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PubMed1997

Casein kinase 1 is tightly associated with paired-helical filaments isolated from Alzheimer's disease brain.

The protein kinase activity tightly associated with paired helical filaments (PHFs) purified from the brain tissue of individuals with Alzheimer's disease has been characterized in vitro. The activity is shown to phosphorylate casein, an exogenous substrate, with a maximal velocity of approximately 2 nmol/min/mg, suggesting it comprises a significant component of the total protein in the PHF preparation. On the basis of substrate selectivity, isoquinoline sulfonamide inhibitor selectivity, in-gel renaturation assays, and western analysis, the activity consists of closely related members of the alpha branch of the casein kinase 1 family of protein kinases. Because of its tight association with PHFs and its phosphate-directed substrate selectivity, casein kinase 1 is positioned to participate in the pathological hyperphosphorylation of tau protein that is observed in neurodegenerative diseases such as Alzheimer's disease.

Adult↗
PubMed2002

Estradiol reverses TGF-beta1-induced mesangial cell apoptosis by a casein kinase 2-dependent mechanism.

BACKGROUND: The slower rate of progression of chronic renal disease in women than in men is explained in part by the ability of estradiol to reverse the stimulatory effect of transforming growth factor-beta1 (TGF-beta1) on collagen IV synthesis at the level of casein kinase 2 activation. Casein kinase 2 also phosphorylates and activates the pro-apoptotic protein, p53. We hypothesized that estradiol would reverse TGF-beta1-induced mesangial cell apoptosis by antagonizing the stimulatory effects of TGF-beta1 on casein kinase 2 activity, thereby preventing p53 activation. METHODS: The effects of TGF-beta1 on mesangial cell apoptosis, p53 phosphorylation, Bax and Bcl-2 levels, caspase 9 activity, and cleavage of PARP were examined. The abilities of estradiol and a specific inhibitor of CK2 (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) (DRB) to modulate the effects of TGF-beta1 on these processes were also examined. RESULTS: TGF-beta1 (2 ng/mL), which up-regulates CK2 activity, induces apoptosis in murine mesangial cells together with p53 serine389 phosphorylation, up-regulation of Bax, suppression of Bcl-2, destabilization of mitochondrial permeability transition pores, stimulation of caspase 9 activity and activation of PARP. TGF-beta1-induced p53 activation and all the intermediary steps leading to mesangial cell apoptosis were reversed by estradiol (10-9 mol/L) and by DRB, potent inhibitors of CK2 activity, but not by inhibitors of the p38 MAPK, ERK or JNK signaling cascades. In contrast, TGF-beta1 failed to induce apoptosis in p53 knockout mesangial cells. CONCLUSIONS: Our data suggest that CK2 mediates the stimulatory effects of TGF-beta1 on mesangial cell apoptosis via a p53-dependent mechanism. The ability of estradiol to reverse TGF-beta1-induced apoptosis may contribute to the protective effects of female gender on the course of chronic renal disease.

Animals↗
PubMed1982

Effects of insulin, cortisol and prolactin on lipid, protein and casein syntheses in goat mammary tissue in organ culture.

The actions of insulin, prolactin and cortisol on protein, lipid and casein syntheses were examined in goat mammary explants in culture. The synthesis of the three products was stimulated by insulin and prolactin associated or not with cortisol. Casein synthesis was stimulated only when prolactin was present in the culture medium. Prolactin alone was able to significantly support the induction of casein synthesis. After preculture in the presence of insulin alone, the tissue lost part of its capacity to respond to the prolactin stimulus but, when precultured in the presence of insulin + cortisol, it retained its total sensitivity. These data suggest that insulin, prolactin and cortisol are involved in the maintenance of goat mammary tissue in culture but that prolactin essentially stimulates milk synthesis.

Adipose Tissue↗
PubMed1991

Exogenous and endogenous contributions to nitrogen fluxes in the digestive tract of pigs fed a casein diet. II. Ileal and faecal digestibilities and absorption of amino acids.

The present work aimed at quantifying nitrogen (N) and amino acid (AA) fluxes in the digestive tract of growing pigs fed a casein diet. In this paper we report on digesta passage at the terminal ileum, on apparent balances at the ileal and faecal levels, and on nutrients appearance in the portal vein. Digesta flow-rate at the terminal ileum was maximum between 6 and 12 h after the meal. About 10% of N and 5% of total AA ingested were recovered within 24 h. AA absorption started 30 min after the meal, and was measurable until 13 to 14 h. The total AA absorbed in 24 h accounted for 128% of the AA ingested. The AA composition of ileal digesta was very different from that of casein, closely resembling that of endogenous proteins. The AA composition of faeces was very close to that of bacterial proteins. The ileal digestibilities of AA, though lower than their faecal values, were very high. This was confirmed by AA absorption balances greater than 100%. These data suggest that casein was almost totally digested by the terminal ileum, and that endogenous AA were substantially reabsorbed. These findings are supported by data on endogenous N recycling (15N), reported in a following paper.

Amino Acids↗
PubMed2004

Structure and rheological behavior of casein micelle suspensions during ultrafiltration process.

The stability and mechanism underlying the formation of deposits of casein micelles during ultrafiltration process were investigated by small-angle and ultra small-angle x-ray scattering (SAXS and USAXS). The casein micelle dispersions consisted of phospho-caseinate model powders and the measurements probed length scales ranging from 1 to 2000 nm. Rheometric and frontal filtration measurements were combined with SAXS to establish the relationship between the rheological behavior of deposits (shear and/or compression) and the corresponding microstructure. The results revealed two characteristic length scales for the equilibrium structure with radius of gyrations R(g), about 100 and 5.6 nm pertaining to the globular micelles and their non-globular internal structure, respectively. The SAXS measurements further indicated that the increase of temperature from 20 to 70 degrees C or the decrease of pH from 6.6 to 6 lead to agglomeration of the globular micelles. In situ scattering measurements showed that the decrease of permeation flows is directly related to the deformation and compression of the micelles in the immediate vicinity of the membrane.

Caseins↗
PubMed1980

Absolute requirement of glucocorticoid for expression of the casein gene in the presence of prolactin.

Second thoracic mammary glands of immature BALB/c female mice were stimulated to pregnancy-like lobuloalveolar (LA) development aftr 6 days of incubation in a corticosteroid-free step I culture medium containing insulin, prolactin, estradiol, progesterone, and growth hormone. A low basal level (0.0009%) of casein mRNA (mRNAcsn) sequences was detectable in the LA glands by a specific cDNA probe. Subsequent incubation of the LA glands for 3 days in medium containing insulin and prolactin or insulin and cortisol failed to elicit mRNAcsn above the basal level, indicating that neither prolactin nor cortisol alone can support casein gene expression. However, an increase in mRNAcsn levels was observed when the 3-day incubation with insulin and cortisol or insulin and prolactin was followed by 3 days of culture in presence of insulin, prolactin, and cortisol. When a 3-day incubation with insulin and prolactin was followed by 3 days in insulin and cortisol medium, mRNAcsn levels in the gland remained similar to the basal level. However, a 20-fold increase in the mRNAcsn levels ensued when the LA glands were sequentially incubated for 3 days in insulin and cortisol and then for another 3 days in insulin and prolactin medium. After a preincubation in insulin and cortisol medium, the LA glands retained residual cortisol during subsequent incubation in insulin and prolactin medium, and the mRNAcsn levels in these glands were related to the level of residual cortisol present. When mRNAcsn and the residual cortisol level reached a minimum, addition of fresh cortisol to the medium caused a 20-fold increase in the mRNAcsn levels. This indicates that cortisol is a limiting factor in insulin and prolactin medium and its presence is absolutely required for casein gene expression.

Animals↗
PubMed1981

Prolactin-like activity of anti-prolactin receptor antibodies on casein and DNA synthesis in the mammary gland.

Prolactin receptors were partially purified from rabbit mammary gland membranes by using an affinity chromatography technique. Antibodies against this prolactin receptor preparation were obtained in guinea pig and sheep. Both antisera were able to inhibit the binding of 125I-labeled ovine prolactin to rabbit mammary gland membranes. When added to culture media of rabbit mammary explants, the anti-prolactin receptor antiserum inhibited the capacity of prolactin to initiate casein synthesis and casein mRNA accumulation as a function of the antiserum concentration. However, in the absence of prolactin, both antisera (guinea pig and sheep) at moderate concentrations were capable of mimicking prolactin action on casein gene expression and on DNA synthesis. At higher concentrations, the anti-prolactin receptor antibodies inhibited their own actions. Several characteristics of the prolactin effect were also observed with the anti-prolactin receptor antibody: the stimulatory effect of the antibody was amplified by glucocorticoids; colchicine, which was capable of blocking prolactin action, also prevented the induction by the antibody. Lysosomotropic agents, which do not interfere with prolactin action, did not alter the response observed with the antibody. These results indicate that an anti-prolactin receptor antibody can mimic two major actions of prolactin obtained in mammary explant culture and suggests that the prolactin molecule is not required beyond the initial binding to its receptor.

Animals↗
PubMed1991

Rapid activation of hippocampal casein kinase II during long-term potentiation.

Several studies suggest that protein kinase C and type II Ca2+/calmodulin-dependent protein kinase are activated during induction of long-term potentiation (LTP). We now report that casein kinase II (CK-II), which is present in high concentration in the hippocampus, is also activated in the CA1 region during LTP. CK-II activity increased within 2 min after a train of high-frequency electrical stimulations and reached a maximum (2-fold increase) 5 min later before returning to baseline value. The stimulated protein kinase activity, which was blocked by a selective antagonist of N-methyl-D-aspartate receptors, exhibited specific properties of CK-II, including phosphorylation of the specific substrates of CK-II, marked inhibition by a low heparin concentration, and the use of GTP as a phosphate donor. CK-II activity was also selectively and rapidly augmented in another form of LTP produced by bath application of tetraethylammonium; this LTP (called LTPk) is Ca2+ dependent but N-methyl-D-aspartate independent. Phosphorylation of casein that was not inhibited by heparin (i.e., casein kinase I) remained unchanged. We suggest that an increase in CK-II activity is important in LTP induction.

Adenosine Triphosphate↗
PubMed1992

Phosphorylation by cellular casein kinase II is essential for transcriptional activity of vesicular stomatitis virus phosphoprotein P.

We have previously shown that phosphorylation of vesicular stomatitis virus (VSV) phosphoprotein P by cellular protein kinase activity is an essential prerequisite for its transcriptional function. We have now purified this protein kinase by monitoring its ability to phosphorylate bacterially expressed, unphosphorylated P protein. Biochemical studies showed that the kinase is indistinguishable from casein kinase II, a ubiquitous cyclic AMP-independent protein kinase present in a wide variety of eukaryotic cells and tissues. Functional VSV transcription could be reconstituted with viral L protein, N-RNA template, and P protein phosphorylated by either purified cellular protein kinase or purified casein kinase II. The unusual role of casein kinase II in the transcription process of a nonsegmented negative-strand RNA virus would have important implications in host-virus interactions and antiviral therapy.

Capsid↗
PubMed1995

CCAAT/enhancer-binding protein isoforms beta and delta are expressed in mammary epithelial cells and bind to multiple sites in the beta-casein gene promoter.

Lactogenic hormone-dependent expression of the rat beta-casein gene in mammary epithelial cells is controlled via a complex regulatory region in the promoter. The sequence between -176 and -82 is the minimal region to confer the response to glucocorticoid hormone and prolactin on a heterologous promoter. The response is further enhanced by the region between -282 and -176. DNase I footprinting experiments and electromobility shift assays revealed the presence of four binding sites for CCAAT/enhancer-binding protein (C/EBP) isoforms in the hormone response region between -220 and -132. In nuclear extracts from mammary epithelial cells, the prevalent C/EBP isoform binding to these sites is beta (C/EBP-beta). C/EBP-delta is also present in mammary epithelial cells, whereas C/EBP-alpha is not detectable. The C/EBP sites are located in close proximity to the previously characterized binding sites for the prolactin-inducible mammary gland factor/signal transducer and activator of transcription-5, the nuclear factor YY1, and the glucocorticoid receptor. The importance of the two proximal C/EBP binding sites at the 5' border of the minimal region was tested by mutational analysis. Mutations of each site were found to inhibit strongly both the basal and the lactogenic hormone-induced transcription of a beta-casein gene promoter chloramphenicol acetyltransferase construct. The results implicate C/EBPs as important regulators of beta-casein gene expression in the mammary epithelium.

Animals↗
PubMed1995

Role of COOH-terminal phosphorylation in the regulation of casein kinase I delta.

Casein kinase I delta is a member of the casein kinase I (CKI) family, a group of second messenger independent protein kinases. We present evidence that the COOH-terminal domain of CKI delta has regulatory properties. CKI delta expressed in Escherichia coli was activated by heparin, as found previously, and by treatment with the catalytic subunit of type-1 protein phosphatase (CS1). Concomitant with activation by CS1, there was a reduction in the apparent molecular weight of CKI delta from 55,000 to 49,000 as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Truncation of CKI delta by removal of the COOH-terminal 110 amino acids eliminated the ability of CS1 to activate or to increase electrophoretic mobility. Casein kinase I alpha, a 37-kDa isoform that lacks an extended COOH-terminal domain, was not activated by CS1 or the presence of heparin. However, a chimeric enzyme consisting of CKI alpha fused to the COOH-terminal domain of CKI delta was activated by both heparin and CS1. Analysis of the effects of CS1 on a series of CKI delta COOH-terminal truncation mutants identified an inhibitory region between His317 and Pro342, which contained six potential phosphorylation sites. From analysis of the specific activites of these truncation mutants, removal of the same region resulted in enzyme with a specific activity nearly 10-fold greater than wild-type. Thus, CKI delta activity can be regulated by phosphorylation of its COOH terminus, which may serve to create an autoinhibitory domain. This mechanism of regulation could have important consequences in vivo.

Amino Acid Sequence↗
PubMed1996

Hormonally regulated double- and single-stranded DNA-binding complexes involved in mouse beta-casein gene transcription.

Transcription of the 252-base pair-long mouse beta-casein gene promoter is induced by the synergistic action of insulin, prolactin, and glucocorticoid in a primary mammary epithelial cell culture. The promoter contains a region termed block C having a highly conserved sequence and position among many casein genes. Mutation of block C reduced the response of the promoter to lactogenic hormones 84%. Nuclear extracts from lactating mouse mammary glands contained both a double-stranded and a single-stranded DNA binding protein complex (DS1 and SS), which specifically bind to the sequences AAATTAGCATGT and CCACAA of block C, respectively. The DS1 and the SS protein complexes were approximately 400 and 280 kDa, respectively. Each complex contained a DNA-binding component(s) having a molecular mass of approximately 120 kDa for DS1 and 80 and 65 kDa for SS. Deoxycholate, which interferes with the protein-protein interactions, inhibited the binding activities of DS1 and SS. The maximal increase in the binding activity of DS1 and SS in the mammary gland occurred during pregnancy and during lactation, respectively. In organ culture, the DS1 activity is increased by epidermal growth factor or prolactin in combination with insulin, whereas the SS activity is enhanced by insulin, prolactin, and glucocorticoid. These results suggest that multiprotein complexes binding to the double- and single-stranded DNA of block C mediate hormonal induction of beta-casein gene transcription.

Animals↗
PubMed1997

Stimulus-dependent phosphorylation of G-protein-coupled receptors by casein kinase 1alpha.

We have previously demonstrated that the phospholipase C-coupled m3-muscarinic receptor is phosphorylated in an agonist-sensitive manner by a protein kinase of approximately 40 kDa purified from porcine cerebellum (Tobin, A. B., Keys, B., and Nahorski, S. R. (1996) J. Biol Chem. 271, 3907-3916). This kinase, called muscarinic receptor kinase (MRK), is distinct from second messenger-regulated protein kinases and from beta-adrenergic receptor kinase and other members of the G-protein-coupled receptor kinase family. In the present study we propose that MRK is casein kinase 1alpha (CK1alpha) based on the following evidence: 1) the amino acid sequence from two proteolytic peptide fragments derived from purified MRK corresponded exactly to sequences within CK1alpha. 2) Casein kinase activity co-eluted with MRK activity from the final two chromatography steps in the purification of porcine brain MRK. 3) Recombinant CK1alpha expressed in Sf9 cells is able to phosphorylate both casein and the bacterial fusion protein, Ex-m3, that contains a portion of the third intracellular loop of the m3-muscarinic receptor downstream of glutathione S-transferase. 4) Partially purified CK1alpha increased the level of muscarinic receptor phosphorylation in an agonist-sensitive manner when reconstituted with membranes from Chinese hamster ovary-m3 cells expressing the human recombinant m3-muscarinic receptor. 5) Partially-purified CK1alpha phosphorylated rhodopsin, contained in urea-treated bovine rod outer segment membranes, and the extent of phosphorylation was increased in the presence of light. These data demonstrate that the kinase previously called MRK is CK1alpha, and that CK1alpha offers an alternative protein kinase pathway from that of the G-protein-coupled receptor kinase family for the stimulus-dependent phosphorylation of the m3-muscarinic receptor, rhodopsin, and possibly other G-protein-coupled receptors.

Animals↗
PubMed1999

Molecular chaperone-like properties of an unfolded protein, alpha(s)-casein.

All molecular chaperones known to date are well organized, folded protein molecules whose three-dimensional structure are believed to play a key role in the mechanism of substrate recognition and subsequent assistance to folding. A common feature of all protein and nonprotein molecular chaperones is the propensity to form aggregates very similar to the micellar aggregates. In this paper we show that alpha(s)-casein, abundant in mammalian milk, which has no well defined secondary and tertiary structure but exits in nature as a micellar aggregate, can prevent a variety of unrelated proteins/enzymes against thermal-, chemical-, or light-induced aggregation. It also prevents aggregation of its natural substrates, the whey proteins. alpha(s)-Casein interacts with partially unfolded proteins through its solvent-exposed hydrophobic surfaces. The absence of disulfide bridge or free thiol groups in its sequence plays important role in preventing thermal aggregation of whey proteins caused by thiol-disulfide interchange reactions. Our results indicate that alpha(s)-casein not only prevents the formation of huge insoluble aggregates but it can also inhibit accumulation of soluble aggregates of appreciable size. Unlike other molecular chaperones, this protein can solubilize hydrophobically aggregated proteins. This protein seems to have some characteristics of cold shock protein, and its chaperone-like activity increases with decrease of temperature.

Alcohol Dehydrogenase↗
PubMed2001

The development-associated cleavage of lens connexin 45.6 by caspase-3-like protease is regulated by casein kinase II-mediated phosphorylation.

Gap junctions are important in maintaining lens transparency and metabolic homeostasis. In this paper, we report that the gap junction-forming protein, connexin (Cx) 45.6, was specifically truncated during lens development and that the majority of the truncated fragments were located in the differentiated lens fibers. When isolated lens membranes were treated by caspase-3, the truncated fragments of Cx45.6 were reproduced, and this truncation occurred at the COOH terminus of Cx45.6. Moreover, when primary lens cells were treated with apoptosis-inducing reagents, Cx45.6 was cleaved similarly as the in vitro treatment by caspase-3, and this cleavage was blocked by a caspase-3 inhibitor. These results suggest that caspase-3 is responsible for the development-associated cleavage of Cx45.6. The cleavage site of Cx45.6 was identified between amino acid residues Glu(367) and Gly(368). We have shown previously that Ser(363) is an in vivo phosphorylated site by casein kinase II, and this specific phosphorylation leads to a rapid turnover of Cx45.6. Interestingly, we found here that when Ser(363) was phosphorylated by casein kinase II, the cleavage of Cx45.6 catalyzed by caspase-3 was inhibited. This study, for the first time, demonstrates that a connexin can be a direct target of an apoptotic protease and that cleavage by caspase-3-like protease leads to the development-associated truncation of a lens connexin. Finally, caspase-3-mediated cleavage can be regulated by casein kinase II-mediated phosphorylation, suggesting that Cx45.6 turnover and specific cleavage by caspase-3-like protease is alternatively modulated.

Animals↗
PubMed2004

Casein kinase II-mediated phosphorylation of NF-kappaB p65 subunit enhances inducible nitric-oxide synthase gene transcription in vivo.

Nitric oxide (NO) produced by inducible nitric-oxide synthase (NOSII) is mainly regulated at the transcriptional level by the nuclear factor-kappaB (NF-kappaB). In the present study, we further analyzed the role of NF-kappaB in the in vivo transcriptional regulation of NOSII gene by comparing two clones isolated from the EMT-6 mouse mammary cancer cell line. In response to interleukin (IL)-1beta or lipopolysaccharide (LPS), EMT-6 clone J (EMT-6J) cells produce 3-fold more NO than EMT-6 clone H (EMT-6H) cells, an effect correlated with enhanced activation of NF-kappaB in EMT-6J cells. In response to IL-1beta, the kinetics of degradation of NF-kappaB inhibitors IkappaB-alpha and IkappaB-beta, the nucleo-cytoplasmic shuttling of the transcription factor and its binding to a specific DNA sequence were similar in both clones. In contrast, an IL-1beta-induced phosphorylation of serine residues in NF-kappaB p65 subunit was observed in EMT-6J, but not in EMT-6H, cells. This IL-1beta-induced phosphorylation of p65 was specifically prevented by pretreatment of EMT-6J cells with the casein kinase II inhibitor DRB. Small interfering RNA-mediated depletion of casein kinase II-alpha subunit also decreased NF-kappaB transcriptional activity and NOSII gene transcription in IL-1beta and LPS-stimulated EMT-6J cells to the levels observed in EMT-6H cells treated in the same conditions. Altogether, these data indicate that casein kinase II-mediated phosphorylation of p65 subunit can enhance the transcriptional activity of NF-kappaB in vivo. This post-translational modification of the transcription factor can be responsible for increased NOSII gene transcription and NO production in tumor cells exposed to either IL-1beta or LPS.

Animals↗
PubMed1980

Phosphopeptides and soluble calcium in the small intestine of rats given a casein diet.

1. Semi-synthetic diets containing 200 g protein/kg were meal-fed for 1.5 h to groups of rats. The contents of the whole small intestinal tract were collected and the amount of soluble calcium was determined. 2. In the rats given 200 g casein/kg diet, formation of a fraction containing macrophosphopeptide in the small intestine was confirmed by gelfitration of the intestinal contents on Sephadex G-25. However, this macrophosphopeptide fraction was not found when casein alone was fed. 3. In the intestinal contents at 2.5 h after ingestion, the amounts of both soluble Ca and phosphorus were significantly higher in rats fed the casein diet than in those fed diets containing egg albumin, isolated soya-bean protein or an amino acid mixture. However, the amount of insoluble Ca was least in rats fed the casein diet.

Animals↗
PubMed1982

Protein-polyphenol reactions. 1. Nutritional and metabolic consequences of the reaction between oxidized caffeic acid and the lysine residues of casein.

1. Studies were made on the lysine content of casein reacted with caffeic acid oxidized aerobically under alkaline conditions of enzymically with tyrosinase (EC 1. 14. 18. 1). 2. Loss of fluorodinitrobenzene (FDNB)-reactive lysine was rapid at pH 10 and increased with time and the temperature of the reaction, with concentration of caffeic acid and with the oxygenation of the mixture. In presence of the enzyme mushroom tyrosinase, maximum reduction of reactive lysine occurred at pH 7 and was dependent on the reaction time and on the concentration of caffeic acid. 3. Reaction of alpha-formyl-LO-[U-14C]lysine with caffeic acid at pH 10 showed the rapid formation of five reaction products which appeared to polymerize gradually as the reaction progressed. 4. The nutritionally available lysine content of the casein-caffeic acid mixtures, as assayed with rats, was reduced after both alkaline and enzyme reactions, as were faecal digestibility, net protein ratio and net protein utilization. Biological value however was not reduced. 5. In metabolic studies using goat milk casein labelled with L-[3H]lysine and reacted with caffeic acid in the same way, the lysine-caffeoquinone reaction products were not absorbed by the rat but were excreted directly in the faeces. 6. The importance of the reaction of proteins with caffeoquinone and chlorogenoquinone (formed by the oxidation of caffeic and chlorogenic acids respectively) is discussed in relation to the production of sunflower protein, leaf protein and other vegetable-protein concentrates.

Animals↗