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At least 343 records · Page 19Linked to original sources
PubMed1985

Purification of protein kinase C from bovine rod outer segments.

Rod outer segments (ROS) from bovine retinae were found to have high levels of calcium/phospholipid dependent protein kinase (protein kinase C). Protein kinase C behaves as an extrinsic membrane protein and phosphorylates rhodopsin in a calcium-dependent manner. The abundance of protein kinase C in ROS is similar to that of rhodopsin kinase. Its ability to phosphorylate rhodopsin in ROS membranes suggests protein kinase C may play an important role in the regulation of signal transduction in the ROS. The limited set of extrinsic membrane proteins and abundance of protein kinase C makes this tissue an extremely useful source to purify protein kinase C. The extrinsic membrane protein fraction has 6-7 U protein kinase C activity per mg protein, and the enzyme is quite stable apparently due to the lack of proteases in the preparation. A procedure was developed using phosphatidylserine- and calcium-dependent binding of protein kinase C to phenyl-Sepharose in low ionic strength buffer to resolve protein kinase C and other calcium-binding proteins from the majority of extrinsic membrane proteins. Protein kinase C was eluted using EGTA, and peak fractions directly loaded onto a DEAE-cellulose column. The protein kinase C peak eluted from the ion-exchange column was pooled and had a specific activity greater than 1,000 nmol phosphate transferred to histone per min per mg protein with a recovery of 25 percent of the starting activity. The procedure to purify protein kinase C from ROS is simple and can be completed in one day.

Animals↗
PubMed1986

Histone H1 kinase in exponential and synchronous populations of Chinese hamster fibroblasts.

Nuclear histone kinase activity, specifically histone H1 phosphotransferase activity, was shown to increase in synchronous Chinese hamster cells from the G1/S boundary to late G2/early M phase. Chromatin extracts purified by DEAE-Sephacel chromatography showed a cAMP-independent kinase activity that demonstrated cell cycle dependence and high specificity for histone H1 as the phosphate acceptor in the presence of [gamma-32P] ATP. This activity was purified approximately 40-fold. Using as substrates calf thymus histone H1 subfractions resolved by Bio-Rex 70 ion exchange chromatography, phosphorylation by the nuclear histone H1 kinase indicated that 32P incorporation into H1-2 was at least twice that for H1-1 and H1-3 subfractions. Both amino- and carboxy-terminal fragments generated by N-bromosuccinimide cleavage were phosphorylated. Phosphoamino acid analysis showed phosphothreonine to be approximately twice as abundant as phosphoserine. Histone H1 kinase activity was not activated by cyclic nucleotides, nor inhibited by cAMP-dependent protein kinase inhibitors or regulatory subunits. There was no effect on activity by Ca2+ alone or in the presence of calmodulin or diacylglycerol. Kinase activity was inhibited by nonhydrolyzable analogs of ATP such as adenyl-5'-yl imidodiphosphate, by 5'-p-fluorosulfonylbenzoyladenosine which binds to the ATP binding site of the enzyme, and by quercetin. Column fractions enriched in histone H1 kinase were labeled with 5'-p-fluorosulfonylbenzoyl[8-14C]adenosine, and peptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One band, Mr 67,000, was specifically labeled and may represent the H1 kinase catalytic subunit.

Animals↗
PubMed1989

A post-ribosomal supernatant from activated Xenopus eggs that displays post-translationally regulated oscillation of its cdc2+ mitotic kinase activity.

A cell-free extract prepared from activated Xenopus eggs by high-speed centrifugation displays one spontaneous cycle of activation and inactivation of histone H1 kinase and MPF activity that is largely attributable to Xenopus p32cdc2. The timing of the oscillation closely follows that observed in intact eggs, is associated with large changes in endogenous protein phosphorylation and depends entirely on post-translational events. The extract can be fractionated into soluble and particulate material, both of which components are required for the oscillatory behaviour. Kinase activation does not require Mg+ ATP, but its rapid inactivation, which coincides with the destruction of cyclin, is inhibited both by EDTA and the protein kinase inhibitor 6-dimethylaminopurine. This suggests that protein phosphorylation is required for cyclin destruction and kinase inactivation.

Adenine↗
PubMed1993

Transforming growth factor beta 1-mediated inhibition of smooth muscle cell proliferation is associated with a late G1 cell cycle arrest.

The effect of transforming growth factor beta 1 (TGF beta 1) on the proliferative response of aortic smooth muscle cells (SMC) in vitro was investigated. TGF beta 1 substantially inhibited the growth of human and bovine SMC. Rapidly growing SMC and quiescent serum-stimulated SMC were inhibited by TGF beta 1 with an ID50 of approximately 0.5 ng/ml and maximal inhibition was observed at 10 ng/ml TGF beta 1. In the presence of TGF beta 1, quiescent serum-stimulated SMC progress into the G1 phase of the cell cycle, but become reversibly arrested at a point temporally located 1-2 hours from S phase. Release from this late G1 TGF beta 1 arrest point results in S phase entry within 2 hours. Associated with this inhibitory effect is a decrease in the histone H1 kinase activity of p34cdc2 protein kinase while TGF beta 1 has no effect on the transcription or translation of p34cdc2. Under these growth inhibitory conditions, TGF beta 1 is still capable of upregulating the expression of fibronectin mRNA. These results suggest that TGF beta 1 growth inhibition in SMC is associated with the regulation of p34cdc2 activity in late G1.

Animals↗
PubMed1993

Molecular cloning and identification of two types of hamster cyclin-dependent kinases: cdk2 and cdk2L.

We isolated two types of hamster cyclin-dependent kinase 2 (cdk2) cDNAs from BHK21 cells derived from Golden hamsters. One type of cdk2 (cdk2hm) encodes the 32 kDa protein consisting of 298 predicted amino acids and shows strong homology to the cdk2 cDNAs of humans and Xenopus. The other cdk2 (cdk2Lhm) encodes the 38 kDa protein containing the insertion of 48 amino acids in the cdk2hm protein. Immunoblotting analysis suggested that these two types of cdk2 protein exist in mammalian cells. The cdk2hm has the activity of protein kinase, while the cdk2Lhm does not, however, both bind with cyclin E.

3T3 Cells↗
PubMed1984

Nuclear protein kinases.

Nuclear protein kinases include enzymes that transfer the gamma-phosphate of ATP to serine, threonine, lysine or histidine in proteins. Nuclear kinases with a preference for basic proteins are known as histone kinases; those preferring acidic protein substrates are casein kinases. Histone kinases include both cyclic AMP-independent protein kinases and cyclic AMP-dependent protein kinases. The best-characterized cyclic AMP-independent nuclear protein kinase is associated with cell proliferation and is activated (or transported to the nucleus) in G2 phase of the cell cycle. It phosphorylates specific serine and threonine residues in the non globular domains of histone H1 and appears to promote chromosome condensation. The cyclic AMP-dependent protein kinase has unknown nuclear function(s), although it may be translocated from cytoplasm to nucleus in response to specific hormonal stimuli which are also associated with changes in transcriptional activity. There is a massive peak of nuclear cyclic AMP-dependent protein kinase activity in G2 phase of the cell cycle. Nuclear casein kinases are apparently very heterogeneous. Two of these enzymes have been purified to homogeneity. They phosphorylate non-histone chromosomal proteins, including RNA polymerase and ornithine decarboxylase. Phosphorylated ornithine decarboxylase is inactive enzymatically but, in Physarum, it binds to the rDNA minichromosome and stimulates rRNA transcription. Kinases forming phosphoramidate bonds occur in a variety of rat tissues and form phosphohistide in histone H4 and phospholysine in histone H1.

Casein Kinases↗
PubMed1991

Isolation of the human cdk2 gene that encodes the cyclin A- and adenovirus E1A-associated p33 kinase.

Cyclins are regulatory subunits which associate with kinases to form complexes that control many of the important steps in cell-cycle progression. The best characterized of the cyclin-containing complexes is the association of cyclin B with the p34cdc2 kinase. The p34cdc2/cyclin B complex is required for the G2 to M transition (see refs 1-4 for review), but the physiological role of other cyclin complexes is unclear. Human cyclin A binds independently to two kinases, associating with either p34cdc2 or a related protein, p33 (refs 5-7). In adenovirus-transformed cells, the viral E1A oncoprotein seems to associate with p33/cyclin A but not with p34cdc2/cyclin A (B. Faha, M.M., L-H.T. and E.H., manuscript submitted). To isolate the gene for p33, we have cloned several novel human cdc2-related genes. The protein product of one of these genes, cdk2 (cyclin-dependent kinase 2), shares 65% sequence identity with p34cdc2 (ref. 8) and 89% identity with the Xenopus Eg-1 gene product. Immunochemical characterization and partial proteolytic mapping show that the cdk2 gene product is the cyclin A-associated p33. Immunoprecipitations of the p33cdk2 protein suggest that it can act as a protein kinase in vitro. As p33cdk2 is bound to cyclin A and is targeted by the viral E1A protein, we suggest that the p33cdk2/cyclin A complex has a unique role in cell-cycle regulation of vertebrate cells.

Adenovirus Early Proteins↗
PubMed1996

Leishmania mexicana p12cks1, a homologue of fission yeast p13suc1, associates with a stage-regulated histone H1 kinase.

We have isolated a Leishmania mexicana homologue of the fission yeast suc1 gene using PCR with oligonucleotides designed to conserved regions of cdc2 kinase subunits (cks). The product of cks1 is a 12 kDa polypeptide, which has 70% identity with human p9cks1 and 44% identity with fission yeast p13suc1.p12cks1 was detected in the three life-cycle stages of L. mexicana by immunoblotting. Recombinant p12cks1 (p12cks1his) bound to agarose beads was used as a matrix to affinity-select histone H1 kinase complexes from Leishmania, yeast and bovine extracts. Immunoblotting showed that yeast and bovine cdc2 kinase bound to p12cks1his, thus demonstrating functional homology between L. mexicana p12cks1 and yeast p13suc1. Histone H1 kinase activity was found at a high level in the proliferative promastigote and amastigote forms of L. mexicana, but at a low level in the non-dividing metacyclic form. These activities are likely to be the same as the leishmanial p13suc1 binding kinase (SBCRK) described previously [Mottram, Kinnaird, Shiels, Tait and Barry (1993) J. Biol. Chem. 268, 21044-21051]. A distinct cdc2-related kinase, L. mexicana CRK1, was also found to associate with p12cks1his but affinity-depletion experiments showed that CRK1 was not responsible for the histone H1 kinase activity associating with p12cks1his in promastigote cell extracts. The finding that p12cks1 associates with at least two cdc2-related kinases, SBCRK and CRK1, is consistent with the presence of a large gene family of cdc2-related kinases in trypanosomatids, a situation thought to be more similar to higher eukaryotes than yeast.

Amino Acid Sequence↗
PubMed1990

Cell division in higher plants: a cdc2 gene, its 34-kDa product, and histone H1 kinase activity in pea.

The mitotic cell cycle of yeast and animal cells is regulated by the cdc2 gene and its product, the p34 protein kinase, and by other components of the MPF or histone H1 kinase complex. We present evidence that cdc2, p34, and a histone H1 kinase also exist in higher plants. Protein extracts from 10 plant species surveyed display a 34-kDa component recognized by a monoclonal antibody directed against an evolutionarily conserved epitope of fission yeast p34. Nondenatured protein extracts of mitotic Pisum sativum (garden pea) tissues were fractionated by gel filtration, electrophoretically separated under denaturing conditions, and immunoblotted. p34 crossreactive material was apparent in both low and high molecular mass fractions, indicating that pea p34 occurs as both a monomer and as part of a high molecular mass complex. Histone H1 kinase activity was found predominantly in the higher molecular mass fractions, those to which the least phosphorylated form of pea p34 was confined. We also report the cloning of the pea homologue of cdc2 by polymerase chain reaction. DNA sequence analysis reveals perfect conservation of the hallmark "PSTAIR" sequence motif found in all cdc2 gene products analyzed to date.

Amino Acid Sequence↗
PubMed1994

Chromatin condensation during apoptosis is accompanied by degradation of lamin A+B, without enhanced activation of cdc2 kinase.

Chromatin condensation paralleled by DNA fragmentation is one of the most important criteria which are used to identify apoptotic cells. However, comparable changes are also observed in interphase nuclei which have been treated with cell extracts from mitotic cells. In this respect it is known that in mitosis, the lamina structure is broken down as a result of lamin solubilization and it is possible that a similar process is happening in apoptotic cells. The experiments described in this study have used confluent cultures of an embryonic fibroblast cell line which can be induced to undergo either apoptosis at low serum conditions or mitosis. Solubilization of lamin A+B was analyzed by immunoblotting and indirect immunofluorescence. These studies showed that in mitotic cells lamina breakdown is accompanied by lamin solubilization. In apoptotic cells, a small amount of lamin is solubilized before the onset of apoptosis, thereafter, chromatin condensation is accompanied by degradation of lamin A+B to a 46-kD fragment. Analysis of cellular lysates by probing blots with anti-PSTAIR followed by anti-phosphotyrosine showed that in contrast to mitosis, dephosphorylation on tyrosine residues did not occur in apoptotic cells. At all timepoints after the onset of apoptosis there was no significant increase in the activation of p34cdc2 as determined in the histone H1 kinase assay. Coinduction of apoptosis and mitosis after release of cells from aphidicolin block showed that apoptosis could be induced in parallel with S-phase. The sudden breakdown of chromatin structure may be the result of detachment of the chromatin loops from their anchorage at the nuclear matrix, as bands of 50 kbp and corresponding multimers were detectable by field inversion gel electrophoresis (FIGE). In apoptotic cells all of the DNA was fragmented, but only 14% of the DNA was smaller than 50 kbp. DNA strand breaks were detected at the periphery of the condensed chromatin by in situ tailing (ISTAIL). Chromatin condensation during apoptosis appears to be due to a rapid proteolysis of nuclear matrix proteins which does not involve the p34cdc2 kinase.

Animals↗
PubMed1996

Radiation with 1 Gy prevents the activation of the mitotic inducers mitosis-promoting factor (MPF) and cdc25-C in HeLa cells.

The mechanism of the transient G2 arrest induced by small doses of ionizing radiation involves the failure to activate the correctly formed pre-mitosis-promoting factor (MPF) complex of cyclin B and p34cdc2 by dephosphorylation at Tyr15 of the latter, as recent studies of other laboratories have indicated. Similar data were obtained with the G2 arrest-inducing agents epidermal growth factor and the phorbol ester 12-0- tetradecanoylphorbol-13-acetate (H. Barth and V. Kinzel, Exp. Cell Res., 212: 383-388, 1994, and H. Barth and V. Kinzel, J. Cell. Physiol., 162: 44-51, 1995). To differentiate the radiation consequences in synchronized HeLa cells from those of 12-0-tetradecanoylphorbol-13-acetate and epidermal growth factor, experiments with a very small dose (1 Gy) have been carried out in cells close to the G2-M border and, for comparison, in mitotic cells. We show that in addition to the failure of p34cdc2 dephosphorylation at Tyr15, radiation with 1 Gy also prevents the activation of the phosphatase cdc25-C, the enzyme catalyzing the MPF activation. In contrast, irradiation of mitotic cells with 1 Gy did not influence that fraction of either MPF or cdc25-C already activated. Moreover, the gain in MPM-2 antigenicity of cdc25-C, usually indicative of an activating phosphorylation, is shown to be prevented by 1 Gy. The data indicate that the initiation of the proposed autocatalytic loop between MPF and cdc25-C becomes interrupted by radiation, but they give no hint at which point.

CDC2 Protein Kinase↗
PubMed1986

The assay of the activity of protein kinase C with the synthetic oligopeptide substrate designed for histone kinase II.

The activity of histone kinase II was determined on the basis of its ability to phosphorylate the nonapeptide Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide designed previously as a specific substrate for this enzyme. Histone kinase II was purified from calf thymus extract by DEAE-cellulose chromatography followed by hydroxylapatite chromatography and high-performance liquid chromatography on a Protein Analysis column (I-125). The Mr value of histone kinase II estimated by the latter method was 50,000-55,000, but several observations indicated that histone kinase II was a product of a proteolytic process. Since the substrate specificity determinants for histone kinase II known from our previous investigations are very similar to those for protein kinase C, it was presumable that histone kinase II was the proteolytic fragment of protein kinase C. Therefore, the nonapeptide was tested as a substrate for protein kinase C prepared from rabbit brain extract by DEAE-cellulose chromatography. The activity of histone kinase II was also detected in brain extract. Histone kinase II was eluted from the DEAE-cellulose in the known position of the proteolytic fragment of protein kinase C. The nonapeptide Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide proved to be a better substrate than H1 histone for the detection of the activity of protein kinase C because it was not phosphorylated by the cAMP-dependent protein kinase and the Vmax of protein kinase C was about one order of magnitude higher with the peptide than with H1 histone. The apparent Km of protein kinase C for the peptide was identical with that of histone kinase II (0.2 mM).

Animals↗
PubMed1983

Protein kinase patterns in hyperplastic rat thyroids and in human non-toxic nodular goitres.

Patterns of cAMP-dependent protein kinases and cAMP-independent histone and casein kinases of hyperplastic rat thyroid glands and of human nodular non-toxic goitres have been analysed in two subcellular compartments, cytosol and particulate fraction. In hyperplastic rat glands the different compartmentalization of the two cAMP-dependent isoenzymes was preserved and the pattern of cAMP-independent protein kinases was not changed qualitatively, but the activities of the three classes of protein kinases were enhanced to different degrees: the highest increase was observed for the cAMP-dependent enzymes and the lowest for the cAMP-independent casein kinases. Analysis of individual peaks of cAMP-dependent kinases showed selective stimulation of the cytosolic Type II form and independent control of the Type I activity in the two subcellular compartments. Among cAMP-independent protein kinases only two histone kinase peaks were selectively enhanced; other kinase entities were changed to a lesser degree. During the involution of hyperplastic glands, a transient but differential enhancement of nearly all kinases was noted at first, which was followed later by a strong decrease, more or less rapid, of almost all kinase activities. In the regressed glands, neither the thyroid weight nor the pattern of different kinase entities corresponded to those of control, untreated glands, indicating that some of the protein kinase alterations in hyperplastic tissues are partly irreversible. In spite of great similarities, human and rat goitre tissues are not identical. The most important difference was the loss of compartmentalization of the cAMP-dependent isoenzymes in human tissue. The different ratios of the light and heavy peaks of cytosolic cAMP-independent histone kinases was the second characteristic which distinguished human and rat glands.

Animals↗
PubMed1978

Multiple forms of cytosol and membrane-bound protein kinase activity in human erythrocytes.

Both cytosol and membranes of human erythrocytes display protein kinase activity towards exogenous protein substrates such as casein, phosvitin and histones. The histone kinase activity, unlike casein kinase, of both cytosol and membranes is increased by cyclic AMP. The protein kinase forms removed from the membranes with 0.7 M NaCl, phosphorylate only serine residues of both casein and histones through a mechanism cyclic AMP-independent. The protein kinase activity located in the cytosol (hemolysate) is due also to enzyme forms phosphorylating both serine and threonine residues of casein, in addition to forms phosphorylating only serine residues of casein and histones. Also the cytosol kinase forms, once partially purified by Sepharose 6B filtration, appear to be cyclic AMP-independent.

Caseins↗
PubMed1986

Distribution of a seminal plasma-associated protein kinase inhibitor in normal, oligozoospermic, and vasectomized men.

Human sperm-free seminal plasma contains an inhibitor, which is protein in nature, of the histone kinase present in seminal plasma. Since protein kinase inhibitors have been observed to be present in spermatozoa, the objective of the present study was to determine whether this seminal plasma-associated enzyme inhibitor originates from the sperm, or whether it is a component of accessory secretion(s) comprising the seminal plasma. Sperm-free seminal plasma from normospermic (greater than 20 X 10(6) sperm/ml), oligozoospermic (less than or equal to 20 X 10(6) sperm/ml), and vasectomized donors was obtained, and inhibitor-enriched fractions were prepared by (NH4)2SO4 fractionation and gel filtration. Contamination of the sperm-free seminal plasma by spermatozoa or spermatozoan components was negligible as assessed by light microscopy, polyacrylamide gel electrophoresis, and measurement of the activity of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase. Specific (inhibitory units/mg protein) and total inhibitory activities were determined in each of the donors by constructing linear inhibition curves using various concentrations of inhibitor. The results were correlated with the initial sperm concentration. There was no apparent relationship between the amount of inhibitory activity present and the initial sperm concentration. The histone kinase inhibitor also did not appear to be associated with testicular or epididymal secretions since it was observed in the seminal plasma of vasectomized donors. It is concluded that this inhibitor of histone kinase originates from the accessory secretions comprising the human ejaculate.

Carrier Proteins↗