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W Pyerin

Publications and source records attributed to W Pyerin.

At least 37 records · Page 2Linked to original sources
PubMed1996

Promoter of the gene encoding the bovine catalytic subunit of cAMP-dependent protein kinase isoform C beta 2.

Genomic sequences flanking the 5' end of the cDNA encoding isoform C beta 2 of the catalytic subunit of bovine cAMP-dependent protein kinase were cloned, sequenced and analyzed for promoter activity and transcription initiation sites. A region of 913 bp upstream the translation initiator ATG was amplified from genomic DNA by vectorette polymerase chain reaction. In primer extension reactions and RNase protection assays, residues C (at position -91), T (-71) and G (-70) were found to serve as transcription initiation sites of the gene. Amplification products and sub-fragments thereof were ligated upstream of the reporter gene chloramphenicol acetyltransferase to test for promoter activity. Constructs were transiently transfected into a Chinese hamster ovary cell line which was shown to express endogenous C beta 2 mRNA. The genomic sequence upstream the C beta 2 cDNA does have promoter activity. The region from position -51 to -292 proved sufficient to drive efficient transcription of the reporter gene. The promoter is AT rich (68%), does not contain a TATA box within 50 bp upstream of the first initiation site and possesses putative binding sites for several transcription factors such as PEA-3 and a glucocorticoid receptor.

Animals↗
PubMed1996

Interaction sites between catalytic and regulatory subunits in human protein kinase CK2 holoenzymes as indicated by chemical cross-linking and immunological investigations.

Protein kinase CK2, a heterotetramer composed of two catalytic subunits (alpha and/or alpha') and two regulatory subunits (beta), has been examined for intermolecular contact sites by methods that allow investigation of the native, unaltered proteins. Antibodies were raised against a series of 11 subunit peptides, affinity purified, and ensured for site specific binding by peptide competition. Chemical cross-linking of CK2 subunits with a hydrophilic carbodiimide and analysis of fused subunits and of CNBr-digested fusion products by immunoblotting with the sequence specific antibodies identified a tight interaction between positions beta55-70 and alpha65-80 (alpha'66-81) of subunits beta and alpha (alpha'), respectively. This was corroborated by cross-linking of subunits with peptides alpha65-80 and beta55-70 by a peptide-based enzyme-linked immunosorbent assay in which peptides bound to wells via C-10 spacer arms are probed for complexing individual subunits and immunoprecipitation with antibodies anti-alpha65-80 and anti-beta55-70, resulting in precipitation but not coprecipitation of subunits. This alpha-beta (alpha'-beta) interaction site obviously is also of functional importance since subunits with attached antibodies cannot reconstitute to the fully active holoenzyme. Indeed, sites beta55-70 and alpha65-80 (alpha'66-81) correspond to an acidic (beta) and a basic (alpha or alpha') domain involved in activity and stability control and in substrate and cosubstrate binding (kinase domain II/III), respectively. By contrast, a number of suspected contact sites were found to be rather loose and not essential for enzyme control as concluded from precipitation behavior of respective antibodies and the toleration of attached antibodies when active holoenzymes were being constituted. At subunit beta, these include the terminal positions beta2-14 and beta204-213, the positions beta97-105 and beta140-156, and, surprisingly, also beta171-186 which have been shown by deletion mutation and peptide replacement studies to represent a positively affecting interaction site. At subunits alpha and alpha', these are the C-terminal positions alpha329 -343 and alpha'336-350. Binding of antibodies to the positions alpha15-27 (alpha'16-28) and position alpha151-166(alpha'152-167), on the other hand, inhibits activity.

Amino Acid Sequence↗
PubMed1996

Induced release of cell surface protein kinase yields CK1- and CK2-like enzymes in tandem.

Several types of cell exhibit cell surface protein kinase (ecto-PK) activities with Ser/Thr-specificity. Ecto-PK sharing certain characteristics of protein kinase CK2 can be detached from intact cells by interaction with exogenous substrates (Kübler, D., Pyerin, W., Burow, E., and Kinzel, V. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4021-4025). However, a detailed molecular analysis of this ecto-PK was hampered by the vanishingly small amounts of labile enzyme protein obtained by substrate-inducible enzyme release. We now describe the stabilization and enrichment of released ecto-PK by precipitation with polyethylene glycol followed by affinity chromatography on heparin-agarose. Ecto-PK is shown to consist of two separate forms released in tandem, ecto-PK I and ecto-PK II. Comparison with cell homogenates as well as cell surface biotinylation experiments excluded contamination with intracellular PK. Purified ecto-PK I and ecto-PK II exhibit respectively selective phosphorylation of CK1- and CK2-specific peptide substrates, a complementary sensitivity to inhibitory agents and a differential use of the cosubstrates ATP and GTP. Ecto-PK I consists of a 40-kDa moiety; the ecto-PK II is an ensemble of three components of 43- and 40-kDa (catalytic subunits) and a noncatalytic 28-kDa subunit. In addition, components of the ecto-PK II react with CK2-specific antibodies. Further, comparative peptide mapping and the results of mass spectrometry in combination with assignment of amino acid sequences confirmed that ecto-PK II is closely related if not identical to the protein kinase CK2. Assays with intact cells that result in the phosphorylation of a variety of endogenous membrane proteins showed that both ecto-PKs participate, and further, certain ecto-PK substrates become preferentially labeled by one or another of the enzymes, whereas others are phosphorylated by both ecto-PK activities.

Amino Acid Sequence↗
PubMed1994

Inhibition of cyclic AMP-triggered aromatase gene expression in human choriocarcinoma cells by antisense oligodeoxynucleotide.

Aromatase, an endomembrane-bound cytochrome P450, is the key enzyme of estrogen biosynthesis. Aromatase inhibitors, therefore, are clinically important tools in the treatment of estrogen-dependent tumor growth. To improve the specificity of these tools, inhibition at the nucleic acid level was examined. An antisense oligodeoxynucleotide complementary to the translation start region of human aromatase transcripts (antisense-arom) was synthesized and used to inhibit cyclic AMP-triggered aromatase gene expression in a human choriocarcinoma cell line (JEG-3), both as occurring in an autocrine fashion by secreted human chorionic gonadotropin or as induced by application of the membrane-permeating dibutyryl cyclic AMP. Significant inhibition was obtained in both cases, reaching 70% and 60%, respectively. In addition, the antisense-arom treatment led to accelerated mRNA degradation. The inhibition at the nucleic acid level was accompanied by a decrease of both the aromatase protein and microsomal aromatase activity. The data appear to indicate the antisense strategy to be a most promising approach for the development of a novel type of specific aromatase inhibitor.

Aromatase↗
PubMed1994

Casein kinase II is required for transition of G0/G1, early G1, and G1/S phases of the cell cycle.

Casein kinase II (CKII) is a ubiquitous serine/threonine protein kinase with many cellular functions, including participation in mitogenic signaling by cytoplasmic nuclear translocation (Lorenz, P., Pepperkok, R., Ansorge, W., and Pyerin, W. (1993) J. Biol. Chem. 268, 2733-2739). To examine whether cell compartment-specific availability is a requirement for CKII function during cell cycle progression, antibodies against CKII beta, the regulatory subunit of CKII, were microinjected into the cytoplasm or the nucleus of G0-synchronized human primary fibroblasts (IMR-90) at the time of mitogenic stimulation or at various intervals thereafter. Significant inhibition of the stimulation was obtained with both cytoplasmic and nuclear injections. The inhibition was reversible, was not observed with control antibodies, and was abolished by co-injection of purified CKII holoenzyme. The inhibition differed, however, in extent, duration, and cell cycle phase between cytoplasmic and nuclear injections. After cytoplasmic injection, inhibition reached 45-50% and was effective at two intervals within the first 2 h and at 12-16 h post-stimulation, i.e. at G0/G1 phase transition and at the G1/S phase boundary of the cell cycle. After injection into the nucleus, the inhibition was considerably stronger, reaching 80-85%, and was effective for the first 6 h post-stimulation, i.e. for the transition of G0/G1 phase and the adjoining first part of G1 phase. Cytoplasmic or nuclear injections within S phase affected neither DNA synthesis nor cell division. The data suggest that cell cycle transition from G0 to S phase requires the presence of a certain functional level of CKII at defined times and at defined cellular locations as follows: for transition of G0/G1 at both the nucleus and the cytoplasm, for transition of early G1 at the nucleus, and for transition of G1/S at the cytoplasm.

Animals↗
PubMed1994

Recombinant human casein kinase II. A study with the complete set of subunits (alpha, alpha' and beta), site-directed autophosphorylation mutants and a bicistronically expressed holoenzyme.

Human casein kinase II (CKII) is a ubiquitous and multipotential Ser/Thr kinase involved in the regulation of cell growth and differentiation. Biochemically, two characteristics are particularly notable; first, the tetrameric composition of two catalytic subunits (alpha and/or alpha') and two regulatory subunits (beta); second, the autophosphorylation of the holoenzyme at the N-terminus of CKII beta, suspected to be involved in tuning of the kinase activity. Whether CKII alpha and CKII alpha' reconstitute comparably with CKII beta to form holoenzyme is unclear. For a systematic investigation, the complete set of recombinant CKII subunits and of autophosphorylation mutants of CKII beta were expressed in Escherichia coli and comparative reconstitutions carried out. At 1:1 molar ratio, CKII beta stimulated both catalytic subunits roughly fivefold with phosvitin as a substrate. The level of activity reached with both of the reconstituted CKII isoforms was of the same order of magnitude as that of holoenzyme isolated from human placenta. It was also similar to a recombinant alpha 2 beta 2 holoenzyme whose expression had been attained in E. coli with a bicistronic construct containing the coding regions of CKII beta and CKII alpha in a tandem arrangement. Both Ser2 and Ser3 were identified as the autophosphorylation sites; replacement of one of these with Ala by oligonucleotide-mediated site-directed mutagenesis influenced only the extent of CKII beta autophosphorylation, replacement of both resulted in a loss of autophosphorylation. Despite these differences, the stimulatory effect of all the CKII beta mutants was comparable both to each other and to that of wild-type CKII beta. This was also obtained when substrates other than phosvitin were employed such as tubulin, or upstream-binding factor (UBF). However, the degree of stimulation was substrate specific and ranged from 2-5-fold with no major differences between CKII alpha and CKII alpha' stimulation. Calmodulin phosphorylation by both CKII alpha and CKII alpha' was decreased similarly by CKII beta and the CKII beta mutants. Proteins such as cAMP-responsive-element-binding protein (CREB), HPV16 E7 or Jun were not phosphorylated by either catalytic subunit but became substrates of both in the presence of CKII beta or CKII beta mutants. The data suggest that CKII alpha and CKII alpha' form similar CKII holoenzymes and that the tuning of holoenzyme activity is independent of the autophosphorylation status of CKII beta.

Base Sequence↗
PubMed1994

The human gene (CSNK2A1) coding for the casein kinase II subunit alpha is located on chromosome 20 and contains tandemly arranged Alu repeats.

We have isolated and characterized a 18.9-kb genomic clone representing a central portion of the human casein kinase II (CKII) subunit alpha gene (CSNK2A1). Using the whole clone as a probe, the gene was localized on chromosome 20p13. The clone contains eight exons whose sequences comprise bases 102 to 824 of the coding region of the human CKII alpha. The exon/intron splice junctions conform to the gt/ag rule. Three of the nine introns are located at positions corresponding to those in the CKII alpha gene of the nematode Caenorhabditis elegans. The introns contain eight complete and eight incomplete Alu repeats. Some of the Alu sequences are arranged in tandems of two or three, which seem to originate from insertions of younger Alu sequences into the poly(A) region of previously integrated Alu sequences, as indicated by flanking direct repeats.

Animals↗
PubMed1994

Human casein kinase II: structures, genes, expression and requirement in cell growth stimulation.

Casein kinase II (CKII) is an ubiquitous Ser/Thr protein phosphotransferase in control of a variety of crucial cellular functions including metabolism, signal transduction, transcription, translation and replication. CKII levels are consistently higher in neoplastic tissues. The human CKII is composed of subunits alpha, alpha', and beta with molecular masses of 43, 38 and 28 kDa, respectively, that form heterotetrameric holoenzymes (alpha 2 beta 2; alpha alpha' beta 2, alpha'2 beta 2) showing autophosphorylation particularly at subunit beta and hence suspected to play a regulatory role. The amino acid sequences of subunits indicate high evolutionary conservation. Employing the complete set of tissue-derived (placenta) and recombinant (expressed in E. coli) subunits and CKII holoenzymes, the catalytic function of alpha and alpha' and the several-fold stimulation by beta is shown to occur comparably in tissue-derived and recombinant CKII and the autophosphorylation of beta is shown by site-directed mutagenesis to be not decisive for the tuning of CKII activity. The human genome contains two genes encoding CKII alpha. First, there is a processed (pseudo)gene which is 99% homologous to the CKII alpha cDNA and which possesses a promoter-like region adjacently upstream with TATA and CAAT boxes so that transcription cannot be excluded. Second, there is an active gene of which we have characterized so far a 18.9 kb long central fragment which contains 8 exons comprising bases 102-824 of the CKII alpha coding region. The gene fragment contains repetitive elements, most prominently 16 Alu repeats. The genome further contains one as yet uncharacterized CKII alpha' gene and one gene encoding CKII beta. The CKII beta gene has been characterized as a 4.2 kb spanning gene composed of seven exons which possesses three transcription start sites and the translation start site in the second exon. The first intron harbors an Alu repeat also. The promoter region of the CKII beta gene contains elements such as multiple GC boxes, a CpG island, and nonstandard-positioned CAAT boxes but lacks a TATA box thus characterizing the gene as a housekeeping gene. The CKII genes are not clustered at a certain chromosome but rather are distributed over the whole human genome. Using the genomic clones as the probes for in situ hybridization, the active CKII alpha gene was mapped to chromosome 20p13, the processed CKII alpha (pseudo)gene to chromosome 11p15, and the CKII beta gene to chromosome 6p21. (The CKII alpha' gene has been localized on chromosome 16 with a cDNA probe.).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Human protein kinase CK2 genes.

We have analyzed the genomic structure of human protein kinase CK2. Of the presumably four genes, the gene encoding the regulatory subunit beta and a processed (pseudo)gene of the catalytic subunit alpha have been characterized completely. In addition, a 18.9 kb-long central part of the gene encoding the catalytic subunit alpha has been characterized. The subunit beta gene spans 4.2 kb and is composed of seven exons. Its promoter region shows several features of a "housekeeping gene" and shares common features with the promoter of the regulatory subunit of cAMP-dependent protein kinase. Conforming to the genomic structure, the beta gene transcripts form a band around 1.1 kb. The central part of the subunit alpha gene contains eight exons comprising bases 102 to 824 of the translated region. Within the introns, 16 Alu repeats were identified, some of which arranged in tandems. The structure of both human CK2 coding genes, alpha and beta, is highly conserved. Several introns are located at corresponding positions in the respective genes of the nematode Caenorhabditis elegans. The processed alpha (pseudo)gene has a complete open reading frame and is 99% homologous to the coding region of the CK2 alpha cDNA. Although the gene has a promoter-like upstream region, no transcript could be identified so far. The genomic clones were used for localization in the human genome. The beta gene was mapped to locus 6p21, the alpha gene to locus 20p13 and the alpha (pseudo)gene to locus 11p15. There is no evidence for additional alpha or beta loci in the human genome.

Amino Acid Sequence↗
PubMed1994

Requirement of casein kinase 2 for entry into and progression through early phases of the cell cycle.

Requirement of protein kinase CK2 during cell cycle was examined by specific perturbation of CK2 in the intact cell by antisense-oligodeoxynucleotides and microinjection of antibodies. When quiescent human primary lung fibroblasts (IMR-90) were exposed before growth stimulation to oligodeoxynucleotides complementary to the translation start region of mRNAs encoding subunit alpha or beta, a significant inhibition of growth stimulation by epidermal growth factor or serum was observed. The inhibition was reversible and decreased or abolished with mutated antisense-oligodeoxynucleotides. The inhibitory effect coincided with a decrease of CK2 protein (immunostaining with beta subunit antibody) at entry into and during the first several hours of the cell cycle. Injection of beta-specific monoclonal and polyclonal antibodies into IMR-90 cells caused significant inhibition of growth stimulation. The inhibition was reversible, not observed with control antibodies, and strongly reduced by coinjection of CK2 holoenzyme. Cytoplasmic injection inhibited up to 50-60% and was effective at two intervals within the first 2 h and at 12-16 h poststimulation, i.e., at G0/G1 phase transition and at G1/S boundary, respectively. The inhibition at G0/G1 transition is paralleled by an inhibition of cytoplasmic-nuclear translocation of beta subunit protein. Injection of beta antibodies into the nucleus inhibited growth stimulation by as much as 80-85% and was effective for the first 6 h poststimulation, i.e., at G0/G1 phase transition and progression through the adjoining early G1 phase. Nuclear as well as cytoplasmic injections performed during S phase affected neither DNA synthesis nor cell division.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1993

Human casein kinase II. The subunit alpha protein activates transcription of the subunit beta gene.

Casein kinase II (CKII), an ubiquitous serine/threonine protein kinase in control of a variety of crucial cellular functions, is composed of catalytic subunits (alpha and alpha') and regulatory subunits (beta). The adjusted activity of CKII is determined by the actual conformational state of CKII beta and the stoichiometry of the CKII subunits. Thus, the expression control of CKII beta is of particular concern. Carrying out gel shifts and footprints with affinity-purified proteins and cellular extracts in combination with mutational analysis we find that aside NF1 and Sp1, two out of the many factors predicted to bind to the upstream promoter region of the human CKII beta gene (Voss, H., Wirkner, U., Jakobi, R., Hewitt, N. A., Schwager, C., Zimmermann, J., Ansorge, W., and Pyerin, W. (1991) J. Biol. Chem. 266, 13706-13711), CKII alpha protein is able to complex with the CKII beta gene promoter. The complex of CKII beta-DNA/CKII alpha-protein is shown to occur within the 170-239-base pair (bp) segment upstream of the first transcription start site of the gene. The DNA motif contains, in a distance of 44 bp, two GC-rich boxes, 5'-GGGGCCC and 5'-CCCCTGGGC, and represents a novel cis-acting element; the binding of the CKII alpha protein activates the CKII beta gene promoter. This is manifested by driving the expression of the indicator gene luciferase or of CKII alpha-cDNA in HeLa cells. The binding of the CKII alpha protein is inhibited due to CKII beta protein addition or by mimicking the corresponding situation in vivo by overexpression of the CKII subunits. The data suggest that cells may maintain a certain CKII subunit stoichiometry via transcriptional control; excess of nuclear CKII alpha protein could activate the CKII beta gene transcription causing CKII beta protein to increase which, in turn, could feed back to abolish the action of CKII alpha at the CKII beta gene promoter.

Base Sequence↗
PubMed1993

Cell biological studies with monoclonal and polyclonal antibodies against human casein kinase II subunit beta demonstrate participation of the kinase in mitogenic signaling.

Casein kinase II (CKII) is a highly conserved ubiquitous serine/threonine kinase composed of two catalytically active (alpha and/or alpha') and two regulatory (beta) subunits. It has been suspected that, among numerous other cellular functions, CKII might play a role in the control of mitogenic signaling. To test for such a role and its mechanism in intact cells, monoclonal antibodies (mAbs) were generated against CKII beta using a recombinant protein containing amino acids 20-200 of human CKII beta. The CKII beta-specific mAb with the highest reactivity, mAb IVG6 (classified as IgG1 with kappa light chains), was purified to homogeneity. It recognized a CKII beta epitope comprising the amino acids 140-156, a basic and highly conserved region. In addition, polyclonal antibodies (pAbs) were raised and made monospecific by affinity purification. pAbs-mediated quantitative immunofluorescence microscopy of human IMR-90 fibroblasts and/or Western blots of cell fractions revealed (i) CKII beta was present in exponentially growing cells at a 2-3-fold higher level than in quiescent cells, (ii) CKII beta was localized predominantly in the nucleus of cells (3-15-fold cytoplasmic level depending on cellular state and assay used), and (iii) the nuclear/cytoplasmic ratio of CKII beta was higher by a factor of 2 in exponentially growing cells. Consequently, mitogenic stimulation of quiescent cells by fetal calf serum doubled the nuclear/cytoplasmic ratio of CKII beta. The increase occurred within the 1st h of stimulation. The translocation of CKII beta into the nucleus was inhibited when mAb IVG6 was injected into the cytoplasm at the time of mitogenic stimulation. This microinjection also significantly inhibited the cell proliferation. The data imply that cytoplasmic CKII participates in the transmission of mitogenic signals by translocation into the nucleus.

Antibodies, Monoclonal↗
PubMed1993

System for quantitation of gene expression in single cells by computerized microimaging: application to c-fos expression after microinjection of anti-casein kinase II antibody.

A system which allows sensitive and fast automated analysis of weakly labeled fluorescent specimens is described. It is tested in the analysis of c-fos expression stimulated by fetal calf serum and calibrated by quantitation of defined solutions injected into cells with the automated microinjection system. Low light level imaging technology combined with quantitative image processing methods and computer control of the hardware allows fully automated analysis of fluorescent molecules in single living or fixed cells. Reliable methods for subtraction of fluorescent background and automated identification of objects of interest in double-stained cells are described. The accuracy of quantitation is considerably improved by normalizing the fluorescence intensities of respective fluorophores in the same object by the method of ratio imaging. The error rate in determining the relative protein content in single cells is less than 15%. The method is applied to microinjection studies with a monoclonal antibody against casein kinase II subunit beta. Microinjection of this antibody into synchronized cells specifically inhibits c-fos expression stimulated by fetal calf serum. In combination with the computer-automated capillary microinjection system, the technique will become a useful tool in experiments requiring quantitative single cell analysis.

Antibodies↗
PubMed1993

Antibodies to casein kinase II in sera of patients with mixed connective tissue disease: evaluation with recombinant proteins.

In this study we determined the prevalence of autoantibodies against casein kinase II (CKII) in patients positive for anti-70K marker antibodies, which is indicative of mixed connective tissue disease. An anti-CKII ELISA was established with the use of bacterially expressed recombinant CKII proteins. Eight out of 52 anti-70K-positive sera (15%) were positive for anti-CKII antibodies, which recognized preferentially the CKII alpha subunit. All control sera (n = 52) were anti-CKII negative. Thus, the occurrence of anti-CKII antibodies may be of value for differential diagnosis.

Antibody Specificity↗
PubMed1992

Cloning of the C alpha catalytic subunit of the bovine cAMP-dependent protein kinase.

The bovine C alpha type catalytic subunit of the cAMP-dependent protein kinase was cloned. A partial cDNA was isolated from a bovine heart cDNA library. This clone contained 120 bp of the coding sequence and the entire 3' untranslated region of 1431 bp. The complete coding region was cloned by PCR amplification from total bovine heart and skeletal muscle RNA. The sequence of the 3' oligonucleotide was taken from the partial cDNA clone whereas the 5' oligonucleotide was chosen by comparison of sequences of published C alpha subunits from other species. In the deduced amino acid sequence there is one deviation from the published bovine C alpha protein sequence, aspartic acid 286 is exchanged by an asparagine. The C alpha mRNA was found to be expressed differentially in various bovine tissues.

Amino Acid Sequence↗