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PubMed1995

Identification of a casein kinase activity found elevated in human cytomegalovirus transformed cells.

Protein phosphorylation plays an important role in the regulation of cellular growth and proliferation and is thus thought to play a role in tumorigenesis. It has previously been reported that cells transformed by human cytomegalovirus (HCMV) contain two to four fold higher than normal levels of protein phosphorylation on serine and threonine residues, and two to six fold higher than normal levels of a casein kinase activity. We have now identified the major casein kinase activity found elevated in HCMV transformed cells as casein kinase type II; identification of this kinase was necessary in order to begin to define its role in HCMV mediated morphological transformation. Most of the differences in casein kinase II activity between normal and HCMV transformed cells were explained by differences in casein kinase II protein levels. This represents the first report concerning the elevation of casein kinase II activity in cells transformed by human cytomegalovirus.

Amino Acid Sequence↗
PubMed1993

Renaturation of casein kinase II from recombinant subunits produced in Escherichia coli: purification and characterization of the reconstituted holoenzyme.

Casein kinase II is a heterotetrameric protein kinase with an alpha 2 beta 2 composition; the subunits can be separated only under harsh denaturing conditions. In this study, the optimal conditions for renaturation of denatured casein kinase II have been established. Purified casein kinase II from rabbit reticulocytes was denatured with 8 M urea and 0.1 M dithiothreitol at 25 degrees C. Various parameters, including arginine, oxidized glutathione/dithiothreitol, substrate, and temperature were optimized for renaturation. Under optimal conditions, the denatured protein kinase was successfully renatured with a recovery of 75% activity and eluted around 160,000 Da upon gel filtration, indicating the tetrameric structure. When the alpha (catalytic) and beta (regulatory) subunits of casein kinase II from Drosophila were cloned and overexpressed with the pET3a vector in Escherichia coli, the majority of the subunits were in an insoluble form. The optimal conditions for denaturation/renaturation of the recombinant casein kinase II from Drosophila were identical to those developed for the holoenzyme, except for the redox state and temperature. When the alpha subunit was solubilized and renatured in an approximate 1:1 ratio with the beta subunit, the catalytic activity was stimulated fourfold over that of the alpha subunit alone. The reconstituted enzyme was purified to apparent homogeneity in one step by chromatography on heparin--TSK. Gel filtration indicated the formation of an alpha 2 beta 2 tetramer. The reconstituted recombinant casein kinase II exhibited characteristics of the native holoenzyme in subunit composition, inhibition by heparin, stimulation by basic compounds, and the KCl concentration required for optimal activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Phosphorylation of membrane proteins by cytosolic casein kinases in human erythrocytes. Effect of monovalent ions, 2,3-bisphosphoglycerate and spermine.

Membrane proteins of human erythrocytes can be phosphorylated not only by membrane casein kinase (MS) but also by cytosolic casein kinases CS and CTS, resembling casein kinase I and II, respectively. Casein kinase CS, like membrane casein kinase MS, preferentially phosphorylates membrane proteins such as band 2 (spectrin, beta-subunit) and band 3, which are the major phosphate-acceptor proteins in the endogenous phosphorylation of isolated ghosts in the presence of [gamma-32P]ATP. By contrast, cytosolic casein kinase CTS phosphorylates, in addition to band 2, some membrane proteins, whose endogenous phosphorylation in isolated ghosts under the same conditions is negligible, if any. The CS- and CTS-catalyzed phosphorylations exhibit different response to increasing NaCl (or KCl) concentrations up to physiological levels (140 mM KCl, 20 mM NaCl); i.e. CS- and MS-catalyzed phosphorylations are strongly inhibited by 75-150 mM KCl (or NaCl), while CTS-catalyzed phosphorylation is practically unaffected. In the absence of added NaCl, CS- and MS-catalyzed phosphorylations are markedly inhibited by 1.5-3 mM 2,3-bisphosphoglycerate, whereas CTS-catalyzed phosphorylation appears to be practically unaffected. Finally, CS- and MS-catalyzed phosphorylations are slightly inhibited also by 1 mM spermine, while CTS-catalyzed phosphorylation is enhanced by this polycation concentration.

2,3-Diphosphoglycerate↗
PubMed1975

Immunofluorescence detection of casein in human mammary dysplastic and neoplastic tissues.

To detect and localize casein in human mammary dysplastic and neoplastic tissues, an indirect immunofluorescence method has been devised. Anti-casein antibodies have been obtained from rabbits immunized with casein isolated from human milk. Cryostat sections post-fixed in alcohol and paraffin sections from routinely formalin-fixed tissues proved suitable. The immuno-fluorescence method revealed the presence of casein in epithelial cells in mammary dysplasia and in some, but not all, cases of carcinoma of the breast. Well differentiated (Grade I) carcinomas were shown to contain casein, mainly localized at the inner border of the epithelial cells; the milk protein was present also in cells metastatic in lymph nodes. In Paget's disease of the nipple, casein could be detected in neoplastic cells in the ducts and also in cells identifiable with the typical intra-epidermal Paget cells. This finding is evidence of a functional differentiation of Paget cells along lines proper to the mammary epithelium.

Animals↗
PubMed1977

Casein-mediated neutrophil chemotaxis: a parallel between surface binding and chemotaxis.

This study was directed toward demonstrating an interaction between human polymorphonuclear leukocytes (PMNs) and the chemotactic factor,casein. Experiments conducted using fluorescein-labeled casein indicated that PMNs have a membrane receptor for this chemotactic factor. This receptor was specific for casein and was not found on lymphocytes. A direct parallel was shown between the binding of FITC--casein and the PMN chemotactic response to this protein. Binding to the receptor was optimal at 25 degrees C, unaffected by sodium azide, and partially reduced by iodoacetate. Enzymatic treatment of PMNs with pronase and trypsin resulted in a loss of both FITC--casein-mediated PMN fluorescence and chemotaxis. Our data indicate that human PMNs have a membrane receptor for casein and that interaction with this receptor results in a chemotactic response.

Binding, Competitive↗
PubMed1975

Localization of casein-rich, fat-rich and DNA-synthesizing cells in monolayer cultures of mid-pregnant mouse mammary epithelium.

Monolayers of 16-day pregnant BALB/cfC3H/Crl mouse mammary epithelial cells were examined for the occurrence and distribution of cells which contain large amounts of casein and fat and those which synthesize DNA. Cells within the central portions of epithelial cells appeared rich in casein and fat, whereas cells on the peripheral edges of the colonies synthesized DNA almost exclusively. Casein deposits and DNA synthesis were mutually exclusive phenomena, since only 2% of the cells synthesizing DNA also stained for casein. Of the casein-rich cells, 74% were also rich in fat, suggesting that cells wich contain large deposits of casein almost always contain large amounts of fat. These results indicate that a specialization of function exists between cells on the growing edge and those centrally located within a single colony of mammary epithelial cells.

Animals↗
PubMed1992

Reexamination of the polymeric distributions of kappa-casein isolated from bovine milk.

kappa-Casein the stabilizing protein of the colloidal milk protein complex was purified from bovine skim milk by the method of McKenzie and Wake (Biochim, Biophys. Acta. 47, 240, 1961). The preparations were examined by sodium dodecyl sulfate gel electrophoresis in the presence and absence of a reducing agent. In the presence of a reducing agent, the kappa-casein migrates as a single low molecular weight band. However, in the absence of a reducing agent, a characteristic pattern of aggregates of varying molecular weight was observed with components ranging from monomer to octamer in integer steps. Densitometry of the Coomassie blue stained gels showed an almost equal distribution of components in each band; carbohydrate staining showed preferential location of sugar residues in lower molecular weight components. Treatment with chymosin (rennin) caused a downward shift in apparent molecular weight for each band with no change in the relative intensity of the Coomassie blue stained bands. Similar gel patterns were observed in whole caseins and partially purified kappa-caseins, indicating that this size distribution is a natural disulfide-linked reporter for the distribution of kappa-casein in casein colloids (micelles).

Animals↗
PubMed1989

Detection of adulteration of pasteurised milk with whey by determination of the casein-bound phosphorus and protein nitrogen content.

A new method to detect the adulteration of pasteurised milk with whey is described. The method is based on the determination of casein-bound phosphorus (Pcas) and the protein nitrogen content in milk. By using a mean Pcas content (0.85 g P/100 g casein), a Kjeldahl factor of 6.34 and protein N, the relative casein-N content (casein N/protein N x 100) can be estimated. Over a period of one year the variation in the phosphorus factor of dried skimmed milk samples amounted to 2.9%. In order to calculate the percentage of added whey, a standard curve has been prepared in which the relative casein content is plotted against the corresponding whey percentage. The results of 26 analyses on genuine pasteurised milk (Pcas = 21.7 mg/100 ml; casein N/protein N = 80.9%) and on 25 whey samples were used for the standard curve. In laboratory-made blends of pasteurised milk and cheese whey (5%, 10%, 20% and 30%), the estimated percentage of whey varied between -1.2% and +2.9% from the actual value. The analysis of nine samples from the Brazilian market showed that four samples were clearly adulterated with cheese whey. Sweet and/or acid whey addition will be detected by the proposed method.

Animals↗
PubMed1978

Effect of polypeptide hormones on stimulation of casein secretion by mouse mammary epithelial cells grown on floating collagen gels.

The in vitro effects of protein hormones on the stimulation of casein secretion by mouse mammary epithelial cells were studied. Mouse mammary glands were enzymatically dissociated and used immediately or were stored frozen and thawed just before use. Cells were cultured on floating collagen gels in the presence of insulin, cortisol and a pituitary or placental polypeptide hormone. Casein, released into the medium, was assayed by a radioimmunoassay against one of the components of mouse casein. Mammary cells released casein into the medium in the presence of as little as 10 ng of ovine prolactin per ml of medium. Human growth hormone stimulated the casein secretion to the same extent as prolactin. Human placental lactogen, ovine and bovine growth hormones were less stimulatory. Luteinizing hormone, follicle-stimulating hormone and thyroid-stimulating hormone had no effect on the stimulation of casein secretion.

Animals↗
PubMed1981

Simultaneous occurrence of pregnancylike lobuloalveolar morphogenesis and casein-gene expression in a culture of the whole mammary gland.

Entire second thoracic mammary glands of estrogen- and progesterone-treated immature virgin BALB/c mice were stimulated to pregnancylike lobuloalveolar morphogenesis after 6 days of incubation with insulin (5 micrograms/ml), aldosterone (1 micrograms/ml), growth hormone (5 micrograms/ml), cortisol (5 micrograms/ml), and prolactin (80 ng/ml, present as a contaminant in 5 micrograms/ml growth hormone). The alveolar growth in the glands, as judged by morphological studies, was accompanied by an increase in cell number as a function of incubation time in the hormonal medium. Hybridization of the total RNA from these glands to the casein mRNA specific complementary DNA probe (cDNAcsn) revealed that the level of casein mRNA rises from 0.00012 to 0.005% between 1 and 6 days of incubation. Estimates showed that the concentration of casein mRNA per cell rises 17-fold from 70 molecules on Day 1 to 1200 molecules on Day 6, whereas the number of epithelial cells increases only twofold during the same incubation time. When the growth hormone preparation was totally replaced by 80 ng of prolactin during the 6-day incubation, casein-mRNA levels were found to be 0.0083%. These results demonstrate that a pregnancy-like morphogenesis and concurrent expression of the casein gene in vitro can be achieved in a controlled hormone environment containing high cortisol and low prolactin concentrations. This one-step mammogenesis-lactogenesis culture model should be useful for studying the mechanisms of hormonal regulation of casein-gene expression observed in prepartum mammary gland in vivo.

Aldosterone↗
PubMed2001

Functional effects of casein kinase I-catalyzed phosphorylation on lens cell-to-cell coupling.

The functional consequence of the casein kinase I-catalyzed phosphorylation of the lens gap junctional protein connexin49 was investigated using a sheep primary lens cell culture system. To determine whether the phosphorylation of connexin49 catalyzed by endogenous casein kinase I results in an altered junctional communication between lens cells, the effect of the casein kinase I-specific inhibitor CKI-7 on Lucifer Yellow dye transfer between cells in the lens culture was examined. Dye transfer was analyzed in cultures of different ages because we have demonstrated previously that the expression of connexin49 increases as the cultures age while that of connexin43, which is likely not a substrate for casein kinase I, has been shown to decrease [Yang & Louis (1999) Invest. Ophthalmol. Vis. Sci. 41: 2568-2564]. In 9-day old lens cultures, in which gap junctions are composed primarily of connexin43, CKI-7 had little effect on the rate of dye transfer between lens cells. In contrast, treatment of 15-day and 28-day old cultures with CKI-7 resulted in a significant increase in the rate of dye transfer. Thus, the extent of this CKI-7-dependent increase in cell-to-cell communication was positively correlated with the level of expression of connexin49, the major casein kinase I substrate in lens plasma membranes. These results suggest that the casein kinase I-catalyzed phosphorylation of connexin49 decreases cell communication between connexin49-containing gap junctions in the lens.

Animals↗
PubMed1988

Determination of the quaternary structural states of bovine casein by small-angle X-ray scattering: submicellar and micellar forms.

Whole casein occurs in milk as a spherical colloidal complex of protein and salts called the casein micelle, with approximate average radii of 650 A as determined by electron microscopy. Removal of Ca2+ is thought to result in dissociation into smaller noncolloidal protein complexes called submicelles. Hydrodynamic and light scattering studies on whole casein submicelles suggest that they are predominantly spherical particles with a hydrophobic core. To investigate whether the integrity of a hydrophobically stabilized submicellar structure is preserved in the electrostatically stabilized colloidal micellar structure, small-angle X-ray scattering (SAXS) experiments were undertaken on whole casein from bovine milk under submicellar (without Ca2+) and micellar (with added 10 mM CaCl2) conditions. All SAXS results showed multiple Gaussian character and could be analyzed best by nonlinear regression in place of the customary Guinier plot. Analysis of the SAXS data for submicellar casein showed two Gaussian components which could be interpreted in terms of a particle with two concentric regions of different electron density, designated as a "compact" (subscript C) core and a "loose" (subscript L) shell, respectively. The submicelle was found to have an average molecular weight of 285,000 +/- 14,600 and a mass fraction of higher electron density core, k, of 0.212 +/- 0.028. The radius of gyration of the core, RC, was 37.98 +/- 0.01 A with an electron density difference, delta rho C, of 0.0148 +/- 0.0014 e-/A3, while the loose region had values of RL = 88.2 +/- 0.8 A with delta rho L = 0.0091 +/- 0.0003 e-/A3. Calculated distance distribution functions and normalized scattering curves also were consistent with an overall spherical particle with a concentric spherical inner core of higher electron density. These results, and in particular the remarkably low electron densities of the shells, can be interpreted in terms of a loosely packed spherical aggregate stabilized by a hydrophobic inner core and surrounded by an even more loosely packed hydrophilic region, in agreement with the results of other studies. The SAXS data for the colloidal micellar casein, which yield only cross-sectional information related to a window of scattered intensity, were analyzed by a sum of three Gaussians with no residual function. The two Gaussians with the lower values of the radius of gyration were interpreted again as an indication of an inhomogeneous spherical particle of two electron densities with the same centroid.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Purification and properties of a protamine kinase and a type II casein kinase from bovine kidney mitochondria.

Bovine kidney mitochondrial extracts contain an inactive protamine kinase and an inactive casein kinase. The protamine kinase was activated by chromatography on poly(L-lysine)-agarose. Two forms of this soluble mitochondrial protamine kinase were separated by chromatography on protamine-agarose. Both forms were purified about 80,000-fold to apparent homogeneity. Both forms of the protamine kinase consist of a single polypeptide chain with an apparent Mr approximately 45,000. Both enzyme forms underwent autophosphorylation without significant effect on activity, and both forms exhibited identical substrate specificities. The protamine kinase showed little activity toward branched-chain alpha-keto acid dehydrogenase (less than 3%), and it was essentially inactive (less than 0.1%) with pyruvate dehydrogenase, casein, and ovalbumin. The enzyme was active with histone H1 and with bovine serum albumin. Protamine kinase activity was unaffected by heparin (up to 100 micrograms/ml), by the protein inhibitor of cyclic AMP-dependent protein kinase, by Ca2+ and calmodulin, and by monoclonal antibody to the catalytic domain of protein kinase C from rat brain. The casein kinase was activated in the presence of spermine or by chromatography of the extract on DEAE-cellulose or poly(L-lysine)-agarose. The enzyme was purified about 80,000-fold to apparent homogeneity. It exhibited an apparent Mr 130,000 as determined by gel-permeation chromatography on Sephacryl S-300 in the presence of 0.5 M NaCl. Two subunits, with apparent Mr's 36,000 (alpha) and 28,000 (beta) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The kinase underwent autophosphorylation of its beta-subunit, without significant effect on activity. Casein kinase activity was inhibited 50% by 1.5 micrograms/ml of heparin. Spermine (1.0 mM) stimulated activity of the purified kinase two- to three-fold at 1.5 mM Mg2+. Half-maximal stimulation occurred at 0.1 mM spermine. The kinase utilized both ATP and GTP as substrates. The casein kinase showed little activity (less than 1%) toward pyruvate dehydrogenase and branched-chain alpha-keto acid dehydrogenase from kidney mitochondria, and the kinase was essentially inactive with glycogen synthase a. The properties of this soluble mitochondrial kinase indicate that it is a type II casein kinase.

Animals↗
PubMed1992

Casein kinase II-catalysed phosphorylation of calmodulin is altered by amino acid deletions in the central helix of calmodulin.

Calmodulin is phosphorylated by casein kinase II on Thr-79, Ser-81, Ser-101 and Thr-117. To determine the consensus sequences for casein kinase II in intact calmodulin, we examined casein kinase II-mediated phosphorylation of engineered calmodulins with 1-4 deletions in the central helical region (positions 81-84). Total casein kinase II-catalyzed phosphate incorporation into all deleted calmodulins was similar to control calmodulin. Neither CaM delta 84 (Glu-84 deleted) nor CaM delta 81-84 (Ser-81 to Glu-84 deleted) has phosphate incorporated into Thr-79 or Ser-81, but both exhibit increased phosphorylation of residues Ser-101 and Thr-117. These data suggest that phosphoserine in the +2 position may be a specificity determinant for casein kinase II in intact proteins and/or secondary structures are important in substrate recognition by casein kinase II.

Amino Acid Sequence↗
PubMed1992

Posttranscriptional regulation of alpha-casein mRNA accumulation by laminin.

We have utilized primary cultures of rat mammary epithelial cells to study mechanisms by which laminin regulates the prolactin-dependent accumulation of alpha-casein mRNA. Mammary cells accumulate approximately fivefold more alpha-casein mRNA when cultured on laminin than when cultured on tissue plastic and the accumulation of alpha-casein mRNA is prolactin dependent. On the basis of transcription assays there is approximately a twofold increase in the alpha-casein mRNA transcription rate in cells cultured on laminin over that of tissue culture plastic. Measurements on the turnover of alpha-casein mRNA show that this mRNA is stabilized fourfold more on laminin than on tissue culture plastic, while there was no significant difference in the turnover of poly(A) RNA on either substratum. These data indicate that laminin regulates the cytoplasmic levels of alpha-casein mRNA accumulation primarily at the post-transcriptional level by increasing the stabilization of this mRNA.

Animals↗
PubMed1988

Casein kinase 2 activity increases in the prereplicative phase of liver regeneration.

Cytosolic casein kinase activity increased up to 2-fold in the first 6 h after partial hepatectomy and then decreased to control values. This increase was due mainly to casein kinase 2, which reached maximal values at 6-8 h of liver regeneration. In contrast, casein kinase 1 showed a smaller increase at 4 h and then started to decrease reaching values of about 70% of control at 16 h. The increase in total casein kinase 2 was accompanied with an activation of the enzyme, as determined by the low/high beta-casein activity ratio assay. Administration of an acute dose of glucagon to control rats also increased the activity ratio but failed to cause any rise in total casein kinase 2 activity.

Animals↗
PubMed1982

The effect of semipurified diets containing different proportions of either casein or soybean protein on the concentration of cholesterol in whole serum, serum lipoproteins and liver in male and female rats.

Male and female lean Zucker strain rats were fed cholesterol-enriched semipurified diets containing 2 levels (20% and 50%, w/w) of either casein or soybean protein for a period of 14 weeks. In the female rats, the feeding of casein diets resulted in significantly higher levels of serum cholesterol than when diets containing soybean protein were fed. In addition, the hypercholesterolemic effect of dietary casein could be enhanced by increasing the proportion of this protein in the diet. Modulations in the proportion of dietary soybean protein did not significantly affect the serum cholesterol levels. In the male rats, however, no such differential effects were observed, indicating a difference between male and female rats in susceptibility to the induction of changes in serum cholesterol levels by dietary means. Upon feeding casein diets, both the male and female rats exhibited a shift of cholesterol from the high density lipoproteins to the lipoproteins with a lower density. This effect was more pronounced in the female than in the male rats. Liver cholesterol concentrations were markedly affected by modulations both in the type and proportion of dietary protein in both sexes. The concentration of cholesterol in the liver of the rats was highest in those fed the 50% casein diet and progressively lower in the animals on diets containing 20% casein, 20% soybean protein and 50% soybean protein.

Animals↗
PubMed1985

Effects of casein and soy protein on hepatic and serum lipids and lipoprotein lipid distributions in the rat.

Rats fed a semipurified diet containing casein developed higher levels of circulating triglycerides and cholesterol than animals fed a soy protein-containing diet. The increased serum lipid levels in non-fasted rats were associated largely with the d less than 1.006 g/ml lipoprotein particles (e.g. chylomicrons or very low density-like lipoproteins). In addition, casein-fed rats exhibited higher levels of circulating insulin and depressed hepatic 7 alpha-hydroxylase levels compared to soy-fed rats. Supplementation of the casein diet with arginine, to give an arginine/lysine ratio comparable to that in the soy diet, resulted in a reduction of d less than 1.006 g/ml lipids, a reduction in serum insulin levels and an elevation in hepatic 7 alpha-hydroxylase activity. Supplementation of the soy diet with lysine also resulted in modification of these parameters toward those observed with casein diets, albeit the effects were less dramatic. The results suggest that the hyperlipidemia associated with feeding casein-based diet is associated with decreased rates of clearance of chylomicron-like lipoproteins and their component triglycerides and cholesterol. Furthermore, this is largely prevented by addition of arginine to diets containing casein as the sole protein source.

Animals↗