Early cell growth stimulation is inhibited by casein kinase II antisense oligodeoxynucleotides.
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Casein Kinase II↗
Biomedical subjects
W Pyerin
Publications and source records attributed to W Pyerin.
Human casein kinase II subunit alpha: sequence of a processed (pseudo)gene and its localization on chromosome 11.
A human 4.3 kb genomic DNA fragment, containing the information of a processed (pseudo)gene of casein kinase II subunit alpha (CKII alpha) was isolated and sequenced. The genomic CKII alpha sequence is 99% homologous to the CKII alpha cDNA, carries several nucleotide exchanges, a poly(A) stretch at its 3' end and is flanked at both ends by a 16 bp repeat. It has a promoter-like region including two TATA boxes and a CAAT box. Although translation of transcripts would be terminated by a stop codon after two third of the coding region, the resulting protein would still contain the catalytic domains. However, so far Northern blots with a 3' specific probe were negative. The 4.3 kb genomic fragment containing the processed CKII alpha (pseudo)gene was mapped by in situ hybridization to chromosome 11p15.
Base Sequence↗
Atrial natriuretic peptide is phosphorylated by intact cells through cAMP-dependent ecto-protein kinase.
Recently we demonstrated the presence of cell-surface-located cAMP-dependent protein kinase (ecto-PK A) activity in a number of different cell types [Kübler, D., Pyerin, W., Bill, O., Hotz, A., Sonka, J. and Kinzel, V. (1989) J. Biol. Chem. 264, 14549-14555]. The question of the physiological role of externally directed kinase activity prompted a search for potential natural substrates present in the intercellular fluid. In the present study we have investigated the phosphorylation by ecto-PK A of the human atrial natriuretic peptide ANP99-126, a hormone released by cardiac cells. This 28-amino-acid peptide carries the phosphorylation consensus sequence Arg-Arg-Ser-Ser for the PK A. Incubation of various cell lines (including epithelial, epidermal, myoblast and lymphoma cells) or freshly isolated blood cells (macrophages, erythrocytes and platelets) with ANP in the presence of low micromolar concentrations of ATP resulted in the phosphorylation of ANP at Ser residues. The ANP phosphorylation reaction proved strictly dependent on cAMP; cAMP could not be replaced by cGMP. The phosphorylation was inhibited by the PK A-specific inhibitory peptide and increased linearily for up to 15 min and with a Km value of 3-5 microM for ANP. At higher ATP concentrations (greater than 100 microM) the incorporation rates amounted to about 0.3 mmol P (mol ANP)-1 min-1. The rise of intracellular cAMP in HEL30 (an epidermal cell line) after application of the beta-adrenergic receptor agonist isoproterenol led to an approximately three-fold stimulation of ANP phosphorylation which appears to be brought about by an efflux of intracellular cAMP. Employing cell supernatant fluids and cell sonicates, it could be shown that the phosphorylation of ANP results from the ecto-PK A. Comparison of ANP with ANP phosphorylated in vitro using purified catalytic subunit of PK A showed that phosphorylation is accompanied by certain changes in the average solution conformation of the peptide, consistent with the changes known to occur in its biological activity. Our results demonstrate cAMP-dependent phosphorylation of the peptide hormone analogue ANP99-126 by intact cells through ecto-PK A, an intriguing mechanism for post-translational processing of ANP.
Adenosine Triphosphate↗
Structure of the gene encoding human casein kinase II subunit beta.
Casein kinase II (CKII) is a ubiquitous serine/threonine protein kinase with numerous key functions in cell metabolism and growth. The human CKII has a tetrameric structure; two catalytic subunits (alpha and alpha') form the holoenzyme together with two presumably regulatory subunits (beta). The gene encoding CKII subunit beta was isolated from human genomic DNA and analyzed for its primary structure using exclusively nonradioactive procedures. The gene was found to span 4.2 kilobase pairs and to be composed of seven exons. Exon sizes range from 76 (exon 5) to 329 base pairs (bp) (exon 1), intron sizes from 145 (intron V) to 965 bp (intron II). All exon-intron junctional sequences conform to the canonical GT-AG rule. Primer extension analysis determined three transcription initiation sites, at 951, 919, and (minor) 840 bp upstream of the translation start site. The translation start is located early in the second exon; exon 1 is untranslated. The 3'-cleavage/polyadenylation signal sequence (AA-TAAA) is in the last exon at position 4173 bp relative to the first transcription initiation site. The coding sequence for CKII beta comprises 648 nucleotides identical to the published CKII beta-cDNA sequence (Jakobi, R., Voss, H., and Pyerin, W. (1989) Eur. J. Biochem. 183, 227-233). The upstream promoter region of the CKII beta gene contains multiple potential gene regulatory sequence elements, noticeable DNA structures, and the characteristics of a housekeeping gene (more than one transcription initiation site, lack of a TATA-box, presence of a CpG island, occurrence of multiple GC boxes and of nonstandard positioned CCAAT boxes). The CKII beta gene promoter shares common features with that of mammalian protein kinases and is closely related to the regulatory subunit gene promoter of cAMP-dependent protein kinase.
Amino Acid Sequence↗
Rat C alpha catalytic subunit of the cAMP-dependent protein kinase: cDNA sequence and evidence that it is the only isoform expressed in myoblasts.
A full length cDNA clone encoding the C alpha type catalytic subunit of cAMP-dependent protein kinase was isolated from a cDNA library of differentiated rat myoblast L6 cell line. The 2137 bp clone codes for a protein of 351 amino acid residues having more than 90% sequence identity to C alpha subunits of other mammalians. The C alpha isoform was found to be the only isoform of catalytic subunits expressed in myoblast cells as was determined in Northern blot analysis.
Amino Acid Sequence↗
Isoform C beta 2, an unusual form of the bovine catalytic subunit of cAMP-dependent protein kinase.
The catalytic (C) subunit is the phosphorylating component of the cAMP-dependent protein kinase, a key element in a multitude of hormonally controlled cellular functions. The C-subunit, thought to be a solitary protein until several years ago, is now known to be a group of isoforms comprising as yet C alpha, C beta, and C gamma. We report here the isolation of a full-length cDNA clone coding for a hitherto undiscovered isoform of the bovine C-subunit. The end parts of the 5'-coding region and the 5'-noncoding region of this 3365-base pair clone are unique, whereas the rest of the coding region and the 3'-noncoding region are identical to those of isoform C beta. The clone has therefore been named C beta 2. The deduced amino acid sequence of C beta 2 has a length of 397 amino acid residues and a calculated molecular mass of 46.1 kDa, thus being some 6 kDa higher than that of any known C-subunit. In vitro translation of clone C beta 2 resulted in a single 46-kDa protein. The unique amino-terminal sequence of C beta 2 lacks the usual myristoylation site of C-subunits. It contains a stretch of hydrophobic residues (residues 7-19) and a stretch which may fold into an amphiphilic alpha-helix (residues 16-27) conceivably serving targeting functions. The existence of isoform C beta 2 is confirmed by: (i) the isolation of a second independent C beta 2 clone, (ii) the development of products of expected size and sequence upon amplification from total RNA of various bovine tissues with the polymerase chain reaction using C beta 2-specific primers, and (iii) Northern blots probed with a cDNA fragment containing exclusively C beta 2 sequence. C beta 2 mRNA has a size of 4.4 kilobases and is expressed in various bovine tissues, mainly in heart and brain. Both the size and tissue distribution are indistinguishable from those of C beta mRNA, thus explaining the failure of previous investigations to distinguish it from C beta 2. Southern blotting and polymerase chain reaction with genomic DNA indicate that intron sequence(s) exist at the C beta 2/C beta deviation site (bases 267/268). The deviation site is equivalent to the exon 1/exon 2 splice site of the mouse C-subunit. Since splice sites are highly conserved and since not a single mutation is found downstream of the deviation site, it is tempting to suppose that C beta 2 and C beta are coded by one gene which possesses two alternatively spliced exons 1.
Amino Acid Sequence↗
Cell growth stimulation by EGF: inhibition through antisense-oligodeoxynucleotides demonstrates important role of casein kinase II.
Casein kinase II (CKII) is a highly conserved ubiquitous serine/threonine kinase composed of two catalytically active subunits (alpha and/or alpha') and two presumably regulatory subunits (beta). CKII has numerous cellular functions including a possible role in mitogenic signaling. To address this question, growth-arrested primary human fibroblasts (IMR-90) were exposed prior growth stimulation by epidermal growth factor (EGF) to oligodeoxynucleotides complementary to the translation start region of mRNAs coding for CKII alpha and beta subunits. A significant inhibition of growth stimulation (up to 60%) was observed with both antisense-alpha and antisense-beta. The inhibition was reversible, became decreased with mutated antisense-oligodeoxynucleotides, and neutralized by simultaneous presence of respective sense-oligodeoxynucleotides. The expected down-regulation of CKII protein due to hybrid formation of antisense-oligodeoxynucleotides with target mRNAs was investigated by determination of the intracellular protein level of CKII beta-subunit by immunofluorescence and quantitative image analysis. The protein was revealed to be localized predominantly in the nucleus and to become significantly decreased due to antisense-beta treatment of cells. The maximum decrease coincided with the early phase (first several hours) of growth stimulation by EGF when antisense-beta incubation was started 6-2 h before growth stimulation, the period within which application of antisense-alpha and antisense-beta caused the maximum of inhibition of growth stimulation. Thus CKII obviously plays, with both subunit alpha and subunit beta, an important role in the early phase of mitogenic stimulation.
Base Sequence↗
Evidence for ecto-protein kinase activity that phosphorylates Kemptide in a cyclic AMP-dependent mode.
The heptapeptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) is a synthetic construct of a substrate for cAMP-dependent protein kinase (PK). In this work we show that Kemptide has all the properties of a cytophilic substrate, i.e. it is a molecule preserving cell membrane intactness when added to cultured cells. Kemptide thus satisfies the prerequisites for employment in assays for cell surface-located ecto-PK activity. Different types of intact cells catalyze the phosphorylation of Kemptide in the presence of extracellular ATP and cAMP with Km values of 3-4 microM for Kemptide. Kemptide phosphorylation was influenced by PKI, the inhibitory protein specific for cAMP-PK. The results of comparative experiments with intact cells and with cell extracts demonstrate the ectoenzyme nature of this cAMP-PK. Further, the possibility was ruled out of a transfer of enzyme activity from damaged cells to the surface of intact cells. The anchorage of the surface cAMP-PK activity to the plasma membrane appears to be relatively stable since (i) cell supernatants, obtained after preincubation of intact cells with cAMP or Kemptide, did not show Kemptide phosphorylation, and (ii) the cAMP-dependent PK activity remained with cells even after five consecutive washes with cAMP or Kemptide. This is in contrast to the ecto-cAMP-independent phosvitin/casein type PK (Kübler, D., Pyerin, W., Burow, E., and Kinzel, V. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4021-4025) which is released from intact cells through the addition of substrate. Data are presented which show that both ectokinase activities are exhibited independently. In conjunction with published evidence for an active export of cAMP from cells as well as for the appearance of extracellular ATP the demonstration of an ecto-cAMP-PK further supports the potential of PK for intercellular regulation. The potential of ecto-cAMP-PK is demonstrated by its ability to phosphorylate biologically active forms of atrial natriuretic peptide, the atrial natriuretic peptide, which possesses the specific sequence for a cAMP-PK-catalyzed phosphorylation.
Amino Acid Sequence↗
Fast liquid chromatographic assay of androgen aromatase activity.
A rapid, sensitive nonradiometric assay method for the enzymatic aromatization of androgens has been developed using reversed-phase fast liquid chromatography. The use of a 3-microns-particle, 5-cm-long column allowed analysis of androstenedione, estrone, testosterone, and estradiol within 1 min. The reliability of the method was demonstrated by measuring the aromatase activity of human placental microsomes and that of the purified reconstituted aromatase system using both substrates. The rapid analysis enabled the processing of a large number of samples in a short time, which makes the present method especially suitable for the analysis of chromatographic fractions obtained during enzyme purifications and for enzyme kinetic studies.
Androstenedione↗
Glutathione S-transferase is an in vitro substrate of Ca++-phospholipid-dependent protein kinase (protein kinase C).
Glutathione S-transferase was found to be a good substrate of Ca++-phospholipid-dependent protein kinase in vitro. Of 6 isozymes of glutathione transferase purified from rat liver cytosol (1-1, 1-2, 2-2, 3-3, 3-4, 4-4), only isozymes 1-1, 1-2 and 2-2 were significantly phosphorylated by the kinase purified from rabbit brain. Phosphorylation was more pronounced in subunit 1 than in subunit 2, and the degree of the phosphorylation was similar in all three homo- and heterodimers, where 1 mol of phosphoryl group per mol subunit was transferred to the subunit 1. The phosphorylated transferase 1-1 showed decreased affinity for bilirubin, suggesting that the phosphorylation affects the function of glutathione S-transferase in an isozyme-specific manner.
Animals↗
Separation and purification of component proteins of the cytochrome P-450-dependent microsomal monooxygenase system by high-performance liquid chromatography.
The component proteins of the hepatic microsomal monooxygenase system, including various cytochrome P-450 isozymes, were separated and isolated from liver microsomes of untreated rabbits by Aminohexyl Sepharose and high-performance liquid chromatography (TSK preparative DEAE 5-PW, Bio-Rad HPHT). In addition to the known cytochrome P-450 isozymes, two new isozymes and one variant of the major isozyme were isolated. The monooxygenase activity was reconstituted by incorporating the purified proteins into liposomal membranes.
Animals↗
Human phosvitin/casein kinase type II. Molecular cloning and sequencing of full-length cDNA encoding subunit beta.
Phosvitin/casein kinase type II is an ubiquitous, highly conserved enzyme consisting of subunits alpha, alpha' and beta. Subunit beta, presumably serving regulatory functions, was prepared from human placenta and the amino acid sequence of a protease digestion peptide was determined. The deduced nucleotide sequence was employed for the synthesis of a mixture of 20mers as a hybridization probe to screen a lambda gt10 HeLa cell cDNA library for clones encoding subunit beta. A full-length clone consisting of 1013 bp was isolated and the sequence of both strands determined. The deduced amino acid sequence of the largest open reading frame encodes 215 amino acid residues predicting a maximum Mr of 24925. The nucleotide sequence of the human beta subunit shows a similarity of 75% to that of Drosophila melanogaster and the deduced amino acid sequence a similarity of 88% and 97% to that of D. melanogaster and bovine lung, respectively. No further protein sequence currently known exhibits any notable similarity. Using a 537-bp restriction fragment of the cDNA clone representing 80% of the coding region, a 1-kb subunit-beta transcript was detected by Northern hybridization in total RNA prepared from human epithelial cells and placenta as well as from bovine heart suggesting a single transcript of the beta-subunit gene and also demonstrating high similarity also between the human and bovine nucleotide sequences. The same restriction fragment was used as the probe to indicate in Southern hybridizations that the corresponding genomic DNA contains at least one intron of roughly 2.5 kb in length and presumably is a single copy gene.
Amino Acid Sequence↗
Phosphorylation of hepatic phenobarbital-inducible cytochrome P-450.
The major phenobarbital-inducible cytochrome P-450 purified from rat liver, a member of family II of the cytochrome P-450 gene superfamily, is rapidly phosphorylated by cAMP-dependent protein kinase. The phosphorylation reaches greater than 0.5 mol phosphate/mol P-450 after 5 min and is accompanied by a decrease in enzyme activity. The serine residue in position 128 was shown to be the sole phosphorylation site and a conformational change of the protein was indicated by a shift of the carbon monoxide difference spectrum of the reduced cytochrome from 450 to 420 nm. Comparison of amino acid sequences of various cytochrome P-450 families revealed a highly conserved arginine residue in the immediate vicinity of the phosphorylated serine residue which constitutes the kinase recognition sequence. It also revealed that only the members of the cytochrome P-450 family II carry this kinase recognition sequence. To find out whether this phosphorylation also occurs in vivo, the exchangeable phosphate pool of intact hepatocytes derived from phenobarbital-pretreated rats was labeled with 32Pi followed by an incubation of the cells with the membrane-permeating dibutyryl-cAMP or with the adenylate cyclase stimulator glucagon to activate endogenous kinase. As a result, a microsomal polypeptide with the same electrophoretic mobility as cytochrome P-450 became strongly labeled. Peptide mapping and immunoprecipitation with monospecific antibodies identified this protein as the major phenobarbital-inducible cytochrome P-450. It becomes phosphorylated at the same serine residues as in the cell-free phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Filter-supported preparation of lambda phage DNA.
A rapid and simple method is described for the isolation of DNA from phage lambda which requires neither special equipment nor expensive material such as cesium chloride for ultracentrifugation nor extractions with organic solvents or ethanol precipitation. Microgram quantities of lambda DNA are obtained in less than 2 h from 90-mm plate lysates or 5-ml liquid cultures. The method allows the simultaneous isolation of large numbers of probes, e.g., clones from phage libraries. Lambda phages are precipitated by polyethylene glycol/sodium chloride and recovered by low speed centrifugation onto glass fiber filters positioned in disposable syringes. The DNA of phages is released by a 50% formamide/4 M sodium perchlorate solution, washed in filter-bound form, eluted with a small volume of low-salt buffer or water, and finally recovered by centrifugation. Comparison of the DNA isolated by this method with that obtained by two conventional procedures reveals both a similar recovery and a similar suitability for restriction enzyme digestion and subcloning.
Bacteriophage lambda↗
Phospholipid bilayer membranes play decisive roles in the cytochrome P-450-dependent monooxygenase system.
Hepatic microsomal monooxygenase was reconstituted by incorporating cytochrome P-450 and NADPH-cytochrome P-450 reductase, which had been purified from phenobarbital-pretreated rabbit liver microsomes, into phospholipid liposomal membranes. The NADPH-dependent monooxygenase activity of the reconstituted system was found to be dependent on the phospholipid-to-protein ratio, i.e., the two-dimensional concentration of the two proteins on the plane of the membranes. A similar concentration dependence was also observed in the cytochrome b5 and NADH-cytochrome b5 system, which had been incorporated into liposomal membranes. The diffusion process of the proteins in the membrane, therefore, plays an important role in the monooxygenase system. When the fluidity of the membrane was changed by utilizing a synthetic dimyristoylphosphatidylcholine, which shows a well-defined gel to liquid crystalline phase transition, the activation energy of the monooxygenase reaction was changed at around the phase transition temperature, suggesting a conformational change of cytochrome P-450 caused by the fluidity change of the membrane. The incorporation of P-450 into liposomes was also found to affect the binding of substrates to cytochrome P-450. The decrease in the apparent dissociation constant of substrates upon incorporation into membranes suggests that the lipid membrane acts as a pool for hydrophobic substrates, which are concentrated in the lipid phase, and that cytochrome P-450 takes substrates directly from the membrane phase. Phospholipid membranes, therefore, play very important roles in various phases of the reaction of cytochrome P-450-dependent monooxygenase.
Animals↗
The effects of phosphatase on the components of the cytochrome P-450-dependent microsomal monooxygenase.
Incubation of rabbit liver microsomes with alkaline phosphatase resulted in a marked decrease of NADPH-dependent monooxygenase activities. This decrease was found to be correlated with the decrease of NADPH-cytochrome c reductase activity catalyzed by NADPH-cytochrome P-450 reductase. Neither the content of cytochrome P-450, as determined from its CO difference spectrum, nor the peroxide-supported demethylase activity catalyzed by cytochrome P-450 alone was affected by the phosphatase treatment. NADH-cytochrome b5 reductase and cytochrome b5 were not affected by the phosphatase either. NADPH-cytochrome P-450 reductase purified from rabbit liver microsomes lost its NADPH-dependent cytochrome c reductase activity upon incubation with phosphatase in a way similar to that of microsome-bound reductase. Flavin analysis showed that the phosphatase treatment caused a decrease of FMN with concomitant appearance of riboflavin. Alkaline phosphatase, therefore, inactivates the reductase by attacking its FMN, and the inactivation of the reductase, in turn, leads to a decrease of the microsomal monooxygenase activities.
7-Alkoxycoumarin O-Dealkylase↗
Isoenzyme-specific phosphorylation of cytochromes P-450 and other drug metabolizing enzymes.
A series of fourteen cytochrome P-450 isoenzymes was treated with three different protein kinases and found to divide into isoenzymes phosphorylated by both the cyclic AMP-dependent kinase and the calcium-phospholipid-dependent kinase (P-450 PB 3a and PB 2e), by none of these kinases (P-450 PB 1b, MC 1b, UT 1, and thromboxane synthase), and by either the cyclic AMP-dependent kinase (P-450 LM 2, PB 2d, and PB 3b) or the calcium-phospholipid-dependent kinase (P-450 PB 1a, PB 2a, MC 1a, LM 3c, and LM 4). Other components of the monooxygenase system, cytochrome P-450 reductase, cytochrome b5, cytochrome b5 reductase as well as microsomal epoxide hydrolase, were poor substrates for the kinases employed. On the other hand, glutathione transferases 1-2 and 4-4, but not 3-3, were relatively good substrates for the calcium-phospholipid-dependent kinase.
Animals↗
Posttranslational modifications of the cytochrome P-450 monooxygenase system.
Two forms of enzymatic posttranslational modifications of the monooxygenase system are described: modification by phosphatase and modification by protein kinase. Phosphatase treatment of microsomes isolated from phenobarbital-pretreated rabbits and rats caused a marked decrease of monooxygenase activity which was paralleled by a comparable decrease of NADPH-cytochrome P-450 reductase activity while the second essential component of the system, cytochrome P-450, remained unaltered. Thus phosphatase attacks monooxygenase via reductase. Protein kinases showed the opposite preference; while cytochrome P-450 was phosphorylated, NADPH-cytochrome P-450 reductase was not. Thus the kinase affects monooxygenase via cytochrome P-450. The phosphorylation of cytochrome P-450 turned out to be a specific reaction observed only with certain cytochrome P-450 isoenzymes and certain protein kinases.
Animals↗