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PubMedDate not supplied

[Identification of bacterial clones that encode cow's caseins by direct immunological screening of the cDNA library].

A sensitive immunoassay was used to identify recombinant DNA plasmids carrying cDNA fragments of bovine caseins in the cDNA library from rRNA of bovine mammary glands. Colonies grown on nitrocellulose filters were lysed in situ and proteins from the lysates were blotted onto CNBr-activated filter paper. Antigens covalently bound to the CNBr-activated paper or bound to the nitrocellulose filters were detected by reaction with antiserum to caseins, followed by 125I-labeled protein A from Staphylococcus aureus and autoradiography. Four clones were positive among 5400 bacterial clones of the cDNA library--al, b2, b5, h7. Molecular weights of chimeric proteins pre-beta-lactamase:casein synthesized in Escherichia coli were determined by immunoblotting. Colony hybridization and DNA sequence analysis showed that clone b5 contained cDNA fragment of bovine kappa-caseins and clone h7 cDNA fragment of beta-casein. The last clone was designated pKcas beta-7.

Animals↗
PubMed1984

Synergistic phosphorylation and activation of ATP-Mg-dependent phosphoprotein phosphatase by F A/GSK-3 and casein kinase II (PC0.7).

The ATP-Mg-dependent phosphoprotein phosphatase is believed to consist of a catalytic subunit and a regulatory component identified as phosphatase inhibitor-2. It was found in this study that isolated inhibitor-2 was phosphorylated in serine residues by casein kinase II to at least 3 mol of phosphate per mol of inhibitor-2 while another protein kinase, F A/GSK-3, introduced no more than 0.3 mol of phosphate per mol exclusively in threonine residues. Analysis of tryptic digests by high performance liquid chromatography indicated that casein kinase II action resulted in two major (peaks 1 and 2) and two minor phosphopeptides whereas F A/GSK-3 action generated only peak 2. Combined action of the two protein kinases introduced an additional 0.4-0.6 mol of phosphate per mol over that predicted for simple additive behavior. This synergistic phosphorylation was associated with increased phosphate in peak 2 and correlated with unchanged phosphoserine but increased phosphothreonine, to a level approaching 1 mol/mol. ATP-Mg-dependent protein phosphatase was either reconstituted from purified inhibitor-2 and low molecular weight type 1 phosphatase or isolated as an inactive complex (Fc). Both phosphatase complexes were activated by F A/GSK-3 which caused a transient phosphorylation of the inhibitor-2 component. Casein kinase II alone phosphorylated the inhibitor-2 in both phosphatase complexes without affecting the enzyme activity. Exposure to the combination of F A/GSK-3 and casein kinase II resulted in a synergistic phosphorylation. Furthermore, the combined action of the two protein kinases caused a synergistic activation of the phosphatase at submaximal F A/GSK-3 levels. The results suggest that interactions between phosphorylation sites may play a role in the activation of the ATP-Mg-dependent phosphatase, in particular that phosphorylation by casein kinase II at serine can potentiate the phosphorylation of threonine by F A/GSK-3 with subsequent influence on phosphatase activation.

Animals↗
PubMed1983

Isolation and characterization of casein-producing and -nonproducing cell populations from 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinomas.

Previous studies from our laboratory have demonstrated that hormone-dependent 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinomas possess only a limited ability to synthesize the mammary gland-specific milk proteins, and this expression of normal differentiated function is limited to a small subpopulation of tumor cells (S. C. Supowit and J. M. Rosen, Cancer Res., 42: 1355-1360, 1982). In an effort to define the molecular defect in hormone-regulated casein gene expression in these tumors, a method has been developed which separates the 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinoma into two subpopulations of cells. Collagenase-dissociated tumor cell suspensions were fractionated by both continuous and discontinuous Percoll gradient centrifugation, and the separated cell populations were analyzed following plating on collagen-coated dishes. Two fractions of differing densities were isolated. The top fraction (rho = 1.05), containing approximately 75% of the dissociated tumor epithelial cells, is depleted in casein-producing cells, while the bottom fraction (rho = 1.07) is enriched 5- to 10-fold in casein-producing cells as assayed by both indirect immunofluorescence, using a specific anticasein antibody, and direct measurement of casein messenger RNA levels. These two subpopulations were further characterized by scanning and transmission electron microscopy, with respect to their cell morphology and ultrastructure, for the presence of hormone receptors; the arrangement of the epithelial cell marker, keratin; and their cell cycle distributions and ploidy. Prolactin, insulin, and estradiol binding were found to be uniform throughout each cell subpopulation and qualitatively similar between fractions. Both fractions appeared to be epithelial-like in morphology, containing numerous surface microvilli as assessed by scanning electron microscopy and containing numerous surface microvilli as assessed by scanning electron microscopy and contained keratin filaments as determined by indirect immunofluorescence. Furthermore, they had similar cell cycle distributions and ploidies of 2n. Differences in cell ultrastructure were observed between these two tumor epithelial cell fractions, reflecting an increased development of rough endoplasmic reticulum in the bottom fraction and a higher concentration of lipid-containing vesicles and free polysomes in the top fraction. The failure of the majority of the cells within the 7,12-dimethylbenz(a)anthracene-induced mammary tumors to produce casein in response to prolactin is not due to a receptor-negative phenotype or general cell type difference.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1983

Identity of the in vivo phosphorylation site in high mobility group 14 protein in HeLa cells with the site phosphorylated by casein kinase II in vitro.

The pattern and sites of phosphorylation of high mobility group (HMG) chromosomal proteins in HeLa cells labeled in vivo with [32P]orthophosphate have been compared with those of isolated HeLa HMG protein labeled in vitro by purified protein kinase enzymes. In synchronized HeLa cells there is phosphorylation of two HMG proteins designated hHMG 14 alpha 1 and alpha 2. hHMG 14 alpha 1 and alpha 2 are phosphorylated in a single identical tryptic phosphopeptide which runs toward the anode with electrophoresis at pH 4.7. The specific activity of phosphorylation at this site increased 2.5-fold in both hHMG 14 alpha 1 and alpha 2 in metaphase compared to interphase cultures. In vitro only casein kinase II specifically catalyzed phosphorylation of hHMG 14 alpha 1 and alpha 2 among a mixture of hHMG proteins; phosphorylation occurred at the site which was phosphorylated in vivo. The site of phosphorylation catalyzed by casein kinase II is distinct from sites in HMG proteins phosphorylated by cyclic nucleotide-dependent protein kinases or by casein kinase I. Casein kinase I specifically catalyzed phosphorylation of histone H1. These results indicate that casein kinase II is the enzyme which catalyzes the major phosphorylation of hHMG protein which occurs in vivo.

Amino Acids↗
PubMed1982

Type II regulatory subunit of cAMP-dependent protein kinase. Phosphorylation by casein kinase II at a site that is also phosphorylated in vivo.

A study of the phosphorylated sites on the regulatory subunit of type II cyclic AMP-dependent protein kinase is described. The site of autophosphorylation, previously reported (Takio, K., Walsh, K. A., Neurath, H., Smith, S. B., Krebs, E. G., and Titani, K. (1980) FEBS Lett. 114, 83-88), is found to contain the only residue phosphorylated by the catalytic subunit. Endogenous protein-bound phosphate, present at a second site, is observed in the NH2-terminal region of purified type II regulatory subunit. A serine residue in this same domain of type II regulatory subunit is shown to be phosphorylated in vitro by casein kinase II. The stoichiometry of phosphate incorporation by casein kinase II indicates a single site of phosphorylation that is equivalent to the site containing the endogenous phosphate. The apparent Km and Vmax of casein kinase II for the phosphorylation of purified type II regulatory subunit containing 0.55 molar equivalents of endogenous phosphate are 36 microM and 3.5 mumol/min/mg, respectively. Phosphatase treatment of type II regulatory subunit decreased the determined Km to 13 microM but does not change the Vmax consistent with the suggestion that casein kinase II is phosphorylating a site partially occupied by endogenous phosphate. Comparison of peptide maps of type II regulatory subunit phosphorylated in vitro by casein kinase II with in vivo modified type II regulatory subunit indicates that the site modified in vitro is also phosphorylated in vivo.

Amino Acid Sequence↗
PubMedDate not supplied

Kinetic behaviour of human erythrocyte casein kinases. Effect of 2, 3-bisphosphoglycerate and heparin.

The predominant cAMP-independent casein kinase CS isolated from human erythrocyte hemolysate and membrane casein kinase MS are inhibited to the same extent by 2,3-bisphosphoglycerate (2,3-DPG). However, under some conditions, both enzymes may appear to be unaffected or even stimulated by this prominent red cell metabolite, depending on casein and monovalent salt (NaCl, KCl) concentrations employed in the assay. In addition, both casein kinases CS and MS exhibit identical response to heparin, being unaffected by increasing concentrations of this mucopolysaccharide up to 400 ng/ml. On the contrary, the minor cytosolic casein kinase CTS, is inhibited by both 2,3-DPG and heparin under all conditions tested in the assay.

2,3-Diphosphoglycerate↗
PubMed1982

Hormonal control of casein synthesis in mammary explants from pregnant goats.

The effects of insulin, cortisol, prolactin, 3,3',5-triiodo-L-thyronine (L-T3) and progesterone on the synthesis of total protein and casein in mammary explants from pregnant goats were studied. In the absence of hormones and in the presence of insulin plus cortisol the rate of incorporation of 14C-leucine into proteins that were precipitated with the anti-casein antibody decreased during culture. The addition of prolactin to hormonal combination of insulin and cortisol caused large stimulation of rates of casein synthesis. Maximum incorporation of leucine was attained between 3 and 5 days of culture in the presence of 0.5 microgram ml-1 of prolactin. Prolactin stimulated-casein and total protein synthesis were not consistently affected by the addition of L-T3 or progesterone. The inhibition of DNA synthesis by hydroxyurea or cytosine-arabinofuranoside had no effect on casein synthesis in mammary explants from pregnant goats.

Animals↗
PubMed1981

Calf thymus RNA polymerases I and II do not contain subunits structurally related to casein kinases I and II.

Both calf thymus RNA polymerases I and II contain small subunits of molecular weight nearly identical with the subunits of casein kinases II and I, respectively. Antibodies prepared against calf thymus casein kinase II react with the Mr = 44,000 and 26,000 subunits of protein kinase but do not react with the Mr = 44,000 and 25,000 subunits of RNA polymerase I. These RNA polymerase I and casein kinase II subunits were purified by polyacrylamide gel electrophoresis, labeled with 125I and peptide maps generated. The tryptic peptide map of neither the Mr = 44,000 nor the 25,000 subunit of RNA polymerase I resemble the map obtained for the subunits of similar size in casein kinase II. The peptide maps generated from the Mr = 25,000 subunits of RNA polymerases I and II are, however, identical. Calf thymus RNA polymerase I, prepared by standard procedures is contaminated with casein kinase II which can be removed by rechromatography on DEAE-Sephadex. Antibodies prepared against calf thymus protein kinase I also fail to interact with the RNA polymerase II subunit of comparable size. Furthermore, peptide maps indicate that these subunits are not structurally related.

Amino Acid Sequence↗
PubMed1994

[Polymorphism of alphas-casein in white short-haired polled goats].

Distribution of alleles of the alphas-casein complex was studied by isoelectric focusing in the White Shorthaired Polled goat according to the method of Krause et al. (1988) and Mahé et al. (1993). In addition to the common alleles alpha s1-CnA and B, the alpha s1-CnE allele is described; it has not yet been observed in this breed. We are not able to confirm the occurrence of the alpha s1-CnC allele. At least one out of three defective mutants (alpha s1-CnD,F and 0) was found, nevertheless their identification was not discussed. The following percentile occurrence (Tab. I) of the above-mentioned alleles of alpha s1-casein was determined: alpha s1-CnA = 11.30; alpha s1-CnB = 38.26; alpha s1-CnC = 0; alpha s1-CnE = 17.39; alpha s1-CnX = 33.04 (the letter X indicates the defective mutants without specifying their type and number). Our results differ significantly from those of Boulamger et al. (1984), Grosclaude et al. (1987) and Trakovická (1992). Higher (in contrast to the French authors) and lower alpha s1-casein allele rates (as compared to Trakovická, 1992) were observed. The higher occurrence of the alpha s2-CnB is also conspicuous. However, our observations correspond to those according to which the "strong" alleles have higher frequencies in European breeds than in the French ones (Grosclaude et al., 1992-cit. Mahé et al., 1993). The alleles of alpha s2-casein were also investigated. The electrophoretic variant of the alpha s2-casein was observed next to the type B and located closer to the anode (suspected mutation); on that account it was named B-. This variant was also seen in a homozygous form.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1994

Association of casein kinase 2 with nuclear matrix. Possible role in nuclear matrix protein phosphorylation.

The nuclear matrix is thought to play a fundamental role in the nuclear structure and functions relating to cell proliferation and regulation of gene activity. Phosphorylation of nuclear matrix proteins has been observed, and multiple protein kinases may be involved in this process. Casein kinase 2 is a ubiquitous messenger-independent serine/threonine protein kinase that has been implicated in cellular growth and proliferation. The presence of immunoreactive casein kinase 2 in purified nuclear matrix preparations was demonstrated in rat liver and prostate tissues by employing specific antibodies. The enzyme was active toward the specific decapeptide substrate Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu and exhibited other properties characteristic of casein kinase 2. Phosphorylation of nuclear matrix proteins catalyzed by intrinsic protein kinase activity in intact nuclei was also observed, and the results suggested that this reaction was at least in part catalyzed by casein kinase 2. The profile of nuclear matrix proteins labeled with 32Pi in situ in intact liver cells was similar to that observed in nuclear matrix proteins isolated from nuclei labeled with [gamma-32P]ATP in vitro. Association of casein kinase 2 with the nuclear matrix may be of significance in the post-translational modification of certain proteins in this fraction.

Amino Acid Sequence↗
PubMed1993

Copurification of casein kinase II with 20 S proteasomes and phosphorylation of a 30-kDa proteasome subunit.

The 20 S proteasome is a multicatalytic protease that has been implicated in several processes including ATP/ubiquitin-dependent proteolysis. However, the ATP requirement(s) related to proteasome function is undefined. We demonstrate that a protein kinase activity copurifies through multiple steps utilized to isolate latent 20 S proteasomes from human erythrocytes. The kinase phosphorylates serine residues within a single 30-kDa proteasome subunit. The activity is not sensitive to cyclic AMP or protein kinase inhibitor, indicating that it is not a cyclic AMP-dependent kinase. It is sensitive to nanomolar levels of heparin and is able to utilize both ATP and GTP as phosphodonors, similar to casein kinase II activity. Moreover, a polyclonal antibody specific for casein kinase II recognizes the alpha' subunit of casein kinase II in the 20 S preparation and specifically immunoprecipitates the proteasome-phosphorylating activity. These characteristics suggest that the proteasome kinase is similar or identical to casein kinase II. It is suggested that phosphorylation of the 30-kDa proteasome subunit by casein kinase II may be involved in regulating the activity and/or assembly of proteasome complexes.

Blotting, Western↗
PubMed1993

Interleukin 1 and tumor necrosis factor stimulate two novel protein kinases that phosphorylate the heat shock protein hsp27 and beta-casein.

We have partially purified and characterized two protein kinases that were strongly activated by interleukin-1 (IL-1) or tumor necrosis factor (TNF) in MRC-5 fibroblasts. The kinases were separated by anion exchange chromatography of cytosolic fractions. They phosphorylated in vitro the small heat shock protein (hsp27) or beta-casein and were stimulated 3- and 4.5-fold, respectively, in cells that had been exposed to IL-1 or TNF for 10 min. They were distinct from the mitogen-activated protein kinases, whose activation by IL-1 or TNF has been reported recently. The hsp27 kinase phosphorylated its substrate on serine residues. Its molecular mass was estimated to be 45-kDa by gel filtration. It is probably involved in the increase in hsp27 phosphorylation seen in intact cells. The beta-casein kinase behaved as a 65-kDa protein. It phosphorylated its substrate on serine and threonine residues and had little activity on alpha-casein. The hsp27 and beta-casein kinases were not activated after stimulation of the cells with phorbol myristate acetate (PMA). In contrast, the MAP kinases were activated to a similar extent (2-3-fold) by the cytokines and by PMA. The hsp27- and beta-casein kinases probably correspond to novel enzymes whose mechanisms of activation may be independent of protein kinase C or MAP kinases.

Caseins↗
PubMed1993

Phosphorylation of synaptotagmin I by casein kinase II.

Synaptotagmin I is an abundant synaptic vesicle protein that binds Ca2+ in a phospholipid-dependent manner and is thought to function in synaptic vesicle exocytosis. We have now studied the phosphorylation of synaptotagmin I. Synaptotagmin I is one of the major substrates in brain for casein kinase II, which phosphorylates synaptotagmin at a single threonine. The phosphorylation site was mapped using recombinant proteins to threonine 128 of synaptotagmin I, which is located in the sequence between the transmembrane region and the C2 domain repeats of synaptotagmin I. The phosphorylation site of synaptotagmin I is also present in synaptotagmin II and is evolutionarily conserved between different species. Preceding the phosphorylation site, synaptotagmins I and II contain a lysine-rich sequence. Casein kinase II phosphorylation of many substrates is strongly stimulated by the addition of polylysine, but phosphorylation of synaptotagmin I by casein kinase II is not. In recombinant proteins, removal of the lysine-rich sequence of synaptotagmin I makes its phosphorylation dependent on exogenous polylysine, suggesting that the lysine-rich sequence in synaptotagmin serves as an endogenous polylysine stimulation signal for casein kinase II. Our data demonstrate that synaptotagmin I is an efficient substrate for casein kinase II at a conserved site with a possible modulatory role in nerve terminal function.

Amino Acid Sequence↗
PubMed1996

[Polymorphism of the kappa-casein gene in populations of the subfamily Bovinae].

Polymorphism of the 5'-untranslated region and exon 4 of kappa-casein (kappa-casein) gene was studied in Yakutian and Black Pied cattle, yak, European bison, and buffalo by means of a polymerase chain reaction and subsequent restriction fragment length polymorphism analysis (PCR-RFLP). In the species studied, restriction polymorphism by the endonucleases AluI and Bg/II in the 5'-untranslated region of the gene is absent. Four restriction endonucleases testing nucleotide substitutions in 136 codon (TaqI), 148 codon (HinfI and HindIII), and 167 and 168 codons (PstI) were used to study polymorphism of exon 4. The use of several restriction endonucleases allowed three alleles of kappa-casein (kappa-CnA, kappa-CnB, kappa-CnF) to be typed and new allele variants in yak, European bison, and buffalo to be revealed. Nucleotide sequences of the fragments of exon 4 studied were determined for two new alleles of the gene: kappa-CnG in yak and European bison and kappa-CnH in buffalo. Nucleotide substitutions determining new alleles were localized. In kappa-CnG, 148 and 168 codons coincide with the corresponding codons of kappa-CnB, and 136 and 167 codons correspond to kappa-CnA. Stop codons of kappa-CnG in yak are different from stop codons of other alleles of the gene: TGA, instead of TAA. The nucleotide sequence of exon 4 of kappa-CnH differs from bovine kappa-CnA by 15 nucleotide substitutions, causing 10 amino acid changes in the protein sequence, which coincide with the corresponding known amino acid sequence of kappa-casein in buffalo. Interbreed and interspecies differences in the profile of allele frequencies of the species studied were revealed. Aspects connected with evolution of the alleles of kappa-casein are discussed.

Alleles↗
PubMedDate not supplied

Construction of phosphorylatable monoclonal antibody CC49 with a casein kinase II recognition site.

A phosphorylation site for casein kinase II was introduced into chimeric monoclonal antibody CC49 (MAb-chCC49) by site-specific mutation of the coding sequence. The phosphorylation site for the casein kinase II was positioned at the carboxyl terminus of the heavy chain constant region of the MAb-chCC49. The resultant modified MAb-chCC49CKII was expressed in NS0 cells and purified. The MAb-chCC49CKII protein was phosphorylated by casein kinase II with [gamma-32P]ATP to high radiospecific activity. The 32P-labeled MAb-chCC49CKII binds to cells expressing TAG-72 antigens. The introduction of the phosphorylation sites for casein kinase II into monoclonal antibodies (MAb) provides a new reagent for the diagnosis and treatment of cancers. This demonstrates that the casein kinase II recognition site can also be used to introduce phosphorylation sites into proteins.

Amino Acid Sequence↗
PubMed1996

Golgi apparatus mammary gland casein kinase: monitoring by a specific peptide substrate and definition of specificity determinants.

The casein kinase from the Golgi apparatus of lactating mammary gland (GEF-CK) is distinct from ubiquitous 'casein kinases' termed protein kinases CK1 and CK2 and appears to define a family of secretory pathways protein kinases that phosphorylate seryl residues followed by an acidic residue at position +2. In this report we show that a new synthetic peptide substrate derived from beta-casein (beta[28-40]) is suitable for the fast, efficient and selective monitoring of GEF-CK, being unaffected by CK1 and CK2, and we define the consensus sequence of this protein kinase as being Ser-Xaa-Glu/SerP, distinct from that of CK2 (Ser/Thr-X-X-Glu/Asp/SerP/TyrP). In particular, the failure to recognize Asp as crucial specificity determinant prevents the phosphorylation of the specific CK2 peptide substrate RRRADDSDDDDD by GEF-CK. Thus, peptide substrates are now available for the fast and specific monitoring of all the three classes of 'casein kinases', CK1, CK2 and GEF-CK.

Amino Acid Sequence↗
PubMed1997

Creaming and Flocculation of Oil-in-Water Emulsions Containing Sodium Caseinate

The influence of protein content on the stability of concentrated oil-in-water emulsions (35 or 45 vol% oil, droplet diameter approximately 0.5 &mgr;m, pH 6.8) containing sodium caseinate as the sole emulsifying agent has been investigated. Time-dependent creaming profiles were determined at 30°C using an ultrasound velocity scanning technique with data analysis based on a Urick equation renormalization technique. The results indicate that creaming kinetics has a complex dependence on caseinate content. At low protein content (1 wt%), corresponding to less than half that required for saturation monolayer coverage, the emulsion is destabilized by bridging flocculation (accompanied by some coalescence). At higher protein content (2 wt%), where individual droplets are fully protected against protein bridging or coalescence by the thick adsorbed protein layer, the unflocculated emulsion has good stability over a period of several weeks. With further increase of protein content (>/=3 wt%), the observed creaming stability is reduced again, with the rate of serum separation at the bottom of the sample now greatly increased. This is attributed here to depletion flocculation by unadsorbed caseinate, probably in the form of small particles called "casein submicelles." Light microscopy has confirmed that the visually observable extent of reversible depletion flocculation in concentrated emulsions of this type is very sensitive to overall protein content. Once the caseinate concentration reaches a high value (6 wt%), the strength of the depletion interaction is such that it produces a very strong emulsion droplet network which can reorganize only slowly, and is hence much more stable to creaming and serum separation.

Journal Article↗
PubMed1984

Phosphorylation and inactivation of rabbit skeletal muscle glycogen synthase: distinction between kinase Fa-, phosphorylase kinase-, and glycogen synthase (casein) kinase-1-catalyzed reactions.

Rabbit skeletal muscle glycogen synthase was phosphorylated by kinase Fa, phosphorylase kinase, and cAMP-independent synthase (casein) kinase-1 to determine the differences among these kinase-catalyzed reactions. The stoichiometry of phosphate incorporation, the extent of inactivation, and the sites of phosphorylation were compared. Synthase (casein) kinase-1 catalyzes the highest level of synthase phosphorylation (4 mol/subunit) and inactivation (reduction of the activity ratio to below 0.05). The sites, defined by characteristic tryptic peptides, phosphorylated by synthase (casein) kinase-1 are distinguishable from those by kinase Fa and phosphorylase kinase. In addition, synthase (casein) kinase-1, unlike kinase Fa, does not activate ATP X Mg2+-dependent protein phosphatase. These results demonstrate that synthase (casein) kinase-1 is a distinct glycogen synthase kinase.

Animals↗