Histone H1 Kinase Research Articles

Studies on fertilization of the teleost. II. Nuclear behavior and changes in histone H1 kinase.

In order to understand the dynamic responses of gamete nuclei upon fertilization in the fish, Oryzias latipes, the relationship between changes in the activity of histone H1 kinase and nuclear behavior was examined during fertilization. Kinase activity rapidly decreased concomitant with the initiation of the propagative exocytosis of cortical alveoli following sperm attachment to the egg plasma membrane post-insemination (PI). Activity again increased 30 min PI. Similar changes in kinase activity, migration and syngamy of pronuclei, and subsequent cleavage were observed with aphidicolin or actinomycin D treatment, except that formation of abnormal metaphase chromosomes was retarded in aphidicolin-treated zygotes. Pretreatment of unfertilized eggs with cycloheximide or 6-dimethylaminopurine (6-DMAP) caused no nuclear changes. The activity of histone H1 kinase in these eggs rapidly declined following sperm penetration and exocytosis, but did not undergo subsequent increase in the presence of these inhibitors. In these eggs with low histone H1 kinase activity, the fertilization process from sperm penetration to syngamy occurred normally, but the pronuclear membrane did not break down and the chromosomes did not condense. The present data suggest that in fish eggs, DNA replication as well as the synthesis and phosphorylation of proteins, especially cyclin B, are required for normal formation of metaphase chromosomes at the first cleavage, but not for fertilization events from sperm penetration through to nuclear migration resulting in syngamy.

A Maternal Form of the Phosphatase Cdc25A Regulates Early Embryonic Cell Cycles in Xenopus laevis

In mammalian cells the Cdc25 family of dual-specificity phosphatases has three distinct isoforms, termed A, B, and C, which are thought to play discrete roles in cell-cycle control. In this paper we report the cloning of Xenopus Cdc25A and demonstrate its developmental regulation and key role in embryonic cell-cycle control. Northern and Western blot analyses show that Cdc25A is absent in oocytes, and synthesis begins within 30 min after fertilization. The protein product is localized in the nucleus in interphase and accumulates continuously until the midblastula transition (MBT), after which it is degraded. Upon injection into newly fertilized eggs, wild-type Cdc25A shortened the cell cycle and accelerated the timing of cleavage, whereas embryos injected with phosphatase-dead Cdc25A displayed a dose-dependent increase in the length of the cell cycle and a slower rate of cleavage. In contrast, injection of the phosphatase-dead Cdc25C isoform had no effect. Western blotting with an antibody specific for phosphorylated tyr15 in Cdc2/Cdk2 revealed a cycle of phosphorylation/dephosphorylation in each cell cycle in control embryos, and in embryos injected with phosphatase-dead Cdc25A there was a twofold increase in the level of p-tyr in Cdc2/Cdk2. Consistent with this, the levels of cyclin B/Cdc2 and cyclin E/Cdk2 histone H1 kinase activity were both reduced by approximately 50% after phosphatase-dead Cdc25A injection. The phosphatase-dead Cdc25A could be recovered in a complex with both Cdks, suggesting that it acts in a dominant-negative fashion. These results indicate that periodic phosphorylation of Cdc2/Cdk2 on tyr15 occurs in each pre-MBT cell cycle, and dephosphorylation of Cdc2/Cdk2 by Cdc25A controls at least in part the length of the cell cycle and the timing of cleavage in pre-MBT embryos. The disappearance of Cdc25A after the MBT may underlie in part the lengthening of the cell cycle at that time.

Enhancement of G2 Checkpoint Function by Gelsolin Transfection in Human Cancer Cells

We have previously reported that human gastric (TMK1) and urinary bladder (UMUC2) cancer cell lines show markedly reduced expression of an actin-regulatory protein, gelsolin [S. Moriya et al., (1994), Int. J. Oncol. 5, 1347–1351, M. Tanaka et al. (1995), Cancer Res. 55, 3228–3232]. When gelsolin expression is restored by transfection, cancer cells lost tumorigenicity in vivo [M. Tanaka et al. (1995), Cancer Res. 55, 3228–3232]. Here, we show that gelsolin-overexpressing TMK1 and UMUC2 cells are more resistant to UVC irradiation. Increased resistance is associated with increases in the proportion of cells in the G2 phase of the cell cycle compared to similarly treated control neotransfectants. After UVC irradiation, synchronized gelsolin-overexpressing UMUC2 cells had a prolonged S phase followed by delayed G2 accumulation compared to neotransfected UMUC2 cells as determined by cell cycle analysis. The levels of cyclin B1 and cdk1 histone H1 kinase activity in gelsolin transfectants remained low during S and early G2 phase and the production of diacylglycerol induced by UVC was reduced in gelsolin transfectants compared to neotransfectants. These observations suggest that gelsolin enhances G2 checkpoint function of cells through lipid metabolism, leading to UVC resistance. Considered together with recent evidence that radiation clastogenesis and chemical carcinogenesis are cell-cycle-dependent, down regulation of gelsolin may lead to the malignant transformation of human gastric or urinary bladder cancers by attenuating G2 checkpoint function.

Effects of hormones and osmolarity in the culture medium on germinal vesicle breakdown of porcine oocytes

The present study was conducted to examine effects of hormones and osmolarity on germinal vesicle breakdown (GVBD) and histone H1 kinase (HIK) activity in porcine oocytes cultured in vitro. The basic medium used for culture of oocytes was modified Tyrode's solution in which the osmolarity was adjusted to 134 to 495 mOsm by changing the concentration of sodium chloride (NaCl). When the hormones were present, osmolarity of medium that allows GVBD of oocytes was less than 400 mOsm. However, the range of osmolarity of medium that allows meiotic maturation of oocytes was 210 to 362 mOsm. On the other hand, without hormonal supplement, the incidence of GVBD in oocytes decreased as the osmolarity of the medium increased in the rage of 210 to 362 mOsm. By increasing the osmolarity of the medium from 210 to 362 mOsm by addition with sorbitol instead of NaCl, the incidence decreased from 89.1 % to 13.3 %. In oocytes cultured in medium of 210 mOsm without hormones, the percentage of oocytes that underwent GVBD and had increased H1K activity 20 h after culture was significantly higher (P<0.05) than those of oocytes cultured in the same medium supplemented with hormones or medium of 362 mOsm. These results indicate that in vitro induction of GVBD in porcine oocytes is strongly affected by osmolarity of the medium in the absence of hormones. The results also suggest that, under low osmolarity (210 mOsm), GVBD is accelerated with rapid increase of H1K activity.

Interplay of maturation-promoting factor and mitogen-activated protein kinase inactivation during metaphase-to-interphase transition of activated bovine oocytes.

The objective of the present study was to examine the activity changes in histone H1 kinase (also known as maturation-promoting factor [MPF]) and mitogen-activated protein kinase (MAPK) and their constituent proteins in in vitro-matured bovine oocytes after in vitro fertilization (IVF) or after parthenogenetic activation induced by calcium ionophore A23187 alone or by the ionophore followed by either 6-dimethylaminopurine (6-DMAP) or cycloheximide (CHX). Inactivation of both H1 kinase and MAPK occurred after both A23187+6-DMAP treatment and IVF; inactivation of H1 kinase preceded inactivation of MAPK. However, MAPK was inactivated much earlier in 6-DMAP-treated oocytes. Further analysis of constituent cell cycle proteins of these kinases by Western blot showed that A23187 alone could not induce changes in cdc2, cdc25, or ERK2 but induced reduction of cyclin B1. IVF and A23187+CHX induced similar changes: cyclin B1 was destroyed shortly after activation followed by accumulation of cyclin B1, phosphorylation of cdc2, and dephosphorylation of ERK2 at pronuclear formation 15 h after activation. No change in cdc25 was observed at this time. In contrast, A23187+6-DMAP treatment resulted in earlier phosphorylation of cdc2 and dephosphorylation of ERK2 at 4 h after treatment when the pronucleus formed. Moreover, accumulation of both cdc25 and cyclin B1 was detected at 15 h. Microinjection of ERK2 antibody into A23187-treated oocytes resulted in pronuclear formation. In conclusion, activation of bovine oocytes with 6-DMAP led to earlier inactivation of MAPK, while CHX induced inactivation of MAPK parallel to that following sperm-induced oocyte activation. Destruction of cyclin B is responsible for inactivation of MPF, while phosphorylation of cdc2 is likely responsible for maintaining its low activity. Inactivation of MAPK is closely associated with pronuclear development regardless of the activation protocol used.

Cytoplasmic maturation for activation of pig follicular oocytes cultured and arrested at metaphase I.

A large population (62-90%) of pig follicular oocytes can mature to metaphase II after culture for 48 h. However, a proportion (6-22%) remain in an immature stage at metaphase I (metaphase I-arrested). The main objective of this study was to determine whether the cytoplasm of metaphase I-arrested pig oocytes is capable of being activated by sperm penetration or parthenogenetic stimulation. After culture for 48 h, oocytes without a polar body (73% were shown to be at metaphase I after staining) and those with a polar body (94% were at metaphase II) were fertilized in vitro. A total of 69% and 62% of the oocytes were activated to form a female pronucleus, respectively, and the rate of polar body extrusion induced by fertilization in the activated oocytes was 90% (the first polar body) and 95% (the second polar body), respectively. When oocytes without and with a polar body were stimulated with an electric pulse, 53% and 81% of the oocytes were activated, respectively. The rate of polar body extrusion in the activated oocytes was 73% (the first polar body) and 79% (the second polar body), respectively. In contrast, young metaphase I oocytes cultured for 24 h had low (6%) or zero activation rate after in vitro fertilization or electric pulse stimulation. However, about one-third of the young metaphase I oocytes penetrated by spermatozoa after in vitro fertilization responded to electric pulse 12 h after insemination, and almost all (93%) were activated when they were stimulated 24 h after insemination. Patterns of polypeptide synthesis and histone H1 kinase activity were similar in metaphase I-arrested and metaphase II oocytes, and were characterized by increase in a 25 kDa polypeptide and by decrease in kinase activity. Although the first step of meiotic division is impaired, these results indicate that metaphase I-arrested oocytes are mature cytoplasmically.

Induction of apoptosis in cancer cells by tumor necrosis factor and butyrolactone, an inhibitor of cyclin-dependent kinases.

Induction of apoptosis by tumor necrosis factor (TNF) is modulated by changes in the expression and activity of several cell cycle regulatory proteins. We examined the effects of TNF (1-100 ng/ml) and butyrolactone I (100 microM), a specific inhibitor of cyclin-dependent kinases (CDK) with high selectivity for CDK-1 and CDK-2, on three different cancer cell lines: WEHI, L929 and HeLa S3. Both compounds blocked cell growth, but only TNF induced the common events of apoptosis, i.e., chromatin condensation and ladder pattern of DNA fragmentation in these cell lines. The TNF-induced apoptosis events were increased in the presence of butyrolactone. In vitro phosphorylation assays for exogenous histone H1 and endogenous retinoblastoma protein (pRb) in the total cell lysates showed that treatment with both TNF and butyrolactone inhibited the histone H1 kinase (WEHI, L929 and HeLa) and pRb kinase (WEHI) activities of CDKs, as compared with the controls. The role of proteases in the TNF and butyrolactone-induced apoptosis was evaluated by comparing the number and expression of polypeptides in the cell lysates by gel electrophoresis. TNF and butyrolactone treatment caused the disappearance of several cellular protein bands in the region between 40-200 kDa, and the 110-90- and 50-kDa proteins were identified as the major substrates, whose degradation was remarkably increased by the treatments. Interestingly, the loss of several cellular protein bands was associated with the marked accumulation of two proteins apparently of 60 and 70 kDa, which may be cleavage products of one or more proteins. These findings link the decrease of cyclin-dependent kinase activities to the increase of protease activities within the growth arrest and apoptosis pathways induced by TNF.

Dual Specificity Protein Kinase Activity of Testis-specific Protein Kinase 1 and Its Regulation by Autophosphorylation of Serine-215 within the Activation Loop

TESK1 (testis-specific protein kinase 1) is a protein kinase with a structure composed of an N-terminal protein kinase domain and a C-terminal proline-rich domain. Whereas the 3.6-kilobase TESK1 mRNA is expressed predominantly in the testis, a faint 2.5-kilobase TESK1 mRNA is expressed ubiquitously. The kinase domain of TESK1 contains in the catalytic loop in subdomain VIB an unusual DLTSKN sequence, which is not related to the consensus sequence of either serine/threonine kinases or tyrosine kinases. In this study, we show that TESK1 has kinase activity with dual specificity on both serine/threonine and tyrosine residues. In an in vitro kinase reaction, the kinase domain of TESK1 underwent autophosphorylation on serine and tyrosine residues and catalyzed phosphorylation of histone H3 and myelin basic protein on serine, threonine, and tyrosine residues. Site-directed mutagenesis analyses revealed that Ser-215 within the "activation loop" of the kinase domain is the site of serine autophosphorylation of TESK1. Replacement of Ser-215 by alanine almost completely abolished serine autophosphorylation and histone H3 kinase activities. In contrast, replacement of Ser-215 by glutamic acid abolished serine autophosphorylation activity but retained histone H3 kinase activity. These results suggest that autophosphorylation of Ser-215 is an important step to positively regulate the kinase activity of TESK1.

Binding and catalytic properties of the Cdc2 and Crp proteins of Dictyostelium.

Dictyostelium expresses at least two proteins of the cyclin-dependent kinase (Cdk) family, Cdc2 and Crp. Cdc2 levels remain relatively constant during differentiation, whereas the levels of Crp increase dramatically as differentiation progresses. Crp is highly related to the mammalian Cdk5, and p25 (a truncated form of p35, the activating subunit of Cdk5 from mammalian brain) stimulates the histone H1 kinase activity of GST-Crp by several fold. In contrast, p25 does not stimulate the histone H1 kinase activity of GST-Cdc2 or the Cdc2 activity present in cell extracts from vegetative Dictyostelium cells. GST-Cdc2, in vitro translated Cdc2 and Cdc2 from all stages of differentiation bind to p13suc1. In contrast, GST-Crp, in vitro translated Crp and the Crp protein present in cell extracts do not bind to p13suc1. We have confirmed a previous report by Arakane and Maeda [J. Plant Res. (1997) 110, 81-85] that there is a peak of p13suc1 bound histone H1 kinase activity during late development, but we found that there was no corresponding peak of p13suc1 bound Cdc2 protein that corresponds to this activity. Taken together, these data suggest that neither Cdc2 nor Crp is responsible for the late developmental peak of histone H1 kinase activity that binds to p13suc1.

Functions of cyclin A1 in the cell cycle and its interactions with transcription factor E2F-1 and the Rb family of proteins.

Human cyclin A1, a newly discovered cyclin, is expressed in testis and is thought to function in the meiotic cell cycle. Here, we show that the expression of human cyclin A1 and cyclin A1-associated kinase activities was regulated during the mitotic cell cycle. In the osteosarcoma cell line MG63, cyclin A1 mRNA and protein were present at very low levels in cells at the G0 phase. They increased during the progression of the cell cycle and reached the highest levels in the S and G2/M phases. Furthermore, the cyclin A1-associated histone H1 kinase activity peaked at the G2/M phase. We report that cyclin A1 could bind to important cell cycle regulators: the Rb family of proteins, the transcription factor E2F-1, and the p21 family of proteins. The in vitro interaction of cyclin A1 with E2F-1 was greatly enhanced when cyclin A1 was complexed with CDK2. Associations of cyclin A1 with Rb and E2F-1 were observed in vivo in several cell lines. When cyclin A1 was coexpressed with CDK2 in sf9 insect cells, the CDK2-cyclin A1 complex had kinase activities for histone H1, E2F-1, and the Rb family of proteins. Our results suggest that the Rb family of proteins and E2F-1 may be important targets for phosphorylation by the cyclin A1-associated kinase. Cyclin A1 may function in the mitotic cell cycle in certain cells.

Calcium/Calmodulin-dependent Phosphorylation and Activation of Human Cdc25-C at the G2/M Phase Transition in HeLa Cells

The human tyrosine phosphatase (p54(cdc25-c)) is activated by phosphorylation at mitosis entry. The phosphorylated p54(cdc25-c) in turn activates the p34-cyclin B protein kinase and triggers mitosis. Although the active p34-cyclin B protein kinase can itself phosphorylate and activate p54(cdc25-c), we have investigated the possibility that other kinases may initially trigger the phosphorylation and activation of p54(cdc25-c). We have examined the effects of the calcium/calmodulin-dependent protein kinase (CaM kinase II) on p54(cdc25-c). Our in vitro experiments show that CaM kinase II can phosphorylate p54(cdc25-c) and increase its phosphatase activity by 2.5-3-fold. Treatment of a synchronous population of HeLa cells with KN-93 (a water-soluble inhibitor of CaM kinase II) or the microinjection of AC3-I (a specific peptide inhibitor of CaM kinase II) results in a cell cycle block in G2 phase. In the KN-93-arrested cells, p54(cdc25-c) is not phosphorylated, p34(cdc2) remains tyrosine phosphorylated, and there is no increase in histone H1 kinase activity. Our data suggest that a calcium-calmodulin-dependent step may be involved in the initial activation of p54(cdc25-c).

Sodium Butyrate Induces G2 Arrest in the Human Breast Cancer Cells MDA-MB-231 and Renders Them Competent for DNA Rereplication

When exposed to sodium butyrate (NaBut), exponentially growing cells accumulate in G1 and G2 phases of the cell cycle. In the human breast cancer cell line MDA-MB-231, an arrest in G2 phase was observed when the cells were released from hydroxyurea block (G1/S interface) in the presence of NaBut. The inhibition of G2 progression was correlated with increased contents both of total p21Waf1and of p21Waf1associated with cyclin A and with an inhibition of cyclin A- and B1-associated histone H1 kinase activities measured in cell lysates, as well as with dephosphorylation of the RB protein. A decrease in the cell contents of cyclins A and B1 was also observed but this decrease was preceded by p21Waf1accumulation. When NaBut was removed from the culture medium of cells blocked in G2 phase, p21Waf1level decreased and, instead of proceeding to mitosis, these cells resumed a progression toward DNA rereplication. These results suggest that the induction of p21Waf1by NaBut leads to the inhibition of the sequential activation of cyclin A- and B1-dependent kinases in this cell line, resulting in the inhibition of G2 progression and rendering the cells competent for a new cell division cycle.

Effects of Phosphorylation of Threonine 160 on Cyclin-dependent Kinase 2 Structure and Activity

We have prepared phosphorylated cyclin-dependent protein kinase 2 (CDK2) for crystallization using the CDK-activating kinase 1 (CAK1) from Saccharomyces cerevisiae and have grown crystals using microseeding techniques. Phosphorylation of monomeric human CDK2 by CAK1 is more efficient than phosphorylation of the binary CDK2-cyclin A complex. Phosphorylated CDK2 exhibits histone H1 kinase activity corresponding to approximately 0.3% of that observed with the fully activated phosphorylated CDK2-cyclin A complex. Fluorescence measurements have shown that Thr160 phosphorylation increases the affinity of CDK2 for both histone substrate and ATP and decreases its affinity for ADP. By contrast, phosphorylation of CDK2 has a negligible effect on the affinity for cyclin A. The crystal structures of the ATP-bound forms of phosphorylated CDK2 and unphosphorylated CDK2 have been solved at 2.1-A resolution. The structures are similar, with the major difference occurring in the activation segment, which is disordered in phosphorylated CDK2. The greater mobility of the activation segment in phosphorylated CDK2 and the absence of spontaneous crystallization suggest that phosphorylated CDK2 may adopt several different mobile states. The majority of these states are likely to correspond to inactive conformations, but a small fraction of phosphorylated CDK2 may be in an active conformation and hence explain the basal activity observed.

Effects of male accessory sex glands on sperm decondensation and oocyte activation during in vivo fertilization in golden hamsters

Removal of paternal male accessory sex glands (ASG) could cause a delay in DNA synthesis in hamster zygotes fertilized in vivo. In view of the fact that this process is closely related to pronuclear development which, in part, depends on sperm nuclear decondensation and oocyte activation during fertilization, we carried out a series of experiments were undertaken to determine whether ASG also has an effect on these early events. (1) Oocytes were collected from females mated with SH (sham-operated control), AGX (bilateral excision of ampullary glands), VPX (bilateral excision of ventral prostates) or TX (excision of all ASG) males (n = 8 per group) at 4, 5 and 6 h post coitus. (2) Epididymal spermatozoa were incubated with total ventral prostate (VP) secretion to study its effect on dithiothreitol-induced sperm decondensation. (3) Histone H1 kinase activity in oocytes collected as described in (1) was determined. (4) Exocytosed cortical granules on oocytes were labelled with FITC-LCA and quantified by a Metamorph Imaging System. Results showed that sperm decondensation and resumption of meiosis in oocytes in VPX and TX groups were significantly slower compared with SH. VP secretion augmented sperm decondensation in vitro. At 4 h post coitus, the relative activity of histone H1 kinase in the TX and VPX groups was significantly higher than that in the SH group (p < 0.01). Cortical granule exocytosis in the AGX group was consistently weaker at all time points studied and was significantly lower than that of the control at 4 h post coitus (p < 0.05), while the percentage of polyspermic fertilization in the AGX group was significantly higher compared with that in the SH group (p < 0.05). Taken together, these results show that the lack of exposure of spermatozoa to secretions of the ASG does not jeopardize their ability to penetrate ova, although other aspects of their function in the early stages of gamete interaction and subsequent initiation of embryonic development are affected.

Inhibition of S6 Kinase by Rapamycin Blocks Maturation ofRana dybowskiiOocytes

Studies were carried out to define the hormone-induced signal transduction pathway during maturation ofRana dybowskiioocytes. Rapamycin, a specific inhibitor of S6 kinase, blocked progesterone-induced oocyte germinal vesicle breakdown (GVBD) in a dose-dependent manner indicating that S6 kinase is required for meiotic maturation ofRanaoocytes. Addition of rapamycin within 3 h, but not 6 h, of progesterone treatment inhibited GVBD. In contrast, cycloheximide, a general protein synthesis inhibitor, blocked GVBD even when added 9 h after progesterone addition. A twofold increase in S6 kinase activity occurred within 1 h of progesterone stimulation and rapamycin inhibited this activity. Rapamycin also suppressed, in a dose-dependent manner, progesterone-induced protein synthesis during the first 12 h of culture but less effectively later. Histone H1 kinase activity (maturation-promoting factor, MPF) was observed in oocyte extracts at two different times (between 6 and 9 h and at 24 h) following progesterone stimulation. Rapamycin blocked H1 kinase activity between 6 and 9 h of culture but not that observed at 24 h. In contrast, cycloheximide suppressed progesterone-induced H1 kinase activity as well as protein synthesis throughout the course of incubation. Such results indicate that rapamycin and cycloheximide have common and unique effects on oocyte maturation and suggest that progesterone-induced S6 kinase activity is closely associated with induction of protein synthesis and activation of MPF during oocyte maturation. Results inRanacontrast with those obtained inXenopuswhere rapamycin inhibited S6 kinase but failed to inhibit GVBD or protein synthesis. Differences in the response ofRanaandXenopusoocytes to rapamycin are discussed in relation to seasonal, biochemical, and species variations.

Functional association of TGF-beta receptor II with cyclin B.

Utilizing the cytoplasmic tail of Transforming Growth Factor Receptor Type II (TGFbeta RII) as bait in a yeast two hybrid system, we have identified human cyclin B2 as a direct physical partner of TGFbeta RII. Analysis of deletion mutants of glutathione-S-transferase (GST)-cyclin B2 mapped its binding domain for TGFbeta RII to the C-terminal and revealed a negative regulatory region immediately upstream of the cyclin box. Using recombinant proteins, Cdc2 was demonstrated to indirectly interact with TGFbeta RII via cyclin B2. This interaction was reproduced in THP-1 monocytic cells, where TGFbeta treatment markedly enhanced the ability of cyclin B2 and, correspondingly, Cdc2 from TGFbeta-treated THP-1 cells, to bind the GST-TGFbeta RII fusion protein. More importantly, TGFbeta RII co-precipitated with cyclin B2 in TGFbeta-treated THP-1 cells. TGFbeta treatment also caused threonine phosphorylation of Cdc2 in the TGFbeta RII-cyclin B2-Cdc2 complex in THP1 cells, in parallel with down regulation of Cdc2 function as measured by histone H1 kinase activity. Cyclin B1 had the same capacity to bind TGFbeta RII and mediate indirect Cdc2 binding. These results suggest an alternative mechanism that cell cycle arrest in the G1/S phase caused by TGFbeta may, in part, be due to inactivation of cyclin B/Cdc2 kinase, which is needed for entry into the G2/M phase.

Studies on fertilization in the teleost. III. The relationship between nuclear behavior and the histone H1 kinase activity in anesthetized medaka eggs

To investigate the mechanisms of fertilization in the teleostean egg, the relationship between the nuclear behavior and the activity of histone H1 kinase was examined in medaka, Oryzias latipes, eggs that were anesthetized at sperm penetration. Inseminated in the anesthetized state, most eggs failed to undergo the propagative waves of increase in cytoplasmic Ca(2+) and exocytosis of cortical alveoli (CABD). The sperm-penetrated eggs that exhibited no or partial CABD only around the animal pole underwent a transient contraction of the cortical cytoplasm toward the animal pole region and were designated nonactivated eggs. Temporary compaction of the second meiotic metaphase (MII) chromosomes was accompanied by contractile movement of the cortical cytoplasm, but not by completion of the second meiotic division. The activity of histone H1 kinase in nonactivated eggs remained high, although it decreased slightly concurrent with sperm penetration. Cyclin B and cdc2 levels remained unchanged as well. The nonactivated eggs began to transform the penetrated sperm nucleus into metaphase chromosomes in the cortical cytoplasm facing the inner end of micropylar canal within 20 min postinsemination (PI). Two figures of typical metaphase chromosomes were found in the animal pole area at </=40 min PI. Chromosome condensation in nonactivated eggs was not inhibited by actinomycin D, nor was the high activity of histone H1 kinase reduced. In the presence of cycloheximide or 6-dimethylaminopurine (6-DMAP), however, the compact sperm nucleus and the MII chromosomes transformed to interphase nuclei without CABD or extrusion of the polar body, although the activity of histone H1 kinase remained high. These results suggest that in the fish egg, transformation of MII chromosomes to an interphase nucleus may not be caused by loss of MPF activity, but rather than by the loss of activity of a short-lived protein kinase(s), sensitive to 6-DMAP that is independent of CABD in the cascade reactions triggered by increased cytoplasmic calcium. Copyright 1999 Wiley-Liss, Inc.

Acquisition of competence to condense metaphase I chromosomes during spermatogenesis.

Little is known about the timing of meiotic prophase events during spermatogenesis in the mouse or how these events are related to cell-cycle progression. This work was designed to test hypotheses about the timing and biochemical correlates of developmental acquisition of competence to condense bivalent pairs of homologous chromosomes held together by chiasmata. The experimental approach takes advantage of the fact that okadaic acid (OA) treatment of pachytene spermatocytes causes precocious entry into metaphase I (MI) of meiosis. Leptotene and zygotene (L/Z) spermatocytes are not competent to respond to OA with condensation of chiasmate bivalent chromosomes. Competence for MI condensation of chiasmate bivalents is acquired by the middle of the pachytene stage of meiotic prophase, several days after homologous chromosomes become fully synapsed. The acquisition of MI competence is paralleled by the accumulation of histone H1t in the nuclei of mid-pachytene spermatocytes. Biochemical differences also exist between the incompetent L/Z spermatocytes and the competent pachytene spermatocytes. Both have the molecular components of metaphase promoting factor, CDC2 and CYCLIN B1; however, the histone H1 kinase activity of metaphase promoting factor of incompetent L/Z spermatocytes is not activated by OA, as it is in competent pachytene spermatocytes. Additionally, the CDC25C protein phosphatase is present in competent pachytene spermatocytes, but not in incompetent L/Z or early pachytene spermatocytes. Both incompetent and competent spermatocytes accumulate MPM-2 phosphoepitopes and phosphorylated histone H3 in response to OA treatment, indicating that presence of these antigens is not sufficient to promote condensation of meiotic chromosomes. These data demonstrate that meiotic competence of spermatocytes is acquired after homologous chromosome pairing is established and is coincident with first appearance of histone H1t and CDC25C protein phosphatase in spermatocytes.

Differential expression of genes for cyclin-dependent protein kinases in rice plants.

Cyclin-dependent protein kinases (CDKs) play key roles in regulating the eukaryotic cell cycle. We have analyzed the expression of four rice (Oryza sativa) CDK genes, cdc2Os1, cdc2Os2, cdc2Os3, and R2, by in situ hybridization of sections of root apices. Transcripts of cdc2Os1, cdc2Os2, and R2 were detected uniformly in the dividing region of the root apex. cdc2Os1 and cdc2Os2 were also expressed in differentiated cells such as those in the sclerenchyma, pericycle, and parenchyma of the central cylinder. By contrast, signals corresponding to transcripts of cdc2Os3 were distributed only in patches in the dividing region. Counterstaining of sections with 4', 6-diamidino-2-phenylindole and double-target in situ hybridization with a probe for histone H4 transcripts revealed that cdc2Os3 transcripts were abundant from the G2 to the M phase, but were less abundant or absent during the S phase. The levels of the Cdc2Os3 protein and its associated histone H1-kinase activity were reduced by treatment of cultured cells with hydroxyurea, which blocks cycling cells at the onset of the S phase. Our results suggest that domains other than the conserved amino acid sequence (the PSTAIRE motif) have important roles in the function of non-PSTAIRE CDKs in distinct cell-cycle phases.

A link between cell cycle and cell death: Bax and Bcl-2 modulate Cdk2 activation during thymocyte apoptosis.

Resting thymocytes undergoing apoptosis in response to specific stimuli degrade the cdk inhibitor p27(Kip1) and upregulate Cdk2 kinase activity. Inhibition of Cdk2 kinase activity efficiently blocks cell death via certain apoptosis pathways whereas overexpression of Cdk2 accelerates such cell death, suggesting its involvement in the signal transduction pathways activated by certain apoptotic stimuli. We found that Cdk2 activation during thymocyte apoptosis can be regulated by p53, Bax and Bcl-2. The highly elevated Cdk2 kinase activity in the apoptosing thymocytes is not associated with its canonical cyclins, cyclin E and cyclin A, and requires de novo synthesis of proteins for activation to take place. We therefore propose Cdk2 activation to be a crucial event in distinct pathways of apoptosis and the point at which the cell cycle and cell death pathways interact.