PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “PHOSPHOPEPTIDES”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 379 records · Page 21Linked to original sources
PubMed2005

Anti-phosphopeptide antibody, P-STM as a novel tool for detecting mitotic phosphoproteins: identification of lamins A and C as two major targets.

A polyclonal, phospho-epitope-specific antibody (P-STM) was generated to detect the activated p21-activated kinase 2 (PAK2), based on the regulatory autophosphorylation site Thr(402) of PAK2 [Yu et al., 1998]. In this report, we show that this antibody can also recognize many phosphoproteins in mitotic HeLa and A431 cells. Signal of these phosphoproteins emerged after treating the cells with nocodazole and okadaic acid, and was highly detected in G2-M phase transition of HeLa cells released from double thymidine block. Immunofluorescence analysis revealed that P-STM strongly stained HeLa cells at prometaphase and metaphase, but not at interphase and anaphase. Interestingly, this staining pattern was almost identical to that obtained by staining with MPM2, a monoclonal antibody known to react with phosphoproteins in mitotic HeLa cells. However, the phosphoproteins detected by the two antibodies are quite different. Two-dimensional gel electrophoresis (2DE) and tryptic peptide fingerprint analysis by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry were employed to identify lamins A and C as two of the mitotic cell-specific phosphoproteins recognized by P-STM. Lamins A and C immunoprecipitated from nocodazole-treated cells, but not from untreated cells showed strong reactivity to P-STM, and this reactivity lost completely after protein phosphatase 2A treatment. In summary, our results show that P-STM represents a novel tool for detecting mitotic phosphoproteins, which are different from those recognized by MPM2, and that lamins A and C are the two prominent mitotic phosphoproteins detected by P-STM.

Amino Acid Sequence↗
PubMed2002

Synthesis of complex phosphopeptides as mimics of p53 functional domains.

A complete set of mono-, di- and triphosphorylated peptides comprising amino acids 10-27, the Mdm2 and p300 binding site(s) of p53, with and without a fluorescein label at the N-terminus, was synthesized by step-by-step solid phase synthesis. Fluorescence polarization analysis revealed that phosphorylation at Thr18 decreased binding to recombinant Mdm2 protein compared with the unphosphorylated and the two other single phosphorylated analogues. Unlabelled multiply phosphorylated peptides corresponding to this amino-terminal transactivation domain proved to be powerful tools in analysing the phosphate specificity of existing anti-p53 monoclonal and polyclonal antibodies using direct ELISA. The tetramerization domain of human p53 protein was modelled with a 53 residue-long unlabelled unphosphorylated and Ser315-phosphorylated peptide pair. CD analysis showed similar alpha-helical structures for both peptides and no major difference in the secondary structure could be observed upon phosphorylation. Size-exclusion HPLC indicated that these synthetic oligomerization domain mimics underwent a pH-dependent tetramerization process, but the presence of a phosphate group at Ser315 did not modify the oligomeric state of the 308-360 p53 fragments. Nevertheless, the fluorescein-labelled Ser315 phosphorylated peptide bound to the downstream signalling ligand DNA topoisomerase I protein with slightly higher affinity than did the unphosphorylated analogue.

Amino Acid Sequence↗
PubMed2003

Stable isotope labeling of phosphopeptides for multiparallel kinase target analysis and identification of phosphorylation sites.

An approach for multiparallel target identification and relative quantification of in vitro kinase activities in two different biological samples, using liquid chromatography/mass spectrometry (LC/MS), is described. Synthetic target peptides, containing the putative regulatory phosphorylation sites of sucrose-phosphate synthase (SPS) isoenzymes from Arabidopsis thaliana, were simultaneously in vitro phosphorylated and their phosphorylation states determined. Quantification was achieved by stable isotope labeling of the phosphoserine moiety with ethanethiol and [(2)D(5)]-ethanethiol. This revealed different kinase activities in extracts of wild-type (WT) plants and mutant plants lacking plastidic phosphoglucomutase (PGM). The multiparallel assay allowed the determination of favored substrate specificities among the putative phosphorylation sites in SPS. Additionally, we extended the method to unambiguously identify phosphorylation sites in peptides via differential labeling.

Amino Acid Sequence↗
PubMed2000

A strategy for identification and quantitation of phosphopeptides by liquid chromatography/tandem mass spectrometry.

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) is a state-of-the-art method of structural analysis of peptides/proteins. Here, using activating transcription factor-2 (ATF2) as an example, we report how LC/MS/MS data were processed to generate selected ion tracings for identification of phosphorylated peptides based on their parallel elution behavior with their nonphosphorylated analogs. Via this approach, we verified that amino acid residues Thr-69, Thr-71, and Ser-90 of ATF2 were the in vitro targets for c-Jun kinase. Selected ion tracing method was also used to quantitatively determine phosphorylation states of peptides. We demonstrated that the phosphorylation of Thr-69/Thr-71 was increased in response to ultraviolet irradiation specifically in subconfluent but not in confluent cultures. About 24% of Thr-69/Thr-71-containing segment were singly phosphorylated in subconfluent cultures, while minimal phosphorylation occurred in confluent cultures. In contrast, Ser-112 phosphorylation remained unaffected by cell densities. This strategy could be applied to the studies of a variety of modifications seen in various regulated cellular processes.

Activating Transcription Factor 2↗
PubMed1998

Structural basis for the high affinity of amino-aromatic SH2 phosphopeptide ligands.

An anthranyl moiety placed at the N terminus of a phosphotyrosine peptide potentiates the inhibitory effect of this small peptide on the binding of the Grb2 SH2 domain to the EGF receptor. Using molecular modeling procedures based on the Lck SH2 domain structure, this observation was rationalized in terms of a suitably favorable pi-pi stacking interaction between the anthranyl moiety and the arginine alphaA2 (ArgalphaA2) residue side-chain of Grb2 SH2. The crystal structure of the Grb2 SH2 domain in complex with the inhibitor 2-Abz-EpYINQ-NH2 (IC50 26 nM) has been solved in two different crystal forms at 2.1 and 1.8 A resolution. This structure confirms the modeling based on the Lck SH2 domain. The ArgalphaA2 residue is conserved in most SH2 domains. Thus, as expected, the anthranyl group also confers high affinity to small peptide ligands of other SH2 domains such as Lck-, PLC-gamma-amino-terminal and p85 amino-terminal SH2 domains as demonstrated by structure affinity relationships (SAR) data. These potent peptides with an amino-terminal surrogate group and the structure of Grb2 SH2 domain in complex with one such peptide represent good starting points for the design and optimization of new inhibitors of many SH2 domains.

Adaptor Proteins, Signal Transducing↗
PubMed1979

Phosphopeptides and gamma-carboxyglutamic acid-containing peptides in calcified turkey tendon: their absence in uncalcified tendon.

Uncalcified samples of turkey tendon obtained prior to calcification, and other samples from areas of tendon that never calcify, contain little or no O-phosphoserine [Ser(P)], O-phosphothreonine [Thr(P)] and gamma-carboxyglutamic acid (Gla). Significant amounts of all three of these Ca2+-binding amino acids, which are found in EDTA-extractable, non-collagenous proteins, are detected coincident with the onset of mineralization of a tendon and increase in concentration as mineralization proceeds.

1-Carboxyglutamic Acid↗