Histone H1 Kinase Research Articles

MSK2 and MSK1 mediate the mitogen- and stress-induced phosphorylation of histone H3 and HMG-14.

Cells respond to mitogenic or stress stimuli by the rapid induction of immediate-early (IE) genes, which occurs concomitantly with the phosphorylation of histone H3 and the high-mobility-group protein HMG-14. In mammalian cells this response is mediated via ERK and p38 MAP kinase pathways, but the identity of the downstream kinase that phosphorylates histone H3 has been contentious. One study, based on Coffin- Lowry cells defective in RSK2, reported that RSK2 was the histone H3 kinase, while a second study, based on the efficiency of RSKs and MSKs as in vitro histone H3 kinases, and their relative susceptibility to kinase inhibitors, suggested that MSKs were responsible. We show here that the histone H3 phosphorylation response is normal in Coffin-Lowry cells. Further more, we show that histone H3 and HMG-14 phosphorylation is severely reduced or abolished in mice lacking MSK1 and MSK2. We also show that, despite this, histone H3 acetylation is unimpaired in these cells and that IE genes can be induced, although at a reduced efficiency. We conclude that MSKs are the major kinases for histone H3 and HMG-14 in response to mitogenic and stress stimuli in fibroblasts.

Differential effects of low-dose docosahexaenoic acid and eicosapentaenoic acid on the regulation of mitogenic signaling pathways in mesangial cells

Although dietary fish oil supplementation has been used to prevent the progression of kidney disease in patients with IgA nephropathy, relatively few studies provide a mechanistic rationale for its use. Using an antithymocyte (ATS) model of mesangial proliferative glomerulonephritis, we recently demonstrated that fish oil inhibits mesangial cell (MC) activation and proliferation, reduces proteinuria, and decreases histologic evidence of glomerular damage. We therefore sought to define potential mechanisms underlying the antiproliferative effect of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the predominant ω-3 polyunsaturated fatty acids found in fish oil, in cultured MC. DHA and EPA were administered to MC as bovine serum albumin fatty-acid complexes. Low-dose (10-50 μmol/L) DHA, but not EPA, inhibited basal and epidermal growth factor (EGF)–stimulated [ 3H]-thymidine incorporation in MCs. At higher doses (100 μmol/L), EPA and DHA were equally effective in suppressing basal and EGF-stimulated MC mitogenesis. Low-dose DHA, but not EPA, decreased ERK activation by 30% ( P < .01), as assessed with Western-blot analysis using phosphospecific antibodies. JNK activity was increased by low-dose DHA but not by EPA. p38 activity was not significantly altered by DHA or EPA. Cyclin E activity, as assessed with a histone H1 kinase assay, was inhibited by low-dose DHA but not by EPA. DHA increased expression of the cell cycle inhibitor p21 but not p27; EPA had no effect on p21 or p27. We propose that the differential effect of low-dose DHA vs EPA in suppressing MC mitogenesis is related to down-regulation of ERK and cyclin E activity and to induction of p21.

CDK2 is involved in the S-phase lengthening induced by glucocorticoids in normal human lymphocytes

Involvement of CDK2 in glucocorticoid-mediated S-phase lengthening was analyzed in this work. Dexamethasone (DXM) treatment of PHA-stimulated lymphocytes induced a decrease in CDK2 mRNA expression without any change in mRNA stability. This glucocorticoid-induced decrease in CDK2 mRNA expression could be suppressed by cycloheximide treatment. These results support the hypothesis of an indirect effect of DXM at the transcriptional level. Furthermore, CDK2 protein expression also decreased while the rate of protein synthesis and stability remained unchanged, suggesting a second post-translational level of regulation with the preferential degradation of a CDK2 protein stock fraction. The analysis of co-precipitated proteins showed that glucocorticoids induced modifications of protein complex composition. We found i) a preservation of the linkage capability of CDK2 for cyclin E and A, ii) a relative increase in p27kip1 linkage, and iii) a decrease in p21waf1 complexed with CDK2. As a consequence of CDK2 decrease and modifications of protein complex composition, pRb and histone H1 kinase activity of CDK2 was profoundly decreased. All these results pinpoint the role of CDK2 in glucocorticoid-induced S-phase lengthening and the potential activator role of p21waf1 for CDK2 in human lymphocytes.

The alga Chlamydomonas reinhardtii UVS11 gene is responsible for cell division delay and temporal decrease in histone H1 kinase activity caused by UV irradiation

The aim of the present work was to study the possible role of the UVS11 gene of the alga Chlamydomonas reinhardtii, in regulation of the cell cycle. To characterize the defect of a uvs11 mutant in respect to DNA damage-dependent cell cycle arrest, we examined first the influence of the tubulin-destabilizing drug methyl benzimidazole-2-yl-carbamate (MBC) on inhibition of mitosis in response to UV 254 nm. Then the growth and reproductive processes and activity of cyclin-dependent kinases (CDK)-like kinases during the cell cycle of C. reinhardtii were investigated. In both, the wild type and the uvs11 mutant strain were compared under standard conditions and after DNA damage caused by UV 254 nm. We assume the green alga C. reinhardtii possesses control mechanisms allowing to stop the cell cycle progression before mitosis in response to DNA damage. The results indicate that the uvs11 mutant is not able to stop the cell cycle after UV irradiation. We suggest that a product of the UVS11 gene affects cell response to DNA damage and influences a decrease in histone H1 kinase activity.

Monoclonal antibody against dnmt1 arrests the cell division of xenopus early-stage embryos

DNA methylation plays a crucial role in embryogenesis, and Dnmt1 is known to be a key enzyme in the maintenance of DNA methylation. Dnmt1 is highly accumulated in mature oocytes and eggs. To analyze the function of the maternally accumulated Dnmt1, we injected monoclonal antibodies that specifically recognize the amino terminus of Xenopus Dnmt1 into Xenopus laevis embryos. The monoclonal antibodies inhibited the cell division of the embryos before the midblastula transition. Monoclonal antibody neither inhibited DNA methylation activity of Dnmt1 in vitro nor affected its stability in embryos. In addition, injection of α-amanitin, an inhibitor of transcription, did not rescue the cell division arrest. The results suggest that the inhibition of cell division by monoclonal antibodies was due neither to the direct inhibition of DNA methylation activity of Dnmt1 nor to aberrant transcription before the midblastula transition. The morphology of chromatin of the arrested cells showed that the cell cycle was arrested at interphase. This was supported by the biochemical analysis in which the arrested cells demonstrated low histone H1 kinase activity, which indicated that the cells had not entered M phase. Dnmt1 may have an important function other than DNA methylation activity for early embryogenesis in Xenopus laevis.

Sip2, an N-Myristoylated β Subunit of Snf1 Kinase, Regulates Aging in Saccharomyces cerevisiae by Affecting Cellular Histone Kinase Activity, Recombination at rDNA Loci, and Silencing

Saccharomyces cerevisiae has evolved a number of mechanisms for sensing glucose. In the present study we examine the mechanism by which one of these pathways, involving Snf1, regulates cellular aging. Snf1 is a heterotrimer composed of a catalytic alpha subunit (Snf1p) that phosphorylates target proteins at Ser/Thr residues, an activating gamma subunit (Snf4p), and a beta subunit (Sip1p, Sip2p, or Gal83). We previously showed that forced expression of Snf1p or loss of Sip2p, but not the other beta subunits, causes accelerated aging, while removal of Snf4p extends life span (Ashrafi, K., Lin, S. S., Manchester, J. K., and Gordon, J. I. (2000) Genes Dev. 14, 1872-1885). We now demonstrate that in wild type cells, there is an age-associated shift in Sip2p from the plasma membrane to the cytoplasm, a prominent redistribution of Snf4p from the plasma membrane to the nucleus, a modest increase in nuclear Snf1p, and a concomitant increase in cellular Snf1 histone H3 kinase activity. Covalent attachment of myristate to the N-terminal Gly of Sip2p is essential for normal cellular life span. When plasma membrane association of Sip2p is abolished by a mutation that blocks its N-myristoylation, Snf4p is shifted to the nucleus. Rapidly aging sip2 Delta cells have higher levels of histone H3 kinase activity than their generation-matched isogenic wild type counterparts. Increased Snf1 activity is associated with augmented recombination at rDNA loci, plus desilencing at sites affected by Snf1-catalyzed Ser(10) phosphorylation of histone H3 (the INO1 promoter plus targets of the transcription factor Adr1p). The rapid-aging phenotype of sip2 Delta cells is fully rescued by blocking recombination at rDNA loci with a fob1 Delta allele; rescue is not accompanied by amelioration of an age-associated shift toward gluconeogenesis and glucose storage. Together, these findings suggest that Sip2p acts as a negative regulator of nuclear Snf1 activity in young cells by sequestering its activating gamma subunit at the plasma membrane and that loss of Sip2p from the plasma membrane to the cytoplasm in aging cells facilities Snf4p entry into the nucleus so that Snf1 can modify chromatin structure.

The effect of PD98059 on MAPK regulation in cumulus-enclosed and cumulus-free mouse oocytes.

The effect of the p42/44 mitogen-activated kinase (MAPK) inhibitor, PD98059, on MAPK activation and meiosis resumption in mouse oocytes was studied. When germinal vesicle (GV)-stage denuded oocytes (DOs) were cultured continuously in 50 microM PD98059, germinal vesicle breakdown (GVBD) was postponed for 2-3 h. MAPK phosphorylation and activation was delayed as well. However, PD98059 did not impair histone H1 kinase activation. After 14 h of culture there was no significant difference in the rate of DOs reaching metaphase II (MII) arrest in either control or experimental conditions. The effect of PD98059 on MAPK inhibition was further tested in epidermal growth factor (EGF)-treated oocytecumulus complexes (OCCs). Exposure of GV-stage OCCs for 5 min to EGF (10 ng/ml) induced a considerable increase in MAPK phosphorylation. After OCCs were further cultured in 50 microM PD98059 a rapid dephosphorylation of MAPK was induced. Already after 1 min of treatment the non-phosphorylated form of MAPK dominated, indicating the high effectivity of PD98059. This result indicates that short EGF/PD98059 treatment of OCCs induced MAPK phosphorylation/dephosphorylation in cumulus cells only. As only a transient delay in MAPK phosphorylation and activation was observed in PD98059-treated DOs we conclude that there is also another PD98059-nonsensitive pathway(s) leading to MAPK activation in mouse oocytes. The data obtained suggest that meiosis resumption in mouse oocytes is somehow influenced by the MEK/MAPK activation pathway.

Complex role of histone H1 in transactivation of MMTV promoter chromatin by progesterone receptor

Transcription from the mouse mammary tumor virus (MMTV) promoter can be induced by glucocorticoids or progestins. The corresponding receptors bind to a cluster of hormone responsive elements (HREs) and activate the promoter by synergistic interactions with ubiquitous transcription factors, in particular nuclear factor 1 (NF1). Synergism between hormone receptors and NF1 depends on the precise positioning of the promoter sequences on the surface of a histone octamer in chromatin, but how linker histones participate in the process is unclear. Asymmetric binding of histone H1 to chromatin organized MMTV promoter sequences compacts the nucleosomal structure and leads to repression of basal transcription and to reduced binding of NF1. In contrast, H1 containing MMTV chromatin binds PR with higher affinity and is transcribed more efficiently in the presence of PR and NF1 than chromatin free of linker histone. Thus histone H1 represses hormone independent transcription and enhances the synergism between PR and NF1 resulting in tighter hormonal regulation. This positive effect of H1 is likely due to a better defined nucleosome positioning over the MMTV promoter. Upon binding of PR to the promoter chromatin a hitherto unidentified kinase is recruited or activated that phosphorylates H1. This is not sufficient for transcriptional activation but is likely a requisite for the action of ATP-dependent chromatin remodelling complexes. Following remodelling and in the presence of NF1, which maintains the open nucleosome conformation, additional PR molecules bind, transactivation takes place and H1 is displaced from the promoter during transcription initiation. Therefore, H1 plays a key role during the initial hormonal activation of the MMTV promoter in native chromatin, which includes recruitment by PR of a histone H1 kinase and an ATP-dependent chromatin remodelling complex, followed by NF1 binding, increased PR binding, transcription initiation and H1 displacement.

The effect of activation of Mammalian oocytes on remodeling of donor nuclei after nuclear transfer.

Activation of bovine oocytes by experimental procedures that closely mimic normal fertilization is essential both for intracytoplasmic sperm injection and for nuclear transfer (NT). Therefore, with the goal of producing haploid activated oocytes, we evaluated whether butyrolactone I and bohemine, either alone or in combination with ionomycin, are able to activate young matured mammalian oocytes. Furthermore, the effect on the patterns of DNA synthesis after pronuclear formation as well as changes in histone H1 kinase and MAP kinase activities during the process of activation were studied. Our results with bohemine show that the specific inhibition of cyclin-dependent kinases (CDKs) in metaphase II bovine oocytes induces parthenogenetic activation in a dose dependent manner (25, 50, and 100 microM, respectively), either alone (3%, 30%, and 50%) or in combination with ionomycin (30%, 70%, and 87.5%). The effect of two activation protocols on nuclear remodeling, DNA synthesis during the first cell cycle, chromosome segregation after first mitosis, and development to blastocyst of embryos produced by somatic nuclear transfer were studied. Pronuclear formation was significantly higher when activation lasted 5 h compared to 3 h for both ethanol-cycloheximide and ionomycin-bohemine treatment. Initiation of DNA synthesis was delayed in ethanol-cycloheximide group, however, after 12-h labeling 100% of embryos synthesized DNA in both groups. Analysis of two-cell embryos with DNA probes for chromosome 6, 7, and 15 by fluorescence in situ hybridization showed that at least 50% of NT embryos were of normal ploidy, independent of the activation protocol.

Interaction of p58PITSLRE, a G2/M-specific Protein Kinase, with Cyclin D3

The p58(PITSLRE) is a p34(cdc2)-related protein kinase that plays an important role in normal cell cycle progression. Elevated expression of p58(PITSLRE) in eukaryotic cells prevents them from undergoing normal cytokinesis and appears to delay them in late telophase. To investigate the molecular mechanism of p58(PITSLRE) action, we used the yeast two-hybrid system, screened a human fetal liver cDNA library, and identified cyclin D3 as an interacting partner of p58(PITSLRE). In vitro binding assay, in vivo coimmunoprecipitation, and immunofluorescence cell staining further confirmed the association of p58(PITSLRE) with cyclin D3. This binding was observed only in the G(2)/M phase but not in the G(1)/S phase of the cell cycle; meanwhile, no interaction between p110(PITSLRE) and cyclin D3 was observed in all the cell cycle. The overexpression of cyclin D3 in 7721 cells leads to an exclusively accumulation of p58(PITSLRE) in the nuclear region, affecting its cellular distribution. Histone H1 kinase activity of p58(PITSLRE) was greatly enhanced upon interaction with cyclin D3. Furthermore, kinase activity of p58(PITSLRE) was found to increase greatly in the presence of cyclin D3 using a specific substrate, beta-1,4-galactosyltransferase 1. These data provide a new clue to our understanding of the cellular function of p58(PITSLRE) and cyclin D3.

Studies on fertilization in the teleost IV. Effects of aphidicolin and camptothecin on chromosome formation in fertilized medaka eggs.

To clarify the mechanisms of fish fertilization, the effects of inhibitors of DNA polymerase-alpha and DNA topoisomerases on nuclear behavior before and after fertilization were examined in eggs of the medaka, Oryzias latipes. Eggs underwent the fertilization process from sperm penetration to karyogamy of pronuclei, even when inseminated and incubated in the continuous presence of aphidicolin (DNA polymerase alpha inhibitor), camptothecin (DNA topoisomerase I inhibitor), etoposide, or beta-lapachone (DNA topoisomerase II inhibitor). However, continuous treatment with aphidicolin or camptothecin during fertilization inhibited the formation of sister chromosomes that were normally separated into blastomeres at the time of the subsequent cleavage. Sister chromosome formation appeared concomitantly with an increase in histone H1 kinase activity at the end of DNA synthesis, 30 min post insemination. However, non-activated eggs that were inseminated in saline containing anesthetic MS222 and aphidicolin had high levels of histone H1 kinase and MAP kinase activities, and transformation of the penetrated sperm nucleus to metaphase chromosomes occurred even in the presence of aphidicolin or camptothecin. The male chromosomes were normally separated into two anaphase chromosome masses upon egg activation. These results suggest that DNA polymerase alpha or DNA topoisomerase I, but not DNA topoisomerase II, may be required for the process by which the mitotic interphase nucleus transforms to separable metaphase chromosomes while the activity of MAP kinase is low, unlike the situation in meiotic division, during which MAP kinase activity is high and DNA replication is not required.

Acute signaling by the LH receptor is independent of protein kinase C activation.

LH receptor activation leads to the phosphorylation/activation of p42/44 MAPK in preovulatory granulosa cells. As the LH receptor can activate both adenylyl cyclase and phospholipase C, we hypothesized that the LH receptor could elicit phosphorylation of p42/44 MAPK through activation of protein kinase A (PKA) and/or protein kinase C (PKC). Preovulatory granulosa cells in serum-free primary cultures were treated with ovulatory concentrations of human chorionic gonadotropin (hCG), an LH receptor agonist, with or without various inhibitors. The PKA inhibitor H89 as well as the myristoylated PKA inhibitor peptide PKI strongly inhibited hCG-stimulated p42/44 MAPK phosphorylation, whereas the PKC inhibitor GF109203X had no effect on p42/44 MAPK phosphorylation. LH receptor-stimulated phosphorylation of cAMP response element-binding protein (CREB), histone H3, and MAPK kinase (MEK) was also strongly inhibited by H89 and not by GF109203X. The extent of PKC activation was assessed in preovulatory granulosa cells using three criteria: translocation of PKC isoforms to the membrane fraction, phosphorylation of a known PKC substrate, and autophosphorylation of PKC delta on an activation-related site. By all three criteria PKCs were partially activated before hCG stimulation, and hCG treatment failed to elicit further PKC activation, in vitro or in vivo. Taken together, these results indicate that, under primary culture conditions where physiological levels of signaling proteins are present, hCG signals to activate MEK, p42/44 MAPK, CREB, and histone H3 in a predominantly PKA-dependent and PKC-independent manner. Unexpectedly, PKCs were partially activated in the absence of LH receptor activation, and LH receptor activation did not elicit further detectable PKC activation.

Anti-Epidermal Growth Factor Receptor Monoclonal Antibody 225 Upregulates p27KIP1 and p15INK4B and Induces G1 Arrest in Oral Squamous Carcinoma Cell Lines

Epidermal growth factor receptor (EGFR) regulates the growth and progression of human oral squamous cell carcinoma (SCC). Recently, the link between EGFR signaling and the cell cycle has been identified. Some reports have described that EGFR-blocking monoclonal antibody 225 (mAb225) induced G1 arrest and inhibited the growth of various cancer cells. The purpose of this study was to evaluate the effect of mAb225 on human oral SCC cell lines. Exposure to mAb225 in culture inhibited the growth of oral SCC cell lines in an EGFR number-independent manner, with the percent inhibition ranging from 13.8 to 76.6%. Flow-cytometric analysis demonstrated that treatment with mAb225 induced cell accumulation in G1 phase, accompanied by a decrease in the percentage of cells in the S phase. Apoptosis was not seen in this study. G1 arrest was accompanied by a decrease in CDK2-, CDK4-, and CDK6-associated histone H1 kinase activities, and an increase in the expression levels of cell cycle inhibitors p27^KIP1 and p15^INK4B. These results suggested that the antiproliferative effect of EGFR blockade by mAb225 in oral SCC may be mediated by p27^KIP1 and p15^INK4B.

Phosphorylation of retinoblastoma-related protein by the cyclin D/cyclin-dependent kinase complex is activated at the G1/S-phase transition in tobacco.

In mammals, D-type cyclin-associated kinases mainly regulate the G1/S transition by phosphorylating the retinoblastoma (Rb) protein. We previously demonstrated that in tobacco, cyclin D (Nicta; CycD3;3) is complexed with the PSTAIRE-containing cyclin-dependent kinase (CDKA) from tobacco. Here, we show that Nicta; CycD3;3-associated kinases phosphorylate both the tobacco Rb-related protein (NtRb1) and histone H1. Although NtRb1 kinase activity was detected only during the middle G1- to early S-phase, histone H1 kinase activity was observed as two peaks in G1- to S-phase and G2/M- to M-phase. Importantly, we show that the proportion of cells in the G1-phase was reduced in transgenic Bright Yellow-2 cells overexpressing Nicta; CycD3;3-GFP. Mutational analyses revealed that phosphorylation of Thr-191 in Nicta; CycD3;3 possibly is required for both full kinase activity and localization predominantly to the nucleus. These data suggest that Nicta; CycD3;3 acts as a rate-limiting regulator in the G1/S transition by forming active complexes with CDKA or its related kinases to phosphorylate Rb-related protein and potentially plays a novel role during G2/M and mitosis.

A cyclin-dependent protein kinase homologue associated with the basal body domains in the ciliate Tetrahymena thermophila

The tight coupling between cell cycle progression and morphogenetic development in the unicellular ciliates presents a unique model system for examination of the roles of Cdks in developmental processes. We here describe the isolation and characterization of the first cyclin-dependent kinase (Cdk) homologue, TtCdk1, from Tetrahymena thermophila. TtCdk1 corresponds to the larger of the two polypeptides recognized by anti-PSTAIRE antibody in a whole cell lysate, which differ from each other in their affinity for yeast p13 suc1 protein. In contrast to the constant protein expression levels of typical eukaryotic Cdks, the TtCdk1 protein level fluctuates periodically over the vegetative cell cycle, reaching a maximum at the end of the cell cycle, correlating with its histone H1 kinase activity. Its association with the membrane-skeletal domains that surround mature, but not nascent, basal bodies in the cell cortex suggests that TtCdk1 plays a role in the regulation of cortical morphogenesis in T. thermophila. A partial TtCDK1 knockout cell line constructed through somatic biolistic transformation resulted in a reduction of the regularity of the rows of basal bodies plus an additional effect on chromatin condensation in both macro- and micronuclei. Unlike the situations in higher eukaryotic cells, no apparent effect on basal body duplication was found upon disruption of the TtCDK1 gene.

Effector mechanisms of norcantharidin-induced mitotic arrest and apoptosis in human hepatoma cells.

NCTD is a demethylated form of cantharidin with antitumor properties, which is now in use as a routine anticancer drug against hepatoma. However, there is limited information on the effect of NCTD on human cancer cells. In the present study, NCTD inhibited proliferation, caused mitotic arrest, then progressed to apoptosis within 96 hr in 3 human hepatoma cell lines: HepG2, Hep3B and Huh-7. NCTD treatment (5 microg/ml) enhanced the expression of Cdc25C and p21(Cip1/Waf1), increasing the phosphorylation of these 2 proteins. In addition, NCTD treatment induced an earlier increase in cyclin B1-associated histone H1 kinase activity within 48 hr, but an approximately 70% reduction of both protein level and kinase activity of cyclin B1 was observed at 72 hr. Treatment with NCTD significantly decreased the expression of p53 protein but did not affect the expression of Cdk1 and p27(Kip1). Moreover, NCTD treatment also increased the phosphorylation of Bcl-2 and Bcl-X(L) but did not affect the expression of Bax or Bad. Bcl-2 phosphorylation appears to inhibit its binding to Bax since less Bax was detected in immunocomplex with Bcl-2 in NCTD-treated HepG2 cells. In addition, NCTD treatment caused activation of caspase-9 and caspase-3, preceding DNA fragmentation and morphologic features of apoptosis. Pretreatment with the broad-spectrum caspase inhibitor z-VAD-fmk markedly inhibited NCTD-induced caspase-3 activity and cell death. These results suggest that phosphorylation of p21(Cip1/Waf1) and Cdc25C and biphasic regulation of cyclin B1-associated kinase activity may contribute to NCTD-induced M-phase cell-cycle arrest. Furthermore, the increase of p21(Cip1/Waf1), phosphorylation of Bcl-2 and Bcl-X(L), activation of caspase-9 and caspase-3 may be the molecular mechanism through which NCTD induces apoptosis.

Effect of 6‐dimethylaminopurine on electrically activated in vitro matured porcine oocytes

The effect of the protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), on the maturation promoting factor (MPF) activity, pronuclear formation, and parthenogenetic development of electrically activated in vitro matured (IVM) porcine oocytes was investigated. Oocytes were activated by exposure to two DC pulses, each of 1.5 kV/cm field strength and 60 microsec duration, applied 1 sec apart. In the first experiment, subsequent incubation with 2 or 5 mM 6-DMAP for 3 hr increased the incidence of blastocyst formation compared with no treatment, whereas incubation with 2 or 5 mM 6-DMAP for 5 hr did not. In the proceeding experiments, oocytes exposed to 6-DMAP were incubated with 2 mM of the reagent for 3 hr. Assaying histone H1 kinase activity in the second experiment revealed that the levels of active MPF in electrically activated oocytes treated with 6-DMAP were depleted more rapidly and remained depleted for longer compared with electrical activation alone. The kinetics of MPF activity following 6-DMAP treatment were similar to that found in inseminated oocytes in the third experiment. The effect of 6-DMAP was correlated with an increased incidence of parthenogenetic blastocyst formation. A fourth experiment was undertaken to examine the diploidizing effect of 6-DMAP. Electrically activated oocytes treated with 6-DMAP and cytochalasin B, either alone or in combination, displayed a higher incidence of second polar body retention compared with those that were untreated or treated with cycloheximide alone. After 6 days of culture in vitro, parthenotes exposed to 6-DMAP, either alone or in combination with cytochalasin B, formed blastocysts at a greater rate compared with those exposed to cytochalasin B alone, cycloheximide alone or no treatment. The combined 6-DMAP and cytochalasin B treatment induced the highest rate of blastocyst formation (47%), but the numbers of trophectoderm and total cells in these blastocysts were lower compared with those obtained following exposure to 6-DMAP alone. These results suggest that the increased developmental potential of 6-DMAP-treated parthenotes may be attributable to the MPF-inactivating effect of 6-DMAP, rather than the diploidizing effect of 6-DMAP.

Cell cycle-dependent dynamic association of cyclin/Cdk complexes with human DNA replication proteins.

We have previously described the isolation of a replication competent (RC) complex from calf thymus, containing DNA polymerase alpha, DNA polymerase delta and replication factor C. Here, we describe the isolation of the RC complex from nuclear extracts of synchronized HeLa cells, which contains DNA replication proteins associated with cell-cycle regulation factors like cyclin A, cyclin B1, Cdk2 and Cdk1. In addition, it contains a kinase activity and DNA polymerase activities able to switch from a distributive to a processive mode of DNA synthesis, which is dependent on proliferating cell nuclear antigen. In vivo cross-linking of proteins to DNA in synchronized HeLa cells demonstrates the association of this complex to chromatin. We show a dynamic association of cyclins/Cdks with the RC complex during the cell cycle. Indeed, cyclin A and Cdk2 associated with the complex in S phase, and cyclin B1 and Cdk1 were present exclusively in G(2)/M phase, suggesting that the activity, as well the localization, of the RC complex might be regulated by specific cyclin/Cdk complexes.

Heterologous expression and catalytic properties of the C-terminal domain of starfish cdc25 dual-specificity phosphatase, a cell cycle regulator.

The 3'-terminal region of starfish Asterina pectinifera cdc25 cDNA encoding the C-terminal catalytic domain was overexpressed in Escherichia coli. The C-terminal domain consisted of 226 amino acid residues containing the signature motif HCxxxxxR, a motif highly conserved among protein tyrosine and dual-specificity phosphatases, and showed phosphatase activity toward p-nitrophenyl phosphate. The enzyme activity was strongly inhibited by SH inhibitors. Mutational studies indicated that the cysteine and arginine residues in the conserved motif are essential for activity, but the histidine residue is not. These results suggest that the enzyme catalyzes the reaction through a two-step mechanism involving a phosphocysteine intermediate like in the cases of other protein tyrosine and dual-specificity phosphatases. The C-terminal domain of Cdc25 activated the histone H1 kinase activity of the purified, inactive form of Cdc2.cyclin B complex (preMPF) from extracts of immature starfish oocytes. Synthetic diphosphorylated di- to nonadecapeptides mimicking amino acid sequences around the dephosphorylation site of Cdc2 still retained substrate activity. Phosphotyrosine and phosphothreonine underwent dephosphorylation in this order. This is the reverse order to that reported for the in vivo and in vitro dephosphorylation of preMPF. Monophosphopeptides having the same sequence served as much poorer substrates. As judged from the results with synthetic phosphopeptides, the presence of two phosphorylated residues was important for specific recognition of substrates by the Cdc25 phosphatase.

Identification and characterization of Nek6 protein kinase, a potential human homolog of NIMA histone H3 kinase

In Aspergillus nidulans, the kinase activity of NIMA (never in mitosis, gene A) is critical for the initiation of mitosis. NIMA regulates mitotic chromatin condensation through phosphorylation of histone H3 at serine 10. In the present study, we identified human Nek6 (hNek6), a member of the mammalian NIMA-related kinases. The predicted hNek6 protein is comprised of 338 amino acids. Northern blot analysis revealed that hNek6 transcripts are ubiquitously expressed with the highest expression found in the heart and skeletal muscle. Lower cell cycle-dependent expression of hNek6 transcripts was observed in the early G1 phase. GFP-fused hNek6 protein showed both nuclear and cytoplasmic localizations in HeLa cells. Fluorescence in situ hybridization using full-length hNek6 cDNA as a probe showed that the hNek6 gene is localized to human chromosome 9q33–34, a region at which the loss of heterozygosity is associated with transitional cell carcinomas. Importantly, recombinant hNek6 protein produced in insect cells effectively phosphorylated histones H1 and H3, but not casein. Thus, these results suggest that, unlike other mammalian NIMA-related kinases, Nek6 is a mitotic histone kinase which regulates chromatin condensation in mammalian cells.