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At least 145 records · Page 8Linked to original sources

PubMed1998

A core-shell model of calcium phosphate nanoclusters stabilized by beta-casein phosphopeptides, derived from sedimentation equilibrium and small-angle X-ray and neutron-scattering measurements.

Calcium phosphate nanoclusters were prepared under standardised conditions using 10 mg ml(-1) of the 25-amino-acid N-terminal tryptic phosphopeptide of bovine beta-casein as a stabilising agent. The Mr determined by sedimentation equilibrium was 197,600+/-13,700 and the apparent radius of gyration determined by X-ray scattering was 2.80+/-0.05 nm. A small-angle neutron scattering contrast variation study in 1H2O/2H2O mixtures was performed and gave radii of gyration at the calculated match points for the calcium phosphate (88.2% 2H2O) and phosphopeptide (41.3% 2H2O) of 3.39+/-0.08 nm and 1.85+/-0.05 nm, respectively. Measurements at larger scattering wave vector showed a subsidiary maximum at about Q = 1.6 nm(-1). The results are consistent with a model of the nanoclusters comprising a spherical core of 355+/-20 CaHPO4 x 2 H2O units, density 2.31 g ml(-1) and radius 2.30+/-0.05 nm surrounded by 49+/-4 peptide chains with a partial specific volume of 0.7 cm3 g(-1), forming a tightly packed shell with an outer radius of 4.04+/-0.15 nm. This model suggests that the phosphopeptide is able to arrest the process of growth of the precipitating phase of calcium phosphate at its earliest stages. A similar role for whole casein could be vital to the normal functioning of the mammary gland during milk secretion.

Calcium Phosphates↗

PubMed1992

Synthetic phosphopeptide immunogens yield activation-specific antibodies to the c-erbB-2 receptor.

We inoculated rabbits with synthetic phosphopeptides, duplicating a major autophosphorylation site of the c-erbB-2 protooncogene product. The rabbits produced antisera that, after reverse immunoaffinity purification, selectively recognize the erbB-2 protein in its enzymatically active configuration. These anti-phosphopeptide antisera identify a subset of erbB-2-positive human cell lines wherein the protein is constitutively active as a tyrosine kinase. Synthetic phosphopeptides incorporating informative protein phosphorylation sites may prove useful for generating antibodies that indicate the activation state of additional tyrosine kinases and perhaps other proteins phosphorylated on serine and threonine residues.

Animals↗

PubMed1995

Phosphopeptide mapping of cholecystokinin receptors on agonist-stimulated native pancreatic acinar cells.

The cholecystokinin (CCK) receptor on the rat pancreatic acinar cell is a G protein-coupled receptor that is phosphorylated in response to homologous and heterologous agonist stimulation. In this work we have studied the stoichiometry of receptor phosphorylation and have utilized one-dimensional phosphopeptide mapping after cyanogen bromide cleavage to demonstrate that the third intracellular loop is the predominant domain of phosphorylation of this receptor in response to these treatments. Of the average 5 mol of phosphate/mol of receptor, greater than 95% was on the third loop, with the remainder residing on the carboxyl-terminal tail. Serine residues were the site of greater than 95% of phosphorylation, with threonine representing the remainder, and no phosphotyrosine was detected. Further, we have utilized two-dimensional phosphopeptide mapping after subtilisin cleavage to identify differing sites of CCK receptor phosphorylation which are dependent on the agonist utilized to stimulate this cell. Both qualitative and quantitative differences in phosphorylation sites were observed after acinar cell stimulation with different protein kinase C agonists. Further, distinct phosphopeptides on the map were identified as representing substrate(s) of a staurosporine-insensitive kinase activity stimulated only by receptor occupation with native CCK and were felt to represent site(s) of action of a member of the G protein-coupled receptor kinase family. This represents a sensitive and powerful approach that is applicable to sparse receptors residing in their native cellular environment to assess possible differences in patterns of phosphorylation which may be important in agonist-specific receptor regulation.

Adenosine Triphosphate↗

PubMed2006

Reconstructing the regulatory kinase pathways of myogenesis from phosphopeptide data.

Multiple kinase activities are required for skeletal muscle differentiation. However, the mechanisms by which these kinase pathways converge to coordinate the myogenic process are unknown. Using multiple phosphoprotein and phosphopeptide enrichment techniques we obtained phosphopeptides from growing and differentiating C2C12 muscle cells and determined specific peptide sequences using LC-MS/MS. To place these phosphopeptides into a rational context, a bioinformatics approach was used. Phosphorylation sites were matched to known site-specific and to site non-specific kinase-substrate interactions, and then other substrates and upstream regulators of the implicated kinases were incorporated into a model network of protein-protein interactions. The model network implicated several kinases of known relevance to myogenesis including AKT, GSK3, CDK5, p38, DYRK, and MAPKAPK2 kinases. This combination of proteomics and bioinformatics technologies should offer great utility as the volume of protein-protein and kinase-substrate information continues to increase.

Animals↗

PubMed1997

pp60v-src transformation of rat cells but not chicken cells strongly correlates with low-affinity phosphopeptide binding by the SH2 domain.

Substrates critical for transformation by pp60v-src remain unknown, as does the precise role of the src homology 2 (SH2) domain in this process. To continue exploring the role of the SH2 domain in pp60v-src-mediated transformation, site-directed mutagenesis was used to create mutant v-src alleles predicted to encode proteins with overall structural integrity intact but with reduced ability to bind phosphotyrosine-containing peptides. Arginine-175, which makes critical contacts in the phosphotyrosine-binding pocket, was mutated to lysine or alanine. Unexpectedly, both mutations created v-src alleles that transform chicken cells with wild-type (wt) efficiency and are reduced for transformation of rat cells; these alleles are host dependent for transformation. Additionally, these alleles resulted in a round morphological transformation of chicken cells, unlike 12 of the 13 known host-dependent src SH2 mutations that result in a fusiform morphology. Analysis of phosphopeptide binding by the mutant SH2 domains reveal that the in vitro ability to bind phosphopeptides known to have a high affinity for wt src SH2 correlates with wt (round) morphological transformation in chicken cells and in vitro ability to bind phosphopeptides known to have a low affinity for wt src SH2 correlates with rat cell transformation. These results suggest that the search for critical substrates in rat cells should be among proteins that interact with pp60v-src with low affinity.

Animals↗

PubMed1980

Enhanced absorption of calcium by casein phosphopeptides in rachitic and normal chicks.

The present studies were undertaken to investigate the effect of casein phosphopeptides on calcium absorption in normal and rachitic chicks. Phosphopeptides were produced by tryptic hydrolysis of casein and the crude peptide mixture was further purified, using gel filtration. The chromatographic fractions were pooled according to their protein and phosphorus content. Some of these fractions, when introduced into the ligated duodenal loop at a concentration of 1 mg/ml or added to the mucosal solution in vitro at a concentration of 0.1 mg/ml, were capable of increasing the intestinal transport of radiocalcium. This effect was seen both in the normal and rachitic chicks, indicating that the stimulatory response was not dependent upon molecular changes induced by vitamin D. Furthermore, this response was not shared by peptides from albumin, not likely due to phosphate generated from the phosphopeptides, nor related to the phosphate content of the fractions. These experiments demonstrate that a component of milk, in addition to lactose, might account for the high availability of calcium in milk.

Animals↗

PubMed1990

Characterisation of human and murine snRNP proteins by two-dimensional gel electrophoresis and phosphopeptide analysis of U1-specific 70K protein variants.

The proteins of the major human snRNPs U1, U2, U4/U6 and U5 were characterised by two-dimensional electrophoresis, with isoelectric focussing in the first dimension and SDS-polyacrylamide gel electrophoresis in the second. With the exception of protein F, which exhibits an acidic pl value (pl = 3.3), the snRNP proteins are basic. Post-translational modification was found among the proteins associated specifically with the U1 and U2 particles. The most complex modification pattern was observed for the U1-specific 70K protein. This was found in at least 13 isoelectric variants, with pl values ranging from 6.7 to 8.7; these variants differed also in molecular weight. All of the 70K variants are phosphorylated in the cell. Thin-layer analysis of their tryptic phosphopeptides revealed that the 70K variants have four major phosphopeptides in common, in addition to which at least four additional serine residues are phosphorylated to different extents. The comparative phosphopeptide analysis shows that differential phosphorylation alone is not sufficient to explain the occurrence of the many isoelectric variants of 70K, so that the final charge of the 70K variants is determined both by phosphorylation and by other, as yet unidentified posttranslational modifications. By two-dimensional separation of snRNP proteins obtained from mouse Ehrlich ascites tumour cells, it was shown that the pattern of pl values of the mouse proteins was almost identical with the corresponding pattern for human proteins. Even the complex modification patterns of the 70K protein are identical in mouse and man, indicating that the presence in the cell of so many variants of this protein may have functional importance. The major difference between murine and human snRNP proteins is the absence of protein B' from mouse snRNPs. This suggests that the homologous protein B may be able to carry out the task of protein B'.

Animals↗

PubMed1989

Synthesis of a substrate of protein kinase C and its corresponding phosphopeptide.

The syntheses of a protein kinase C (PKC) peptide substrate, H-Lys-Arg-Thr-Leu-Arg-OH, and a phosphopeptide analog of the synthetic substrate, H-Lys-Arg-Thr(P)-Leu-Arg-OH, are reported. PKC phosphorylates the peptide with an apparent KM of 0.30 +/- 0.04 mM and an apparent Vmax equal to one-tenth that of histone III-S. The synthesis of the phosphopeptide features a recently developed convenient phosphorylation procedure for serine and threonine using N,N-diethylamino-dibenzylphosphoramidite. A complete characterization of the PKC substrate and its corresponding phosphopeptide by C-H COSY 2D n.m.r. is included.

Chemical Phenomena↗

PubMed1994

Dephosphorylation of phosphopeptides by calcineurin (protein phosphatase 2B).

38 (6-32 residues) enzymically phosphorylated synthetic peptides have been assayed as substrates for calcineurin, a Ca2+/calmodulin-dependent protein phosphatase (PP-2B) belonging to the family of Ser/Thr-specific enzymes but also active on phosphotyrosine residues. Many peptides reproduce, with suitable modifications, naturally occurring phosphoacceptor sites. While protein phosphatases 2A and 2C are also very active on short phosphopeptides, an extended N-terminal stretch appears to be a necessary, albeit not sufficient, condition for an optimal dephosphorylation, comparable to that of protein substrates, of both phosphoseryl and phosphotyrosyl peptides by calcineurin. This finding corroborates the view that higher-order structure is an important determinant for the substrate specificity of calcineurin. However, a number of shorter peptides are also appreciably dephosphorylated by this enzyme, their efficiency as substrates depending on local structural features. All the peptides that are appreciably dephosphorylated by calcineurin contain basic residue(s) on the N-terminal side. A basic residue located at position -3 relative to the phosphorylated residue plays a particularly relevant positive role in determining the dephosphorylation of short phosphopeptides. Acidic residue(s) adjacent to the C-terminal side of the phosphoamino acid are conversely powerful negative determinants, preventing the dephosphorylation of otherwise suitable peptide substrates. However, calcineurin displays an only moderate preference for phosphothreonyl peptides which are conversely strikingly preferred over their phosphoseryl counterparts by the other classes of Ser/Thr-specific protein phosphatases. Moreover calcineurin does not perceive as a strong negative determinant the motif Ser/Thr-Pro in peptides where this motif prevents dephosphorylation by the other classes of Ser/Thr protein phosphatases. Whenever tested on phosphotyrosyl peptides, calcineurin exhibits a specificity which is strikingly different from that of T-cell protein tyrosine phosphatase, a bona fide protein tyrosine phosphatase. In particular while the latter enzyme is especially active toward a number of phosphopeptides reproducing the phosphoacceptor sites of src products and of calmodulin whose N-terminal moieties are predominantly acidic, the artificial substrate phospho-angiotensin II, bearing an arginine residue at position -2, is far preferred by calcineurin over all phosphotyrosyl peptides of similar size. Collectively taken these results show that the specificity of calcineurin, rather than resting on a given consensus sequence, is determined by a variety of primary and higher-order structural features conferring to it an overall selectivity that is different from those of any other known protein phosphatase.

Amino Acid Sequence↗

PubMed1995

Comparison of the specificity of bacterially expressed cytoplasmic protein-tyrosine phosphatases SHP and SH-PTP2 towards synthetic phosphopeptide substrates.

SHP and SH-PTP2 are related cytoplasmic protein-tyrosine phosphatases having two tandem amino-terminal src homology 2 domains linked to a single catalytic domain. There is growing evidence that these two molecules may exhibit opposing effects within specific signaling pathways. However, the relative contributions of the src homology 2 domains or the catalytic domains to these opposing effects are not well known. To evaluate the potential contribution of the catalytic domains, we compared the substrate specificity of the two phosphatases. As seen previously, the catalytic activities of bacterially expressed SHP and SH-PTP2 were regulated by the presence of the linked src homology 2 domains. In addition, we characterized a cryptic thrombin cleavage site within the carboxy-terminus of SHP that led to a striking increase in the activity of the catalytic domain. Employing a panel of phosphopeptide substrates whose sequences were modeled after intracellular phosphorylation sites, both SHP and SH-PTP2 demonstrated a similar specificity pattern. Similar to SH-PTP2, SHP failed to elicit detectable phosphate release from several phosphopeptide substrates, while displaying catalytic efficiencies that ranged over approximately 40-1.6 x 10(3) M-1 s-1 towards other substrates. In contrast, the PTP-1B phosphatase dephosphorylated all of the phosphopeptide substrates tested with approximately equal ease. The overall similarity demonstrated by the catalytic domains of SHP and SH-PTP2 suggested that differences in the in vivo behavior of these two molecules might not stem from differences in the substrate specificity of the catalytic domains, suggesting instead that the specificity of the src homology 2 domains is more important in this regard.

Amino Acid Sequence↗

PubMed2003

BRCT repeats as phosphopeptide-binding modules involved in protein targeting.

We used a proteomic approach to identify phosphopeptide-binding modules mediating signal transduction events in the DNA damage response pathway. Using a library of partially degenerate phosphopeptides, we identified tandem BRCT (BRCA1 carboxyl-terminal) domains in PTIP (Pax transactivation domain-interacting protein) and in BRCA1 as phosphoserine- or phosphothreonine-specific binding modules that recognize substrates phosphorylated by the kinases ATM (ataxia telangiectasia-mutated) and ATR (ataxia telangiectasia- and RAD3-related) in response to gamma-irradiation. PTIP tandem BRCT domains are responsible for phosphorylation-dependent protein localization into 53BP1- and phospho-H2AX (gamma-H2AX)-containing nuclear foci, a marker of DNA damage. These findings provide a molecular basis for BRCT domain function in the DNA damage response and may help to explain why the BRCA1 BRCT domain mutation Met1775 --> Arg, which fails to bind phosphopeptides, predisposes women to breast and ovarian cancer.

Amino Acid Motifs↗

PubMed1995

Anticariogenicity of calcium phosphate complexes of tryptic casein phosphopeptides in the rat.

Casein phosphopeptides (CPP) stabilize calcium phosphate through the formation of casein-phosphopeptide amorphous calcium-phosphate complexes (CPP-CP). The ability of CPP-CP to reduce caries activity was investigated by use of specific-pathogen-free rats inoculated with Streptococcus sobrinus. The animals consumed a defined cariogenic diet free of dairy products. Solutions (100 microL) of the CPP-CP (0.1, 0.2, 0.5, 1.0% w/v) were applied to the animals' molar teeth twice daily. Other groups of animals received solutions containing 500 ppm F, the non-phosphorylated peptides of a casein tryptic digest (0.5% w/v), or the calcium-phosphate complex of a synthetic octapeptide, Ac-Glu-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-NHMe, corresponding to the common sequence in the CPP. The CPP-CP significantly reduced caries activity in a dose-response fashion, with 1.0% CPP-CP producing 55% and 46% reductions in smooth surface and fissure caries activity, respectively, being similar to that of 500 ppm F. The anticariogenic effects of CPP-CP and fluoride were additive, since animals receiving 0.5% CPP-CP plus 500 ppm F had significantly lower caries activity than those animals receiving either CPP-CP or fluoride alone. The tryptic digest of casein with the phosphopeptides selectively removed showed no anticariogenic activity. The synthetic octapeptide-calcium phosphate complex significantly reduced caries activity, confirming that this calcium-phosphate-stabilizing portion of the casein phospho-peptides is associated with anticariogenicity. The CPP-CP did not significantly affect the level of S. sobrinus in fissure plaque.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2001

Phosphopeptides derived from hen egg yolk phosvitin: effect of molecular size on the calcium-binding properties.

Two different phosphopeptide (PPP) fragments derived from partially dephosphorylated hen egg yolk phosvitin were prepared by tryptic digestion, and their Ca2+ binding property compared with that of commercial casein phosphopeptides (CPP). The smaller fragment of less than 1 kDa and O-phospho-1-serine did not bind Ca2+ to any significant extend, while PPP of 1-3 kDa showed a higher ability than CPP to render soluble calcium. The results show that not only the phosphoserine residues are critical for Ca2+ binding, but also the molecular size of the phosphopeptides.

Animals↗

PubMed1999

Phosphorylated peptides can be transported by TAP molecules, presented by class I MHC molecules, and recognized by phosphopeptide-specific CTL.

CTL recognize short peptide fragments presented by class I MHC molecules. In this study, we examined the effect of phosphorylation on TAP transport, binding to class I MHC molecules, and recognition by CTL of peptide fragments from known phosphorylated oncogene proteins or virus phosphoproteins. We show that phosphopeptides can be efficiently transported from the cytosol to the endoplasmic reticulum by the TAP. Furthermore, we show that phosphorylation can have a neutral, negative, or even a positive effect on peptide binding to class I MHC. Finally, we have generated phosphopeptide-specific CTL that discriminate between the phosphorylated and the nonphosphorylated versions of the peptide. We conclude that phosphopeptide-specific CTL responses are likely to constitute a subset of the class I MHC-restricted CTL repertoire in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Direct binding of visual arrestin to a rhodopsin carboxyl terminal synthetic phosphopeptide.

PURPOSE: The phosphorylated carboxyl terminus of rhodopsin is required for the stable binding of visual arrestin to the full length rhodopsin molecule. Phosphorylation of the carboxyl terminus has been shown to induce conformational changes in arrestin, which promote its binding to the cytoplasmic loops of rhodopsin. However, it has not been determined whether phosphorylation is also responsible for the direct binding of the rhodopsin carboxyl terminus to arrestin. To further investigate the role of rhodopsin phosphorylation on arrestin binding, surface plasmon resonance was used to measure the interaction between a synthetic phosphopeptide corresponding to the carboxyl terminus of rhodopsin and visual arrestin in real time. METHODS: Synthetic peptides were generated that correspond to the phosphorylated and nonphosphorylated carboxyl terminus of bovine rhodopsin. These peptides were immobilized on a biosensor chip and their interaction with purified visual arrestin was monitored by surface plasmon resonance on a BIAcore 2000 or 3000. RESULTS: A synthetic peptide phosphorylated on residues corresponding to Ser-338, Thr-340, Thr-342 and Ser-343 of bovine rhodopsin was sufficient for direct binding to visual arrestin. In contrast, a second phosphopeptide phosphorylated on Thr-340 and Thr-342 and a nonphosphorylated synthetic peptide were not able to bind arrestin. A peptide fully substituted at all serine and threonine residues with glutamic acid was unable to substitute for phosphorylation. CONCLUSIONS: Surface plasmon resonance is a sensitive method for detecting small differences in affinity. We were successful in using this technique to detect differences in the affinity of phosphorylated and nonphosphorylated rhodopsin peptides for visual arrestin. The data suggest that these are low-affinity interactions and indicate that phosphorylation is responsible for the direct binding of the rhodopsin carboxyl terminus to visual arrestin. Four phosphorylated residues are sufficient for this interaction. Because the affinity of the synthetic phosphopeptide for arrestin is substantially lower than the full length rhodopsin molecule, the cytoplasmic loops and rhodopsin carboxyl terminus appear to interact in a cooperative manner to stably bind arrestin.

Animals↗

PubMed1981

HeLa ribosomal protein S6. Insulin and dibutyryl cyclic AMP affect different phosphopeptides.

Tryptic phosphopeptides from HeLa ribosomal protein S6 have been separated by chromatography, followed by electrophoresis. Three phosphopeptides were found in S6 from cells treated in vivo with dibutyryl cAMP and at least four different phosphopeptides were found from cells treated with insulin plus the essential amino acids. Only phosphoserine was found in S6 isolated from insulin-treated cells.

Amino Acids↗

PubMed1982

[Isolation and study of some properties of new phosphopeptides from nerve tissue].

The phosphopeptides were extracted by organic solvents from the lipid-deleted homogenate of rat brain tissue. The latter was obtained by means of trichloroacetic acid from rats injected with 32P-NaH2PO4 into brain ventricles. Chromatography of the brain extract on DEAE-Sephadex A-25 and gel filtration through Sephadex G-25 and G-15 resulted in 3 homogenous fractions of the phosphopeptides characterized by the maximal rate of the label incorporation. These fractions appeared to be homogenous during polyacrylamide gel electrophoresis and thin-layer chromatography. The molecular weights of the fractions are equal to 1200, 1100 and 900, respectively; the isoelectric points lie at 3.2, 3.0 and 3.1, respectively. All the phosphopeptides tested contain large amounts of glycine, serine and glutamic acid and have alanine as N-terminal amino acid.

Amino Acids↗

PubMedDate not supplied

[Fractionation of brain phosphopeptides].

Anion-exchange chromatography was used to fractionate the brain phosphopeptides preparations obtained by the common method and the method modified by the author. The data of a comparative analysis show that in fractions obtained from our preparation the content of amide nitrogen as well as of phosphate residues is higher than in the analogous preparations of Ledig and Mandel. Changes are found in the spectrum of the phosphopeptides fractions isolated during incubation of the brain tissue homogenate under aerobic conditions (1 h; 37 degrees C). It is shown that the amount of phosphopeptide fractions decreases after incubation, and the total content of nitrogen, content of amide nitrogen and phosphorus in eluted fractions undergo redistribution.

Aerobiosis↗