Chromatin-associated protein phosphatase 1 regulates aurora-B and histone H3 phosphorylation

Proper chromosome condensation requires the phosphorylation of histone and nonhistone chromatin proteins. We have used an in vitro chromosome assembly system based on Xenopus egg cytoplasmic extracts to study mitotic histone H3 phosphorylation. We identified a histone H3 Ser(10) kinase activity associated with isolated mitotic chromosomes. The histone H3 kinase was not affected by inhibitors of cyclin-dependent kinases, DNA-dependent protein kinase, p90(rsk), or cAMP-dependent protein kinase. The activity could be selectively eluted from mitotic chromosomes and immunoprecipitated by specific anti-X aurora-B/AIRK2 antibodies. This activity was regulated by phosphorylation. Treatment of X aurora-B immunoprecipitates with recombinant protein phosphatase 1 (PP1) inhibited kinase activity. The presence of PP1 on chromatin suggested that PP1 might directly regulate the X aurora-B associated kinase activity. Indeed, incubation of isolated interphase chromatin with the PP1-specific inhibitor I2 and ATP generated an H3 kinase activity that was also specifically immunoprecipitated by anti-X aurora-B antibodies. Nonetheless, we found that stimulation of histone H3 phosphorylation in interphase cytosol does not drive chromosome condensation or targeting of 13 S condensin to chromatin. In summary, the chromosome-associated mitotic histone H3 Ser(10) kinase is associated with X aurora-B and is inhibited directly in interphase chromatin by PP1.     

Neither Aurora B activity nor histone H3 phosphorylation is essential for chromosome condensation during meiotic maturation of porcine oocytes

Aurora kinase B (AURKB) is a chromosomal passenger protein that is essential for a number of processes during mitosis. Its activity is regulated by association with two other passenger proteins, INCENP and Survivin, and by phosphorylation on Thr 232. In this study, we examine expression and phosphorylation on Thr-232 of AURKB during meiotic maturation of pig oocytes in correlation with histone H3 phosphorylation and chromosome condensation. We show that histone H3 phosphorylation on Ser-10, but not on Ser-28, correlates with progressive chromosome condensation during oocyte maturation; Ser-10 phosphorylation starts around the time of the breakdown of the nuclear envelope, with the maximal activity in metaphase I, whereas Ser-28 phosphorylation does not significantly change in maturing oocytes. Treatment of oocytes with 50 microM butyrolactone I (BL-I), an inhibitor of cyclin-dependent kinases, or cycloheximide (10 microg/ml), inhibitor of proteosynthesis, results in a block of oocytes in the germinal vesicle stage, when nuclear membrane remains intact; however, condensed chromosome fibers or highly condensed chromosome bivalents can be seen in the nucleoplasm of BL-I- or cycloheximide-treated oocytes, respectively. In these treated oocytes, no or only very weak AURKB activity and phosphorylation of histone H3 on Ser-10 can be detected after 27 h of treatment, whereas phosphorylation on Ser-28 is not influenced. These results suggest that AURKB activity and Ser-10 phosphorylation of histone H3 are not required for chromosome condensation in pig oocytes, but might be required for further processing of chromosomes during meiosis.     

Proper chromatin condensation and maintenance of histone H3 phosphorylation during mouse oocyte meiosis requires protein phosphatase activity

We have shown okadaic acid (OA) and calyculin-A (CLA) inhibition of mouse oocyte phosphoprotein phosphatase 1 (PPP1C) and/or phosphoprotein phosphatase 2A (PPP2CA) results in aberrant chromatin condensation, as evidenced by the inability to resolve bivalents. Phosphorylation of histone H3 at specific residues is thought to regulate chromatin condensation. Therefore, we examined changes in histone H3 phosphorylation during oocyte meiosis and the potential regulation by protein PPPs. Western blot and immunocytochemical analysis revealed histone H3 phosphorylation changed during mouse oocyte meiosis, with changes in chromatin condensation. Germinal vesicle-intact (GV-intact; 0 h) oocytes had no phospho-Ser10 but did have phospho-Ser28 histone H3. Oocytes that had undergone germinal vesicle breakdown (GVBD; 2 h) and progressed to metaphase I (MI; 7 h) and MII (16 h) had phosphorylated Ser10 and Ser28 histone H3 associated with condensed chromatin. To determine whether OA-induced aberrations in chromatin condensation were due to alterations in levels of histone H3 phosphorylation, we assessed phosphorylation of Ser10 and Ser28 residues following PPP inhibition. Oocytes treated with OA (1 microM) displayed increased phosphorylation of histone H3 at both Ser10 and Ser28 compared with controls. To begin to elucidate which OA-sensitive PPP is responsible for regulating chromatin condensation and histone H3 phosphorylation, we examined spatial and temporal localization of OA-sensitive PPPs, PPP1C, and PPP2CA. PPPC2A did not localize to condensed chromatin, whereas PPP1beta (PPP1CB) associated with condensing chromatin in GVBD, MI, and MII oocytes. Additionally, Western blot and immunocytochemistry confirmed presence of the PPP1C regulatory inhibitor subunit 2 (PPP1R2) in oocytes at condensed chromatin during meiosis and indicated a change in PPP1R2 phosphorylation. Inhibition of oocyte glycogen synthase kinase 3 (GSK3) appeared to regulate phosphorylation of PPP1R2. Furthermore, inhibition of GSK3 resulted in aberrant oocyte bivalent formation similar to that observed following PPP inhibition. These data suggest that PPP1CB is the OA/CLA-sensitive PPP that regulates oocyte chromatin condensation through regulation of histone H3 phosphorylation. Furthermore, GSK3 inhibition results in aberrant chromatin condensation and appears to regulate phosphorylation of PPP1R2.     

Chromosome condensation in pig oocytes: lack of a requirement for either cdc2 kinase or MAP kinase activity

In this study, butyrolactone I (BL I), a potent and specific inhibitor of cyclin-dependent kinases (cdk), is shown to inhibit germinal vesicle breakdown (GVBD) in pig oocytes. Oocytes treated with 100 microM BL I were arrested in the germinal vesicle (GV)-stage and displayed low activity of cdc2 kinase and MAP kinase. Nevertheless, chromosome condensation occurred and highly condensed bivalents were seen within an intact GV after a 24-hr culture in the presence of BL I. The inhibitory effect of BL I on MAP kinase activation during culture was likely mediated through a cdk-dependent pathway, since MAP kinase activity present in extracts derived from metaphase II eggs was not inhibited by BL I. The block of GVBD could be released by treating oocytes with okadaic acid (OA), an inhibitor of type 1 and 2A phosphatases; 82% of the oocytes treated with the combination of OA/BL I underwent GVBD, and MAP kinase became activated, while cdc2 kinase remained inhibited. These results suggest that both chromosome condensation and GVBD could occur without activation of cdc2 kinase, whereas an increase in MAP kinase activity may be a requisite for GVBD in pig oocytes in conditions when cdc2 kinase activation is blocked by BL I.     

Regulation of mitogen-activated protein kinase phosphorylation, microtubule organization, chromatin behavior, and cell cycle progression by protein phosphatases during pig oocyte maturation and fertilization in vitro

We used okadaic acid (OA), a potent inhibitor of protein phosphatases 1 and 2A, to study the regulatory effects of protein phosphatases on mitogen-activated protein (MAP) kinase phosphorylation, morphological changes in the nucleus, and microtubule assembly during pig oocyte maturation and fertilization in vitro. When germinal vesicle (GV) stage oocytes were exposed to OA, MAP kinase phosphorylation was greatly accelerated, being fully activated at 10 min. However, MAP kinase was dephosphorylated by long-term (>20 h) exposure to OA. Correspondingly, premature chromosome condensation and GV breakdown were accelerated, whereas meiotic spindle assembly and meiotic progression beyond metaphase I stage were inhibited. OA also quickly reversed the inhibitory effects of butyrolactone I, a specific inhibitor of maturation-promoting factor (MPF), on MAP kinase phosphorylation and meiosis resumption. Treatment of metaphase II oocytes triggered metaphase II spindle elongation and disassembly as well as chromosome alignment disruption. OA treatment of fertilized eggs resulted in prompt phosphorylation of MAP kinase, disassembly of microtubules around the pronuclear area, chromatin condensation, and pronuclear membrane breakdown, but inhibited further cleavage. Our results suggest that inhibition of protein phosphatases promptly phosphorylates MAP kinase, induces premature chromosome condensation and meiosis resumption as well as pronucleus breakdown, but inhibits spindle organization and suppresses microtubule assembly by sperm centrosomes in pig oocytes and fertilized eggs.     

Core histone N-termini play an essential role in mitotic chromosome condensation

We have studied the role of core histone tails in the assembly of mitotic chromosomes using Xenopus egg extracts. Incubation of sperm nuclei in the extracts led to the formation of mitotic chromosomes, a process we found to be correlated with phosphorylation of the N-terminal tail of histone H3 at Ser10. When the extracts were supplemented with H1-depleted oligosomes, they were not able to assemble chromosomes. Selective elimination of oligosome histone tails by trypsin digestion resulted in a dramatic decrease in their ability to inhibit chromosome condensation. The chromosome assembly was also inhibited by each of the histone tails with differing efficiency. In addition, we found that nucleosomes were recruiting through the flexible histone tails some chromosome assembly factors, different from topoisomerase II and 13S condensin. These findings demonstrate that histone tails play an essential role in chromosome assembly. We also present evidence that the nucleosomes, through physical association, were able to deplete the extracts from the kinase phosphorylating histone H3 at Ser10, suggesting that this kinase could be important for chromosome condensation.     

The MAPK pathway triggers activation of Nek2 during chromosome condensation in mouse spermatocytes

Chromosome condensation during the G2/M progression of mouse pachytene spermatocytes induced by the phosphatase inhibitor okadaic acid (OA) requires the activation of the MAPK Erk1. In many cell systems, p90Rsks are the main effectors of Erk1/2 function. We have identified p90Rsk2 as the isoform that is specifically expressed in mouse spermatocytes and have shown that it is activated during the OA-triggered meiotic G2/M progression. By using the MEK inhibitor U0126, we have demonstrated that activation of p90Rsk2 during meiotic progression requires activation of the MAPK pathway. Immunofluorescence analysis indicates that activated Erks and p90Rsk2 are tightly associated with condensed chromosomes during the G2/M transition in meiotic cells. We also found that active p90Rsk2 was able to phosphorylate histone H3 at Ser10 in vitro, but that the activation of the Erk1/p90Rsk2 pathway was not necessary for phosphorylation of H3 in vivo. Furthermore, phosphorylation of H3 was not sufficient to cause condensation of meiotic chromosomes in mouse spermatocytes. Other proteins known to associate with chromatin may represent effectors of Erk1 and p90Rsk2 during chromosome condensation. Nek2 (NIMA-related kinase 2), which associates with chromosomes, plays an active role in chromatin condensation and is stimulated by treatment of pachytene spermatocytes with okadaic acid. We show that inhibition of the MAPK pathway by preincubation of spermatocytes with U0126 suppresses Nek2 activation, and that incubation of spermatocyte cell extracts with activated p90Rsk2 causes stimulation of Nek2 kinase activity. Furthermore, we show that the Nek2 kinase domain is a substrate for p90Rsk2 phosphorylation in vitro. These data establish a connection between the Erk1/p90Rsk2 pathway, Nek2 activation and chromosome condensation during the G2/M transition of the first meiotic prophase.     

Phosphorylation-induced rearrangement of the histone H3 NH2-terminal domain during mitotic chromosome condensation

The NH2-terminal domain (N-tail) of histone H3 has been implicated in chromatin compaction and its phosphorylation at Ser10 is tightly correlated with mitotic chromosome condensation. We have developed one mAb that specifically recognizes histone H3 N-tails phosphorylated at Ser10 (H3P Ab) and another that recognizes phosphorylated and unphosphorylated H3 N-tails equally well (H3 Ab). Immunocytochemistry with the H3P Ab shows that Ser10 phosphorylation begins in early prophase, peaks before metaphase, and decreases during anaphase and telophase. Unexpectedly, the H3 Ab shows stronger immunofluorescence in mitosis than interphase, indicating that the H3 N-tail is more accessible in condensed mitotic chromatin than in decondensed interphase chromatin. In vivo ultraviolet laser cross-linking indicates that the H3 N-tail is bound to DNA in interphase cells and that binding is reduced in mitotic cells. Treatment of mitotic cells with the protein kinase inhibitor staurosporine causes histone H3 dephosphorylation and chromosome decondensation. It also decreases the accessibility of the H3 N-tail to H3 Ab and increases the binding of the N-tail to DNA. These results indicate that a phosphorylation-dependent weakening of the association between the H3 N-tail and DNA plays a role in mitotic chromosome condensation.     

Aurora kinase is required for chromosome segregation in tobacco BY-2 cells

Post-translational modifications of core histone tails play crucial roles in chromatin structure and function. Although phosphorylation of Ser10 and Ser28 (H3S10ph and H3S28ph) of histone H3 is ubiquitous among eukaryotes, the phosphorylation mechanism during the cell cycle remains unclear. In the present study, H3S10ph and H3S28ph in tobacco BY-2 cells were observed in the pericentromeric regions during mitosis. Moreover, the Aurora kinase inhibitor Hesperadin inhibited the kinase activity of Arabidopsis thaliana Aurora kinase 3 (AtAUR3) in phosphorylating both Ser10 and Ser28 of histone H3 in vitro. Consistently, Hesperadin inhibited both H3S10ph and H3S28ph during mitosis in BY-2 cells. These results indicate that plant Aurora kinases phosphorylate not only Ser10, but also Ser28 of histone H3 in vivo. Hesperadin treatment increased the ratio of metaphase cells, while the ratio of anaphase/telophase cells decreased, although the mitotic index was not affected in Hesperadin-treated cells. These results suggest that Hesperadin induces delayed transition from metaphase to anaphase, and early exit from mitosis after chromosome segregation. In addition, micronuclei were observed frequently and lagging chromosomes, caused by the delay and failure of sister chromatid separation, were observed at anaphase and telophase in Hesperadin-treated BY-2 cells. The data obtained here suggest that plant Aurora kinases and H3S10ph/H3S28ph may have a role in chromosome segregation and metaphase/anaphase transition.     

Impaired maturation of the siderophore pyoverdine chromophore in Pseudomonas fluorescens ATCC 17400 deficient for the cytochrome c biogenesis protein CcmC

Here we show that during the meiotic maturation of Xenopus oocytes, histone H3 becomes phosphorylated on serine-10 at about the time of maturation promoting factor activation and meiosis I entry. However, overexpression of cAMP-dependent protein kinase that blocks entry into M phase, also leads to massive serine-10 phosphorylation of histone H3 in intact Xenopus oocytes but does not cause chromosome condensation. We also show that the phosphorylation of histone H3 during oocyte maturation requires the activation of the mitogen-activated protein kinase/p90Rsk pathway. Our results indicate that in G2-arrested oocytes, which are about to enter M phase, histone H3 phosphorylation is not sufficient for chromosome condensation.     

Analysis of the role of Aurora B on the chromosomal targeting of condensin I

During mitosis, chromosome condensation takes place, which entails the conversion of interphase chromatin into compacted mitotic chromosomes. Condensin I is a five-subunit protein complex that plays a central role in this process. Condensin I is targeted to chromosomes in a mitosis-specific manner, which is regulated by phosphorylation by mitotic kinases. Phosphorylation of histone H3at serine 10 (Ser10) occurs during mitosis and its physiological role is a longstanding question. We examined the function of Aurora B, a kinase that phosphorylates Ser10, in the chromosomal binding of condensin I and mitotic chromosome condensation, using an in vitro system derived from Xenopus egg extract. Aurora B depletion from a mitotic egg extract resulted in the loss of H3 phosphorylation, accompanied with a 50% reduction of chromosomal targeting of condensin I. Alternatively, a portion of condensin I was bound to sperm chromatin, and chromosome-like structures were assembled when okadaic acid (OA) was supplemented in an interphase extract that lacks Cdc2 activity. However, chromosomal targeting of condensin I was abolished when Aurora B was depleted from the OA-treated interphase extract. From these results, it is suggested that Aurora B-dependent and Cdc2-independent pathways of the chromosomal targeting of condensin I are present.     

Microtubule assembly after treatment of pig oocytes with taxol: correlation with chromosomes, gamma-tubulin, and MAP kinase.

In this study, taxol was used as a tool to study the correlation of microtubule assembly with chromosomes, gamma-tubulin and phosphorylated mitogen-activated protein (MAP) kinase in pig oocytes at different maturational stages. Taxol treatment did not affect meiotic resumption and chromosome condensation but inhibited/disrupted chromosome alignment at the metaphase plate and bipolar spindle formation and thus meiotic progression. Microtubules were co-localized with chromosomes and were found to emanate from the chromosomes in taxol-treated oocytes, suggesting that chromosomes may serve as a source of microtubule organization. In addition, the concentric emanation of microtubules within the chromosome-surrounded area in taxol-treated oocytes suggests that microtubule emanation from the chromosomes may be directed by other microtubule-organizing material. The formation of one large spindle or >/=2 spindles in oocytes after taxol removal shows that minus end microtubule-organizing material can be normally located on both sides of chromosomes only when the chromosomes are aligned on the metaphase plate. The co-localization of gamma-tubulin and phosphorylated MAP kinase with microtubule assembly in both control and taxol-treated oocytes suggests that these two proteins are associated microtubule-nucleating material in pig oocytes. However, Western blot analysis showed that neither cytoplasmic microtubule aster formation nor extensive microtubule assembly in the chromosome region induced by taxol was caused by super-activation of MAP kinase. Taxol also induced microtubule assembly depending on chromosome distribution in the first polar body. The results suggest that chromosomes are always co-localized with microtubules and that emanation of microtubules from the chromosomes may be regulated/directed by microtubule-organizing material including gamma-tubulin and phosphorylated MAP kinase in pig oocytes.     

Mitotic histone H3 phosphorylation by vaccinia-related kinase 1 in mammalian cells

Mitotic chromatin condensation is essential for cell division in eukaryotes. Posttranslational modification of the N-terminal tail of histone proteins, particularly by phosphorylation by mitotic histone kinases, may facilitate this process. In mammals, aurora B is believed to be the mitotic histone H3 Ser10 kinase; however, it is not sufficient to phosphorylate H3 Ser10 with aurora B alone. We show that histone H3 is phosphorylated by vaccinia-related kinase 1 (VRK1). Direct phosphorylation of Thr3 and Ser10 in H3 by VRK1 both in vitro and in vivo was observed. Loss of VRK1 activity was associated with a marked decrease in H3 phosphorylation during mitosis. Phosphorylation of Ser10 by VRK1 is similar to that by aurora B. Moreover, expression and chromatin localization of VRK1 depended on the cell cycle phase. Overexpression of VRK1 resulted in a dramatic condensation of nuclei. Our findings collectively support a role of VRK1 as a novel mitotic histone H3 kinase in mammals.     

Mitotic histone H3 phosphorylation by the NIMA kinase in Aspergillus nidulans

Phosphorylation of histone H3 serine 10 correlates with chromosome condensation and is required for normal chromosome segregation in Tetrahymena. This phosphorylation is dependent upon activation of the NIMA kinase in Aspergillus nidulans. NIMA expression also induces Ser-10 phosphorylation inappropriately in S phase-arrested cells and in the absence of NIMX(cdc2) activity. At mitosis, NIMA becomes enriched on chromatin and subsequently localizes to the mitotic spindle and spindle pole bodies. The chromatin-like localization of NIMA early in mitosis is tightly correlated with histone H3 phosphorylation. Finally, NIMA can phosphorylate histone H3 Ser-10 in vitro, suggesting that NIMA is a mitotic histone H3 kinase, perhaps helping to explain how NIMA promotes chromatin condensation in A. nidulans and when expressed in other eukaryotes.     

Positive regulation of retinoic acid receptor alpha by protein kinase C and mitogen-activated protein kinase in sertoli cells

Mitogen-activated protein kinase (MAPK) and protein phosphatase 2A (PP2A) regulate oocyte meiosis, yet little is known regarding their mechanisms of action. This study addressed the functional importance of active MAPK and PP2A in regulating oocyte meiosis. Experiments were conducted to identify MAPK activation, PP2A activity, intracellular enzyme trafficking, and ultrastructural associations during meiosis. Questions of requisite kinase and/or phosphatase activity and chromatin condensation, microtubule polymerization, and spindle formation were addressed. At the protein level, MAPK and PP2A were present in constant amounts throughout the first meiotic division. Both MAPK and PP2A were activated following germinal vesicle breakdown (GVBD) in conjunction with metaphase I development. Immunocytochemical studies confirmed the absence of active MAPK in germinal vesicle-intact (GVI) and GVBD oocytes. At metaphase I and during the metaphase I/metaphase II transition, activated MAPK colocalized with microtubules, poles, and plates of meiotic spindles. Protein phosphatase 2A was dispersed evenly throughout the GVI oocyte cytoplasm. Throughout the metaphase I/metaphase II transition, PP2A colocalized with microtubules of meiotic spindles. Both active MAPK and PP2A associated with in vitro-polymerized microtubules, suggesting that active MAPK and PP2A locally regulate spindle formation. Inhibition of MAPK activation resulted in compromised microtubule polymerization, no spindle formation, and loosely condensed chromosomes. Treatment with okadaic acid (OA) or calyculin-A (CL-A), which inhibits oocyte cytoplasmic PP2A, caused an absence of microtubule polymerization and spindles, even though MAPK activity was increased under these treatment conditions. Thus, active MAPK is required, but is not sufficient, for normal meiotic spindle formation and chromosome condensation. In addition, the oocyte OA/CL-A-sensitive PP, presumably PP2A, is essential for microtubule polymerization and meiotic spindle formation.     

Female infertility in PDE3A-/- mice: Polo-like kinase 1 (Plk1) may be a target of protein kinase A (PKA) and involved in meiotic arrest of oocytes from PDE3A-/- mice

Mechanisms of cAMP/PKA-induced meiotic arrest in oocytes are not completely identified. In cultured, G₂/M-arrested PDE3A^-/- murine oocytes, elevated PKA activity was associated with inactivation of Cdc2 and Plk1, and inhibition of phosphorylation of histone H3 (S10) and of dephosphorylation of Cdc25B (S323) and Cdc2 (Thr14/Tyr15). In cultured WT oocytes, PKA activity was transiently reduced and then increased to that observed in PDE3A^-/- oocytes; Cdc2 and Plk1 were activated, phosphorylation of histone H3 (S10) and dephosphorylation of Cdc25B (S323) and Cdc2 (Thr14/Tyr15) were observed. In WT oocytes, PKAc were rapidly translocated into nucleus, and then to the spindle apparatus, but in PDE3A^-/- oocytes, PKAc remained in the cytosol. Plk1 was reactivated by incubation of PDE3A^-/- oocytes with PKA inhibitor, Rp-cAMPS. PDE3A was co-localized with Plk1 in WT oocytes, and co-immunoprecipitated with Plk1 in WT ovary and Hela cells. PKAc phosphorylated rPlk1 and Hela cell Plk1 and inhibited Plk1 activity in vitro. Our results suggest that PKA-induced inhibition of Plk1 may be critical in oocyte meiotic arrest and female infertility in PDE3A^-/- mice.Key words: mice oocytes, PDE3A, cAMP, PKA, polo-like kinase 1, centrosome, prophase arrested     

Visualizing histone modifications in living cells: spatiotemporal dynamics of H3 phosphorylation during interphase

Posttranslational histone modifications regulate both gene expression and genome integrity. Despite the dynamic nature of these modifications, appropriate real-time monitoring systems are lacking. In this study, we developed a method to visualize histone modifications in living somatic cells and preimplantation embryos by loading fluorescently labeled specific Fab antibody fragments. The technique was used to study histone H3 Ser10 (H3S10) phosphorylation, which occurs during chromosome condensation in mitosis mediated by the aurora B kinase. In aneuploid cancer cells that frequently missegregate chromosomes, H3S10 is phosphorylated just before the chromosomes condense, whereas aurora B already accumulates in nuclei during S phase. In contrast, in nontransformed cells, phosphorylated H3S10 foci appear for a few hours during interphase, and transient exposure to an aurora B–selective inhibitor during this period induces chromosome missegregation. These results suggest that, during interphase, moderate aurora B activity or H3S10 phosphorylation is required for accurate chromosome segregation. Visualizing histone modifications in living cells will facilitate future epigenetic and cell regulation studies.     

Checkpoint kinase 1 regulates diallyl trisulfide-induced mitotic arrest in human prostate cancer cells

We have shown previously that diallyl trisulfide (DATS), a constituent of processed garlic, inhibits proliferation of PC-3 and DU145 human prostate cancer cells by causing G(2)-M phase cell cycle arrest in association with inhibition of cyclin-dependent kinase 1 activity and hyperphosphorylation of Cdc25C at Ser(216). Here, we report that DATS-treated PC-3 and DU145 cells are also arrested in mitosis as judged by microscopy following staining with anti-alpha-tubulin antibody and 4',6-diamidino-2-phenylindole and flow cytometric analysis of Ser(10) phosphorylation of histone H3. The DATS treatment caused activation of checkpoint kinase 1 and checkpoint kinase 2, which are intermediaries of DNA damage checkpoints and implicated in Ser(216) phosphorylation of Cdc25C. The diallyl trisulfide-induced Ser(216) phosphorylation of Cdc25C as well as mitotic arrest were significantly attenuated by knockdown of check-point kinase 1 protein in both PC-3 and DU145 cells. On the other hand, depletion of checkpoint kinase 2 protein did not have any appreciable effect on G(2) or M phase arrest or Cdc25C phosphorylation caused by diallyl trisulfide. The lack of a role of checkpoint kinase 2 in diallyl trisulfide-induced phosphorylation of Cdc25C or G(2)-M phase cell cycle arrest was confirmed using HCT-15 cells stably transfected with phosphorylation-deficient mutant (T68A mutant) of checkpoint kinase 2. In conclusion, the results of the present study suggest existence of a checkpoint kinase 1-dependent mechanism for diallyl trisulfide-induced mitotic arrest in human prostate cancer cells.     

Novel phosphorylation of histone H3 at threonine 11 that temporally correlates with condensation of mitotic and meiotic chromosomes in plant cells

A novel mitosis-specific phosphorylation site in histone H3 at threonine 11 has been described for mammalian cells. This modification is restricted to the centromeric region while phosphorylation at the classical H3 sites, Ser10 and Ser28 occurs along the entire chromosomal arms. Using phosphorylation state-specific antibodies we found that phosphorylation at threonine 11 occurs also in plant cells, during mitosis as well as meiosis. However, in contrast to animal cells, ph(Thr11)H3 was distributed along the entire length of condensed chromosomes, whereas H3 phosphorylated at Ser10 and Ser28 appeared to be restricted to centromeric/pericentromeric chromatin. Phosphorylation at Thr11 started in prophase and ended in telophase, it correlated with the condensation of mitotic and meiotic chromosomes and was independent of the distribution of late replicating heterochromatin and Giemsa-banding positive regions. Interestingly, treatment of cells with the phosphatase inhibitor cantharidin revealed a high level of Thr11 phosphorylation in interphase cells, in this case particularly in pericentromeric regions. These data show that histone modifications are highly dynamic. Moreover, animal and plant organisms may have evolved individual histone codes.