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PubMed1996

Spindle pole body separation in Saccharomyces cerevisiae requires dephosphorylation of the tyrosine 19 residue of Cdc28.

In eukaryotes, mitosis requires the activation of cdc2 kinase via association with cyclin B and dephosphorylation of the threonine 14 and tyrosine 15 residues. It is known that in the budding yeast Saccharomyces cerevisiae, a homologous kinase, Cdc28, mediates the progression through M phase, but it is not clear what specific mitotic function its activation by the dephosphorylation of an equivalent tyrosine (Tyr-19) serves. We report here that cells expressing cdc28-E19 (in which Tyr-19 is replaced by glutamic acid) perform Start-related functions, complete DNA synthesis, and exhibit high levels of Clb2-associated kinase activity but are unable to form bipolar spindles. The failure of these cells to form mitotic spindles is due to their inability to segregate duplicated spindle pole bodies (SPBs), a phenotype strikingly similar to that exhibited by a previously reported mutant defective in both kinesin-like motor proteins Cin8 and Kip1. We also find that the overexpression of SWE1, the budding-yeast homolog of wee1, also leads to a failure to segregate SPBs. These results imply that dephosphorylation of Tyr-19 is required for the segregation of SPBs. The requirement of Tyr-19 dephosphorylation for spindle assembly is also observed under conditions in which spindle formation is independent of mitosis, suggesting that the involvement of Cdc28/Clb kinase in SPB separation is direct. On the basis of these results, we propose that one of the roles of Tyr-19 dephosphorylation is to promote SPB separation.

Amino Acid Sequence↗
PubMed1981

[Changes in some properties of 3':5'-AMP-dependent protein kinase from rat skeletal muscles during physical exercise].

During 6-week training of rats the activity of isoenzymes I and II of soluble 3':5'-AMP-dependent protein kinase increases by 22 and 33%, respectively. A long-term physical load does not cause any significant changes in the activity of both isoenzymes. The maximal activity of the isoenzymes from skeletal muscles of the control and experimental rats is observed at the same concentrations of 3':5'-AMP and pH of 6,0-6,5. During training and under physical load the apparent Km values for ATP of both isoenzymes of 3':5'-AMP-dependent protein kinase do not change significantly, whereas that of V shows an increase. The apparent Km and V values for the histone increase for isoenzyme I obtained from skeletal muscles of trained rats both at rest and under physical load. In case of isoenzyme II the Km value for the histone decreases, while that of V remains unchanged. The changes in the properties of isoenzymes I and II of 3':5'-AMP-dependent protein kinase from skeletal muscles suggest the participation of the enzyme in adaptation to systematic muscular activity.

Animals↗
PubMed1977

Cytoplasmic and nuclear protein kinases during the cell cycle.

Nuclear and cytoplasmic protein kinases were measured during the traverse of synchronous CHO cultures through G1 into S phase. Cells were synchronized by selective detachment of cells blocked in metaphase using colcemid. Nuclei were isolated and the protein kinases extracted from the nuclear preparation with 0.6 M NaCl. This procedure solubilized greater than 90% of the total protein kinase activity present in the nuclear preparation. DEAE chromatography of this extract showed 5 apparently different ionic forms of nuclear protein kinases. The nuclear protein kinases preferred casein and phosvitin to histone as substrates and were cyclic AMP-independent. Nuclear protein kinase activities increased greater than two-fold, when expressed as units of activity per cell nucleus, during G1 phase traverse, concomitant with a 70% increase in nuclear non-histone proteins (those soluble in 0.6 M NaCl). This resulted in only a 40% increase in the specific activities (units/microgram protein in 0.6 M NaCl extractable nuclear fraction) of these enzymes as cells progressed through G1 into S phase. This was in contrast to cytoplasmic cyclic AMP-dependent protein kinase activities which also increased two-fold during progression through G1 phase while total cellular protein increased less than 20%. Activation of, as well as synthesis of, cyclic AMP-dependent cytoplasmic protein kinases during G1 phase suggests a regulatory mechanism for precise temporal phosphorylation, whereas the constant specific activity in nuclear kinases during cell cycle is more compatible with the maintenance of bulk phosphorylation processes in the nucleus.

Cell Cycle↗
PubMed1990

6DMAP inhibits chromatin decondensation but not sperm histone kinase in sea urchin male pronuclei.

Treatment of sea urchin eggs for 10 min prior to fertilization with the kinase inhibitor 6DMAP (6-dimethylaminopurine) reversibly inhibits swelling and loss of conical morphology of the male pronucleus. Male pronuclei inhibited with 1 mM 6DMAP for 25 min undergo phosphorylation of Sp H1 and Sp H2B histones as fully as do control nuclei. Therefore, Sp histone kinase, whose target sequences resemble those of the M-phase histone kinase, is not inhibited by 6DMAP, and Sp histone phosphorylation, although it may be necessary, is not sufficient for chromatin decondensation.

Adenine↗
PubMed1982

The site of histone H2b phosphorylated by a cyclic nucleotide independent histone kinase.

A cyclic nucleotide independent histone kinase was demonstrated in bovine thymus extract. This enzyme was very similar to that found previously in the cytoplasm and nucleus of human tonsillar lymphocytes (Faragó et al., 1973, Biochim. Biophys. Acta 297, 517 and 1974, ibid 370, 459). High-performance liquid chromatography of the tryptic phosphopeptides of calf thymus histones H1 and H2b phosphorylated by the histone kinase or by the catalytic subunit of cylic AMP dependent protein kinase showed that the intrinsic substrate specificity of these enzymes differed significantly. Under our experimental conditions the histone kinase phosphorylated preferentially the Ser-32 residue, and it did not phosphorylate the Ser-36 residue of histone H2b, while Ser-36 was phosphorylated preferentially by the cyclic AMP dependent protein kinase. A peptide containing the amino acid sequence of histone H2b from Gly-26 to Lys-34 (Gly-Lys-Lys-Arg-Lys-Arg-Ser-Arg-Lys-Ala) was synthesized. This peptide was a competitive inhibitor of histone H1 phosphorylation by the histone kinase and it was also a substrate for this enzyme.

Amino Acid Sequence↗
PubMed1976

Role of histone kinases as mediators of corticotropin-induced steroidogenesis.

In an attempt to determine the role of protein (histone) kinases as mediators of corticotropin-induced corticosterone formation, the ability of homogenates, prepared from adrenals treated with various doses of corticotropin to catalyse the phosphorylation of calf thymus histones was measured. Although corticotropin promoted an increase in histone kinase activity, much more of the hormone was required to induce this response than to stimulate steroidogenesis maximally. In addition, a derivative, nitrophenylsulphenyl-corticotropin, which inhibits the stimulatory effect of corticotropin on cyclic AMP accumulation, stimulated corticosterone synthesis without altering histone kinase activity. Very high doses of nitrophenylsulphenyl-corticotropin were capable of stimulating histone kinase activity. In contrast, when dibutyryl cyclic AMP was used to stimulate steroidogenesis under the same conditions, any dose of the nucleotide which increased adrenal corticosteroid content also increased histone kinase activity. Assuming that histones serve as useful substrates for measurement of total adrenal protein kinase activity, the role of protein kinases as mediators of steroidogenesis is not supported by these studies.

Adrenal Glands↗
PubMed1985

Phosphorylation of five histone H1 subtypes of L5178Y cells at the exponential growth and mitotic phases.

Histone H1 of cells of L5178Y, a mouse lympholeukemic cell line, consists of five molecular species designated as H1-I, II, III, IV, and V. The phosphorylation of these H1 subtypes was examined at the exponential growth phase and during mitosis, by BioRex 70 column chromatography and two-dimensional polyacrylamide gel electrophoresis. In exponentially growing cells, the degree of phosphorylation was different for each subtype. H1-II was the most highly phosphorylated, 1.8 phosphate residues per molecule, followed by H1-IV/V, 1.4, I, 0.8, and III, 0.5. In the mitotic phase, H1-II was also the most highly phosphorylated 6.0 phosphate residues per molecule, H1-IV/V, 3.5, I, 2.7, and III, 1.2. The phosphorylation started simultaneously among the subtypes after colcemid addition, and phosphorylated H1 subtypes accumulated linearly. The rate of incorporation of 32P into each H1 subtype was almost constant during colcemid treatment. During 4 h after colcemid addition, the phosphate residues incorporated into H1 did not dephosphorylated. The H1 kinase activities increased to six times higher during the colcemid treatment.

Animals↗
PubMed1995

Differential regulation of p34cdc2 and p33cdk2 by transforming growth factor-beta 1 in murine mammary epithelial cells.

Cyclin-dependent kinases (cdks) are a family of proteins whose function plays a critical role in cell cycle traverse. Transforming growth factor-beta 1 (TGF-beta 1) is a potent growth inhibitor of epithelial cells. Since cdks have been suggested as possible biochemical markers for TGF-beta growth inhibition, we investigated the effect of TGF-beta 1 on cdc2 and cdk2 in a normal mouse mammary epithelial cell line (MME) and a TGF-beta-resistant MME cell line (BG18.2). TGF-beta 1 decreases newly synthesized cdc2 protein levels within 6 h after addition. Coincident with this decrease in newly synthesized cdc2 protein was a marked reduction in its ability to phosphorylate histone H1. This decrease in kinase activity is not due to a change in steady-state levels of cdc2 protein, since mRNA and total protein levels of cdc2 are not reduced until 12 h after TGF-beta 1 addition. This suggests that the kinase activity of cdc2 is dependent on newly synthesized cdc2 protein. Moreover, the protein synthesis of another cyclin-dependent kinase, cdk2, is not effected by TGF-beta 1 addition, but its kinase activity is substantially reduced. Thus, it appears that TGF-beta decreases the kinase activity of both cdc2 and cdk2 by distinct mechanisms.

Amino Acid Sequence↗
PubMed1995

Brefeldin A provokes indirect activation of cdc2 kinase (MPF) in Xenopus oocytes, resulting in meiotic cell division.

Brefeldin A, a fungal metabolite which disrupts protein traffic, provokes indirect activation of cdc2 protein kinase in Xenopus oocytes. Cdc2 protein kinase activation was judged by MPF (M-phase factor) transfer activity, histone H1 kinase activity, and phosphorylation in vivo of the guanine-nucleotide exchange complex EF-1 beta gamma delta. Oocytes resumed complete meiosis upon brefeldin A treatment. Cdc2 protein kinase, MAP kinase, cyclin B, MPF, and protein synthesis changes were all comparable in brefeldin A-treated oocytes and in progesterone-induced oocytes. ED50 for brefeldin A was 0.6 microM. Brefeldin A activation of cdc2 protein kinase occurs with a long time course. Simultaneous treatment of the oocytes at a subthreshold concentration of 1 nM progesterone and 30 microM brefeldin A considerably shortened the kinetics of maturation. Brefeldin A induction of maturation was sensitive to drugs that act on cAMP metabolism. ID50 for IBMX was 0.1 mM, compared to 1 mM for progesterone-treated oocytes. Brefeldin A inhibited protein traffic in oocytes as determined from protein export experiments. ID50 was between 0.1 and 1 microM. Our results give new insights into the possible mechanism of induction of meiotic maturation and further demonstrate that brefeldin A acts on cell cycle regulatory elements.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Regulation of M-phase progression in Chaetopterus oocytes by protein kinase C.

We have examined the presence of protein kinase C in oocytes of Chaetopterus pergamentaceus and its role in the initiation of germinal vesicle breakdown (GVBD). First, we demonstrated that the oocytes contain a phospholipid- and calcium-dependent protein kinase, protein kinase C (PKC). Since PKC is the primary intracellular receptor for phorbol esters, we tested the ability of phorbol 12,13-dibutyrate (PDBu) to induce GVBD and compared several critical events and processes involved in GVBD induced by PDBu to those induced normally (by seawater). Seawater and 100-200 nM PDBu induced chromosome condensation, spindle formation, and spindle migration over a similar time course. Both treatments induced similar alterations in the SDS-PAGE pattern of newly synthesized proteins. The synthesis of polypeptides of approximately 46 and 54 kDa increased specifically. Both treatments increased oocyte protein phosphorylation, especially of proteins of 22, 32, 46, 55, 64, and 84 kDa. Both treatments resulted in the activation of an M-phase-specific histone H1 kinase activity, which demonstrates the appearance of maturation-promoting factor. Staurosporine, a potent protein kinase C inhibitor, blocked GVBD and the activation of M-phase-specific H1 kinase, whereas HA1004, which preferentially antagonizes protein kinase A, had no effect. The results of this study demonstrate that protein kinase C can activate a wide spectrum of essential biochemical and morphological processes involved in GVBD. Further, these studies suggest that protein kinase C elicits GVBD by activating maturation-promoting factor and support the hypothesis that protein kinase C plays an essential role in oocyte maturation in this species.

Alkaloids↗
PubMedDate not supplied

Modification of protein kinase pattern in human placentae during gestation.

The cAMP-dependent and cAMP-independent histone kinases have been studied in the two subcellular compartments (cytosol and particulate fraction) from placentae of different gestational age. The total protein kinase activity, as well as its distribution between the two compartments, changes during the period of gestation. The total activity is significantly increased in full-term placentae. The increase is much greater for the cAMP-dependent (400 per cent), than for the cAMP-independent (270 per cent) protein kinases. It is much higher (400 per cent) in the cytosol than in the particulate fraction (170 per cent); consequently, the particulate fraction of term placentae shows a relatively lower proportion of protein kinase activity (26 per cent of the total activity) than the corresponding fraction of young placentae (37 per cent). DEAE-cellulose chromatography revealed the presence of two cAMP-dependent protein kinase peaks which correspond to Type I and Type II isoenzymes described in many mammalian tissues (Corbin, Keely and Park, 1975). The Type II isoenzyme is predominant in both first- and third-trimester placentae. The increase in protein kinase activity in term placentae is due to the selective activation of the Type II kinase only. The activity of the Type I isoenzyme remained unchanged throughout the period of gestation. The third peak eluted from the DEAE-cellulose column corresponds to a cAMP-independent sucrose-gradient ultracentrifugation into two distinct peaks similar to those already observed in several rat tissues (Toru-Delbauffe, Ohayon and Pavlovic-Hournac, 1983). The protein kinase patterns of both young and term placentae remain stable during the incubation of the tissues 'in vitro' for three hours.

Cyclic AMP↗
PubMed1975

[Meiosis: role of a histone kinase in the condensation of ovarian oocyte chromosomes of Xenopus laevis and Ambystoma mexicanum].

By injecting heterologous histone-kinase preparations into ovarian Axolotl oocytes, it has been possible to speed up the progesterone-induced process of chromosome condensation. Moreover, in some instances, this condensation and even complete maturation have been obtained after injection of protein kinase alone, thus in the absence of hormone stimulation. Two different histone kinase preparations have been used: one was prepared from ascites cell chromatin and the other from in vitro ovulated Xenopus oocytes.

Ambystoma↗
PubMed1976

Phosphorylation in vitro of eukaryotic initiation factors IF-E2 and IF-E3 by protein kinases.

Purified protein synthesis initiation factors IF-E2 and IF-E3 from rabbit reticulocytes were phosphorylated in vitro with protein kinases isolated from the same source. The highest levels of phosphorylation resulted from incubation of the factors with a cyclic nucleotide-independent protein kinase previously shown to have specificity for acidic proteins. The extent of phosphorylation of initiation factor IF-E2 was between 0.3 and 0.4 mol of phosphate per mol of factor complex, with either ATP or GTP as phosphoryl donor. Initiation factor IF-E2 is composed of three nonidentical polypeptides; only the polypeptide with a molecular weight of 52,000 was phosphorylated. The extent of phosphorylation of initiation factor IF-E3 was between 0.7 and 1.0 mol of phosphate per mol of factor complex with GTP as phosphoryl donor; with ATP, less phosphorylation of the factor was obtained. Initiation factor IF-E3 is composed of 9 to 11 nonidentical polypeptides; only 2 of these, with molecular weights of 120,000 and 70,000, were phosphorylated. A lower level of phosphorylation of initiation factor IF-E3 was found with the cyclic AMP-dependent protein kinase; the polypeptide of molecular weight 140,000 was the major site of phosphorylation.

Adenosine Triphosphate↗
PubMed1996

Effects of phosphate on in vitro 2-cell block of AKR/N mouse embryos based on changes in cdc2 kinase activity and phosphorylation states.

This study demonstrated the effects of phosphate on the 2-cell block of AKR/N mouse embryos at the molecular level and focused on changes in the kinase activity and the phosphorylation state of cdc2, which is shown to regulate the cell division cycle. Removal of phosphate from the culture medium dramatically increased developmental rates to the 4-cell (91.8%) and blastocyst (42.6%) stages compared with those of embryos cultured in 1.17 mM phosphate (3.3% and 0%, respectively). The rate of development to the 4-cell stage was significantly inhibited by 0.001 mM phosphate (p < 0.05), and no morula formation was observed at 1.0 mM. The patterns of cdc2 kinase activity during the first cell cycle in AKR/N embryos were similar to those of control MCH embryos, showing the highest activity at M phase and low activity during the interphase. The phosphorylated form of cdc2 increased during the interphase, indicating that the synthesis of cyclin B and accumulation of inactive pre-maturation-promoting factor (pre-MPF) as well as abrupt dephosphorylation of cdc2 at the first cleavage correlated with the activation of cdc2 kinase. When phosphate was absent, the activation pattern of cdc2 kinase during the second cell cycle in AKR/N embryos was similar to that in the first cell cycle. On the other hand, no dephosphorylation of cdc2 was observed and the kinase activity remained at a low level until 56 h after insemination in the presence of phosphate, although an increase in phosphorylated cdc2 was observed as in the phosphate-free group. Treatment of AKR/N embryos arrested at the 2-cell stage with okadaic acid resulted in the dephosphorylation and activation of cdc2, confirming the presence of a sufficient amount of pre-MPF. These results show that phosphate has a deteriorative effect on the in vitro development of AKR/N embryos and suggest that this effect was not on the synthesis of cyclin B but on the dephosphorylation of phosphorylated cdc2.

Animals↗
PubMed1995

Activation-induced T-cell death is cell cycle dependent and regulated by cyclin B.

Developing thymocytes and some T-cell hybridomas undergo activation-dependent programmed cell death. Although recent studies have identified some critical regulators in programmed cell death, the role of cell cycle regulation in activation-induced cell death in T cells has not been addressed. We demonstrate that synchronized T-cell hybridomas, irrespective of the point in the cell cycle at which they are activated, stop cycling shortly after they reach G2/M. These cells exhibit the diagnostic characteristics of apoptotic cell death. Although p34cdc2 levels are not perturbed after activation of synchronously cycling T cells, cyclin B- and p34cdc2-associated histone H1 kinase activity is persistently elevated. This activation-dependent induction of H1 kinase activity in T cells is associated with a decrease in the phosphotyrosine content of p34cdc2. We also demonstrate that transient inappropriate coexpression of cyclin B with p34cdc2 induces DNA fragmentation in a heterologous cell type. Finally, in T cells, cyclin B-specific antisense oligonucleotides suppress activation-induced cell death but not cell death induced by exposure to dexamethasone. We therefore conclude that a persistent elevation of the level of cyclin B kinase is required for activation-induced programmed T-cell death.

Amino Acid Sequence↗
PubMed1998

c-myc-enhanced S phase entry in keratinocytes is associated with positive and negative effects on cyclin-dependent kinases.

The function of the c-myc proto-oncogene in cell cycle progression remains unclear. In order to examine the role c-myc may play in cell cycle progression, we have expressed the hormone-inducible MycER protein in the nontransformed, EGF-dependent mouse keratinocyte cell line BALB/MK. We have found that activation of MycER, but not a mutant MycER, Gal4ER, or FosER, leads to an EGF-dependent and hormone-dependent increased incorporation of labeled thymidine only during the S phase of the cell cycle in BALB/MK cells. A possible explanation for the increase in thymidine incorporation comes from flow cytometric analyses that reveal that activation of MycER leads to an increase in the total number of cells that enter S phase after EGF restimulation. Investigation of the intracellular effects of Myc activation shows that the expression of several putative Myc-sensitive proteins, cyclins A, E, and D1, and the E2F-1 protein are unaffected by Myc induction. Interestingly, we find that the histone H1 kinase activity associated with an E2F-1 complex containing Cyclin A and Cdk-2, but not that associated with Cyclin E, in late G1 and early S phases is increased in cells containing hormone-activated MycER, but not FosER. Although the mechanism for this Myc-dependent effect on E2F-1-associated kinase activity is still unknown, it does not appear to involve dissociation of the Cdk inhibitor p27Kip1 from the complexes as suggested by others. However, we have also found that hormone-treated cells actually show more p16INK4A inhibitor associated with another kinase, Cdk-4, as the cells are entering S phase. Altogether, the data suggest that the presence of excessive Myc protein in keratinocytes can stimulate otherwise noncycling cells to enter the cell cycle, and that this effect of Myc involves both positive effects on E2F-1-associated Cdk-2 and negative effects on Cdk-4 in late G1.

Animals↗
PubMed1982

Inhibition of cerebral protein kinase activity and cyclic AMP-dependent ribosomal-protein phosphorylation in experimental hyperphenylalaninaemia.

Studies were carried out to elucidate the mechanisms underlying the diminished phosphorylation of cerebral ribosomal protein in experimental hyperphenylalaninaemia [Roberts & Morelos (1980) Biochem. J.190, 405-419]. Administration of N(6),O(2)'-dibutyryl cyclic AMP or 3-isobutyl-1-methylxanthine, which increased phosphorylation of the S6 protein of cerebral 40S ribosomal subunits in control infant rats, did not counteract the decreased phosphorylation of this ribosomal protein resulting from intraperitoneal administration of a loading dose of l-phenylalanine. N(2),O(2)'-Dibutyryl cyclic GMP had no effect on cerebral ribosomal-protein phosphorylation in either control or hyperphenylalaninaemic animals. The phenylalanine-induced decrease in ribosomal-protein phosphorylation was associated with decreased protein kinase activity in cerebral cytosolic and microsomal preparations. However, the maximal protein kinase response to cyclic AMP added in vitro was unaltered by prior administration of phenylalanine in vivo. The heat-stable protein inhibitor of cyclic AMP-dependent protein kinases decreased the activity of these enzymes by about 90% and eliminated the phenylalanine-induced difference in protein kinase activity in the absence of added cyclic AMP. Intracisternal administration of doses of dibutyryl cyclic AMP or 3-isobutyl-1-methylxanthine which increased the cyclic AMP-dependent protein kinase activity ratio in control infant rats was without effect on this index in phenylalanine-treated animals. Dibutyryl cyclic GMP had no effect on the protein kinase activity ratio in either group of animals. These results suggest that inhibition of cerebral cyclic AMP-dependent protein kinases by abnormally high concentrations of phenylalanine may contribute to the decrease in cerebral ribosomal-protein phosphorylation in experimental hyperphenylalaninaemia.

Animals↗