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PubMed2004

Quantitation of phosphopeptides using affinity chromatography and stable isotope labeling.

Reversible phosphorylation of proteins represents an important component of cellular signaling pathways. The isolation of phosphoproteins in complex mixtures and the determination of the level of phosphorylation have been and remain a major challenge. It has prompted the development of several strategies, including immobilized metal affinity capture to enrich for phosphorylated peptides. An improved methodology was published (Ficarro, et al., Nature Biotechnology 2002, 20, 301-305) that showed increased selectivity through esterification of amino acid side chain carboxylic groups of enzymatically digested peptides. This method was applied for relative quantitation of phosphopeptides in conjunction with the use of stable isotope labeling. The merits and limits of the approach are discussed and its application to the analysis of the effects of serum starvation on in vitro cultured human lung cells is presented.

Amino Acid Sequence↗

PubMed2004

Infrared multiphoton dissociation (IRMPD) and collisionally activated dissociation of peptides in a quadrupole ion trap with selective IRMPD of phosphopeptides.

Dissociation of protonated peptides via infrared multiphoton dissociation (IRMPD) provides more extensive sequence information than is obtained with collisionally activated dissociation (CAD) in a quadrupole ion trap due to the lack of the CAD low m/z cutoff and the ability to form secondary and higher order fragments with the non-resonant photoactivation technique. In addition, IRMPD is shown to be useful for the selective dissociation of phosphopeptides over those which are not phosphorylated because the greater photon absorption efficiency of the phosphorylated peptides leads to their more rapid dissociation. Finally, the selectivity of the IRMPD technique for phosphorylated species in complex mixtures is confirmed with the analysis of a mock peptide mixture and a tryptic digest of alpha-casein.

Amino Acid Sequence↗

PubMed2003

Sequence, structure and energetic determinants of phosphopeptide selectivity of SH2 domains.

Here, we present an approach for the prediction of binding preferences of members of a large protein family for which structural information for a number of family members bound to a substrate is available. The approach involves a number of steps. First, an accurate multiple alignment of sequences of all members of a protein family is constructed on the basis of a multiple structural superposition of family members with known structure. Second, the methods of continuum electrostatics are used to characterize the energetic contribution of each residue in a protein to the binding of its substrate. Residues that make a significant contribution are mapped onto the protein sequence and are used to define a "binding site signature" for the complex being considered. Third, sequences whose structures have not been determined are checked to see if they have binding-site signatures similar to one of the known complexes. Predictions of binding affinity to a given substrate are based on similarities in binding-site signature. An important component of the approach is the introduction of a context-specific substitution matrix suitable for comparison of binding-site residues. The methods are applied to the prediction of phosphopeptide selectivity of SH2 domains. To this end, the energetic roles of all protein residues in 17 different complexes of SH2 domains with their cognate targets are analyzed. The total number of residues that make significant contributions to binding is found to vary from nine to 19 in different complexes. These energetically important residues are found to contribute to binding through a variety of mechanisms, involving both electrostatic and hydrophobic interactions. Binding-site signatures are found to involve residues in different positions in SH2 sequences, some of them as far as 9A away from a bound peptide. Surprisingly, similarities in the signatures of different domains do not correlate with whole-domain sequence identities unless the latter is greater than 50%. An extensive comparison with the optimal binding motifs determined by peptide library experiments, as well as other experimental data indicate that the similarity in binding preferences of different SH2 domains can be deduced on the basis of their binding-site signatures. The analysis provides a rationale for the empirically derived classification of SH2 domains described by Songyang & Cantley, in that proteins in the same group are found to have similar residues at positions important for binding. Confident predictions of binding preference can be made for about 85% of SH2 domain sequences found in SWISSPROT. The approach described in this work is quite general and can, in principle, be used to analyze binding preferences of members of large protein families for which structural information for a number of family members is available. It also offers a strategy for predicting cross-reactivity of compounds designed to bind to a particular target, for example in structure-based drug design.

Amino Acid Sequence↗

PubMed2005

NMR studies for identifying phosphopeptide ligands of the HIV-1 protein Vpu binding to the F-box protein beta-TrCP.

The human immunodeficiency virus type 1 (HIV-1) Vpu enhances viral particle release and, its interaction with the ubiquitin ligase SCF-beta-TrCP triggers the HIV-1 receptor CD4 degradation by the proteasome. The interaction between beta-TrCP protein and ligands containing the phosphorylated DpSGXXpS motif plays a key role for the development of severe disease states, such as HIV or cancer. This study examines the binding and conformation of phosphopeptides (P1, LIERAEDpSG and P2, EDpSGNEpSE) from HIV protein Vpu to beta-TrCP with the objective of defining the minimum length of peptide needed for effective binding. The screening step can be analyzed by NMR spectroscopy, in particular, saturation transfer NMR methods clearly identify the residues in the peptide that make direct contact with beta-TrCP protein when bound. An analysis of saturation transfer difference (STD) spectra provided clear evidence that the two peptides efficiently bound beta-TrCP receptor protein. To better characterize the ligand-protein interaction, the bound conformation of the phosphorylated peptides was determined using transferred NOESY methods, which gave rise to a well-defined structure. P1 and P2 can fold in a bend arrangement for the DpSG motif, showing the protons identified by STD-NMR as exposed in close proximity at the molecule surface. Ser phosphorylation allows electrostatic interaction and hydrogen bond with the amino acids of the beta-TrCP binding pocket. The upstream LIER hydrophobic region was also essential in binding to a hydrophobic pocket of the beta-TrCP WD domain. These findings are in good agreement with a recently published X-ray structure of a shorter beta-Catenin fragment with the beta-TrCP complex.

Amino Acid Sequence↗

PubMed2003

A homogeneous assay of kinase activity that detects phosphopeptide using fluorescence polarization and zinc.

Homogeneous antibody-free assays of protein kinase activity have great utility in high-throughput screening in support of drug discovery. In an effort to develop such an assay, we have used a pair of fluorescein-labeled peptides of identical amino acid sequence with and without phosphorylation on serine to mimic the substrate and product, respectively, of a kinase. Using fluorescence polarization (FP), we have demonstrated that a mixture of zinc sulfate, phosphate-buffered saline, and bovine serum albumin added to the peptides dramatically and differentially increased the fluorescence polarization of the phosphorylated peptide over its nonphosphorylated derivative. A similar FP differential was observed using different peptide pairs, though the magnitude varied. The FP values obtained using this method were directly proportional to the fraction of phosphopeptide present. Therefore, an FP assay was developed using a proprietary kinase. Using this FP method, linear reaction kinetics were obtained in enzyme titration and reaction time course experiments. The IC(50) values for a panel of inhibitors of kinase activity were determined using this FP method and a scintillation proximity assay. The IC(50) values were comparable between the two methods, suggesting that the zinc FP assay may be useful as an inexpensive high-throughput assay for identifying inhibitors of kinase activity.

Dose-Response Relationship, Drug↗

PubMed2000

Effects of an anticariogenic casein phosphopeptide on calcium diffusion in streptococcal model dental plaques.

Casein phosphopeptides (CPP) stabilize amorphous calcium phosphate (ACP) and may be used to localize ACP in dental plaque, maintaining a state of supersaturation with respect to tooth enamel, reducing demineralization and enhancing remineralization. The aim here was to investigate these effects by measuring the effect of CPP-ACP on calcium diffusion in plaque. Using Dibdin's effusion system, calcium diffusion was measured in streptococcal model plaques. This demonstrated that by providing a large number of possible binding sites for calcium, 0.1% CPP-ACP reduces the calcium diffusion coefficient by about 65% at pH 7 and 35% at pH 5. Hence, CPP-ACP binds well to plaque, providing a large calcium reservoir within the plaque and slowing diffusion of free calcium. This is likely to restrict mineral loss during a cariogenic episode and provide a potential source of calcium for subsequent remineralization. Overall, once in place, CPP-ACP will restrict the caries process.

Analysis of Variance↗

PubMed2002

Effect of the ratio of casein phosphopeptides to calcium (w/w) on passive calcium transport in the distal small intestine of rats.

OBJECTIVE: We investigated the influence of different ratios between casein phosphopeptides and calcium (CPP/Ca) in intestinal lumen on passive Ca transport across the distal small intestine of rats in vitro. METHODS: We measured the amounts of passive Ca absorbed across the everted distal small intestine of rats with the use of radioactive 45Ca as tracer. Four CPP/Ca ratios (5, 10, 15, and 20 w/w) and three mineral concentrations (4, 8, and 12 mmol/L) were tested on groups of six rats. RESULTS: We found a significant effect of the CPP/Ca ratio on mineral absorption (P < 0.001). The amounts of Ca absorbed increased from a ratio of 5 to a ratio of 15 and decreased with a ratio of 20, with a similar trend for every concentration tested. The more evident effect was found with the concentration of 12 mmol/L of Ca (P < 0.001 to 0.05). CONCLUSION: The positive effect of CPPs on passive Ca absorption seems to depend on the relative amounts of both species in intestinal lumen. In this study, the ratio 15 was identified as the most efficient at increasing mineral transport. This evidence might be considered before suggesting CPP as a Ca dietary integrator, although more studies in vitro and in vivo are needed.

Administration, Oral↗

PubMed1997

Effect of casein phosphopeptides on zinc and calcium absorption from bread meals.

Animal studies suggest that casein phosphopeptides (CPP), formed by proteolytic digestion of casein, have a positive effect on Ca availability. The aim of this study was to investigate the ability of CPP to improve Zn and Ca absorption from phytate-containing bread meals. Secondly, it was tested whether the Ca content of the meal influenced the effect of CPP on Zn absorption from a high-phytate meal. Zn and Ca absorption from single model meals was determined by extrinsic labeling with 65Zn and 47Ca and measurement of whole-body retention. Thirty-one healthy adults (19-30 y) divided into three groups received one of three meal types based on white-wheat rolls with different phytate and Ca content: A: low-phytate/high-Ca (n = 10), B: high-phytate/high-Ca (n = 10) and C: high-phytate/low-Ca (n = 11). Each individual received the same meal type at three different occasions with 0, 250 and 1000 mg CPP added. CPP had no effect on Zn absorption: A: 14.3 +/- 4.4% (mean +/- SD) (0 mg CPP), 16.7 +/- 6.6% (250 mg CPP) and 16.0 +/- 8.8% (1000 mg CPP); B: 7.0 +/- 1.6%, 7.7 +/- 3.0% and 8.0 +/- 2.6%; C: 7.7 +/- 2.5%, 7.0 +/- 2.3% and 6.5 +/- 1.6%, respectively. Addition of 1000 mg CPP reduced fractional Ca absorption from meal A: 33.8 +/- 7.1%, 30.5 +/- 6.3% and 25.9 +/- 8.7% (p < 0.05), whereas CPP had no effect in meal B: 29.3 +/- 8.3%, 27.0 +/- 7.4% and 27.6 +/- 11.0%. However, the absolute quantity of Ca absorbed was unaffected. In conclusion, Zn and Ca absorption from these meals were not significantly influenced by the addition of CPP, Ca addition could not explain the failure of CPP to improve Zn absorption.

Absorption↗

PubMed2000

Inhibition of the ras-dependent mitogenic pathway by phosphopeptide prodrugs with antiproliferative properties.

Phosphopeptide prodrugs bearing two S-acyl-2-thioethyl (SATE) biolabile phosphate protections were developed. They are capable to inhibit the Shc/Grb2 interaction and MAP kinases (ERK1 and ERK2) phosphorylation in cellular assay. The S-acetyl-2-thioethyl (MeSATE) analogue showed an IC50 of 1 microM in the inhibition of the colony formation of tumor cell line NIH3T3/HER2.

3T3 Cells↗

PubMed2003

Identification of a high-affinity phosphopeptide inhibitor of Stat3.

Stat3 is a latent transcription factor that exhibits elevated activity in a variety of human cancers. To find a lead peptide for peptidomimetic drug development we synthesized and tested phosphopeptides derived from known receptor docking sites and found Y(p)LPQTV as the optimal sequence. SAR studies showed that each residue from pY to pY+3 provided binding energy.

Amino Acid Sequence↗

PubMed1993

The synthesis and use of pp60src-related peptides and phosphopeptides as substrates for enzymatic phosphorylation studies.

A series of peptides and phosphopeptides corresponding to the auto-phosphorylation site of pp60src, -Asn-Glu-Tyr416-Thr-Ala-, were prepared by either Boc/solution or Fmoc/solid phase peptide synthesis and used as substrates to study their enzymatic phosphorylation by various casein kinases. The Tyr(P)-containing peptide, Asn-Glu-Tyr(P)-Thr-Ala, was prepared by the use of Fmoc-Tyr(PO3Bzl2)-OH in Fmoc/solid phase peptide synthesis followed by acidolytic treatment of the peptide-resin with 5% anisole/CF3CO2H. Both Asn-Glu-Tyr-Thr-Ala and Asn-Glu-Ser(P)-Thr-Ala were prepared by the Boc/solution phase peptide synthesis and employed hydrogenolytic deprotection of the protected peptides. Enzymatic phosphorylation studies established that (A) the Tyr residue acted as an unusual positive determinant for directing phosphorylation to the Thr-residue, (B) the rate of Thr-phosphorylation was markedly facilitated by a change from the Tyr-residue to the Tyr(P)-residue, and (C) a Ser(P)-residue was as effective as the Tyr(P)-residue in facilitating Thr-phosphorylation. A subsequent structure-function study using Asn-Glu-Phe-Thr-Ala, Asn-Glu-Tyr(Me)-Thr-Ala (prepared by Fmoc/solid phase peptide synthesis) and Asn-Glu-Cha-Thr-Ala (prepared by hydrogenation of Asn-Glu-Tyr-Thr-Ala) established that the rate of Thr-phosphorylation was influenced by the extent of hydrophobic-hydrophobic interactions by the aralkyl side-chain group (either aromatic or aliphatic) of the 416-residue with casein kinase-2; the rate of Thr-phosphorylation being decreased by the introduction of methyl or hydroxyl groups at the 4-position of the aromatic group (i.e. Tyr(Me) and Tyr respectively) but enhanced by the introduction of the hydrophilic phosphate group (i.e. as Tyr(P)).

Amino Acid Sequence↗

PubMed1997

Synthetic study of phosphopeptides related to heat shock protein HSP27.

Two kinds of phosphoserine-containing peptides related to HSP27 were synthesized by the Boc- or Fmoc-mode solid-phase method based on prephosphorylation strategy. In the case of the Boc strategy, the O-phosphono group of the phosphoserine residue was protected with the cyclopentyl or cyclohexyl group. On the other hand, N'-Fmoc-O-[(benzyloxy)-hydroxyphosphinyl]serine was employed in case of the Fmoc strategy. Consequently, it has become-feasible to utilize conventional solid-phase methods for synthesizing any phosphopeptides which are required to elucidate biochemical significance of protein phosphorylation.

Chromatography, High Pressure Liquid↗

PubMed1998

Mass spectrometric and thermodynamic studies reveal the role of water molecules in complexes formed between SH2 domains and tyrosyl phosphopeptides.

BACKGROUND: SH2 domains have a fundamental role in signal transduction. These domains interact with proteins containing phosphorylated tyrosine residues and, in doing so, mediate the interactions of proteins involved in tyrosine kinase signalling. The issue of specificity in SH2 domain interactions is therefore of great interest in terms of understanding tyrosine kinase signal-transduction pathways and in the discovery of drugs to inhibit them. Water molecules are found at the interfaces of many complexes, however, to date little attention has been paid to their role in dictating specificity. RESULTS: Here we use a combination of nanoflow electrospray ionization mass spectrometry (ESI-MS), isothermal titration calorimetry and structural data to investigate the effect of water molecules in complexes formed between the SH2 domain of tyrosine kinase Src and tyrosyl phosphopeptides. Binding studies have been performed using a series of different peptides that were selected to allow changes in the water content at the complex interface and demonstrate changes in specificity. ESI-MS enables quantification of the number of water molecules that interact with a higher affinity than those generally found solvating the biomolecular complex. CONCLUSIONS: Comparing the interactions of different peptides, we show that an intricate network of water molecules have a key role in dictating specificity. The use of mass spectrometry to quantify tightly bound water molecules may prove of general use in structural biology, where an independent determination of the water molecules associated with a structure would be advantageous. Furthermore, the ability to assess whether given water molecules are important in high-affinity binding could make this method a precious tool in drug design.

Crystallography, X-Ray↗

PubMed2002

Fragmentation of phosphopeptides by atmospheric pressure MALDI and ESI/Ion trap mass spectrometry.

An investigation of phosphate loss from phosphopeptide ions was conducted, using both atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) and electrospray ionization (ESI) coupled to an ion trap mass spectrometer (ITMS). These experiments were carried out on a number of phosphorylated peptides in order to investigate gas phase dephosphorylation patterns associated with phosphoserine, phosphothreonine, and phosphotyrosine residues. In particular, we explored the fragmentation patterns of phosphotyrosine containing peptides, which experience a loss of 98 Da under collision induced dissociation (CID) conditions in the ITMS. The loss of 98 Da is unexpected for phosphotyrosine, given the structure of its side chain. The fragmentation of phosphoserine and phosphothreonine containing peptides was also investigated. While phosphoserine and phosphothreonine residues undergo a loss of 98 Da under CID conditions regardless of peptide amino acid composition, phosphate loss from phosphotyrosine residues seems to be dependent on the presence of arginine or lysine residues in the peptide sequence.

Amino Acids↗

PubMed2000

The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phospho-dependent signaling mechanisms.

Forkhead-associated (FHA) domains are a class of ubiquitous signaling modules that appear to function through interactions with phosphorylated target molecules. We have used oriented peptide library screening to determine the optimal phosphopeptide binding motifs recognized by several FHA domains, including those within a number of DNA damage checkpoint kinases, and determined the X-ray structure of Rad53p-FHA1, in complex with a phospho-threonine peptide, at 1.6 A resolution. The structure reveals a striking similarity to the MH2 domains of Smad tumor suppressor proteins and reveals a mode of peptide binding that differs from SH2, 14-3-3, or PTB domain complexes. These results have important implications for DNA damage signaling and CHK2-dependent tumor suppression, and they indicate that FHA domains play important and unsuspected roles in S/T kinase signaling mechanisms in prokaryotes and eukaryotes.

14-3-3 Proteins↗

PubMed1999

The FHA domain is a modular phosphopeptide recognition motif.

FHA domains are conserved sequences of 65-100 amino acid residues found principally within eukaryotic nuclear proteins, but which also exist in certain prokaryotes. The FHA domain is thought to mediate protein-protein interactions, but its mode of action has yet to be elucidated. Here, we show that the two highly divergent FHA domains of Saccharomyces cerevisiae Rad53p, a protein kinase involved in cell cycle checkpoint control, possess phosphopeptide-binding specificity. We also demonstrate that other FHA domains bind peptides in a phospho-dependent manner. These findings indicate that the FHA domain is a phospho-specific protein-protein interaction motif and have important implications for mechanisms of intracellular signaling in both eukaryotes and prokaryotes.

Amino Acid Sequence↗

PubMed1999

Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 14-3-3 in ligand binding.

We have solved the high-resolution X-ray structure of 14-3-3 bound to two different phosphoserine peptides, representing alternative substrate-binding motifs. These structures reveal an evolutionarily conserved network of peptide-protein interactions within all 14-3-3 isotypes, explain both binding motifs, and identify a novel intrachain phosphorylation-mediated loop structure in one of the peptides. A 14-3-3 mutation disrupting Raf signaling alters the ligand-binding cleft, selecting a different phosphopeptide-binding motif and different substrates than the wild-type protein. Many 14-3-3: peptide contacts involve a C-terminal amphipathic alpha helix containing a putative nuclear export signal, implicating this segment in both ligand and Crm1 binding. Structural homology between the 14-3-3 NES structure and those within I kappa B alpha and p53 reveals a conserved topology recognized by the Crm1 nuclear export machinery.

14-3-3 Proteins↗

PubMed2000

The effect of high intakes of casein and casein phosphopeptide on calcium absorption in the rat.

The effect of the level or source of dietary protein or protein-derived peptides on Ca absorption is not well understood. We determined, therefore, the influence of habitual dietary casein level, meal casein and meal casein phosphopeptide (CPP) on Ca absorption in the rat. True fractional Ca absorption was investigated in male 7-week-old rats, Wistar strain, in three separate studies using a faecal 47Sc: 47Ca ratio method. In studies A and C, rats (n 8 per group) were fed on a purified diet containing 200 g casein/kg for 2 weeks. Rats were then given a 47Ca-labelled meal (10 g) containing (per kg) either 0, 100, 200, or 300 g casein (study A) or 0, 100, 200, 350 or 500 g CPP (study C). In study B, rats (n 24 per group) were fed on a purified diet containing (per kg) either 200, 350 or 500 g casein for 2 weeks. Each group was then further randomized into three groups (n 8 per group) and given a 47Ca-labelled meal (10 g of the same diet) containing (per kg) either 200, 350 or 500 g casein. Ca absorption from a meal was unaffected by increasing meal casein concentration from 0 to 300 g/kg (study A), but was increased with a meal casein content of 500 g/kg (study B). Fractional Ca absorption decreased with increasing usual dietary casein intake in the range 200-500 g/kg (study B), suggesting intestinal adaptation. Ca absorption was unaffected by inclusion of 100 g CPP/kg in a single meal but was significantly (P < 0.001) reduced by 200, 350 and 500 g CPP/kg meal, with no evident dose-relationship. Thus, while Ca absorption was enhanced by high-casein meals, the mechanism remains unclear.

Animals↗