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At least 19 recordsLinked to original sources
PubMedDate not supplied

[Stimulation with triiodothyronine of the synthesis of DNA, total proteins, nuclear proteins and nuclear matrix proteins in cultured cells of hepatoma Morris 7777].

Intensity of DNA and protein biosynthesis was decreased in Morris hepatoma 7777 cultivated cells in absence of thyroid hormones. Physiological concentrations of triiodothyronine increased synthesis of DNA and total proteins after lag phase within 12-48 hrs, while synthesis of nuclear and nuclear matrix proteins was stimulated already within 2 hrs. This suggests that stimulation of nuclear proteins biosynthesis occurred prior to the hormone effect on proliferation and cell metabolism. Alterations in sensitivity of chromatin to DNAase I were not observed. Response of the Morris hepatoma cells to thyroid hormones may be used in studies of molecular mechanisms of thyroid hormones action.

Amino Acids↗
PubMed1981

Binding of [3H]benzo(a)pyrene metabolites to the AKR mouse embryo cell line nuclear proteins.

Nuclear proteins from AKR-2B mouse embryo cells incubated 24 hr with [3H]benzo(a)pyrene (1 microgram/ml; 10 microCi/ml) were purified to remove nucleic acids and characterized by isoelectric focusing and sodium dodecyl sulfate gel electrophoresis on polyacrylamide gels. Analysis of the nuclear proteins by isoelectric focusing revealed a distribution of protein in the pH gradient with two prominent bands of proteins, one focusing at pH 5 and the other at pH 11, both of which bound significant amounts of the carcinogen. The acid-soluble proteins from the nuclear preparation were analyzed by isoelectric focusing, and the results also revealed two peaks of protein and radioactivity at pH's of 5 and 11. Further analysis of the acid-soluble fraction on acid urea gels revealed that significant radioactivity was associated with proteins that migrated near but slower than histone H1 which may be the high-mobility group proteins 1 and 2. Radioactivity also comigrated with histones H3 and H2B. Low levels of radioactivity were associated with histone H1 and H4.

Animals↗
PubMed1986

Spermine inhibits the phosphorylation of the 11,000- and 10,000-dalton nuclear proteins catalyzed by nuclear protein kinase NI in NB-15 mouse neuroblastoma cells.

The nuclear protein kinase NI (NI kinase) was purified from NB-15 mouse neuroblastoma cells by phosphocellulose column and casein affinity column chromatography. The purified NI kinase exhibited (i) an apparent subunit molecular weight of about 37,000, (ii) autophosphorylation, and (iii) insensitivity to inhibition by heparin. When NI kinase was added to heat-treated neuroblastoma nuclei in the presence of [gamma-32P] ATP, two proteins with apparent subunit molecular weights of 11,000 and 10,000 were prominently phosphorylated. Other protein kinases tested including the nuclear protein kinase NII, Type I cAMP-dependent protein kinase, and protein kinase C did not catalyze the phosphorylation of these two proteins. The NI kinase-catalyzed phosphorylation of these two proteins was completely inhibited by 1 mM spermine. In contrast, 10 mM putrescine, 2 mM spermidine, 5 mM arginine, and 10 mM NH4Cl, had no inhibitory effect on this phosphorylation reaction. Our study also indicated that the phosphorylation of the 11,000- and 10,000-dalton proteins occurred in the nuclear matrix fraction but not in heterogeneous nuclear ribonucleoproteins, high mobility group proteins, or histone fractions. We have previously reported that spermine specifically inhibits the endogenous phosphorylation of an 11,000-dalton nuclear protein in various mammalian cell lines (Chen, K. Y., and Verma, R. (1984) Biochem. Biophys. Res. Commun. 118, 710-716). The present study suggests that the 11,000- and 10,000-dalton nuclear proteins may be native substrates of nuclear protein kinase NI and that their phosphorylation can be affected by physiological concentrations of spermine.

Animals↗
PubMed1988

Identification of the human papillomavirus type 18 E6 and E6 proteins in nuclear protein fractions from human cervical carcinoma cells grown in the nude mouse or in vitro.

We recently reported the transcription patterns of human papillomavirus (HPV) type 18 sequences in human cervical carcinoma cell lines. The open reading frames (ORFs) E6* and E6 represent the 5'-terminal cistrons in HPV18 mRNAs. ORF E6* was assumed to be specific for HPV types associated with genital carcinomas. To identify the predicted gene product, ORF E6* from a HeLa cDNA clone was expressed as an MS2 fusion protein in Escherichia coli. The C-terminal 23 amino acid residues were chemically synthesized. A panel of monoclonal antibodies was generated, recognizing E6* and E6* plus E6, respectively. In human cervical carcinoma cell lines grown in vitro these monoclonal antibodies specifically immunoprecipitate the putative Mr 17,000 and 18,000 HPV18 E6 proteins in nuclear protein fractions. In a HPV18 DNA containing human cervical carcinoma established in nude mice, these monoclonal antibodies specifically immunoprecipitate a polypeptide with a molecular weight of 6500 as predicted for the HPV18 ORF E6* gene product in a nuclear protein fraction.

Animals↗
PubMed1982

Ethionine inhibits in vivo methylation of nuclear proteins.

Nuclear protein methylation was studied in regenerating rat liver by giving [methyl-3H]methionine 45 h after partial hepatectomy. Ethionine, a liver carcinogen, has been shown to alter the methylation patterns in a basic protein (histone) fraction, as well as an acidic protein (non-histone) fraction present in a 0.25 N HCl nuclear extraction. The proteins present in the 0.25 N HCl extraction were separated by chromatography using a Bio-Rex 70 cation exchange column. Polyacrylamide gel electrophoresis and total amino acid analysis showed the first protein fraction contained acidic large molecular weight non-histone proteins, while the second fraction contained basic small molecular weight histone proteins. Both fractions were then hydrolyzed, and the amino acids chromatographed on an Aminex A-5 cation exchange column. The histones were found to contain epsilon-N-mono, di and trimethyllysine derivatives; whereas the non-histone fraction contained these lysine derivatives and additional basic amino acid identified as NG,NG-dimethylarginine. Ethionine (0.5 mg/g body weight) was found to inhibit in vivo methylation of lysine to form epsilon-N-mono, di and trimethyllysine, 46, 52 and 68%, respectively. The formation of NG,NG-dimethylarginine was inhibited by 85%. Ethylation of these proteins was also studied by giving [ethyl-3H]ethionine. After hydrolysis, the non-histones were found to contain a labeled lysine and arginine derivative, but in the histone fraction only labeled lysine was found.

Animals↗
PubMed1979

Comparative aspects of nuclear proteins and nuclear composition of cultivated embryonic, neonatal and neoplastic rat brain cells of glial origin.

Differences in nuclear composition and variations in the types of acid soluble nuclear proteins were identified when cultivated neoplastic and nontransformed rat brain cells were compared. HeLa S-3 cells were used as a biological reference in these studies. The nuclei of anaplastic glioma cells were found to contain more total nuclear protein and greater amounts of nuclear RNA, than the nuclei of nontransformed embryonic and neonatal rat brain cells. Densitometer profiles of samples developed by polyacrylamide gel electrophoresis indicated that all three major histone classes were present in the nuclei of the four different cell types examined. Each type of cell was grown in the presence of 3H-lysine or 3H-lysine plus 14C-arginine to further characterize both the histones and the acid soluble nonhistone proteins. Neoplastic and nontransformed cells contained similar quantities of histones H4, H3, H2A and H2B. In contrast, transformed cells were found to contain two dominant subspecies of lysine rich H1 histone, while only one histone H1 subcomponent was extracted from the nuclei of the embryonic and neonatal rat brain cells. Nuclei isolated from HeLa cells and anaplastic glioma cells also contained increased varieties and larger quantities of acid soluble nonhistone nuclear proteins.

Animals↗
PubMed2000

A method for the large-scale cloning of nuclear proteins and nuclear targeting sequences on a functional basis.

We describe here a selection strategy allowing the cloning of sequences that contain a functional nuclear targeting signal. Our method relies on the use of green fluorescent protein fusion proteins to identify nuclear targeting sequences. Transfected cells expressing nuclear protein fusions were isolated on the basis of their nuclear fluorescence using flow cytometry and the transfected DNAs were recovered after bacterial transformation with total DNA from pools of sorted cells. Starting from a cDNA expression library, in which only 1% of the expressed proteins were nuclear, we obtained a 70-fold enrichment in nuclear protein-encoding clones after a single round of selection. Among the 63 clones that have been partially sequenced to date, 25 (40%) corresponded to known nuclear proteins and 13 (20%) to previously uncharacterized sequences. Despite their ability to target the green fluorescent protein marker to the cell nucleus, about half of the cloned sequences did not encode canonical basic or bipartite nuclear localization signals. The method can thus be applied to the large-scale cloning of functional nuclear targeting sequences, which opens the way to a wide investigation of nuclear import mechanisms and to the identification of previously unknown nuclear proteins.

Animals↗
PubMed1980

Preferential phosphorylation of basic non-histone proteins by nuclear protein kinase NII from rat liver.

Rat liver nuclear protein kinase NII, which is independent of cyclic nucleotides, phosphorylated both the acidic protein casein and the basic histone preparation, soluble in 0.25 M HCl, which was extracted from the cell nuclei. When the isolated nuclei were incubated with [32P]ATP in the presence of protein kinase NII, more than 95% of the 32P-labelled proteins was recovered in the 0.25 M HCl extract. In order to analyze the substrate proteins of protein kinase NII, a histone preparation free from the endogenous protein kinase activities was used as substrate. The results demonstrated that histones were not phosphorylated, except for a faint 32P incorporation into the H3 fraction. Instead, multiple non-histone proteins were highly phosphorylated, which were present in a minor quantity in the histone preparation. The molecular weights of the main phosphorylated proteins were 72000, 68000, 56000, 47000, 46000, 43000, 38000 and 32000. Isoelectric focusing demonstrated the basic nature of the phosphorylated proteins, and as much as 72% of 32P radioactivity was distributed in the pH region higher than 7.0. Furthermore, many proteins phosphorylated in the isolated nuclei with added protein kinase NII were also found to be basic non-histone proteins. These results indicate that protein kinase NII preferentially phosphorylates in vitro a set of nuclear basic non-histone proteins.

Animals↗
PubMed1997

High levels of the GTPase Ran/TC4 relieve the requirement for nuclear protein transport factor 2.

The GTPase Ran/TC4 and the 14-kDa protein nuclear transport factor 2 (NTF2) are two of the cytosolic factors that mediate nuclear protein import in vertebrates. Previous biochemical studies have shown that NTF2 binds directly to the GDP-bound form of Ran/TC4 and to proteins of the nuclear pore complex that contain phenylalanine-glycine repeats. In the present study we have used molecular genetic approaches to study the Saccharomyces cerevisiae homologue of NTF2. The scNTF2 gene encodes a protein that is 44% identical to the human protein. We found that deletion of the scNTF2 gene is lethal and that repression of NTF2p expression by a regulatable promoter results in gross structural distortions of the nuclear envelope. In a screen for high copy number suppressors of a scNTF2 deletion, the only gene we isolated other than scNTF2 itself was GSP1, the S. cerevisiae homologue of Ran/TC4. Furthermore, we found that high levels of Ran/TC4 can relieve the requirement for NTF2 in a mammalian-permeabilized cell assay for nuclear protein import. These data suggest that certain of the nuclear protein import functions of NTF2 and Ran/TC4 are closely linked and that NTF2 may serve to modulate a transport step involving Ran/TC4.

Biological Transport↗
PubMed1976

Nuclear proteins. II. Similarity of nonhistone proteins in nuclear sap and chromatin, and essential absence of contractile proteins from mouse liver nuclei.

High resolution SDS slab gel electrophoresis has been used to examine the distribution of nonhistone proteins (NHP) in the saline-EDTA, Tris, and 0.35 M NaCl washes of isolated mouse liver nuclei. These studies led to the following conclusions: (a) all the prominent NHP which remain bound to DNA are also present in somewhat similar proportions in the saline-EDTA, Tris, and 0.35 M NaCl washes of nuclei; (b) a protein comigrating with actin is prominent in the first saline-EDTA wash of nuclei, but present as only a minor band in the subsequent washes and on washed chromatin; (c) the presence of nuclear matrix proteins in all the nuclear washes and cytosol indicates that these proteins are distributed throughout the cell; (d) a histone-binding protein (J2) analogous to the HMG1 protein of K. V. Shooter, G.H. Goodwin, and E.W. Johns (Eur J. Biochem. 47:236-270) is a prominent nucleoplasmic protein; (e) quantitation of the major NHP indicates that they are present in a range of 2.2 X 10(5)-5.2 X 10(6) copies per diploid nucleus. Most of the electrophoretically visible NHP are probably structural rather than regulatory proteins; (f) actin, myosin, tubulin, and tropomyosin, if present at all, constitute a very minor fraction of the nuclear NHP. Contractile proteins constitute a major portion of the NHP only when the chromatin is prepared from crude cell lysates instead of from purified nuclei. These studies support the conclusion that there are no clear differences between many nucleoplasmic and chromatin-bound nonhistone proteins. Except for the histones, many of the intranuclear proteins appear to be in equilibrium between DNA, HnRNA, and the nucleoplasm.

Actins↗
PubMed1992

A mutant nuclear protein with similarity to RNA binding proteins interferes with nuclear import in yeast.

We have isolated mutants of the yeast Saccharomyces cerevisiae that are defective in localization of nuclear proteins. Chimeric proteins containing the nuclear localization sequence from SV40 large T-antigen fused to the N-terminus of the mitochondrial F1 beta-ATPase are localized to the nucleus. Npl (nuclear protein localization) mutants were isolated by their ability to grow on glycerol as a consequence of no longer exclusively targeting SV40-F1 beta-ATPase to the nucleus. All mutants with defects in localization of nucleolar proteins and histones are temperature sensitive for growth at 36 degrees C. Seven alleles of NPL3 and single alleles of several additional genes were isolated. NPL3 mutants were studied in detail. NPL3 encodes a nuclear protein with an RNA recognition motif and similarities to a family of proteins involved in RNA metabolism. Our genetic analysis indicates that NPL3 is essential for normal cell growth; cells lacking NPL3 are temperature sensitive for growth but do not exhibit a defect in localization of nuclear proteins. Taken together, these results indicate that the mutant forms of Npl3 protein isolated by this procedure are interfering with nuclear protein uptake in a general manner.

Amino Acid Sequence↗
PubMedDate not supplied

Effects of mitogen-activated protein kinases on nuclear protein import.

ERK-2 MAP kinase activation induces inhibitory effects on nuclear protein import in vascular smooth muscle cells. The mechanism and characteristics of this effect of ERK-2 were investigated. An unusual dose-dependent effect of ERK-2 on nuclear protein import was identified. At higher concentrations (1 microg/mL) of ERK-2, nuclear protein import was stimulated, whereas lower concentrations (0.04 microg/mL) inhibited import. Intermediate concentrations exerted intermediate effects. The stimulatory and inhibitory effects at the 2 different ERK-2 concentrations were observed in both conventional, permeabilized cell assays of nuclear protein import and with in situ microinjection of smooth muscle cells. The biphasic effects of ERK-2 on import were also found for the other 2 members of the MAPK family, p38 and JNK. RanGAP was identified by structural analysis as a candidate target protein responsible for mediating the effects of ERK-2. After pretreatment with high concentrations of ERK-2, RanGAP activity was significantly increased by approximately 50%. In contrast, low concentrations of ERK-2 significantly attenuated RanGAP activity. These results demonstrate that all 3 members of the MAPK family can alter nuclear protein import in opposite directions depending upon the concentration of ERK-2 used. RanGAP represents the MAP kinase target whereby nuclear transport can be stimulated or inhibited.

Active Transport, Cell Nucleus↗
PubMed1985

Hormonal control of the phosphorylation of histones, HMG proteins and other nuclear proteins.

Hormone-dependent phosphorylation and dephosphorylation of nuclear proteins may play an important part in regulating nuclear function and specific gene expression. Some progress has been made in identifying specific nuclear proteins whose phosphorylation is affected by specific hormones; however, relatively little is known about the regulatory mechanisms involved, or about the molecular consequences of increased or decreased phosphorylation. It is suspected--but not yet proved--that cAMP-dependent effects on transcription are mediated at least partly by increases in nuclear cAMP-dependent protein kinase (A-kinase) activity, and consequent increases in the phosphorylation of specific chromatin proteins. In several instances, increased phosphorylation has been found to precede or correlate with cAMP-mediated induction of specific gene products. Several chromatin proteins are susceptible to cAMP-dependent phosphorylation in vivo, including histones H1 and H3, the high mobility group protein HMG 14 (which is preferentially associated with actively transcribed chromatin), and at least three other basic nonhistone proteins. The A-kinase phosphorylation sites of the majority of H1, H3 and HMG 14 molecules in chromatin appear to be inaccessible to A-kinase in vivo; nothing is known about the factors determining their accessibility, which may be tightly regulated and may vary significantly from cell to cell and tissue to tissue. Many hormone-induced changes in nuclear protein phosphorylation may be cAMP-independent. cAMP-independent mechanisms could involve a variety of nuclear enzymes including, for example, cGMP-dependent, Ca2+/calmodulin-dependent, Ca2+/phospholipid-dependent and polyamine-dependent protein kinases. So far, however, there is little solid evidence in support of a role for any specific cAMP-independent protein kinase in mediating hormonally induced increases in the phosphorylation of specific, identified nuclear proteins.

Animals↗
PubMed1998

Identification of specific nuclear protein kinase C isozymes and accelerated protein kinase C-dependent nuclear protein phosphorylation during myocardial ischemia.

Protein kinase C (PKC) has been suggested to mediate, at least in part, multiple processes in the pathophysiological sequelae of myocardial ischemia. The present study demonstrates that the epsilon, eta and iota isozymes of PKC are translocated to nuclei in response to brief intervals of global ischemia as well as reperfusion of ischemic rat myocardium. Concomitant with the translocation of PKC isozymes to nuclei during ischemia, increased PKC-mediated nuclear protein phosphorylation was observed. Taken together, the present results demonstrate that nuclear signaling mechanisms are activated during myocardial ischemia that include PKC translocation and PKC-mediated nuclear protein phosphorylation.

Animals↗
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↗
PubMed1986

In vitro transport of a fluorescent nuclear protein and exclusion of non-nuclear proteins.

An in vitro system was developed that provides a quick microscopic assay for nuclear transport. The assay uses an extract of Xenopus eggs, normal or synthetic nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. This in vitro system accurately mimics in vivo nuclear transport, both in exclusivity and in the amount of accumulation observed (up to 17-fold). Selective accumulation of fluorescent nucleoplasmin is observed microscopically within 30 min with rat liver nuclei, Xenopus embryonic nuclei, regrown Xenopus sperm nuclei, or nuclei reconstituted in vitro from bacteriophage lambda DNA. This transport requires the signal domain of nucleoplasmin. Furthermore, the ability of nuclei to accumulate nucleoplasmin directly correlates with their ability to exclude the fluorescent non-nuclear proteins, FITC-immunoglobulin and phycoerythrin. An active transport model would predict that nuclear transport be temperature- and energy-dependent and that inhibition of transport by either low temperature or energy depletion would be reversible. Both predictions were confirmed in our system. Nucleoplasmin accumulation increases with temperature, while the protein is completely excluded at 0 degrees C. The effects of low temperature are reversible. As found for 125I-labeled nucleoplasmin (Newmeyer, D. D., J. M. Lucocq, T. R. Bürglin, and E. M. De Robertis, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:501-510), transport of fluorescent nucleoplasmin is inhibited by ATP depletion. This effect is reversed by later ATP addition. Under ATP-depleted conditions non-nuclear proteins continue to be excluded. These results argue for a direct role of ATP in transport rather than for a simple role in preserving envelope integrity. In a first step towards defining the minimum requirements for a transport medium, egg extracts were depleted of membrane vesicles. Membrane-depleted extracts neither support transport nor maintain the integrity of the nuclear envelope.

Animals↗