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PubMed1994

Phosphopeptide occupancy and photoaffinity cross-linking of the v-Src SH2 domain attenuates tyrosine kinase activity.

Phosphorylation of c-Src at carboxyl-terminal Tyr-527 suppresses tyrosine kinase activity and transforming potential, presumably by facilitating the intramolecular interaction of the C terminus of Src with its SH2 domain. In addition, it has been shown previously that occupancy of the c-Src SH2 domain with a phosphopeptide stimulates c-Src kinase catalytic activity. We have performed analogous studies with v-Src, the transforming protein from Rous sarcoma virus, which has extensive homology with c-Src. v-Src lacks an autoregulatory phosphorylation site, and its kinase domain is constitutively active. Phosphopeptides corresponding to the sequences surrounding c-Src Tyr-527 and a Tyr-Glu-Glu-Ile motif from the hamster polyoma virus middle T antigen inhibit tyrosine kinase activity of baculovirus-expressed v-Src 2- and 4-fold, respectively. To determine the mechanism of this regulation, the Tyr-527 phosphopeptide was substituted with the photoactive amino acid p-benzoylphenylalanine at the adjacent positions (N- and C-terminal) to phosphotyrosine. These peptides photoinactivate the v-Src tyrosine kinase 5-fold in a time- and concentration-dependent manner. Furthermore, the peptides cross-link an isolated Src SH2 domain with similar rates and specificity. These data indicate that occupancy of the v-Src SH2 domain induces a conformational change that is transmitted to the kinase domain and attenuates tyrosine kinase activity.

Amino Acid Sequence↗

PubMed2003

Subunit E of mitochondrial ATP synthase: a bioinformatic analysis reveals a phosphopeptide binding motif supporting a multifunctional regulatory role and identifies a related human brain protein with the same motif.

The mitochondrial adenosine triphosphate (ATP) synthase is located in the inner membrane and consists of at least 16 subunit types in animals, one of which is subunit e, the function of which is not clearly defined. A highly homologous protein is located in the nucleus and named progesterone receptor binding protein (RBF), to designate its role in this organelle. In addition, the expression level of subunit e in mammalian cells fluctuates greatly and is induced by certain carcinogens and elevated in liver cancers. Because these previous observations suggested to us that subunit e may play multifunctional regulatory roles, we employed a bioinformatic approach to test this view. First, from sequence alignment studies, secondary structure analyses, and basic local alignment search tool (BLAST) searches, we concluded that mitochondrial subunit e and the homologous nuclear protein RBF are most likely the same protein. Second, we examined the known sequence and structure of one of the most common multifunctional cell regulatory proteins, the 14-3-3 protein, involved in phosphopeptide binding, and deduced that it has an apparent binding motif (-KX(6)R---RY-). Third, from careful examination of the conserved residues within all subunit e sequences in the database, we discovered that this protein has a comparable binding motif (-RY---KX(6)R-). Finally, in a BLAST search for additional homologs of subunit e, we found a human brain protein, KIAA1578, the C-terminal 30 amino acids of which are identical to those of human subunit e. This protein also contains a potential phosphopeptide binding motif. In summary, these studies provide support for the view that subunit e is a multifunctional cell regulator involved in cell signaling, and implicate the involvement of the KIAA1578 protein in cell signaling as well. These studies suggest also that, while functioning as a subunit of mitochondrial ATP synthases, subunit e may help regulate these complexes by binding to phosphopeptides within one or more of the other subunit types.

14-3-3 Proteins↗

PubMed2004

Structural basis of BACH1 phosphopeptide recognition by BRCA1 tandem BRCT domains.

BRCT tandem domains, found in many proteins involved in DNA damage checkpoint and DNA repair pathways, were recently shown to be phosphopeptide binding motifs. Using solution nuclear magnetic resonance (NMR) spectroscopy and mutational analysis, we have characterized the interaction of BRCA1-BRCT domains with a phosphoserine-containing peptide derived from the DNA repair helicase BACH1. We show that a phenylalanine in the +3 position from the phosphoserine of BACH1 is bound to a conserved hydrophobic pocket formed between the two BRCT domains and that recognition of the phosphate group is mediated by lysine and serine side chains from the amino-terminal BRCT domain. Mutations that prevent phosphopeptide binding abolish BRCA1 function in DNA damage-induced checkpoint control. Our NMR data also reveal a dynamic interaction between BRCA1-BRCT and BACH1, where the bound phosphopeptide exists as an equilibrium of two conformations and where BRCA1-BRCT undergoes a transition to a more rigid conformation upon peptide binding.

BRCA1 Protein↗

PubMed1987

Phosphorylation heterogeneity of tryptic phosphopeptides of chicken riboflavin-binding protein.

The tryptic phosphopeptide of hen egg white riboflavin-binding protein has been found to exist as a mixture of peptides which differ only with respect to the number of covalently bound phosphoryl groups. Anion-exchange chromatography was used to separate homologues of the tryptic phosphopeptide of egg white riboflavin-binding protein. Four peptide peaks were obtained and analyzed using plasma desorption mass spectrometry. Molecular ions obtained agree closely with calculated molecular weight values for phosphopeptides with 8, 7 and 5 phosphoryl groups. Amino acid analyses showed that the octa- and hepta-phosphorylated peptides were pure and had the same amino acid compositions.

Animals↗

PubMed2003

Inhibitory effects of rosmarinic acid on Lck SH2 domain binding to a synthetic phosphopeptide.

In the course of screening inhibitors from the methanol (MeOH) extracts of 168 medicinal plants against lymphocyte cell-specific kinase (Lck) Src -homology 2 (SH2) binding to a synthetic phosphotyrosine-containing peptide (phosphopeptide), we isolated rosmarinic acid from the MeOH extract of Prunella vulgaris, which showed specific inhibitory activity. The IC 50 value for Lck SH2 binding to phosphopeptide (SGSGEEPQpYEEIPI) of hamster polyomavirus middle-sized tumor (hmT pY324) was 7 microM. However, even at concentrations of 0.1 to 1000 microM, no significant inhibitions were observed against other SH2 domains binding such as the growth factor receptor binding protein 2 (Grb2) SH2 domain to phosphopeptide of Shc and phospholipase Cgamma1 (PLCgamma1) SH2 domain to translational elongation factor 1alpha (EF1alpha) C-terminal. Rosmarinic acid inhibited interleukin-2 (IL-2) gene expression by 50 % at a concentration of 8 microM in Jurkat cells stimulated with anti-CD3 and anti-CD4 antibodies. FK506 and cyclosporin A (CsA) employed as positive controls showed less than 30 % inhibition at the same concentration. In addition, rosmarinic acid inhibited the intracellular [Ca 2+] i increase in Jurkat cells after T cell activation in a dose-dependent manner at concentrations of 1.4 to 140 microM of rosmarinic acid, which is one of the earliest responses of antigen-specific T cell receptor (TCR) and of the upstream pathway of IL-2 expression. Taken together, these results suggest that rosmarinic acid has the potential to specifically inhibit Lck SH2 domain binding to its cognate ligand, including ZAP-70, Cbl, HS-1, and PLCgamma1, and Lck-dependent Ca 2+ signaling pathway of its downstream effector and finally to modulate IL-2 gene expression after T cell activation.

Adaptor Proteins, Signal Transducing↗

PubMed1996

A comparative study of the phosphotyrosyl phosphatase specificity of protein phosphatase type 2A and phosphotyrosyl phosphatase type 1B using phosphopeptides and the phosphoproteins p50/HS1, c-Fgr and Lyn.

The phosphotyrosyl phosphatase (PTPase) specificity of phosphotyrosyl-phosphatase-activator-(PTPA)-stimulated protein phosphatase (PP)2A(D) (rabbit muscle) and a bona fide PTP-1B (Xenopus laevis oocytes) were examined in vitro using phosphotyrosine-containing peptides, derived from the phosphorylation sites of p34cdc2, p50/HS1 protein, Abl, c-Src and c-Fgr, as well as the intact phosphoprotein p50/HS1 and the Src-related tyrosine kinases, Lyn and c-Fgr. The local specificity determinants were found to be different for both PTPases. The length of the phosphopeptides is more important for PP2A(D) than for PTP-1B, C-terminal acidic residues adjacent to the phosphotyrosine are detrimental for the PTPase activity of PP2A(D), but they do not affect the PTP-1B activity. Acidic residues at the --2 and --3 position relative to Tyr(P) primarily dictate dephosphorylation by PTP-1B. The higher-order structure of the protein substrates also differentially influences both enzymes: the phospho-octapeptide KDDEYpNPA, which reproduces the autophosphorylation site in c-Fgr (Tyr400), is only dephosphorylated by PP2A(D) if embedded in the intact protein, whereas the opposite is true for PTP-1B. Both the intact p50/HS1 phosphoprotein and the derived phosphopeptide are substrates only for PTP-1B and not for PP2A(D). Lyn and c-Fgr phosphorylated by C-terminal Src kinase (CSK) at their down-regulatory site are resistant to the action of both PTPases while the [Phe6]Src-(514-533) phosphopeptide, representing the highly similar site affected by CSK in c-Src, is readily dephosphorylated by both PTPases, although to a different extent. In vitro dephosphorylation of the c-Fgr Tyr400 site by PP2A(D) is correlated with a decreased tyrosine kinase activity towards exogenous substrates. Under experimental conditions in which both Tyr400 (autophosphorylation site) and Tyr511 (down-regulatory site) of c-Fgr are phosphorylated, PP2A(D) can reverse both phosphorylations.

Amino Acid Sequence↗

PubMed2000

Experimental study of phosphopeptide in promoting tooth remineralization.

OBJECTIVE: To explore a new method for effective and safe prevention and treatment of caries. METHODS: Following the theory of tooth remineralization, we searched for the mechanism and clinical application of tooth remineralization with phosphopeptide. RESULTS: Phosphopeptide, with calcium and phosphorus, had a high affinity for hydroxyapatite and could enhance tooth remineralization. CONCLUSION: Phosphopeptide was effective in the prevention and treatment of tooth decay. Clinically, it could be safely applied.

Animals↗

PubMed1996

Resistance to etoposide in human leukemia HL-60 cells: reduction in drug-induced DNA cleavage associated with hypophosphorylation of topoisomerase II phosphopeptides.

Tumor cell resistance to anthracyclines and epipodophyllotoxins can be due to reduced drug accumulation and/or alterations in the activity of topoisomerase II (TOPO II). HL-60 cells selected in 0.05 micrograms/ml doxorubicin (DOX) are 10-fold and > 20-fold resistant to DOX and etoposide (VP-16), respectively. The accumulation of [3H]VP-16 was 2-3-fold lower in the resistant cells (HL-60/DOX 0.05) than in similarly treated parent-sensitive cells (HL-60/S). However, compared with HL-60/S cells, the HL-60/DOX 0.05 cells required > 20-fold higher concentrations of VP-16 to produce equivalent damage to DNA. The reduced formation of VP-16-stabilized DNA cleavable complex in the HL-60/DOX 0.05 cells was not due to differences in the amount of 170-kDa TOPO (alpha) II protein or enzyme catalytic activity between HL-60/S and HL-60/DOX 0.05 cells. Metabolic labeling with [32P]orthophosphoric acid and immunoprecipitation indicated that the level of phosphorylated 170-kDa TOPO II alpha protein in the HL-60-/S cells was 2.2 +/- 0.4-fold higher than that in HL-60/DOX 0.05 cells. Hypophosphorylation (3-fold) of 170-kDa TOPO II protein in HL-60/S cells treated with the calcium chelator 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester produced a > 2-fold reduction in VP-16-induced TOPO II-mediated DNA cleavable complex formation. Two-dimensional mapping of phosphopeptides in complete tryptic digests demonstrated that the reduced phosphorylation of the 170-kDa TOPO II alpha in HL-60/DOX 0.05 cells was due to the hypophosphorylation of at least three phosphopeptides characteristic of HL-60/S cells. Thus, the attenuated ability of TOPO II to form drug-stabilized DNA cleavable complex is related to the phosphorylated state of 170-kDa TOPO II, and in HL-60/DOX 0.05 cells, resistance may be related to hypophosphorylation of three phosphopeptides characteristic of HL-60/S cells.

Antineoplastic Agents, Phytogenic↗

PubMed2001

The synthesis of phosphopeptides.

Phosphorylation and dephosphorylation are key events in protein expression and regulation and in signal transduction. Phosphopeptides are very useful reagents for the study of these processes, and have been used to great advantage in the study of phosphatase substrate specificity, SH2 domain ligand specificity, and protein-protein interactions. Furthermore, the advent of cell-permeable peptide carriers, such as those from the antennapedia homeodomain and the HIV TAT transcription factor, has allowed the study of intracellular events, thus underscoring the utility of these reagents. In this paper we review methods for the synthesis of phosphopeptides with the emphasis on the preparation of phosphoamino acid building blocks.

Amino Acid Sequence↗

PubMed2001

Designing cell-permeant phosphopeptides to modulate intracellular signaling pathways.

A central theme in intracellular signaling is the regulatable interaction of proteins via the binding of specialized domains on one protein to short linear sequences on other molecules. The capability of these short sequences to mediate the required specificity and affinity for signal transduction allows for the rational design of peptide-based modulators of specific protein-protein interactions. Such inhibitors are valuable tools for elucidating the role of these interactions in cellular physiology and in targeting such interactions for potential therapeutic intervention. This approach is exemplified by the study of the role of phosphorylation of specific sites on signaling proteins. However, the difficulty of introducing large hydrophilic molecules such as phosphopeptides into cells has been a major drawback in this area. This review describes the application of recently developed cell-permeant peptide vectors in the introduction of biologically active peptides into cells, with particular emphasis on the antennapedia/penetratin, TAT, and signal-peptide based sequences. In addition, the modification of such peptides to increase uptake efficiency and affinity for their targets is discussed. Finally, the use of cell-permeant phosphopeptides to both inhibit and stimulate intracellular signaling mechanisms is described, by reference to the PLCgamma, Grb2, and PI-3 kinase pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Quadrupole time-of-flight versus triple-quadrupole mass spectrometry for the determination of phosphopeptides by precursor ion scanning.

An API 3000 triple-quadrupole instrument and a QSTAR Pulsar quadrupole time-of-flight (TOF) mass spectrometer were compared for the determination of phosphopeptides by precursor ion scanning in both the positive and negative nanoelectrospray ionization modes. The limits of detection for synthetic phosphopeptides were similar (500 amol microl(-1)) for both types of instruments when monitoring precursors of -79 Da (PO(3)(-)). However, the quadrupole TOF system was approximately fivefold more sensitive (1 fmol microl(-1)) than the triple-quadrupole instrument (5 fmol microl(-1)) when monitoring precursors of 216 Da (immonium ion of phosphotyrosine). The recently introduced Q(2)-pulsing function, which enhances the transmission of fragment ions of a selected m/z window from the collision cell into the TOF part, improved the sensitivity of precursor ion scans on a quadrupole TOF instrument. The selectivity of precursor ion scans is much better on quadrupole TOF systems than on triple quadrupoles because the high resolving power of the reflectron-TOF mass analyzer permits high-accuracy fragment ion selection at no expense of sensitivity. This minimizes interferences from other peptide fragment ions (a-, b-, and y- type) of the same nominal mass but with sufficient differences in their exact masses. As a result, the characteristic immonium ion of phosphotyrosine at m/z 216.043 can be utilized for the selective detection of tyrosine phosphorylated peptides. Our data suggest that, in addition to their superior performance for peptide sequencing, quadrupole TOF instruments also offer a very viable alternative to triple quadrupoles for precursor ion scanning, thus combining high sensitivity and selectivity for both MS and MS/MS experiments in one instrument.

Amino Acid Sequence↗

PubMed2003

Nanoelectrospray-based detection and sequencing of substoichiometric amounts of phosphopeptides in complex mixtures.

A novel immobilized metal affinity chromatography (IMAC)-based protocol was successfully used for sample preparation prior to nanoelectrospray-based sequencing of phosphopeptides. In a first step, phosphorylated peptides are detected in an unseparated peptide mixture using precursor ion scanning in the negative ion mode on a triple-quadrupole mass spectrometer. Then the phosphorylated peptides are enriched by passing the sample over an IMAC column and sequenced in the positive ion mode. The IMAC-based enrichment allows the sequencing of phosphorylated peptides even if other, much more abundant, peptides are present at the same m/z value in the original mixture. Using this two-step approach, we are able to combine the simplicity, sensitivity and specificity of precursor ion scanning-based detection of phosphopeptides using the nanoelectrospray ion source with the ability to sequence phosphorylated peptides even if they are present in substoichiometric amounts.

Animals↗

PubMed2005

Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins.

Immobilized metal ion affinity chromatography (IMAC) is now a widely accepted technique for the separation of natural or artificial products that is beginning to find industrial applications. Here, we introduce a novel procedure for the separation of phosphopeptides and phosphorylated proteins by immobilized zinc(II) affinity chromatography. The phosphate-binding site of the affinity gel is an alkoxide-bridged dinuclear zinc(II) complex, the 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex (Phos-tag), which is linked to a highly cross-linked 4% (w/v) agarose. The affinity gel (Phos-tag agarose) was prepared by the quantitative reaction of N-hydroxysuccinimide-activated Sepharose and a Phos-tag derivative having a 2-aminoethylcarbamoyl group in dry CH3CN. Phosphopeptides were retrieved in a quantitative and highly selective manner by a spin column method using Phos-tag agarose at room temperature. Furthermore, in this study, we demonstrate a simple, rapid, and reusable affinity column chromatography for the separation of phosphorylated proteins such as ovalbumin, alpha(s1)-casein, and beta-casein at physiological pH.

Animals↗

PubMed2005

Synthesis of linear and cyclic phosphopeptides as ligands for the N-terminal SH2-domain of protein tyrosine phosphatase SHP-1.

Linear and cyclic phosphopeptides related to the pY2267 binding site of the epithelial receptor tyrosine kinase Ros have been synthesized as ligands for the amino-terminal SH2 (src homology) domain of protein tyrosine phosphatase SHP-1. The synthesis was accomplished by Fmoc-based solid-phase methodology using side-chain unprotected phosphotyrosine for the linear and mono-benzyl protected phosphotyrosine for the cyclic peptides. According to molecular modelling, the incorporation of a glycine residue between Lys (position pY-1 relative to phosphotyrosine) and Asp or Glu (position pY+2) was recommended for the cyclic candidates. The preparation of these peptides was successfully performed by the incorporation of a Fmoc-Xxx(Gly-OAll)-OH (Xxx = Asp, Glu) dipeptide building block that was prepared in solution prior to SPPS. The cyclization was achieved with PyBOP following Alloc/OAll-deprotection. This study demonstrates the usefulness of allyl-type protecting groups for the generation of side-chain cyclized phosphopeptides. Alloc/OAll-deprotection and cyclization are compatible with phosphorylated tyrosine.

Chromatography, High Pressure Liquid↗

PubMed1993

Verification of the position of the phosphate group in some synthetic phosphopeptides by fast-atom bombardment and tandem mass spectrometry.

Some synthetically obtained linear and cyclic phosphopeptides of low molecular weight have been studied by fast-atom bombardment and tandem mass spectrometry to verify the position of the phosphate group in these compounds. Based upon the occurrence/non-occurrence of loss of phosphoric acid from low abundance fragment ions induced by low- and high-energy collisions with target gases, it is shown that the position of the phosphate group in the phosphopeptides studied can be determined unequivocally.

Amino Acid Sequence↗

PubMed2006

Selective detection and identification of phosphopeptides by dansyl MS/MS/MS fragmentation.

Protein phosphorylation regulates many cellular processes and pathways, such as cell cycle progression, signal transduction cascades and gene expression. Selective detection of phosphopeptides from proteolytic digests is a challenging and highly relevant task in many proteomics applications. Often phosphopeptides are present in small amounts and need selective isolation or enrichment before identification. Here we report a novel approach to label selectively phospho-Ser/-Thr residues by exploiting the features of a novel linear ion trap mass spectrometer. Using dansyl labelling and MS3 fragmentation, we developed a method useful for the large-scale proteomic profiling of phosphorylation sites. The new residues in the sequence were stable and easily identifiable under general conditions for tandem mass spectrometric sequencing.

Caseins↗

PubMed2001

Phosphopeptide analysis by positive and negative ion matrix-assisted laser desorption/ionization mass spectrometry.

This article describes a simple procedure for the detection of phosphorylated peptides by comparable positive and negative ion mode matrix-assisted laser desorption/ionization mass spectrometry measurements. Based on studies with phosphorylated peptides (EAIXAAPFAK, X = pS, pT, pY) and their corresponding non-phosphorylated analogs, it was found that phosphopeptides, which are characterized by a low ionization efficiency in the positive ion mode, exhibit drastically increased signal intensities in the negative ion mode compared to their non-phosphorylated analogs. The effect was successfully used to identify phosphorylated sequences of the commonly used phosphoprotein standards, protein kinase A and beta-casein, by peptide mass fingerprint analyses of the corresponding Lys C and trypsin digests using both (positive and negative) ion modes. The comparison of positive and negative ion spectra of a given protein digest (relative intensity([M - H]-)/relative intensity([M + H]+)) can be used to identify any phosphopeptides present which may then be separated and analyzed further.

Caseins↗

PubMed2001

Alkaline liquid chromatography/electrospray ionization skimmer collision-induced dissociation mass spectrometry for phosphopeptide screening.

A rapid on-line method for the identification of phosphorylated peptides in enzymatic protein digests by specific marker ion signals is described. In our study we investigated the use of alkaline conditions together with a previously described method for selective and sensitive detection of phosphopeptide ions combining high-performance capillary liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS). Phosphorylation-specific marker ions (m/z 79, PO(3)(-), and m/z 97, H(2)PO(4)(-)) were generated by skimmer collision-induced dissociation (sCID) in the negative-ion mode. The method was evaluated and validated for mono-phosphorylated synthetic peptides using different alkaline pH values and CID offsets. Alkaline conditions (pH 10.5) enhance the generation of phosphopeptide-specific fragment ions from serine- and tyrosine-phosphorylated peptides, and enable the use of m/z 79 (PO(3)(-)) and m/z 97 (H(2)PO(4)(-)) as phosphorylation-specific marker traces. Note that HPLC separation in trifluoroacetic acid containing solvents impairs the use of m/z 97 (C(2)F(3)O(-) fragment ion at m/z 97) as a phosphorylation-specific marker. The optimized method was applied for the detection of phosphorylated peptides in a tryptic beta-casein digest. The expected mono- and tetra-phosphorylated peptides were detected and rapidly identified by (mu)LC/ESI-sCID-MS and (mu)LC/ESI-MS analysis.

Alkalies↗