A cdc2-like kinase phosphorylates histone H1 in the amitotic macronucleus of Tetrahymena.

Genetic and biochemical studies have shown that cdc2 protein kinase plays a pivotal role in a highly conserved mechanism controlling the entry of cells into mitosis. It is generally believed that one function of cdc2 kinase is to phosphorylate histone H1 which in turn promotes mitotic chromosome condensation. However, direct evidence linking H1 phosphorylation to mitotic chromatin condensation is limited and the exact cellular function(s) of H1 phosphorylation remains unclear. In this study, we show that mammalian cdc2 kinase phosphorylates H1 from the amitotic macronucleus of Tetrahymena with remarkable fidelity. Furthermore, we demonstrate that macronuclei from Tetrahymena contain a growth-associated H1 kinase activity which closely resembles cdc2 kinase from other eukaryotes. Using polyclonal antibodies raised against yeast p34cdc2, we have detected a 36 kd immunoactive polypeptide in macronuclei which binds to Suc1 (p13)-coated beads and closely follows H1 kinase activity. Since macronuclei divide without mitotic chromosome condensation, these data demonstrate that H1 phosphorylation by cdc2 kinase may be necessary, but is not sufficient to promote mitotic chromatin condensation. The fact that an activity which strongly resembles mammalian cdc2 kinase is active during cell growth in a nucleus which does not undergo mitosis and chromosome condensation suggests that other factors are needed for a true mitotic division to occur. These data also reinforce the notion that H1 phosphorylation has important functions outside mitosis both in Tetrahymena and in mammalian cells.     

Chromosome condensation induced by fostriecin does not require p34cdc2 kinase activity and histone H1 hyperphosphorylation, but is associated with enhanced histone H2A and H3 phosphorylation.

Chromosome condensation at mitosis correlates with the activation of p34cdc2 kinase, the hyperphosphorylation of histone H1 and the phosphorylation of histone H3. Chromosome condensation can also be induced by treating interphase cells with the protein phosphatase 1 and 2A inhibitors okadaic acid and fostriecin. Mouse mammary tumour FT210 cells grow normally at 32 degrees C, but at 39 degrees C they lose p34cdc2 kinase activity and arrest in G2 because of a temperature-sensitive lesion in the cdc2 gene. The treatment of these G2-arrested FT210 cells with fostriecin or okadaic acid resulted in full chromosome condensation in the absence of p34cdc2 kinase activity or histone H1 hyperphosphorylation. However, phosphorylation of histones H2A and H3 was strongly stimulated, partly through inhibition of histone H2A and H3 phosphatases, and cyclins A and B were degraded. The cells were unable to complete mitosis and divide. In the presence of the protein kinase inhibitor starosporine, the addition of fostriecin did not induce histone phosphorylation and chromosome condensation. The results show that chromosome condensation can take place without either the histone H1 hyperphosphorylation or the p34cdc2 kinase activity normally associated with mitosis, although it requires a staurosporine-sensitive protein kinase activity. The results further suggest that protein phosphatases 1 and 2A may be important in regulating chromosome condensation by restricting the level of histone phosphorylation during interphase, thereby preventing premature chromosome condensation.     

High-mobility-group proteins P1, I and Y as substrates of the M-phase-specific p34cdc2/cyclincdc13 kinase

All dividing cells entering the M phase of the cell cycle undergo the transient activation of an M-phase-specific histone H1 kinase which was recently shown to be constituted of at least two subunits, p34cdc2 and cyclincdc13. The DNA-binding high-mobility-group (HMG) proteins 1, 2, 14, 17, I, Y and an HMG-like protein, P1, were investigated as potential substrates of H1 kinase. Among these HMG proteins, P1 and HMG I and Y are excellent substrates of the M-phase-specific kinase obtained from both meiotic starfish oocytes and mitotic sea urchin eggs. Anticyclin immunoprecipitates, extracts purified on specific p34cdc2-binding p13suc1-Sepharose and affinity-purified H1 kinase display strong HMG I, Y and P1 phosphorylating activities, demonstrating that the p34cdc2/cyclincdc13 complex is the active kinase phosphorylating these HMG proteins. HMG I and P1 phosphorylation is competitively inhibited by a peptide mimicking the consensus phosphorylation sequence of H1 kinase. HMG I, Y and P1 all possess the consensus sequence for phosphorylation by the p34cdc2/cyclincdc13 kinase (Ser/Thr-Pro-Xaa-Lys/Arg). HMG I is phosphorylated in vivo at M phase on the same sites phosphorylated in vitro by H1 kinase. P1 is phosphorylated by H1 kinase on sites different from the sites of phosphorylation by casein kinase II. The three thermolytic phosphopeptides of P1 phosphorylated in vitro by purified H1 kinase are all present in thermolytic peptide maps of P1 phosphorylated in vivo in proliferating HeLa cells. These phosphopeptides are absent in nonproliferating cells. These results demonstrate that the DNA-binding proteins HMG I, Y and P1 are natural substrates for the M-phase-specific protein kinase. The phosphorylation of these proteins by p34cdc2/cyclincdc13 may represent a crucial event in the intense chromatin condensation occurring as cells transit from the G2 to the M phase of the cell cycle.     

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

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

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.     

p13suc1 suppresses the catalytic function of p34cdc2 kinase for intermediate filament proteins, in vitro.

The regulation of p34cdc2 kinase activity controls the entry into and exit from mitosis. Although genetic and biochemical evidence suggested close interactions between cyclins, p13suc1 and p34cdc2 kinase, the roles of p13suc1 on p34cdc2 kinase functions remain unclear. To examine the effects of p13suc1 on p34cdc2 kinase function we developed a simple purification procedure for p34cdc2 kinase, unassociated with p13suc1. The key to the purification procedures we used was buffer containing 0.5 M NaCl and 50% ethylene glycol, as a specific elutant of p34cdc2 kinase from p13suc1-Sepharose. This purified p34cdc2 kinase stoichiometrically phosphorylated vimentin and desmin. Exogenous p13suc1 suppressed the phosphorylation of these filament proteins by the kinase and prevented disassembly, although histone H1 phosphorylation was not affected. Peptide mapping analysis showed a similar extent of inhibition by p13suc1 for all five phosphorylation sites by p34cdc2 kinase of vimentin and desmin, hence these p13suc1-induced inhibitions are probably not site-specific. It thus appears that p13suc1 has a selective effect on the catalytic activity of p34cdc2 kinase for these filament proteins.     

Cyclin promotes the tyrosine phosphorylation of p34cdc2 in a wee1+ dependent manner.

The regulation of p34cdc2 was investigated by overproducing p34cdc2, cyclin (A and B) and the wee1+ gene product (p107wee1) using a baculoviral expression system. p34cdc2 formed a functional complex with both cyclins as judged by co-precipitation, phosphorylation of cyclin in vitro, and activation of p34cdc2 histone H1 kinase activity. Co-production of p34cdc2 and p107wee1 in insect cells resulted in a minor population of p34cdc2 that was phosphorylated on tyrosine and displayed an altered electrophoretic mobility. When p34cdc2 and p107wee1 were co-produced with cyclin (A or B) in insect cells, there was a dramatic increase in the population of p34cdc2 that was phosphorylated on tyrosine and that displayed a shift in electrophoretic mobility. The phosphorylation of p34cdc2 on tyrosine was absolutely dependent upon the presence of kinase-active p107wee1. Tyrosine-specific as well as serine/threonine-specific protein kinase activities co-immunoprecipitated with p107wee1. These results suggest that cyclin functions to facilitate tyrosine phosphorylation of p34cdc2 and that p107wee1 functions to regulate p34cdc2, either directly or indirectly, by tyrosine phosphorylation.     

Activation of p34cdc2 protein kinase activity in meiotic and mitotic cell cycles in mouse oocytes and embryos.

p34cdc2 protein kinase is a universal regulator of M-phase in eukaryotic cell cycle. To investigate the regulation of meiotic and mitotic cell cycle in mammals, we examined the changes in phosphorylation states of p34cdc2 and its histone H1 kinase activity in mouse oocytes and embryos. We showed that p34cdc2 has three different migrating bands (referred to as upper, middle and lower bands) on SDS-PAGE followed by immunoblotting with anti-PSTAIR antibody, and that the upper and middle bands are phosphorylated forms since these two bands shifted to the lower one by alkaline phosphatase treatment. In meiotic cell cycle, only germinal vesicle (GV) stage oocytes had the three forms. The phosphorylated forms decreased gradually in oocytes up to 2 h after isolation from follicles, and thereafter the phosphorylation states did not change significantly until metaphase II. However, the histone H1 kinase activity oscillated, being activated at the first and second metaphase in meiosis and inactivated at the time of the first polar body extrusion. These results suggest that changes in phosphorylation states of p34cdc2 triggered its activation at the first metaphase, but not inactivation and reactivation at the first and second metaphase, respectively. In mitotic cell cycle, phosphorylated forms appeared at 4 h after insemination, increased greatly just before metaphase, and were dephosphorylated in metaphase. Histone H1 kinase activity was high only at metaphase. This kinase activation is probably triggered by dephosphorylation of p34cdc2.     

Mutations of p34cdc2 phosphorylation sites induce premature mitotic events in HeLa cells: evidence for a double block to p34cdc2 kinase activation in vertebrates.

In vertebrates, entry into mitosis is accompanied by dephosphorylation of p34cdc2 kinase on threonine 14 (Thr14) and tyrosine 15 (Tyr15). To examine the role of these residues in controlling p34cdc2 kinase activation, and hence the onset of mitosis, we replaced Thr14 and/or Tyr15 by non-phosphorylatable residues and transfected wild-type and mutant chicken p34cdc2 cDNAs into HeLa cells. While expression of wild-type p34cdc2 did not interfere with normal cell cycle progression, p34cdc2 carrying mutations at both Thr14 and Tyr15 displayed increased histone H1 kinase activity and rapidly induced premature mitotic events, including chromosome condensation and lamina disassembly. No phenotype was observed in response to mutation of only Thr14, and although single-site mutation at Tyr15 did induce premature mitotic events, effects were partial and their onset was delayed. These results identify both Thr14 and Tyr15 as sites of negative regulation of vertebrate p34cdc2 kinase, and they suggest that dephosphorylation of p34cdc2 represents the rate-limiting step controlling entry of vertebrate cells into mitosis.     

The retinoblastoma protein is phosphorylated on multiple sites by human cdc2.

The retinoblastoma gene product (pRB) is a nuclear phosphoprotein that is thought to play a key role in the negative regulation of cellular proliferation. pRB is phosphorylated in a cell cycle dependent manner, and studies in both actively dividing and differentiated cells suggest that this modification may be essential for cells to progress through the cell cycle. Using tryptic phosphopeptide mapping we have shown that pRB is phosphorylated on multiple serine and threonine residues in vivo and that many of these phosphorylation events can be mimicked in vitro using purified p34cdc2. Using synthetic peptides corresponding to potential cdc2 phosphorylation sites, we have developed a strategy which has allowed the identification of five sites. S249, T252, T373, S807 and S811 are phosphorylated in vivo, and in each case these sites correspond closely to the consensus sequence for phosphorylation by p34cdc2. This and the observation that pRB forms a specific complex with p34cdc2 in vivo suggests that p34cdc2 or a p34cdc2-related protein is a major pRB kinase.     

Calyculin A causes the activation of histone H1 kinase and condensation of chromosomes in unfertilized sea urchin eggs independently of the maturation-promoting factor

Calyculin A is known to inhibit the type-1 and type-2A phosphatases. We previously reported that calyculin A induces contractile ring formation in unfertilized sea urchin eggs, an increase in histone H(1) kinase activity, and chromosome condensation in the calyculin A-treated unfertilized eggs, and the changes induced by calyculin A are not affected by emetine, an inhibitor of protein synthesis. These observations suggest that the mechanism by which histone H(1) kinases are activated by calyculin A is different from that of maturation-promoting factor (MPF), which is activated by a molecular modification of existed cdc2 and newly synthesized cyclin B. We report here that no cyclin B was detected by immunoblotting of unfertilized calyculin A-treated eggs. In addition, no DNA synthesis was induced by calyculin A. As well, butyrolactone I (an inhibitor of cdc2 and cdk2 kinase) had no effect on the increase in histone H(1) kinase activity nor the chromosome condensation, both of which were induced by calyculin A. Thus, we conclude that calyculin A induces histone H(1) phosphorylation in an MPF-independent manner through inhibition of type-1 phosphatase, and that the chromosome condenses as a result of histone H(1) phosphorylation.     

Comprehensive phosphoprotein analysis of linker histone H1 from Tetrahymena thermophila

Linker histone H1 is highly phosphorylated in normal growing Tetrahymena thermophila but becomes noticeably dephosphorylated in response to certain conditions such as prolonged starvation. Because phosphorylation of H1 has been associated with the regulation of gene expression, DNA repair, and other critical processes, we sought to use mass spectrometry-based approaches to obtain an in depth phosphorylation "signature" for this linker histone. Histone H1 from both growing and starved Tetrahymena was analyzed by nanoflow reversed-phase HPLC MS/MS following enzymatic digestions, propionic anhydride derivatization, and phosphopeptide enrichment via IMAC. We confirmed five phosphorylation sites identified previously and detected two novel sites of phosphorylation and two novel minor sites of acetylation. The sequential order of phosphorylation on H1 was deduced by using mass spectrometry to define the modified sites on phosphorylated H1 isoforms separated by cation-exchange chromatography. Relative levels of site-specific phosphorylation on H1 isolated from growing and starved Tetrahymena were obtained using a combination of stable isotopic labeling, IMAC, and tandem mass spectrometry.     

Mitogen-activated protein kinase kinase 1 (MKK1) is negatively regulated by threonine phosphorylation.

Mitogen-activated protein kinase kinase 1 (MKK1), a dual-specificity tyrosine/threonine protein kinase, has been shown to be phosphorylated and activated by the raf oncogene product as part of the mitogen-activated protein kinase cascade. Here we report the phosphorylation and inactivation of MKK1 by phosphorylation on threonine 286 and threonine 292. MKK1 contains a consensus phosphorylation site for p34cdc2, a serine/threonine protein kinase that regulates the cell division cycle, at Thr-286 and a related site at Thr-292. p34cdc2 catalyzes the in vitro phosphorylation of MKK1 on both of these threonine residues and inactivates MKK1 enzymatic activity. Both sites are phosphorylated in vivo as well. The data presented in this report provide evidence that MKK1 is negatively regulated by threonine phosphorylation.     

Activation of p34(cdc2) kinase around the meiotic resumption in bovine oocytes cultured in vitro

The p34(cdc2) kinase has been identified as a protein factor that is a regulator of meiotic maturation in mammalian oocytes. To investigate the regulatory function of the meiotic resumption in bovine oocytes cultured in vitro, the changes in the phosphorylation states of p34(cdc2) kinase and the histone H1 kinase activity were examined around germinal vesicle breakdown (GVBD). All bovine oocytes just after isolation from their follicles were arrested at the germinal vesicle (GV) stage, and these extracts exhibited two (upper and lower) bands of p34(cdc2) kinase on SDS-PAGE followed by immunoblotting with an antibody against C-terminal peptide of p34(cdc2). When these oocytes were cultured for 24 h in a medium supplemented with 100 microg/ml genistein, tyrosine phosphorylation inhibitor, GVBD was induced in 85% of oocytes, indicating that the upper band of p34(cdc2) kinase in bovine oocytes at the GV stage was already fully phosphorylated tyrosine residue prior to culture. Another (middle) band of p34(cdc2) kinase between the upper and lower bands appeared in the extracts of the oocytes cultured for 4 h, and significant activation of the histone H1 kinase was found in these oocytes (67 +/- 18 fmol/h/oocyte) as compared to that in oocytes cultured for 0 h (46 +/- 11 fmol/h/oocyte). The staining intensity of the middle band and the activity of the histone H1 kinase were further increased after the initiation of GVBD at 6 h of culture, but the quantitative changes of upper and lower bands were not detected throughout the 12 h of culture. Thus, it is concluded that the dephosphorylation of p34(cdc2) kinase followed by activation of the histone H1 kinase after the onset of culture plays a key role in the resumption of meiosis in bovine oocytes.     

Phosphorylation of p97(VCP) and p47 in vitro by p34cdc2 kinase.

The hexameric ATPase p97/yeast Cdc48p has been implicated in a number of cellular events that are regulated during mitosis, including homotypic membrane fusion, spindle pole body function, and ubiquitin-dependent protein degradation. p97/Cdc48p contains two conserved consensus p34cdc2 kinase phosphorylation sites within its second ATP binding domain. This domain is likely to play a role in stabilising the hexameric form of the protein. We therefore investigated whether p97 could be phosphorylated by p34cdc2 kinase in vitro, and whether phosphorylation might influence the oligomeric status of p97. Monomeric, but not hexameric, p97 was phosphorylated by p34cdc2 kinase, as was the p97-associated protein p47. However, phosphorylation by p34cdc2 kinase did not impair subsequent re-hexamerisation of p97, implying that the phosphorylated residue(s) are not critical for interaction between p97 monomers. Moreover, p97 within both interphase and mitotic cytosols was almost exclusively hexameric, suggesting that the activity of p97 is not regulated during mitosis by influencing the extent of oligomerisation.