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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

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

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

Fluctuation of histone H1 kinase activity during meiotic maturation in porcine oocytes.

Porcine oocytes cultured in follicular fluid for various periods of up to 48 h were stained with Hoechst-33342 and classified according to maturation before assaying. Histone H1 kinase activity at metaphase I was approximately 10 times that at the germinal vesicle stage. An abrupt reduction in activity was observed in oocytes emitting the first polar body; then the activity increased again to the same level as at metaphase I. This pattern is similar to those reported in non-mammalian species and supports the concepts that histone H1 kinase is ubiquitous in eukaryotes and controls the meiotic cell cycle in mammals.

Animals

[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

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