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PubMed2003

Charged casein-derived oligopeptides competitively inhibit the transport of a reporter oligopeptide by Lactococcus lactis.

AIM: To study the effect of casein-derived peptides, accumulated during growth of Lactococcus lactis in milk, on its oligopeptide transport (Opp) function. METHODS AND RESULTS: This effect was estimated by analysing the ability of casein-derived peptides to compete for the transport of a reporter peptide by whole L. lactis cells. The transport of the reported peptide was monitored by determining the intracellular concentrations of the corresponding amino acids by means of reverse-phase high-performance liquid chromatography (HPLC). Uptake of the reporter peptide was competitively inhibited by casein-derived peptides. The competition was only because of charged casein-derived peptides, including anionic peptides. The design of specific pure peptides made it possible to evidence for a positive (or negative) influence exerted by the positively (or negatively) charged side chain of the N-terminal amino acid on the competition. CONCLUSIONS: Charged casein-derived peptides impaired the oligopeptide transport function of L. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate an inhibition of Opp when too many peptides are produced by the proteinase. Peptide transport by Opp therefore represents a bottleneck for increasing the growth rate of L. lactis in milk.

Animals↗
PubMed2003

The phosphorylation pattern of human alphas1-casein is markedly different from the ruminant species.

Caseins are highly phosphorylated milk proteins assembled in large colloidal structures termed micelles. In the milk of ruminants, alphas1-casein has been shown to be extensively phosphorylated. In this report we have determined the phosphorylation pattern of human alphas1-casein by a combination of matrix-assisted laser desorption mass spectrometry and amino acid sequence analysis. Three phosphorylation variants were identified. A nonphosphorylated form, a variant phosphorylated at Ser18 and a variant phosphorylated at Ser18 and Ser26. Both phosphorylation sites are located in the amino acid recognition sequence of the mammary gland casein kinase. Notably, no phosphorylations were observed in the conserved region covering residues Ser70-Glu78, which is extensively phosphorylated in the ruminant alphas1-caseins.

Amino Acid Sequence↗
PubMed1984

In vivo milk digestion in the calf abomasum. I. Whole-casein digestion.

Peptide products insoluble in 12% TCA and obtained in the proximal duodenum of calves at different times after ingestion of casein solutions were characterized by polyacrylamide gel electrophoresis, electrofocusing and SDS pore gradient gel electrophoresis. With a 3% whole-casein solution in water the disappearance of electrophoretic bands corresponding to alpha s1 and beta caseins was observed after about 1 h 30. After 3 h and up to 7 h very acidic peptides appeared. With a 3% whole-casein solution in simulated milk ultrafiltrate, comparable patterns were obtained. Nevertheless, the acidic peptides appeared sooner, i.e. they were already detected in the first sample collected after meal ingestion. With the 2 diets, the importance of gastric proteolysis was demonstrated by the appearance of a great number of peptides of various sizes, charges and pHi. On the other hand, an effect of salts on casein proteolysis was detected.

Abomasum↗
PubMed1992

The possible involvement of protein kinase C(s) and inositol phosphate metabolism in the basal but not in the prolactin stimulated casein release by the lactating rabbit mammary epithelial cell.

The secretagogue effect of prolactin (PRL) on casein release by epithelial mammary cells has been previously related to stimulation of the phospholipase A2-arachidonic acid cascade. In order to determine whether other intracellular pathways are implicated in this secretagogue effect, different agents acting on protein kinase C (PKC) and phospholipase C (PLC) activity have been assessed in vitro in lactating rabbit mammary gland fragments. Phorbol ester (20 nm TPA and 1-oleoyl-2-acetyl-sn-glycerol (10 microM (OAG) stimulated newly synthesized casein secretion and potentiated the PRL secretatogue effect. However, 100 microM quercetin, 100 microM H-7 and 5 and 20 nM staurosporine did not inhibit the latter effect. Exogenous PLC did not stimulate casein secretion. PRL did not affect production of inositol phosphates (IPs) during 10 or 60 min exposure. These results show that PKC activation may increase basal levels of casein secretion, and demonstrate that PRL does not act primarily via PKC activation or by PLC activation to stimulate casein secretion.

Alkaloids↗
PubMed1981

Bovine alpha-lactalbumin C and alpha S1-, beta- and kappa-caseins of Bali (Banteng) cattle, bos (Bibos) javanicus.

An electrophoretic examination is made of mild samples taken from eight Bali (banteng) cattle, Bos (bibos) javanicus, at Beatrice Hills, Northern territory, Australia. Starch-gel electrophoresis at pH 9.5 (NaOH-H3BO3 buffer) and filter-paper electrophoresis at pH 8.6 (diethylbarbiturate buffer) indicate that all samples contain a new alpha-lactalbumin variant, designated alpha-lactalbumin C. The order of mobility for bovine variants is / greater than B greater than C. The C variant differs from the common B variant in having one more amide residue (substitution or Gln for Glu). Examination of milk samples by urea-starch-gel electrophoresis at alkaline pH indicates that there is a new alpha S1-casein variant, designated alpha S1-casein EBali, present in some samples. No new kappa-casein variant is detected by this method (all samples typing as kappa-casein B). A new variant of beta-casein, designated A4, is detected by urea-starch-gel electrophoresis at low pH. The variants of milk proteins observed in this paper and in Bell et al. (1981) are discussed in relation to those of other members of the Bovinae, especially the yak, bos (Poephagus) grunniens.

Amino Acids↗
PubMed1977

Hormonal effects on intracellular and secreted casein in cultures of mouse mammary epithelial cells on floating collagen membranes.

Cultured on floating collagen membranes in the presence of lactogenic hormones, dissociated normal mammary epithelial cells from prelactating mice acquire the ultrastructural and biochemical characteristics of differentiated mammary secretory cells in vivo. The cells on floating collagen membranes in medium containing insulin alone have sparse secretory organelles, and a small amount of casein can be detected in these cells with a sensitive radioimmunoassay. These cells resemble counterpart cells in early-pregnant mice. When the cells are exposed to insulin, cortisol, and prolactin, the secretory apparatus is elaborated and significant increases in intracellular and extracellular casein are observed. In this environment, the intracellular casein content is generally four to eight times greater than in freshly dissociated cells or cells cultured in insulin alone. The amount of casein secreted into the medium by floating-collagen-membrane cultures in the three hormones is from 25 to 200 times greater than that secreted by cultures in insulin alone. Cells cultured on plastic substrates in either hormone combination fail to show any increase in intracellular or extracellular casein. On floating collagen membranes, the cells differentiate in response to hormones as they do in vivo and in organ culture. This cell-culture system provides an opportunity to study direct effects of environmental factors on mammary differentiation at the cellular level.

Animals↗
PubMed1979

Glucocorticoid modulation of casein gene transcription in mouse mammary gland.

The influence of cortisol and prolactin on casein gene expression in the mammary gland of lactating BALB/c mice was measured by using a specific cDNA probe to 15S casein mRNA (cDNAcsn). Casein mRNA (mRNAcsn) level in the mammary gland was decreased by 85% 5 days after adrenal ablation, but then was increased 4.4-fold 12 hr after a single injection of hydrocortisone-21-acetate. An 80% decrease in serum prolactin level, induced by the prolactin inhibitor 2-bromo-alpha-ergocryptin (CB-154), did not alter the level of mRNAcsn in the gland. Specific transcription of the casein gene in nuclei isolated from lactating mammary glands was measured by cDNAcsn hybridization to the in vitro synthesized Hg-CTP-containing RNA (Hg-RNA), which was purified by SH-agarose chromatography. The level of the mRNAcsn in Hg-RNA synthesized in the isolated nuclei was 0.09% and this was decreased 85% by alpha-amanitin, indicating that the mRNAcsn sequences in the Hg-RNA were the products of RNA polymerase II-directed DNA-dependent RNA synthesis. Transcription of the mRNAcsn in isolated nuclei was decreased by 70% 5 days after adrenalectomy and a single injection of the glucocorticoid then increased the transcription level 2-fold at 6 hr. Essentially no alteration of the level of transcription was detectable in mammary nuclei isolated from lactating mice with 80% decreased serum prolactin level, induced by CB-154 treatment. The results thus demonstrate a glucocorticoid involvement on the modulation of casein gene expression at the transcriptional level of control.

Adrenalectomy↗
PubMed1985

A synthetic peptide substrate specific for casein kinase II.

A synthetic peptide having the sequence Arg-Arg-Arg-Glu-Glu-Thr-Glu-Glu-Glu was found to serve as a convenient substrate for the protein kinase generally referred to as casein kinase II. The enzyme exhibited an apparent Km of 500 microM for the peptide, as compared to an apparent Km of 50 microM for casein. The maximum velocities for phosphorylation of the peptide and of casein were similar. The peptide was not phosphorylated by any of eight other protein kinases, all of which were shown to be active toward their known substrates. The peptide was used to monitor activity during steps in the purification of casein kinase II from bovine liver. These experiments demonstrated that with this peptide it is now possible to obtain specific measurements of casein kinase II activity in crude enzyme preparations.

Animals↗
PubMed1990

Transfection of beta-casein chimeric gene and hormonal induction of its expression in primary murine mammary epithelial cells.

To study the regulatory sequence elements responsible for casein gene expression, we constructed a chimeric gene containing 5.3 kilobases (kb) of the 5'-flanking sequence and 1.6 kb of the 3'-flanking sequence of the mouse beta-casein gene fused to the bacterial chloramphenicol acetyl-transferase (CAT) gene. The chimeric gene was transfected by the calcium phosphate-precipitation procedure into primary mouse mammary epithelial cells prepared from pregnant mice. The transfection procedure had negligible effect on expression of the endogenous beta-casein gene. Expression of the beta-casein-CAT chimeric gene required the synergistic actions of insulin, hydrocortisone, and prolactin. Expression of the chimeric gene also depended on the appropriate substratum because the degree of hormonal induction of the chimeric gene was much higher in cells cultured on a reconstituted basement membrane (Matrigel) than in cells cultured on either type I collagen gel or plastic. On the other hand, the expression of a simian virus 40-CAT chimeric gene in which the CAT gene was driven by the early promoter of the virus was not influenced by the hormonal milieu and occurred at the highest level in cells cultured on plastic. Additional transfection experiments with a series of beta-casein-CAT constructs suggested the existence of regulatory elements responsible for hormonal induction and negative regulatory elements.

Animals↗
PubMed1990

Stimulation of casein kinase II by epidermal growth factor: relationship between the physiological activity of the kinase and the phosphorylation state of its beta subunit.

To determine relationships between the hormonal activation of casein kinase II and its phosphorylation state, epidermal growth factor (EGF)-treated and EGF-naive human A-431 carcinoma cells were cultured in the presence of [32P]orthophosphate. Immunoprecipitation experiments indicated that casein kinase II in the cytosol of EGF-treated cells contained approximately 3-fold more incorporated [32P]phosphate than did its counterpart in untreated cells. Levels of kinase phosphorylation paralleled levels of kinase activity over a wide range of EGF concentrations as well as over a time course of hormone action. Approximately 97% of the incorporated [32P]phosphate was found in the beta subunit of casein kinase II. Both activated and hormone-naive kinase contained radioactive phosphoserine and phosphothreonine but no phosphotyrosine. On the basis of proteolytic mapping experiments, EGF treatment of A-431 cells led to an increase in the average [32P]phosphate content (i.e., hyperphosphorylation) of casein kinase II beta subunit peptides which were modified prior to hormone treatment. Finally, the effect of alkaline phosphatase on the reaction kinetics of activated casein kinase II indicated that hormonal stimulation of the kinase resulted from the increase in its phosphorylation state.

Alkaline Phosphatase↗
PubMed1990

Extracellular matrix and hormones transcriptionally regulate bovine beta-casein 5' sequences in stably transfected mouse mammary cells.

Milk protein regulation involves synergistic action of lactogenic hormones and extracellular matrix (ECM). It is well established that substratum has a dramatic effect on morphology and function of mammary cells. The molecular mechanisms that regulate the ECM- and hormone-dependent gene expression, however, have not been resolved. To address this question, a subpopulation (designated CID 9) of the mouse mammary epithelial cell strain COMMA-1D has been developed in which more than 35% of the cells express beta-casein, form alveoli-like structures when plated onto a reconstituted basement membrane, and secrete beta-casein unidirectionally into a lumen. These cells were stably transfected with a series of chloramphenicol acetyltransferase (CAT) fusion genes to study transcriptional regulation of the bovine beta-casein gene. The expression of CAT in these lines demonstrated a striking matrix and hormone dependency (greater than 150-fold induction in some cases). This regulation occurred primarily at the transcriptional level and was dependent on the length of the 5' flanking region of the beta-casein promotor. Both matrix and hormonal control of transcription occurred within at least the first 1790 base pairs upstream and/or 42 base pairs downstream of the transcriptional initiation site. The ECM effect was independent of glucocorticoid stimulation. However, prolactin was essential and hydrocortisone further increased CAT expression. Endogenous beta-casein expression in these lines was similar to that of the parent CID 9 cells. Our data indicate the existence of matrix-dependent elements that regulate transcription.

Animals↗
PubMed1995

Phosphorylation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, by casein kinase I in vitro and in vivo.

DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M(r) = 32,000) is a potent inhibitor of protein phosphatase-1 when it is phosphorylated on Thr-34 by cAMP-dependent protein kinase. DARPP-32 is highly enriched in some specific cell populations such as striatonigral neurons and choroid plexus epithelial cells. Here we show that recombinant rat DARPP-32 is phosphorylated by casein kinase I on seryl residues to a stoichiometry of approximately 2 mol of phosphate/mol of protein. DARPP-32 is one of the best known substrates for casein kinase I (Km = 3.4 +/- 0.3 microM), whereas the homologous phosphatase-1 inhibitor, inhibitor-1, is not. Phosphorylation of DARPP-32 by casein kinase I does not alter its ability to inhibit protein phosphatase-1. Residues phosphorylated by casein kinase I were identified as Ser-137 and Ser-189 by site-directed mutagenesis and by protein sequencing. Ser-137 and the preceding stretch of 16-18 acidic residues are conserved in DARPP-32 among all species examined, whereas Ser-189 is not. Phosphorylation of Ser-137 induces an unusual increase in DARPP-32 electrophoretic mobility in polyacrylamide gels in the presence of SDS. In striatonigral neurons, DARPP-32 is phosphorylated on Ser-137 and the stoichiometry of phosphorylation on this residue in vivo appears to be higher in the substantia nigra (axon terminals) than in the striatum (soma and dendrites). These results indicate that casein kinase I is highly active in striatonigral neurons in which it may play important roles, including in protein phosphatase-1 modulation via phosphorylation of DARPP-32.

Amino Acid Sequence↗
PubMed1996

Lactogenic hormone activation of Stat5 and transcription of the beta-casein gene in mammary epithelial cells is independent of p42 ERK2 mitogen-activated protein kinase activity.

HC11 mammary epithelial cells have been used to characterize molecular events involved in the regulation of milk protein gene expression. Treatment of HC11 cells with the lactogenic hormones prolactin, insulin, and glucocorticoids results in transcription of the beta-casein gene. Prolactin induces a signaling event which involves tyrosine phosphorylation of the mammary gland factor, Stat5, a member of the family of signal transducers and activators of transcription (Stat). Here we show that HC11 cells express two Stat5 proteins, Stat5a and Stat5b. Phosphopeptide and phosphoamino acid analysis of Stat5a and Stat5b immunoprecipitated from phosphate-labeled HC11 cells revealed that both proteins were constitutively phosphorylated on serine. Lactogenic hormone treatment resulted in the appearance of a tyrosine-phosphorylated peptide in both Stat5 proteins. Consistent with this observation, a Western blot analysis of Stat5a and Stat5b showed that lactogenic hormones induced a rapid, transient increase in phosphotyrosine which paralleled the binding of Stat5 to its cognate recognition sequence in the beta-casein gene promoter. Lactogenic hormone treatment of the HC11 cells also led to a rapid activation of the mitogen-activated protein (MAP) kinase pathway. We examined the role of this pathway in beta-casein transcription using a specific MAP kinase kinase inhibitor, PD98059. Concentrations of PD98059 which completely abrogated lactogen-induced MAP kinase activation did not affect the phosphorylation state of Stat5, its DNA binding activity, or transcriptional activation of a beta-casein reporter construct. This indicates that the MAP kinase pathway does not contribute to lactogenic hormone induction of the beta-casein gene.

Animals↗
PubMed1999

Transcriptional regulation of endothelial nitric-oxide synthase by an interaction between casein kinase 2 and protein phosphatase 2A.

We previously demonstrated that lysophosphatidylcholine up-regulated endothelial nitric-oxide synthase promoter activity by increasing Sp1 binding via the action of protein serine/threonine phosphatase 2A (Cieslik, K., Zembowicz, A., Tang, J.-L., and Wu, K.K. (1998) J. Biol. Chem. 273, 14885-14890). To characterize the regulation of basal endothelial nitric-oxide synthase promoter activity and the signaling pathway through which lysophosphatidylcholine augments endothelial nitric-oxide synthase transcription, we used a casein kinase 2 inhibitor coupled with immunoprecipitation to demonstrate that basal Sp1 binding and endothelial nitric-oxide synthase promoter activity were controlled by casein kinase 2 complexed with protein serine/threonine phosphatase 2A. Casein kinase 2 catalyzed protein serine/threonine phosphatase 2A phosphorylation thereby inhibiting its activity. Lysophosphatidylcholine selectively activated p42/p44 mitogen-activated protein kinase. Purified extracellular regulated kinase 2 blocked casein kinase 2 activity and increased protein serine/threonine phosphatase 2A activity, resulting in an increased Sp1 binding and endothelial nitric-oxide synthase promoter activity. These results indicate that Sp1 binding to its cognate site on the endothelial nitric-oxide synthase promoter and its transactivation of endothelial nitric-oxide synthase is regulated by post-translational Sp1 phosphorylation and dephosphorylation through a dynamic interaction between casein kinase 2 and protein serine/threonine phosphatase 2A.

Casein Kinase II↗
PubMed2001

Osteoprotegerin ligand induces beta-casein gene expression through the transcription factor CCAAT/enhancer-binding protein beta.

Osteoprotegerin ligand (OPGL, also known as RANKL), a member of the tumor necrosis factor superfamily, is essential for mammary gland development during pregnancy in addition to key roles in the immune system and bone development. Here we show that OPGL induces beta-casein transcription through the CCAAT/enhancer-binding protein beta (C/EBPbeta). In both HC11 cell lines and primary mammary epithelial cells, OPGL stimulation triggers rapid nuclear translocation of C/EBPbeta, which is critical for the expression of the beta-casein gene. Mutation of C/EBbeta binding sites in the beta-casein gene promoter completely abrogated OPGL-induced beta-casein promoter activity. By contrast, OPGL stimulation did not result in STAT5 phosphorylation. In vivo immunohistochemistry studies further demonstrated defective nuclear translocation of C/EBPbeta, but normal STAT5 activation, in OPGL-deficient mice. These data show that OPGL is a critical activator of beta-casein gene expression via the transcription factor C/EBPbeta. Our data provide new insights into the understanding of the molecular events involved in milk protein gene expression.

Active Transport, Cell Nucleus↗
PubMed2006

Casein kinase II phosphorylation of the yeast phospholipid synthesis transcription factor Opi1p.

The transcription factor Opi1p regulates phospholipid synthesis in the yeast Saccharomyces cerevisiae by repressing the expression of several UAS(INO)-containing genes (e.g. INO1). Opi1p repressor activity is most active in inositol-supplemented cells. Regulation of Opi1p repressor activity is mediated by multiple phosphorylations catalyzed by protein kinases A and C. In this work, we showed that Opi1p was also phosphorylated by casein kinase II. Using purified maltose-binding protein-Opi1p as a substrate, casein kinase II activity was dose-and time-dependent and dependent on the concentrations of maltose-binding protein-Opi1p (Km = 25 microg/ml) and ATP (Km = 7 microM). Of three mutations (S10A, S38A, and S239A) in putative phosphorylation sites, 10 only the S10A mutation affected Opi1p phosphorylation. That Ser10 was a specific target of casein kinase II was confirmed by the loss of a phosphopeptide in the S10A mutant protein. The S10A mutation did not affect phosphorylation of Opi1p by either protein kinase A or protein kinase C. Likewise, phosphorylation of Opi1p by casein kinase II was not affected by mutations in protein kinase A (S31A and S251A) and protein (S10A) kinase C (S26A) phosphorylation sites. Expression of the OPI1 allele in an opi1Delta mutant attenuated (2-fold) the repressive effect of Opi1p on INO1 expression, and this effect was only observed when cells were grown in the absence of inositol. These data supported the conclusion that casein kinase II phosphorylation at Ser10 played a role in stimulating the repression of INO1 when Opi1p was not in its most active state (i.e. in inositol-deprived cells).

Adenosine Triphosphate↗
PubMed1978

The effect of the extent of hydrolysis in casein on its specific dynamic action in the rat.

1. Adult female rats given a diet containing 200 g casein/kg showed an increase in heat production which reached a maximum in 10--20 min after completion of food intake. 2. Replacement of casein in the diet by enzymic hydrolysates of casein of different extents of hydrolysis (pepsin for 1 or 3 h and pancreatin for 6, 12 or 24 h) resulted in a decrease in metabolic rate (stimulation) in the rat, reaching a maximum of 61.8% of the control value (non-hydrolysed casein). 3. The specific dynamic action of casein and casein hydrolysate was inversely proportional to the amount of amino-nitrogen released. 4. On the basis of the experimental findings it may be concluded that the synthesis and secretion of digestive enzymes were the main causes of the increase in metabolic rate after protein ingestion.

Animals↗
PubMed1987

The importance of the non-protein components of the diet in the plasma cholesterol response of rabbits to casein. Zinc and copper.

To characterize further the effect of protein-mineral interaction on plasma cholesterol, four groups of rabbits were placed on cholesterol-free, semi-purified diets for 12 weeks. The diets were similar in proximate composition with either casein or soya-bean protein isolate as the protein source (250 g/kg). The dietary zinc content of the casein diets was increased to 22 mg/kg to equal that of the soya-bean diets. The copper content was set at 2 and 6 mg/kg. There were no differences in weight gain; all animals grew at a similar rate. When the dietary Zn content was equalized, the plasma and liver cholesterol responses were similar regardless of the source of protein. Differences in liver Cu and Zn were observed. As dietary Cu increased, Cu decreased and Zn increased in the liver. Hepatic Zn was also higher in soya-bean-fed than in casein-fed animals. No effects on plasma Cu and Zn were seen. The addition of Zn to the casein diet resulted in a similar plasma cholesterol to that of animals fed on the soya-bean diet, suggesting that casein-induced hypercholesterolaemia is due to a marginal Zn intake.

Animals↗