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PubMed1976

Phosphorylation of histone catalyzed by a bovine brain protein kinase.

The use of polyethyleneimine-cellulose thin layer sheets to follow the phosphorylation of histone and decomposition of ATP catalyzed by an adenosine 3':5'-monophosphate (cyclic AMP)-stimulated protein kinase, protein kinase I, has made possible a more detailed analysis of the time course of these reactions than has been achieved previously be observing only recovered phosphorylated protein. When [gamma-32P] ATP was employed, significant error was introduced by the presence of 32Pi at the solvent front on these sheets, and this limited the accuracy of the available information. However, the analysis of assays performed with [U-14C] ATP was straightforward and appeared to have an accuracy comparable to that of the present standard assay. This appears to be the first use of [U-14C] ATP to assay protein kinases. Our physical characterization of protein kinase I showed it to be a homogeneous protein species by polyacrylamide gel electrophoresis, sodium dodecyl sulfate gel electrophoresis and analytical ultracentrifugation. Kinetic studies with protein kinase I indicated the absence of histone phosphatase and cyclic AMP phosphodiesterase activity. Furthermore, the ATPase activity seen is believed to be intimately associated with the protein kinase action, particularly in view of the observed dependence of the rate of Pi production on the presence of cyclic AMP. The kinetic data for the phosphorylation of histone catalyzed by protein kinase I under full stimulation by cyclic AMP are consistent with a double displacement mechanism.

Animals↗
PubMed1996

Mutation in p53 and de-regulation of p53-related gene expression in three human cell lines immortalized with 4-nitroquinoline 1-oxide or 60Co gamma rays.

In vitro models of malignant transformation of human cells may provide considerable insight into the mechanisms of multi-step carcinogenesis. It is well established that normal human cells must be immortalized before they can be malignantly transformed; however, they are stringently destined for aging and are rarely immortalized. The mechanism of cellular aging and immortalization is still unknown. We detected expression of only mutated p53 mRNA by direct sequencing of the reverse-transcribed mRNA in 3 human cell lines immortalized either with 4-nitroquinoline 1-oxide or with 60Co gamma rays. Consequently, only the mutated pS3 protein was expressed in each immortalized cell line. The expression of sdiI/p21 and mdm2, both of which are positively regulated by wild-type p53, was significantly down-regulated in the immortalized cell lines, resulting in over-expression of cdk2 and cdk4. Introduction of the sdiI/p21 gene into these cells was followed by a remarkable decrease in their ability to synthesize DNA. These results indicate that the p53 cascade may play an important role in the immortalization of human cells.

4-Nitroquinoline-1-oxide↗
PubMed1996

Maitotoxin, a calcium channel activator, inhibits cell cycle progression through the G1/S and G2/M transitions and prevents CDC2 kinase activation in GH4C1 cells.

Calcium regulates progression through several checkpoints in the cell cycle, including the G1/S-phase transition, G2/M-phase transition, and exit from mitosis. In the GH4C1 rat pituitary cell line, calcium mobilizing polypeptides and calcium channel activation inhibit cell proliferation. This report examines the effects of maitotoxin (MTX), an activator of type L voltage-dependent calcium channels (L-VDCC), on calcium influx and cell cycle progression in GH4C1 cells. MTX causes both a block from G1 to S-phase and a concentration-dependent accumulation of cells in G2+M. MTX does not increase the mitotic index; thus, sustained calcium channel activation by MTX results in an accumulation of cells in G2. In order to temporally localize the MTX-induced G2 block relative to cell cycle regulatory events at the G2/M transition, we assessed the relative activity of two cell cycle regulatory protein kinases, CDC2 and CDK2, in MTX-treated cells. CDC2-specific histone kinase activity in MTX-treated cells is lower than either in cells blocked in mitosis with the microtubule destabilizing agent demecolcine or in randomly cycling cells. In contrast, the activity of CDK2 is highest in MTX-treated cells, consistent with a G2 block prior to CDC2 activation. Together, these results implicate with a G2 block prior to CDC2 activation. Together, these results implicate calcium as an intracellular signal required for progression through G2 phase of the cell cycle prior to CDC2 kinase activation.

Animals↗
PubMed1995

Cdk1 is a marker of proliferation in human lymphoid cells.

To better understand the relationship between the proliferation of human lymphoid cells and the expression of cdk1, a catalytic subunit of the histone H1 kinase (H1K), we examined its mRNA and protein content in 3 B-cell lines: Ramos, Reh-6 and IARC 963. Cells were elutriated according to their position in the cell cycle. Cell fractions were analyzed for cdk1 mRNA and protein cellular content by Northern blot and immunoblot, respectively, as well as for H1K activity. Both mRNA and protein amounts and H1K activity varied according to cell cycle phase, the lowest values being observed in G1-enriched fractions. For comparison, elutriated fractions were also tested for the expression of cdk2 and cdk4 proteins. Both showed some variations among fractions, but they were less clear than those of cdk1. We also tested 29 samples of lymphoid neoplastic and non-neoplastic tissues for proliferative activity (percentage of S and G2/M cells estimated by flow cytometry) and expression of cdk1, cdk2 and cdk4 proteins. We found a significant correlation between the percentage of cells in S or S + G2/M phases and cdk1 protein content but not cdk2 or cdk4 content. We conclude that cdk1 expression in human lymphoid cells varies during the cell cycle at both mRNA and protein levels.

Biomarkers↗
PubMed1991

Loss of RCC1, a nuclear DNA-binding protein, uncouples the completion of DNA replication from the activation of cdc2 protein kinase and mitosis.

The temperature-sensitive mutant cell line tsBN2, was derived from the BHK21 cell line and has a point mutation in the RCC1 gene. In tsBN2 cells, the RCC1 protein disappeared after a shift to the non-permissive temperature at any time in the cell cycle. From S phase onwards, once RCC1 function was lost at the non-permissive temperature, p34cdc2 was dephosphorylated and M-phase specific histone H1 kinase was activated. However, in G1 phase, shifting to the non-permissive temperature did not activate p34cdc2 histone H1 kinase. The activation of p34cdc2 histone H1 kinase required protein synthesis in addition to the presence of a complex between p34cdc2 and cyclin B. Upon the loss of RCC1 in S phase of tsBN2 cells and the consequent p34cdc2 histone H1 kinase activation, a normal mitotic cycle is induced, including the formation of a mitotic spindle and subsequent reformation of the interphase-microtubule network. Exit from mitosis was accompanied by the disappearance of cyclin B, and a decrease in p34cdc2 histone H1 kinase activity. The kinetics of p34cdc2 histone H1 kinase activation correlated well with the appearance of premature mitotic cells and was not affected by the presence of a DNA synthesis inhibitor. Thus the normal inhibition of p34cdc2 activation by incompletely replicated DNA is abrogated by the loss of RCC1.

Animals↗
PubMed1979

Translocation of cytosol protein kinase into nuclei and the induction of tyrosine hydroxylase in NBD-2 neuroblastoma cells.

Exposure of neuroblastoma cells (NBD-2) to 8-bromo-adenosine 3',5'-cyclic monophosphate (0.2-1.0 mM) (8-Br-cAMP) for 15 min caused a long term increase in the Vmax of tyrosine-3-monooxygenase activity (TH) beginning about 1 day after 8-Br-cAMP application. Cyclic AMP-dependent histone kinase was maximally activated in about 30 min and stayed activated above pretreatment levels for one hour. In cells exposed to 8-Br-cAMP for 15 min, separation of soluble and particle bound histone kinase showed that the total histone kinase activity in the soluble fraction decreased by 40%. This decrease was accompanied by an increase in protein kinase activity in the particulate fraction, suggesting enzyme translocation. After translocation, the enzyme appears to acquire a different substrate affinity because it prefers as a PO43- acceptor, acidic protein rather than histone. In NBD-2 cells this kinase appears to precede, and may be related to, the delayed increase in TH Vmax.

Cell Nucleus↗
PubMed1988

An M-phase-specific protein kinase of Xenopus oocytes: partial purification and possible mechanism of its periodic activation.

The activity of a Ca2+- and cyclic nucleotide-independent protein kinase(s) which catalyzes hyperphosphorylation of a set of endogenous proteins, including a 95-kDa soluble phosphoprotein, is found to fluctuate in both the meiotic and mitotic cell cycles of Xenopus oocytes and activated eggs. The activity is high in M-phase and hardly detectable in interphase. The activity copurifies with a major histone kinase(s) throughout four purification steps: ammonium sulfate precipitation, DEAE-cellulose chromatography, high-performance liquid chromatography on TSK G3000, and CM-Sepharose chromatography. This suggests that a single enzyme shares activity against endogenous proteins and added histones. Changes in the activity of the M-phase-specific protein kinase(s) as assayed in vitro correlate with changes in the extent of protein phosphorylation in oocytes pulse-labeled with 32P-phosphate by microinjection during meiotic maturation and the early embryonic cell cycle. This suggests that the kinase(s) has a broad specificity and plays a key role in the increased protein phosphorylation which occurs at the transition to M-phase. Microinjection of the maturation-promoting factor (MPF) into immature oocytes triggers, after a 10-min lag period, the activation of the M-phase specific kinase(s), even in the absence of protein synthesis. In contrast MPF microinjection does not induce kinase activation in cycloheximide-treated oocytes arrested after completion of the first meiotic cell cycle or in activated eggs arrested in S-phase by incubation in cycloheximide. This suggests that immature oocytes contain an inactive kinase precursor (prokinase) which is synthesized at each of the following cell cycles. In the absence of MPF addition, the prokinase to kinase transition occurs "spontaneously" after a 2-hr lag period in high-speed supernatants prepared from prophase-arrested oocytes if low-molecular-weight metabolites are eliminated by gel filtration. Addition of ATP, but not of AMP-PNP (adenylyl-imidodiphosphate), prevents spontaneous kinase activation in gel-filtered extracts. We propose that MPF activates the M-phase-specific protein kinase in the intact cell by inactivating a factor which requires phosphorylation conditions to inhibit the prokinase to kinase transition.

Adenosine Triphosphate↗
PubMed1982

Microsomal cAMP-independent histone H1 kinase activity in plasmacytoma, Morris hepatoma and normal liver.

A protein kinase activity with high specificity for histone H1 was isolated from mouse plasmacytoma, Morris hepatoma and normal mouse liver and compared by ion exchange chromatography after DEAE-cellulose, hydroxylapatite and Sephadex G-200 chromatography. This cAMP-independent histone H1 kinase is not affected by the heat-stable cAMP-dependent protein kinase inhibitor. It has the following particular properties: it prefers GTP to ATP as substrate and was found to be present with a great activity only in neoplastic tissues. No phosphatase activity was detected in the partially purified histone H1 kinase fraction from normal and neoplastic cells. These results suggest either an increase amount of histone H1 kinase and/or of its activator in neoplastic cells, or the presence of a strong inhibitor in normal cells. This histone H1 kinase appears to be analogous to the chromatin bound kinase which phosphorylates histone H1 at the NH2 and COOH terminal regions. We might suggest an implication of this kinase in the regulation of cell division.

Adenosine Triphosphate↗
PubMed1994

Activation of cyclin A-dependent protein kinases during apoptosis.

Apoptosis was induced in S-phase-arrested HeLa cells by staurosporine, caffeine, 6-dimethylaminopurine, and okadaic acid, agents that activate M-phase-promoting factor and induce premature mitosis in similarly treated hamster cell lines. Addition of these agents to asynchronously growing HeLa cells or to cells arrested in early G1 phase with lovastatin had little or no effect. S-phase arrest also promoted tumor necrosis factor alpha-induced apoptosis, eliminating the normal requirement for simultaneous cycloheximide treatment. For all of the apoptosis-inducing agents tested, the appearance of condensed chromatin was accompanied by 2- to 7-fold increases in cyclin A-associated histone H1 kinase activity, levels approximating the mitotic value. Where examined, both Cdc2 and Cdk2, the catalytic subunits known to associate with cyclin A, were activated. Stable overexpression of bcl-2 suppressed the apoptosis-inducing activity of all agents tested and reduced the amount of Cdc2 and Cdk2 in the nucleus, suggesting a possible mechanism by which bcl-2 inhibits the chromatin condensation characteristic of apoptosis. These findings suggest that at least one of the biochemical steps required for mitosis, activation of cyclin A-dependent protein kinases, is also an important event during apoptosis.

Alkaloids↗
PubMed1977

Stimulatory effect of histones on phosphorylation of nuclear phosphoproteins.

Effect of histones on phosphorylation of nuclear phosphoproteins was studied using two species of phosphoprotein kinases with different kinetic and catalytic properties; namely, protein kinases A1 and A2 (Takeda, M., Matsumura, S., & Nakaya, Y. (1974) J. Biochem. 75, 743-751). The reaction rate for protein kinase A1 was markedly enhanced when histone or polylysine was added to the reaction mixture. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the basic protein served as a stimulator rather than acted as a substrate in this reaction. In contrast, when protein kinase A2 was employed, the stimulatory action of these basic proteins was less marked than for protein kinase A1. It seems likely that the phosphorylation of nuclear phosphoproteins, particularly the reaction catalyzed by protein kinase A1, may be strongly influenced by histones which are integrated in the chromatin structure.

Animals↗
PubMed1978

[Mechanism of action of cyclic AMP-dependent histone kinase. Substrate specificity of the catalytic enzyme unit].

Interaction of several nucleotide derivates with homogenous catalytic subunit of cyclo-AMP-dependent histone kinase from pig brain is studied. Inhibition constants of these compounds are calculated, and the affinity of inhibitors to the enzyme active site is evaluated. The nature of heterocyclic base is found to be the main contribution into binding with substrate. The enzyme specificity with respect to a number of bivalent metal ions is studied, and Mg2+ is demonstrated to be the only efficient enzyme activator. It is shown by means of stationary kinetics that histone kinase-catalysed phosphotransferase reaction has a "ping-pong"-like mechanism.

Animals↗
PubMed1978

[Several properties of 3':5'-AMP-dependent skeletal muscle protein kinases in normal rats and following physical exertion to fatigue].

Three fractions of rat adenosine-3',5'-monophosphate-dependent protein kinase were isolated, partially purified in buffer concentration gradient at normal state and after long-term physical loading and studied. It is found that first two fractions of protein kinases at normal state and after intensive muscular work have similar activities with and without cAMP, apparent Km values for ATP and total histone and half-maximal stimulation by cyclic AMP, but they differed from the third fraction. There are differences in some kinetic parameters and in the cyclic AMP stimulated activities between protein kinases after physical loading. The data obtained suggest the existence of at least two kinases in rat skeletal muscle. The isoenzymes differ in their activities during fatigue.

Animals↗
PubMed1984

Increase in type I cyclic adenosine 3':5'-monophosphate-dependent protein kinase activity and specific accumulation of type I regulatory subunits in adenovirus type 12-transformed cells.

Types I and II cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinases were compared in extracts from untransformed rat 3Y1 cells and 3Y1 cells transformed by the highly oncogenic human adenovirus type 12. Analysis of the kinases through diethylaminoethyl Sepharose column chromatography showed a significantly increased activity of type I, but not of type II, cAMP-dependent protein kinase in two transformed cell clones tested. 8-Azidoadenosine 3':5'-monophosphate, a sensitive photoaffinity-labeling analogue of cAMP, was used to quantitate the regulatory subunits of the type I kinase. A 3- to 6-fold increase in the amount of incorporation of the photoaffinity label into the Mr 48,000 regulatory subunit of type I kinase was obtained in three transformants tested. The increased incorporation was attributed to an increase in the actual amount of the subunit rather than to an increase in the affinity of type I regulatory subunit for the photoaffinity label. These results suggest that type I cAMP-dependent protein kinase activity may be a biochemical marker for adenovirus transformation.

Adenoviruses, Human↗
PubMed1978

Cyclic AMP-dependent histone-specific nucleoplasmic protein kinase from rat liver.

A nucleoplasmic histone kinase activity was isolated from livers of adult rats and purified 39-fold compared with whole nuclei by ultracentrifugation of the nuclear extract and Sephadex G-200 gel filtration in the presence of cyclic AMP. Analysis by polyacrylamide-gel electrophoresis as well as by gel filtration indicates a mol.wt. of approx. 60,000 for the catalytic subunit and 130000-150000 for the cyclic AMP-binding activity. The purified enzyme displays a 20-fold greater preference for histone fractions 1 and 2b than for any non-histone substrate, including alpha-casein. Endogenous protein in the preparation is not appreciably phosphorylated. The unfractioned enzyme is stimulated significantly by cyclic GMP, cyclic IMP and dibutyryl cyclic AMP as well as by cyclic AMP. The catalytic reaction requires Mg2+ (Km 1.9mM) and ATP (Km 15.4 micron). Half-maximal activity of the enzyme is observed with histone 2b at 12micron and histone 1 at a higher substrate concentration. The pH optima are 6.1 and 6.5 with histones 2b and 1 respectively. This nuclear protein kinase appears to be distinct from other nuclear enzymes that have been reported, on the basis of histone specificity, univalent-salt-sensitivity, pH optima and nuclear location. However, the enzyme possesses many properties similar to those of the cytoplasmic kinases, including cyclic AMP-dependence, Mg2+ and ATP affinities and pH optima. It differs from cytoplasmic protein kinase type I, the major form in the liver, with respect to bivalent-cation effects and response to the heat-stable protein kinase inhibitor protein isolated from ox heart.

Animals↗