Isolation and study of cAMP-independent chromatin protein kinases from brain cells]

Two cAMP-independent protein kinases were purified from rat brain neuron chromatin by using extraction with ammonium sulfate with subsequent chromatography on DEAE-Sephadex A-25 and Sephadex G-150. These enzymes were identified as casein kinases NI and NII, respectively. The molecular masses of the proteins as determined by gel filtration are 4500 and 130 Da. Casein kinase NII utilizes ATP (Km = 7.5 mM) and GTP (Km = 8.5 mM) as substrates, while casein kinase NI utilizes only ATP (Km = 6 mM). The activities of the both enzymes are inhibited by Mn2+ and Ca2+, while heparin (1 microgram/ml) inhibits only casein kinase NII. The memory stimulator ethymizol (ethylnorantipheine) increases the activity of casein kinase NII only when brain proteins extracted by 0.35 M NaCl or rat liver HMG-proteins are used as reaction substrates. This substance has no effect on the phosphorylation of casein and histone HI. The role of casein kinase NII of neuronal chromatin in the realization of stimulatory effects of physiologically active substances on RNA synthesis is discussed.     

The 25 kDa protein kinase inhibitor present in liver cell is absent in fast growing HTC cells and is induced in sodium butyrate treated cells

We have recently characterized a cAMP independent protein kinase inhibitor in rat liver. This inhibitor is absent or inactive in fast growing HTC cells and is induced according to exponential kinetics by sodium butyrate, a compound which arrests cell growth at the G1 phase of the cell cycle. It is suggested that the inhibitor could be involved in cell growth regulation.     

Topography of protein kinases and phosphoproteins in the plasma membrane of 3T3 cells

The plasma membrane of 3T3 cells contains at least two different endogenous cyclic AMP-dependent protein kinase systems. One catalyzes the phosphorylation of endogenous protein substrates, i.e., PP24 and PP14, whereas the other catalyzes the phosphorylation of exogenous substrates. In this paper the topography of these cyclic AMP-dependent phosphorylation systems is described. The results show that the kinases which phosphorylate only exogenous substrates are primarily localized to the outer plasma membrane surface whereas the endogenous cyclic AMP-dependent protein kinase and its two endogenous substrates are localized to the cytoplasmic plasma membrane surface. The data also establish that neither the cytoplasmically orientated kinase nor its substrates has a transmembrane orientation even though factors acting on the outer plasma membrane can affect these proteins. This suggests that functional modulation of the cytoplasmically localized cyclic AMP-dependent phosphorylation system can be mediated by a transmembrane regulatory mechanism. The importance of determining the topography of such plasma membrane phosphorylation systems is emphasized by recent studies which show that neoplastic transformation can be mediated at least in part by protein kinases and/or phosphoproteins which are localized on the cytoplasmic surface of the plasma membrane.     

Chromatin protein kinases and phosphoproteins during myoblast growth and differentiation

A photolabile derivative of α-bungarotoxin which binds specifically to $\text{Torpedo}\text{californica}$ acetylcholine receptor has been used to investigate the topography of the membrane associated protein. It is shown that the toxin can be crosslinked to a polypeptide of 40,000 daltons, to which it is known to bind, and in addition to another polypeptide of 65,000 daltons which is a major constituent of the membrane. The results substantiate the notion that this nicotinic acetylcholine receptor is composed of different polypeptides and that some of these interact with each other or are in close proximity on the exterior surface of the post-synaptic membrane.     

Localization of chromatin in "persistent" nucleoli in okadaic acid treated HeLa cells.

In okadaic acid treated HeLa cells, the chromosomes sometimes condense without being accompanied by nuclear envelope breakdown. These cells show "persistent" nucleoli. Within these "persistent" nucleoli the intranucleolar chromatin condenses and can be observed in the region of the dense nucleolar component (DNC) of the nucleoli. Other nucleolar components, namely the fibrillar centre (FC) and the granular component (GC) remain unchanged. These observations strongly speak for the localization of nucleolar chromatin (ribosomal cistrons) within the dense nucleolar component of the interphase nucleolus.     

Ornithine decarboxylase protein diversity and activity modulation in HTC cells

Ornithine decarboxylase of HTC cells was chromatographically separated into three ionically distinct but kinetically similar forms of this protein. The sequential appearance of these ornithine decarboxylase species during enzyme induction, and the accumulation of normally minor species under conditions that stabilize this enzyme, suggest that these represent modifications that are associated with the extremely rapid turnover of this protein $\text{in vivo}$. These forms may also be differentially active or unequally distributed $\text{in vivo}$ as indicated by the selective inactivation of one of the forms by short exposure to α-difluoromethylornithine.     

Glucocorticoid hormones are successively present in two sites with different accessibilities to nucleases in chromatin from HTC cells

Glucocorticoid hormones induce tyrosine aminotransferase synthesis in HTC cells after a lag of approximately 2 h. The question arises whether this lag corresponds to chromatin modifications related to the binding of the hormone. We have analyzed the accessibility to DNases of the hormone binding sites in chromatin from HTC cells incubated for various times in the presence of the hormone. After short incubations the hormone-receptor binding sites in chromatin are accessible to DNases, after longer incubations they become resistant to DNase digestion. The kinetics of the release in vivo of the hormone-receptor complex in an hormone free medium suggests the existence of 2 discrete classes of hormone binding sites in chromatin.     

Rearrangements of chromatin structure in newly repaired regions of deoxyribonucleic acid in human cells treated with sodium butyrate or hydroxyurea

The rate and extent of redistribution of repair-incorporated nucleotides within chromatin during very early times (10-45 min) after ultraviolet irradiation were examined in normal human fibroblasts treated with 20 mM sodium butyrate, or 2-10 mM hydroxyurea, and compared to results for untreated cells. Under these conditions, DNA replicative synthesis is reduced to very low levels in each case. However, DNA repair synthesis is stimulated by sodium butyrate and partially inhibited by hydroxyurea. Furthermore, in the sodium butyrate treated cells, the core histones are maximally hyperacetylated. Using methods previously described by us, it was found that treatment with sodium butyrate had little or no effect on either the rate or the extent of redistribution of repair-incorporated nucleotides during this early time interval. On the other hand, there was a 1.7-2.5-fold decrease in the rate of redistribution of these nucleotides in cells treated with hydroxyurea; the extent of redistribution was unchanged in these cells. Since hydroxyurea has been shown to decrease the rate of completion of "repair patches" in mammalian cells, these results indicate that nucleosome rearrangement in newly repaired regions of DNA does not occur until after the final stages of the excision repair process are completed. Furthermore, hyperacetylation of the core histones in a large fraction of the total chromatin prior to DNA damage and repair synthesis does not appear to alter the rate or extent of nucleosome core formation in newly repaired regions of DNA.     

Myosin-activating protein kinases in human myocardium: Localization and content

Using the immunofluorescence approach, we have determined that the recently detected protein kinases, among which are RhoA-activated kinase, integrin-linked kinase, zipper interacting protein kinase, and death-associated protein kinase, are capable of phosphorylating myosin localized in the Z-lines of human myocardial sarcomeres. Additionally, we studied the content of integrin-linked and zipper interacting protein kinases in human embryonic myocardium, as well as in normal and hypertrophic adult human heart. The content of these protein kinases in adult normal myocardium increases in comparison with the embryonic heart. The content of integrin-linked and zipper interacting protein kinases in hypertrophic myocardium is higher as compared with the normal adult heart. The data obtained suggest the involvement of these protein kinases in the development and hypertrophy of the human heart.     

Protein kinases and their protein substrates associated with chromatin and ribosomes in SV40-transformed rat cells.

The level of endogenous protein phosphorylation in non-histone chromosomal and ribosomal wash proteins is 7--10 times greater in SV40-transformed rat cells than in untransformed parental cells. Protein kinase activity in these proteins was fractionated by either phosphocellulose or DEAE-cellulose chromatography. One major and one minor component were detected in non-histone proteins and only one component in ribosomal wash proteins when the activity in each fraction was measured with an exogenous substrate, casein. These enzymes prefer casein to whole histone as substrate and are cyclic AMP-independent. The enzyme activity in a major peak of non-histone proteins and in ribosomal wash proteins measured with casein as substrate is 3 times greater in transformed cells than in untransformed cells, whereas pH optimum, cation requirements and apparent Km values for casein and ATP are identical or very similar in the two cell types. No significant phosphatase was detected in non-histone and ribosomal wash proteins from the two types of cell. The patterns of endogenous protein phosphorylation in these protein fractions analysed by gel electrophoresis are significantly different between these cells. These results suggest that the high level of endogenous protein phosphorylation in non-histone and ribosomal wash proteins from SV40-transformed cells is caused mainly by the increased activity of protein kinase and the nature of protein substrates.     

The isolation of HTC variant cells which can replicate in butyrate. Changes in histone acetylation and tyrosine aminotransferase induction

We have obtained a number of variant HTC cells which are capable of vigorous replication in the presence of 6 mM sodium butyrate. These cells show characteristic changes in histone acetylation. H2A/H2B are no longer modified and the turnover of histones H3/H4 acetate is about 4-fold greater than in control HTC cells at the same butyrate concentration. Histone deposition continues successfully even though histones H3/H4 become hyperacetylated upon association with the chromatin. Prompt deacetylation of new histones does not appear to be a prerequisite for successful deposition processes. Initial enzymatic studies indicate that not only do the butyrate-resistant cells show an increased deacetylase activity (on a per cell basis), but also the enzyme is less sensitive to sodium butyrate under in vitro assay conditions. In contrast to control HTC cells in 6 mM butyrate in which dexamethasone induction of tyrosine aminotransferase is inhibited, the butyrate-resistant variant cells are capable of tyrosine aminotransferase induction even in the presence of butyrate. The implications of these observations are discussed.     

Localization, solubilization and characterization of plant membrane-associated calcium-dependent protein kinases

Membrane fractions from mature silver beet (Beta vulgaris) deveined leaf and leaf stem homogenates have associated Ca²⁺ -dependent protein kinase. The Ca²⁺ -dependent protein kinase activity is associated with plasma membranes (density 1.14-1.18 grams per cubic centimeter) as determined from copurification on isopycnic centrifugation with plasma membrane markers such as β-glucan synthetase, eosin-5-maleimidelabeling, and specific naphthylphthalamic acid-binding. The Ca²⁺ -dependent protein kinase is not specifically associated with chloroplasts or mitochondria. The membrane-bound Ca²⁺ -dependent protein kinases were solubilized with 0.8% (volume/volume) Nonidet P40. The solubilized enzymes were extensively purified by a protocol involving binding to diethylaminoethyl-cellulose (Whatman DE-52), Ca²⁺ -dependent binding to phenyl-Sepharose CL-4B, gradient elution from diethylaminoethyl-Sephacel (resolving two distinct Ca²⁺ -dependent protein kinases), and gel filtration on Ultrogel AcA 44. These two membrane-derived enzymes have similar molecular weights but differ in protein substrate specificity, in K_m values for ATP, and in Ca²⁺ -independent activation by unsaturated fatty acids. The membrane-bound enzymes correspond closely in these properties to two Ca²⁺ -dependent protein kinases present in the soluble phase.     

Characterization of aldehyde dehydrogenase from HTC rat hepatoma cells

We have proposed developing rat hepatoma cell lines as an in vitro model for studying the regulation of changes in aldehyde dehydrogenase activity occurring duringhepatocarcinogenesis. Aldehyde dehydrogenase purified in a single step from HTC rat hepatoma cells is identical to the aldehyde dehydrogenase isolated from rat hepatocellular carcinomas. HTC aldehyde dehydrogenase is a 110 kDa dimer composed of 54-kDa subunits, prefers NADP⁺ as coenzyme, and preferentially oxidizes benzaldehyde-like aromatic aldehydes but not phenylacetaldehyde. The substrate and coenzyme specificity, effects of disulfiram, pH profile and isoelectric point of HTC aldehyde dehydrogenase are also identical to these same properties of the tumor aldehyde dehydrogenase. In immunodiffusions, both isozymes are recognized with complete identity by anti-HTC aldehyde dehydrogenase antibodies. Having established that HTC aldehyde dehydrogenase is very similar, if not identical, to the aldehyde dehydrogenase found in hepatocellular carcinomas, simplifies the development of molecular probes for examination of the regulation of tumor aldehyde dehydrogenase activity in vivo and in vitro.     

Characterization of aldehyde dehydrogenase from HTC rat hepatoma cells

We have proposed developing rat hepatoma cell lines as an in vitro model for studying the regulation of changes in aldehyde dehydrogenase activity occurring during hepatocarcinogenesis. Aldehyde dehydrogenase purified in a single step from HTC rat hepatoma cells is identical to the aldehyde dehydrogenase isolated from rat hepatocellular carcinomas. HTC aldehyde dehydrogenase is a 100 kDa dimer composed of 54-kDa subunits, prefers NADP+ as coenzyme, and preferentially oxidizes benzaldehyde-like aromatic aldehydes but not phenylacetaldehyde. The substrate and coenzyme specificity, effects of disulfiram, pH profile and isoelectric point of HTC aldehyde dehydrogenase are also identical to these same properties of the tumor aldehyde dehydrogenase. In immunodiffusion, both isozymes are recognized with complete identity by anti-HTC aldehyde dehydrogenase antibodies. Having established that HTC aldehyde dehydrogenase is very similar, if not identical, to the aldehyde dehydrogenase found in hepatocellular carcinomas, simplifies the development of molecular probes for examination of the regulation of tumor aldehyde dehydrogenase activity in vivo and in vitro.     

Localization of catalytic and regulatory subunits of cyclic AMP-dependent protein kinases in mitochondria from various rat tissues.

Observation and quantification of the catalytic subunit C of cyclic AMP-dependent protein kinases by immuno-gold electron microscopy suggested a high concentration of cyclic AMP-dependent protein kinases in mitochondria from liver, kidney, heart and skeletal muscle, pancreas, parotid gland and brain cells. The position of gold particles pointed to a localization in the inner membrane/matrix space. A similar distribution was obtained by immunolocalization of the cyclic AMP-dependent protein kinase regulatory subunits RI and RII in liver, pancreas and heart cells. The results indicated the presence of both the type I and the type II cyclic AMP-dependent protein kinases in mitochondria of hepatocytes, and the preferential occurrence of the type I protein kinase in mitochondria from exocrine pancreas and heart muscle. The immunocytochemical results were confirmed by immunochemical determination of cyclic AMP-dependent protein kinase subunits in fractionated tissues. Determinations by e.l.i.s.a. of the C-subunit in parotid gland cell fractions indicated about a 4-fold higher concentration of C-subunit in the mitochondria than in a crude 1200 g supernatant. Immunoblot analysis of subfractions from liver mitochondria supported the localization in situ of cyclic AMP-dependent protein kinases in the inner membrane/matrix space and suggested that the type I enzyme is anchored by its regulatory subunit to the inner membrane. In accordance with the immunoblot data, the specific activity of cyclic AMP-dependent protein kinase measured in the matrix fraction was about twice that measured in whole mitochondria. These findings indicate the importance of cyclic AMP-dependent protein kinases in the regulation of mitochondrial functions.     

Polyamines alter the phosphorylation pattern of chromatin proteins by endogenous protein kinases

The effect of polyamines on the chromatin phosphorylation by endogenous protein kinases was investigated. Polyamines not only selectively stimulated the phosphorylation of chromatin proteins but also concurrently inhibited the phosphorylation of a number of polypeptides. In particular, a 11,000-dalton polypeptide with pI 4.5–5.0 was highly phosphorylated in the absence of polyamines, despite being a minor component whereas the phosphorylation was strongly inhibited in the presence of polyamines.     

Localization of antigens in the chromatin of normal rat liver cells, not detectable in hepatoma chromatin

Antibodies against rat liver chromatin interact with homologous chromatin as well as with chromatin of Zajdela ascite hepatoma and solid hepatoma 27, but not with the nuclear matrix isolated from these hepatomas. Rat liver chromatin regions hypersensitive to DNAase I and endogenous Mg2+-dependent nuclease are enriched with immunogenic nonhistone proteins. Using antiliver IgG pretreated with chromatin of Zajdela ascite hepatoma and solid hepatoma 27, it was shown that liver chromatin antigens that are not detectable in hepatoma cells are localized in hypersensitive to nucleases chromatin regions buy not in actively transcribed ones.