cycle than the p34cdc2-cyclin B1 kinase. We show that mouse cells possess at least two cdc2-related gene products which form cell cycle regulated histone H1 kinases and we propose that the murine homolog of yeast p34cdc/CDC28 is essential only during the G2-to-M transition in FT210 cells.

Amino Acid Sequence↗
PubMed1992

Phosphorylation of myosin-II regulatory light chain by cyclin-p34cdc2: a mechanism for the timing of cytokinesis.

To understand how cytokinesis is regulated during mitosis, we tested cyclin-p34cdc2 for myosin-II kinase activity, and investigated the mitotic-specific phosphorylation of myosin-II in lysates of Xenopus eggs. Purified cyclin-p34cdc2 phosphorylated the regulatory light chain of cytoplasmic and smooth muscle myosin-II in vitro on serine-1 or serine-2 and threonine-9, sites known to inhibit the actin-activated myosin ATPase activity of smooth muscle and nonmuscle myosin (Nishikawa, M., J. R. Sellers, R. S. Adelstein, and H. Hidaka. 1984. J. Biol. Chem. 259:8808-8814; Bengur, A. R., A. E. Robinson, E. Appella, and J. R. Sellers. 1987. J. Biol. Chem. 262:7613-7617; Ikebe, M., and S. Reardon. 1990. Biochemistry. 29:2713-2720). Serine-1 or -2 of the regulatory light chain of Xenopus cytoplasmic myosin-II was also phosphorylated in Xenopus egg lysates stabilized in metaphase, but not in interphase. Inhibition of myosin-II by cyclin-p34cdc2 during prophase and metaphase could delay cytokinesis until chromosome segregation is initiated and thus determine the timing of cytokinesis relative to earlier events in mitosis.

Amino Acid Sequence↗
PubMed1992

Comparison of histone H1 kinase activity during meiotic maturation between two types of porcine oocytes matured in different media in vitro.

Histone H1 kinase (H1K) activity was assayed during meiotic maturation in porcine oocytes matured in a modified Krebs-Ringer bicarbonate solution (KRB) or in porcine follicular fluid (pFF) in vitro. Oocytes matured in KRB displayed lower male pronucleus formation ability, delayed first polar body emission, and a higher spontaneous activation rate than oocytes matured in pFF. In oocytes matured in pFF, H1K activity was low at the germinal vesicle stage and increased about 8-fold at first and second metaphases, with a transient depression at first anaphase and telophase. The H1K activity at second metaphase in oocytes matured in KRB was significantly lower than that in oocytes matured in pFF. These results suggest that the maturation medium used influences the fluctuation pattern of H1K activity and the biological characteristics of porcine oocytes cultured in vitro.

Animals↗
PubMed1992

Organization and regulation of cortical microtubules during the first cell cycle of Xenopus eggs.

Anti-tubulin antibodies and confocal immunofluorescence microscopy were used to examine the organization and regulation of cytoplasmic and cortical microtubules during the first cell cycle of fertilized Xenopus eggs. Appearance of microtubules in the egg cortex temporally coincided with the outgrowth of the sperm aster. Microtubules of the sperm aster first reached the animal cortex at 0.25, (times normalized to first cleavage), forming a radially organized array of cortical microtubules. A disordered network of microtubules was apparent in the vegetal cortex as early as 0.35. Cortical microtubule networks of both animal and vegetal hemispheres were reorganized at times corresponding to the cortical rotation responsible for specification of the dorsal-ventral (D-V) axis. Optical sections suggest that the cortical microtubules are continuous with the microtubules of the sperm aster in fertilized eggs, or an extensive activation aster in activated eggs. Neither assembly and organization, nor disassembly of the cortical microtubules coincided with MPF activation during mitosis. However, cycloheximide or 6-dimethylaminopurine, which arrest fertilized eggs at interphase, blocked cortical microtubule disassembly. Injection of p13, a protein that specifically inhibits MPF activation, delayed or inhibited cortical microtubule breakdown. In contrast, eggs injected with cyc delta 90, a truncated cyclin that arrest eggs in M-phase, showed normal microtubule disassembly. Finally, injection of partially purified MPF into cycloheximide-arrested eggs induced cortical microtubule breakdown. These results suggest that, despite a lack of temporal coincidence, breakdown of the cortical microtubules is dependent on the activation of MPF.

Animals↗
PubMedDate not supplied

[Oocyte maturation and activation in the common frog and the clawed toad under the action of divalent cations].

Maturation of Rana temporaria and Xenopus laevis oocytes was induced by solutions containing Mn2+ and Co2+ ions. Completion of oocyte maturation was estimated by the following criteria: (1) appearance of the maturation promoting factor (MPF) in the oocyte cytoplasm and (2) oocyte capacity to activation and formation of male pronuclei from the injected sperm nuclei. X. laevis oocytes matured under the effect of Co2+ ions were shown to contain MPF. Oocytes of both species matured under the effect of either ions could not be activated by pricking with a needle and injected sperm nuclei didn't transform into pronuclei. R. temporaria oocytes matured under the effect of ions in late spring, when natural spawning takes place, showed spontaneous activation.

Animals↗
PubMed1992

Staurosporine overrides checkpoints for mitotic onset in BHK cells.

Under normal conditions, mammalian cells will not initiate mitosis in the presence of either unreplicated or damaged DNA. We report here that staurosporine, a tumor promoter and potent protein kinase inhibitor, can uncouple mitosis from the completion of DNA replication and override DNA damage-induced G2 delay. Syrian hamster (BHK) fibroblasts that were arrested in S phase underwent premature mitosis at concentrations as low as 1 ng/ml, with maximum activity seen at 50 ng/ml. Histone H1 kinase activity was increased to approximately one-half the level found in normal mitotic cells. Inhibition of protein synthesis during staurosporine treatment blocked premature mitosis and suppressed the increase in histone H1 kinase activity. In asynchronously growing cells, staurosporine transiently increased the mitotic index and histone H1 kinase activity but did not induce S phase cells to undergo premature mitosis, indicating a requirement for S phase arrest. Staurosporine also bypassed the cell cycle checkpoint that prevents the onset of mitosis in the presence of damaged DNA. The delay in mitotic onset resulting from gamma radiation was reduced when irradiation was followed immediately by exposure to 50 ng/ml of staurosporine. These findings indicate that inhibition of protein phosphorylation by staurosporine can override two important checkpoints for the initiation of mitosis in BHK cells.

Alkaloids↗