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PubMed2002

Endogenous protein phosphorylation and casein kinase activity during seed development in Araucaria angustifolia.

Protein kinases and phosphatases are responsible for several cellular events mediated by protein phosphorylation and dephosphorylation. Among these events are cell growth and differentiation and cellular metabolism. Casein kinase I (CKI) and casein kinase II (CKII) are involved in the phosphorylation of several substrates. Endogenous protein phosphorylation and casein kinase activity were investigated in the megagametophyte of the native Brazilian conifer Araucaria angustifolia, during seed development. It was observed that a number of different polypeptides are phosphorylated in vitro in the three megagametophyte stages of development tested (from globular, cotyledonary and mature embryos, respectively) and the phosphate was incorporated mainly in serine residues. The use of okadaic acid and vanadate in the phosphorylation reactions increased phosphate incorporation in several polypeptides suggesting the presence of serine/threonine as well as tyrosine phosphatases in the megagametophyte. Also, the results obtained in experiments with CKII inhibitor, GTP as phosphate donor, RNA hybridizations, and in-gel kinase assays indicate the presence of CKII in the A. angustifolia megagametophyte.

Casein Kinase II↗
PubMed2002

Biochemical and enzymatic characterization of a partially purified casein kinase-1 like activity from Trypanosoma cruzi.

Two protein kinase activities that use casein as a substrate, Q-I and Q-II, were identified in the epimastigote stage of Trypanosoma cruzi upon chromatography on Q-Sepharose. Q-I was purified further through concanavalin A-sepharose (Q-I*) to remove any trace of the contaminating protease cruzipain. The optimal activity for Q-I* was obtained at pH 8.0, 25 degreesC, 5 mM MgCl(2) and 75 mM NaCl. The size and pI of Q-I* were determined to be 33-36 kDa and 9.6, respectively. When two selective peptide substrates for casein kinases (CKs) (P1: RRKDLHDDEEDEAMSITA for CK1 and P2: RRRADDSDDDDD for CK2) were used, Q-I* was shown to specifically phosphorylate P1. Kinetic studies showed that Q-I* has a K(m) of 5.3 +/- 0.34 mg/ml for casein, 157.6 +/- 5.3 microM for P1 and 35.9 +/- 3.9 microM for ATP. The enzyme was inhibited by N-(2-amino-ethyl)-5-chloroisoquinoline-8-sulfonamide (CKI-7) or 1-(5-chloroisoquinoline-8-sulfonyl) (CKI-8), two inactivators of mammalian CKs. CKI-7 behaved as a competitive inhibitor with respect to ATP, with a K(I) of 75-100 microM. Treatment with high concentrations of polylysine or heparin also resulted in a significant inhibition of Q-I*. Two well-known activators of mammalian CKs, spermine and spermidine, were also tested. Spermine and spermidine activated Q-I* in a dose-dependent manner. Based on the following characteristics: (1) the ionic strength required for elution from anion-exchange resins; (2) its molecular size and monomeric structure; (3) pI; (4) high level of specificity for P1; (5) inactivation by CKI-7 and CKI-8; and (6) insensitivity to GTP and low concentrations of heparin, we conclude that Q-I* belongs to the CK1 family of protein kinases.

Amino Acid Sequence↗
PubMed1965

THE SEPARATION OF MAJOR COMPONENTS OF THE CASEIN OF BOVINE MILK BY ELECTROPHORESIS IN A DENSITY GRADIENT.

1. Zonal electrophoresis in a column stabilized by a density gradient has been applied to the small-scale fractionation of the proteins of the casein complex of cow's milk. 2. The alpha(s)- and beta-fractions from the milk of individual Ayrshire cows have been shown to behave as single homogeneous proteins on electrophoresis at two pH values in starch gels. 3. The alpha(s)-fraction has been found to be indistinguishable from alpha(s)-casein prepared by Ca(2+) fractionation of the same milk samples. 4. On the evidence of their electrophoretic behaviour in starch gels and their elementary analyses, alpha(1)- and alpha(s)-casein are concluded to be substantially the same protein.

Animals↗
PubMed2004

Glucose sensing and signaling in Saccharomyces cerevisiae through the Rgt2 glucose sensor and casein kinase I.

The yeast Saccharomyces cerevisiae senses glucose through two transmembrane glucose sensors, Snf3 and Rgt2. Extracellular glucose causes these sensors to generate an intracellular signal that induces expression of HXT genes encoding glucose transporters by inhibiting the function of Rgt1, a transcriptional repressor of HXT genes. We present the following evidence that suggests that the glucose sensors are coupled to the membrane-associated protein kinase casein kinase I (Yck1). (i) Overexpression of Yck1 leads to constitutive HXT1 expression; (ii) Yck1 (or its paralogue Yck2) is required for glucose induction of HXT1 expression; (iii) Yck1 interacts with the Rgt2 glucose sensor; and (iv) attaching the C-terminal cytoplasmic tail of Rgt2 to Yck1 results in a constitutive glucose signal. The likely targets of Yck1 in this signal transduction pathway are Mth1 and Std1, which bind to and regulate function of the Rgt1 transcription factor and bind to the C-terminal cytoplasmic domain of glucose sensors. Potential casein kinase I phosphorylation sites in Mth1 and Std1 are required for normal glucose regulation of HXT1 expression, and Yck1 catalyzes phosphorylation of Mth1 and Std1 in vitro. These results support a model of glucose signaling in which glucose binding to the glucose sensors causes them to activate Yck1 in the cell membrane, which then phosphorylates Mth1 and Std1 bound to the cytoplasmic face of the glucose sensors, triggering their degradation and leading to the derepression of HXT gene expression. Our results add nutrient sensing to the growing list of processes in which casein kinase I is involved.

Casein Kinase I↗
PubMed1992

Yeast casein kinase I homologues: an essential gene pair.

We report the isolation of an essential pair of Saccharomyces cerevisiae genes that encode protein kinase homologues. The two genes were independently isolated as dosage-dependent suppressors. Increased dosage of YCK1 suppressed defects caused by reduced SNF1 protein kinase activity, and increased dosage of YCK2 relieved sensitivity of wild-type cells to salt stress. The two genes function identically in the two growth assays, and loss of function of either gene alone has no discernible effect on growth. However, loss of function of both genes results in inviability. The two predicted protein products share 77% overall amino acid identity and contain sequence elements conserved among protein kinases. Partial sequence obtained for rabbit casein kinase I shares 64% identity with the two yeast gene products. Moreover, an increase in casein kinase I activity is observed in extracts from cells overexpressing YCK2. Thus YCK1 and YCK2 appear to encode casein kinase I homologues.

Amino Acid Sequence↗
PubMed1993

Casein kinase I-like protein kinases encoded by YCK1 and YCK2 are required for yeast morphogenesis.

Casein kinase I is an acidotropic protein kinase class that is widely distributed among eukaryotic cell types. In the yeast Saccharomyces cerevisiae, the casein kinase I isoform encoded by the gene pair YCK1 and YCK2 is a 60- to 62-kDa membrane-associated form. The Yck proteins perform functions essential for growth and division; either alone supports growth, but loss of function of both is lethal. We report here that casein kinase I-like activity is associated with a soluble Yck2-beta-galactosidase fusion protein in vitro and that thermolabile protein kinase activity is exhibited by a protein encoded by fusion of a temperature-sensitive yck2 allele with lacZ. Cells carrying the yck2-2ts allele arrest at restrictive temperature with multiple, elongated buds containing multiple nuclei. This phenotype suggests that the essential functions of the Yck proteins include roles in bud morphogenesis, possibly in control of cell growth polarity, and in cytokinesis or cell separation. Further, a genetic relationship between the yck2ts allele and deletion of CDC55 indicates that the function of Yck phosphorylation may be related to that of protein phosphatase 2A activity.

Alleles↗
PubMed1998

Engineering of trypsin and its impact on beta-casein processing.

Tryptic processing of beta-casein yields several important nutraceutic and nutritious peptides. However, a final product peptide (1-105) stops the processing, inhibiting the enzyme. In attempt to modulate catalytic properties of this protease, K188 was replaced with aromatic amino acid residues. This aimed amplification of local hydrophobic and electrostatic interactions at the substrate binding site. The catalytic properties of obtained mutants (K188F, K188Y, and K188W) were measured at pH 7, 8, 9, and 10 with synthetic substrates and beta-casein. Kinetic analysis revealed that all the mutants conserve the capacity to split peptide bonds involving arginyl and lysyl residues. However, depending on mutation, the optimum pH of activity changes. As shown only by proteolysis of a natural substrate, produced mutants cleaved near 30 new peptide bonds compared to wild-type trypsin, 8 of them involving asparagine and glutamine amino acids. Some of the new cleavage sites can be related to the nature of the amino acid residue introduced in position 188. Consequently, only the joint use of several methods (synthetic substrate, protein substrate, influence of pH) can help to define better the differences of catalytic properties of wild-type and mutant proteases. Modifications introduced by the mutations are at the origin of the alteration of the specificity of the studied enzymes which are cleaving beta-casein in many places, hydrolysing well the fragment 1-105. Since many tryptic inhibitors contain amidated Glu and Asp, and form amyloid structures, the new mutants could be used in hydrolysing resistant proteic structures.

Amino Acid Sequence↗
PubMed2001

On-membrane digestion of beta-casein for determination of phosphorylation sites by matrix-assisted laser desorption/ionization quadrupole/time-of-flight mass spectrometry.

This article discusses the features of a newly developed matrix-assisted laser desorption/ionization quadrupole/time-of-flight (MALDI-QqTOF) mass spectrometer that is useful in the analysis of phosphorylated peptides. Aliquots of beta-casein, a commonly used phosphorylated protein standard, were digested with trypsin directly on a non-porous polyurethane membrane used as sample support in MALDI-QqTOF mass spectrometry (MS) experiments. Although a complete peptide map was obtained, it was difficult to obtain sequence information for some of the tryptic fragments, in particular T1-2, which bears four phosphate groups and is thus difficult to ionize in positive mode. This article focuses on the sequencing of this particular fragment by comparing MS/MS spectra obtained using different precursor ions. These precursors associated with T1-2 were [M + H](+), [M + H](2+), and [M + H - nH(3)PO(4)](+) ions. Typically, phosphorylated ions showed facile unimolecular losses of phosphoric acid moieties, and produced limited backbone fragmentation. The abundance of [M + H](2+) ions of T1-2 in the full mass spectrum was low relative to that of [M + H](+). [M + H - 4H(3)PO(4)](+) ions as MS/MS precursors underwent backbone fragmentations, with phosphoserine residues transformed into dehydroalanines or serines. Unusual b + 18 u fragments were observed, although only for segments with previously phosphorylated serines. These partly interfered with c-ions, and were noticeable due to overlapping isotopic envelopes. It was possible to establish the sequence of phosphorylated tryptic fragment T1-2 and the location of phosphate groups using the mass of dehydroalanine residues (69 Da) and b + 18 u fragments as markers. All MS and MS/MS spectra obtained with fully phosphorylated beta-casein were compared with spectra acquired with dephosphorylated beta-casein obtained commercially. These comparisons helped assess the spectral differences caused by the presence of phosphate groups. Also, they highlighted the potential usefulness of conducting dephosphorylation directly on the probe prior to MALDI analysis in future studies.

Caseins↗
PubMed2001

Thermodynamic compatibility of sodium caseinate with different pectins. Influence of the milieu conditions and pectin modifications.

Combinations of pectins and caseins are ingredients of many food products. Therefore the thermodynamic compatibility of both components was examined to investigate the influences of environmental factors as well as of the structure of the pectin. High-methoxyl pectin was demethoxylated and amidated, respectively, and tested for the compatibility with sodium caseinate under varying conditions of pH and ionic strength. The compatibility increased with increasing pH and decreasing ionic strength. Demethoxylated pectins were more and amidated pectins less compatible with the caseinate. Changes in the pectin hydrophilicity, solubility and molecular weight and possibly local interactions such as electrostatic attraction, hydrogen bonding and calcium bridges are involved in the compatibility of the components. The type and degree of the pectin modifications as well as the type, composition and properties of the protein were found to be of great importance for the thermodynamic compatibility.

Caseins↗
PubMed1980

Scanning microdensitometry of glycogen zonation in the livers of rats adapted to a controlled feeding schedule and to 30, 60, or 90% casein diets.

The effect of diet composition on diurnal changes in glycogen zonation patterns in rat liver was investigated in individually-caged male Sprague-Dawley rats adapted to the 2 + 22 controlled feeding and lighting schedule and to diets containing 30% casein/55% carbohydrates, 60% casein/25% carbohydrates, or 9.0% casein (30 rats/dietary group). Three rats from each dietary group were killed at the following times relative to the onset of feeding (0 min):--60, --30, 0, 15, 30, 45, 60, 90, 120, and 180 min. Glycogen in cryostat sections from the median and right lateral lobes of the liver was fixed and stained by standard techniques. The optical density of glycogen at points along the path between the central and portal veins of a given lobule was determined, and lobular glycogen gradients of replicate animals were integrated to form a composite lobular glycogen distribution profile. In the period from--60 to 0 min, liver glycogen levels were similar for rats on any of the diets, and the glycogen concentration was similar in periportal (P), midlobular (M), and centrilobular (C) hepatocytes. During the 0- to 45-min period, diet-related glycogen depletion occurred (90 > 60 > 30% casein) by asymmetrical glycogen loss (P > M > C hepatocytes) from the liver lobules. Similar food intake curves occurred for all diets. During the 45- to 180-min period, asymmetrical glycogen accumulation began in lobular parenchymal cells (P > M > C hepatocytes), and rate of accumulation was related to dietary to dietary composition (30 > 60 > 90% casein). The differential responses of parenchymal cells within liver lobules to physiological stimuli resulted in glycogen distribution changes that were rapid and of large magnitude. Our results are consistent with the hypothesis that periportal and midlobular hepatocytes are more metabolically responsive and active than centrilobular hepatocytes

Animals↗
PubMed1995

Developmental regulation of the ovine beta-lactoglobulin/human serum albumin transgene is distinct from that of the beta-lactoglobulin and the endogenous beta-casein genes in the mammary gland of transgenic mice.

We compared the developmental pattern of expression of the sheep beta-lactoglobulin (BLG), the chimeric BLG/human serum albumin (HSA), and the endogenous murine beta-casein genes in the mammary gland of virgin, pregnant and lactating transgenic mice, both at the RNA (expression) and protein (synthesis and secretion) levels. The BLG and casein genes were expressed at very low levels in virgin animals and during early stages of pregnancy. The increase in the expression of these genes started at the second half of pregnancy and reached a peak between the end of pregnancy and day 10 of lactation. The accumulation of their RNA coincided with that of the corresponding proteins, indicating a transcriptional control of expression of these genes. The expression and secretion patterns of the endogenous casein gene in transgenic and nontransgenic mice were indistinguishable. The hybrid BLG/HSA gene constructs displayed distinct patterns of expression in virgin animals and at early stage of pregnancy, from that of the BLG transgene or the endogenous mouse milk protein gene. High levels of expression (17-60% of that on day 18 of pregnancy) were detected in the mammary gland of virgin animals. At day 5 of pregnancy there was a dramatic decrease in HSA synthesis and secretion in all transgenic strains tested. The down-regulation, revealed by immunoprecipitation and immunohistochemical studies, demonstrated that at that stage of pregnancy only 10-18% of ductal structures contained HSA expressing cells in contrast to the majority of ducts expressing HSA in virgin animals. These morphological studies also demonstrated that the down-regulation in HSA synthesis and secretion was correlated with the transition from ducts comprised of a single layer of epithelial cells (characteristic of the virgin state) to ducts composed of multilayers of such cells. In two of the three transgenic strains tested, the down-regulation at the protein level was associated with a similar decrease in HSA transcripts. In the exceptional strain no. 23, HSA transcripts continued accumulating even at this stage. The differences in the control of expression at the RNA level between these transgenic strains were also confirmed by in situ hybridization. Our results suggest the involvement of at least two regulatory mechanisms effective at early stages of gestation in the control of expression/secretion of the HSA transgene targeted for expression in the mammary gland by the BLG milk protein promoter. These putative mechanisms may play key roles in the interplay between normal mammogenesis and lactogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

High performance capillary electrophoresis of casein phosphopeptides containing 2-5 phosphoseryl residues: relationship between absolute electrophoretic mobility and peptide charge and size.

Multiple phosphoseryl-containing peptides from casein containing the cluster sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu- stabilize amorphous calcium phosphate at neutral and alkaline pH and have been shown to be anticariogenic in various in vitro, animal and human experiments. In an approach to obtain insight into the structure and function of these peptides, we previously developed a method for their analysis using high-performance capillary electrophoresis (HPCE). A linear relationship was obtained between absolute electrophoretic mobility and q/M2/3 where q is the net negative charge of the peptide calculated using relevant pKa values and M is the molecular mass. M2/3 is a measure of the surface area of a sphere that has a volume proportional to the M of the peptide and relates to the frictional drag exerted on the peptide during electrophoretic migration. To examine this relationship further, we have analyzed a range of high-performance liquid chromatography (HPLC)-purified cluster, di- and triphosphorylated peptides from casein enzymic (trypsin, alcalase and pancreatin) digests using HPCE and attempted to relate their absolute electrophoretic mobility to various models incorporating peptide size and charge. Absolute electrophoretic mobility of casein phosphopeptides containing the cluster sequence-Ser(P)-Ser(P)-Ser(P)-Glu-Glu- was most closely correlated with q/M2/3. Di- and triphosphorylated peptide absolute electrophoretic mobility correlated with both q/M2/3 and ln(q+l)/n0.43. However, for both the q/M2/3 and In(q+l)/n0.43 relationships with absolute electrophoretic mobility, the di- and triphosphorylated peptides formed a separate linear relationship to that of the cluster peptides. From these relationships, a di- or triphosphorylated peptide exhibited a greater absolute electrophoretic mobility than a corresponding cluster peptide with sam q/M2/3 or In(q+l)/n0.43 value.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1977

[Nutritive-physiological study of sunflower seed protein isolate and spun sunflower seed protein-casein fibers].

Compared with defatted sunflower seeds, in sunflower seed globulin isolates the content of lysine and sulphur containing amino acids is decreased, the content of phenylalanine is increased. The content of the whole essential amino acids of sunflower seed globulin isolate in relation to casein is decreased. The value of the enzymatic invitro available amino acids of sunflower seed globulin is comparable with casein. The digestibility is good, the biological value is in relation to defatted sunflower seed lower. Apart from the lower content of sulphur-containing amino acids the amino acid composition of spun sunflower seed globulin/casein (I:I) fibers corresponds with the calculated value; in relation to sunflower seed globulin isolate the content of lysine and the whole essential amino acids of the spun protein fibers is increased. The enzymatic in-vitro-hydrolysis results altogether in a comparable availability of the amino acids between spun protein fibers and sunflower seed globulin isolates. The digestibility and the biological value of spun protein fibers corresponds with that of casein.

Amino Acids↗
PubMed1990

The p53 tumour suppressor protein is phosphorylated at serine 389 by casein kinase II.

The entire coding sequence of wild-type mouse p53 was expressed in Escherichia coli under control of the PL promoter of bacteriophage lambda. The bacterial p53 protein had identical mobility to p53 from SV3T3 cells on SDS polyacrylamide gels and was recognized in bacterial lysates by three p53-specific monoclonal antibodies, including PAb246 which is specific for wild-type mouse p53. Immunoprecipitates of the bacterial p53 were phosphorylated by a highly purified preparation of rat casein kinase II; the stoichiometry of incorporation was approximately 1 mol of phosphate per mol of p53. The phosphorylated residue was identified by phosphopeptide mapping as serine 389, which is a major site of p53 phosphorylation in vivo. p53 (serine 389) kinase activity was detected on lysates of SV3T3 cells; this activity co-purified with casein kinase II on phosphocellulose and Mono Q columns and was inhibited by heparin. Immunoprecipitates of the p53-T antigen complex from SV3T3 cells also had associated serine 389 kinase activity. Phosphorylation of serine 389 by this kinase was potently inhibited by heparin and quenched by excess unlabelled GTP. The data indicate that p53 is a physiological substrate of casein kinase II, which is stimulated in response to mitogens, phosphorylates nuclear oncoproteins, and may play a role in the transduction of extracellular signals to the nucleus.

Amino Acid Sequence↗
PubMed1992

Casein kinase II phosphorylates the eukaryote-specific C-terminal domain of topoisomerase II in vivo.

The decatenation activity of DNA topoisomerase II is essential for viability as eukaryotic cells traverse mitosis. Phosphorylation has been shown to stimulate topoisomerase II activity in vitro. Here we show that topoisomerase II is a phosphoprotein in yeast and that the level of incorporated phosphate is significantly higher at mitosis than in G1. Comparison of tryptic phosphopeptide maps reveals that the major phosphorylation sites in vivo are targets for casein kinase II. Incorporation of phosphate into topoisomerase II is nearly undetectable at the non-permissive temperature in a conditional casein kinase II mutant. The sites modified by casein kinase II are located in the extreme C-terminal domain of topoisomerase II. This domain is absent in prokaryotic and highly divergent among eukaryotic type II topoisomerases, and may serve to regulate functions of topoisomerase II that are unique to eukaryotic cells.

Amino Acids↗
PubMed1995

Crystal structure of casein kinase-1, a phosphate-directed protein kinase.

The structure of a truncated variant of casein kinase-1 from Schizosaccharomyces pombe, has been determined in complex with MgATP at 2.0 A resolution. The model resembles the 'closed', ATP-bound conformations of the cyclin-dependent kinase 2 and the cAMP-dependent protein kinase, with clear differences in the structure of surface loops that impart unique features to casein kinase-1. The structure is of unphosphorylated, active conformation of casein kinase-1 and the peptide-binding site is fully accessible to substrate.

Adenosine Triphosphate↗
PubMed2001

Trichostatin A inhibits beta-casein expression in mammary epithelial cells.

Many aspects of cellular behavior are defined by the content of information provided by association of the extracellular matrix (ECM) and with cell membrane receptors. When cultured in the presence of laminin-containing ECM and prolactin (Prl), normal mammary epithelial cells express the milk protein beta-casein. We have previously found that the minimal ECM- and Prl-responsive enhancer element BCE-1 was only active when stably integrated into chromatin, and that trichostatin A (TSA), a reagent that leads to alterations in chromatin structure, was able to activate the integrated enhancer element. We now show that endogenous beta-casein gene, which is controlled by a genetic assembly that is highly similar to that of BCE-1 and which is also activated by incubation in ECM and Prl, is instead inhibited by TSA. We provide evidence that the differing response of beta-casein and BCE-1 to TSA is neither due to an unusual effect of TSA on mammary epithelial cells, nor to secondary consequences from the expression of a separate gene, nor to a particular property of the BCE-1 construct. As a component of this investigation, we also showed that ECM mediated rapid histone deacetylation in mammary epithelial cells. These results are discussed in combination with previous work showing that TSA mediates the differentiation of many types of cancer cells but inhibits differentiation of some nonmalignant cell types.

Acetylation↗
PubMed1997

The effect of heparin on the activity of Trichosporon cutaneum casein kinase I.

Casein kinase I from Trichosporon cutaneum ribosome-free extracts was purified. Its molecular mass was calculated for 33 kDa. It was shown that casein, phosvitin and Trichosporon cutaneum ribosomal protein of 15 kDa were preferable substrates for the enzyme. It was found that heparin can stimulate or inhibit CKI activity depending on the substrate used. Stimulation of casein and inhibition of phosvitin phosphorylation was observed. In addition it was shown that ribosomal proteins of 19 kDa and 38 kDa were phosphorylated by CKI only in the presence of heparin.

Casein Kinases↗