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International Journal of Molecular... Oct 2017Drought is one of the major abiotic stresses that negatively affects plant growth and development. is an ecologically important shrub in the mid-Asia desert region and...
Drought is one of the major abiotic stresses that negatively affects plant growth and development. is an ecologically important shrub in the mid-Asia desert region and used as a model for abiotic tolerance research in trees. Protein phosphorylation participates in the regulation of various biological processes, however, phosphorylation events associated with drought stress signaling and response in plants is still limited. Here, we conducted a quantitative phosphoproteomic analysis of the response of roots to short-term drought stress. Data are available via the iProx database with project ID IPX0000971000. In total, 7841 phosphorylation sites were found from the 2019 identified phosphopeptides, corresponding to 1060 phosphoproteins. Drought stress results in significant changes in the abundance of 103 phosphopeptides, corresponding to 90 differentially-phosphorylated phosphoproteins (DPPs). Motif-x analysis identified two motifs, including [pSP] and [RXXpS], from these DPPs. Functional enrichment and protein-protein interaction analysis showed that the DPPs were mainly involved in signal transduction and transcriptional regulation, osmotic adjustment, stress response and defense, RNA splicing and transport, protein synthesis, folding and degradation, and epigenetic regulation. These drought-corresponsive phosphoproteins, and the related signaling and metabolic pathways probably play important roles in drought stress signaling and response in roots. Our results provide new information for understanding the molecular mechanism of the abiotic stress response in plants at the posttranslational level.
Topics: Amino Acid Motifs; Amino Acid Sequence; Computational Biology; Droughts; Fabaceae; Phosphoproteins; Plant Proteins; Plant Roots; Protein Interaction Mapping; Protein Interaction Maps; Proteome; Proteomics; Stress, Physiological; Tandem Mass Spectrometry
PubMed: 29039783
DOI: 10.3390/ijms18102158 -
Journal of Virology Dec 1988Cellular phosphoprotein p53, which seems to be a multifunctional protein, may be assigned to different structural subclasses. Recently established immortalized or...
Cellular phosphoprotein p53, which seems to be a multifunctional protein, may be assigned to different structural subclasses. Recently established immortalized or transformed cell lines that overexpress p53 allowed us to perform a detailed analysis of the quaternary structure of p53. By means of sucrose density gradient centrifugation, we found in simian virus 40-transformed cells that overexpress p53, in addition to high-molecular-weight T-p53 complexes, low-molecular-weight forms. The level of T-p53 complexes within simian virus 40-transformed cells seemed to be determined by the intracellular concentration of p53. However, the presence of uncomplexed T antigen and p53 indicated that an appropriate modification of at least one of the two proteins appears to be necessary for complex formation. Using different monoclonal antibodies that distinguish between (i) p53 associated with T antigen or heat shock proteins and (ii) p53 in apparently free form, we found p53 from transformed cells always in high-molecular-weight forms. p53 from normal and immortalized cells, however, was found mainly in low-molecular-weight forms. Pulse-labeling experiments revealed that oligomerization of p53 is a very rapid process. Monomeric forms of p53 which could be detected only by 2 min of pulse-labeling were rapidly converted to stable, high-molecular-weight oligomers. Furthermore, our data indicate a correlation between the occurrence of p53 in high-molecular-weight forms and the transformation state of the cell.
Topics: Animals; Blotting, Western; Cell Line, Transformed; Centrifugation, Density Gradient; Neoplasm Proteins; Phosphoproteins; Polymers; Precipitin Tests; Proto-Oncogene Proteins; Rats; Tumor Suppressor Protein p53
PubMed: 3054153
DOI: 10.1128/JVI.62.12.4737-4744.1988 -
Nucleic Acids Research Jul 2003The CRP (Cleavage of Radiolabeled Phosphoproteins) program guides the design and interpretation of experiments to identify protein phosphorylation sites by Edman...
The CRP (Cleavage of Radiolabeled Phosphoproteins) program guides the design and interpretation of experiments to identify protein phosphorylation sites by Edman sequencing of unseparated peptides. Traditionally, phosphorylation sites are determined by cleaving the phosphoprotein and separating the peptides for Edman 32P-phosphate release sequencing. CRP analysis of a phosphoprotein's sequence accelerates this process by omitting the separation step: given a protein sequence of interest, the CRP program performs an in silico proteolytic cleavage of the sequence and reports the predicted Edman cycles in which radioactivity would be observed if a given serine, threonine or tyrosine were phosphorylated. Experimentally observed cycles containing 32P can be compared with CRP predictions to confirm candidate sites and/or explore the ability of additional cleavage experiments to resolve remaining ambiguities. To reduce ambiguity, the phosphorylated residue (P-Tyr, P-Ser or P-Thr) can be determined experimentally, and CRP will ignore sites with alternative residues. CRP also provides simple predictions of likely phosphorylation sites using known kinase recognition motifs. The CRP interface is available at http://fasta.bioch.virginia.edu/crp.
Topics: Humans; Internet; Phosphoproteins; Phosphorylation; Phosphoserine; Phosphothreonine; Phosphotyrosine; Radioactive Tracers; Sequence Analysis, Protein; Software
PubMed: 12824437
DOI: 10.1093/nar/gkg513 -
The Journal of Biological Chemistry Apr 2011The Henipavirus genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that recruits the polymerase complex via the phosphoprotein (P). In a...
The Henipavirus genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that recruits the polymerase complex via the phosphoprotein (P). In a previous study, we reported that in henipaviruses, the N-terminal domain of the phosphoprotein and the C-terminal domain of the nucleoprotein (N(TAIL)) are both intrinsically disordered. Here we show that Henipavirus N(TAIL) domains are also disordered in the context of full-length nucleoproteins. We also report the cloning, purification, and characterization of the C-terminal X domains (P(XD)) of Henipavirus phosphoproteins. Using isothermal titration calorimetry, we show that N(TAIL) and P(XD) form a 1:1 stoichiometric complex that is stable under NaCl concentrations as high as 1 M and has a K(D) in the μM range. Using far-UV circular dichroism and nuclear magnetic resonance, we show that P(XD) triggers an increase in the α-helical content of N(TAIL). Using fluorescence spectroscopy, we show that P(XD) has no impact on the chemical environment of a Trp residue introduced at position 527 of the Henipavirus N(TAIL) domain, thus arguing for the lack of stable contacts between the C termini of N(TAIL) and P(XD). Finally, we present a tentative structural model of the N(TAIL)-P(XD) interaction in which a short, order-prone region of N(TAIL) (α-MoRE; amino acids 473-493) adopts an α-helical conformation and is embedded between helices α2 and α3 of P(XD), leading to a relatively small interface dominated by hydrophobic contacts. The present results provide the first detailed experimental characterization of the N-P interaction in henipaviruses and designate the N(TAIL)-P(XD) interaction as a valuable target for rational antiviral approaches.
Topics: Henipavirus; Models, Molecular; Nucleoproteins; Phosphoproteins; Protein Structure, Quaternary; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins; Viral Proteins
PubMed: 21317293
DOI: 10.1074/jbc.M111.219857 -
Journal of Neurochemistry Jun 2012Alzheimer's disease (AD) is the most common form of dementia and cognitive impairment usually characterized by widespread neurodegeneration throughout the association...
Alzheimer's disease (AD) is the most common form of dementia and cognitive impairment usually characterized by widespread neurodegeneration throughout the association cortex, limbic system and hippocampus. Aberrant protein phosphorylation is a defining pathological hallmark of AD and implicated in the dysregulation of major cellular processes through highly dynamic and complex signaling pathways. Here in, we demonstrate 81 proteins, of 600 spots selected, unambiguously identified as phosphorylated, providing a partial phosphoproteome profile of AD substantia nigra and cortex and respective control brain regions. More importantly, abnormal phosphorylation signal intensity of nine physiologically important proteins observed can profoundly affect cell metabolism, signal transduction, cytoskeleton integration, and synaptic function and accounts for biological and morphological alterations. Our studies employed two-dimensional gel electrophoresis for protein separation, Pro-Q(®) Diamond phosphoprotein staining and electrospray ionization quadrupole time of flight tandem MS for protein identification. NetPhosk 1.0 is used for the confirmation of protein modification status as well known/putative phosphoproteins. A further insight into the links among the identified phosphoproteins and functional roles STRING 8.3, KEGG and REACTOME pathway databases were applied. The present quantitative phosphoproteomic analysis can be supportive in establishing a broad database of potential protein targets of abnormal phosphorylation in AD brain.
Topics: Aged; Alzheimer Disease; Cerebral Cortex; Electrophoresis, Gel, Two-Dimensional; Humans; Phosphoproteins; Phosphorylation; Proteome; Proteomics; Spectrometry, Mass, Electrospray Ionization; Substantia Nigra
PubMed: 22436009
DOI: 10.1111/j.1471-4159.2012.07737.x -
Eukaryotic Cell Jul 2009Cilia and flagella are cell organelles that are highly conserved throughout evolution. For many years, the green biflagellate alga Chlamydomonas reinhardtii has served...
Cilia and flagella are cell organelles that are highly conserved throughout evolution. For many years, the green biflagellate alga Chlamydomonas reinhardtii has served as a model for examination of the structure and function of its flagella, which are similar to certain mammalian cilia. Proteome analysis revealed the presence of several kinases and protein phosphatases in these organelles. Reversible protein phosphorylation can control ciliary beating, motility, signaling, length, and assembly. Despite the importance of this posttranslational modification, the identities of many ciliary phosphoproteins and knowledge about their in vivo phosphorylation sites are still missing. Here we used immobilized metal affinity chromatography to enrich phosphopeptides from purified flagella and analyzed them by mass spectrometry. One hundred forty-one phosphorylated peptides were identified, belonging to 32 flagellar proteins. Thereby, 126 in vivo phosphorylation sites were determined. The flagellar phosphoproteome includes different structural and motor proteins, kinases, proteins with protein interaction domains, and many proteins whose functions are still unknown. In several cases, a dynamic phosphorylation pattern and clustering of phosphorylation sites were found, indicating a complex physiological status and specific control by reversible protein phosphorylation in the flagellum.
Topics: Animals; Catalytic Domain; Cell Movement; Chlamydomonas reinhardtii; Chromatography, Affinity; Conserved Sequence; Evolution, Molecular; Flagella; Mass Spectrometry; Peptides; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Phosphotransferases; Protein Structure, Tertiary; Proteome; Proteomics; Species Specificity
PubMed: 19429781
DOI: 10.1128/EC.00067-09 -
BMC Cell Biology Mar 2012Rab11 and its effector molecule, Rab11-FIP3 (FIP3), associate with recycling endosomes and traffic into the furrow and midbody of cells during cytokinesis. FIP3 also...
BACKGROUND
Rab11 and its effector molecule, Rab11-FIP3 (FIP3), associate with recycling endosomes and traffic into the furrow and midbody of cells during cytokinesis. FIP3 also controls recycling endosome distribution during interphase. Here, we examine whether phosphorylation of FIP3 is involved in these activities.
RESULTS
We identify four sites of phosphorylation of FIP3 in vivo, S-102, S-280, S-347 and S-450 and identify S-102 as a target for Cdk1-cyclin B in vitro. Of these, we show that S-102 is phosphorylated in metaphase and is dephosphorylated as cells enter telophase. Over-expression of FIP3-S102D increased the frequency of binucleate cells consistent with a role for this phospho-acceptor site in cytokinesis. Mutation of S-280, S-347 or S-450 or other previously identified phospho-acceptor sites (S-488, S-538, S-647 and S-648) was without effect on binucleate cell formation and did not modulate the distribution of FIP3 during the cell cycle. In an attempt to identify a functional role for FIP3 phosphorylation, we report that the change in FIP3 distribution from cytosolic to membrane-associated observed during progression from anaphase to telophase is accompanied by a concomitant dephosphorylation of FIP3. However, the phospho-acceptor sites identified here did not control this change in distribution.
CONCLUSIONS
Our data thus identify FIP3 as a cell cycle regulated phosphoprotein and suggest dephosphorylation of FIP3 accompanies its translocation from the cytosol to membranes during telophase. S102 is dephosphorylated during telophase; mutation of S102 exerts a modest effect on cytokinesis. Finally, we show that de/phosphorylation of the phospho-acceptor sites identified here (S-102, S-280, S-347 and S-450) is not required for the spatial control of recycling endosome distribution or function.
Topics: Carrier Proteins; Cell Cycle; Cell Division; Cell Line, Tumor; Cytokinesis; Endosomes; HeLa Cells; Humans; Interphase; Phosphoproteins
PubMed: 22401586
DOI: 10.1186/1471-2121-13-4 -
Biophysical Journal Apr 2018The mechanism of interaction of an intrinsically disordered protein (IDP) with its physiological partner is characterized by a disorder-to-order transition in which a...
The mechanism of interaction of an intrinsically disordered protein (IDP) with its physiological partner is characterized by a disorder-to-order transition in which a recognition and a binding step take place. Even if the mechanism is quite complex, IDPs tend to bind their partner in a cooperative manner such that it is generally possible to detect experimentally only the disordered unbound state and the structured complex. The interaction between the disordered C-terminal domain of the measles virus nucleoprotein (N) and the X domain (XD) of the viral phosphoprotein allows us to detect and quantify the two distinct steps of the overall reaction. Here, we analyze the robustness of the folding of N upon binding to XD by measuring the effect on both the folding and binding steps of N when the structure of XD is modified. Because it has been shown that wild-type XD is structurally heterogeneous, populating an on-pathway intermediate under native conditions, we investigated the binding to 11 different site-directed variants of N of one particular variant of XD (I504A XD) that populates only the native state. Data reveal that the recognition and the folding steps are both affected by the structure of XD, indicating a highly malleable pathway. The experimental results are briefly discussed in the light of previous experiments on other IDPs.
Topics: Intrinsically Disordered Proteins; Models, Molecular; Nucleocapsid Proteins; Nucleoproteins; Phosphoproteins; Protein Binding; Protein Domains; Protein Folding; Viral Proteins
PubMed: 29694866
DOI: 10.1016/j.bpj.2018.03.017 -
Biosensors Oct 2022Abnormal protein phosphorylation may relate to diseases such as Alzheimer's, schizophrenia, and Parkinson's. Therefore, the real-time detection of phosphoproteins in...
Abnormal protein phosphorylation may relate to diseases such as Alzheimer's, schizophrenia, and Parkinson's. Therefore, the real-time detection of phosphoproteins in sweat was of great significance for the early knowledge, detection, and treatment of neurological diseases. In this work, anatase/rutile TiO was in situ grown on the MXene surface to constructing the intercalation structure MXene@anatase/rutile TiO ternary heterostructure as a sensing platform for detecting phosphoprotein in sweat. Here, the intercalation structure of MXene acted as electron and diffusion channels for phosphoproteins. The in situ grown anatase/rutile TiO with n-n-type heterostructure provided specific adsorption sites for the phosphoproteins. The determination of phosphoprotein covered concentrations in sweat, with linear range from 0.01 to 1 mg/mL, along with a low LOD of 1.52 μM. It is worth noting that, since the macromolecular phosphoprotein was adsorbed on the surface of the material, the electrochemical signal gradually decreased with the increase of phosphoprotein concentration. In addition, the active sites in the MXene@anatase/rutile TiO ternary heterojunction and synergistic effect of the heterojunction were verified by first-principle calculations to further realize the response to phosphoproteins. Additionally, the effective diffusion capacity and mobility of phosphoprotein molecules in the ternary heterojunction structure were studied by molecular dynamics simulation. Furthermore, the constructed sensing platform showed high selectivity, repeatability, reproducibility, and stability, and this newly developed sensor can detect for phosphoprotein in actual sweat samples. This satisfactory sensing strategy could be promoted to realize the noninvasive and continuous detection of sweat.
Topics: Sweat; Phosphoproteins; Reproducibility of Results; Titanium
PubMed: 36291003
DOI: 10.3390/bios12100865 -
Journal of Virology Mar 1975Oncornaviruses, which contain a virion-associated protein kinase, were found to possess phosphoproteins as virion structural components. One major phosphoprotein common...
Oncornaviruses, which contain a virion-associated protein kinase, were found to possess phosphoproteins as virion structural components. One major phosphoprotein common to strains of laboratory and wild mouse oncornaviruses and a strain of feline leukemia virus was shown to be a polypeptide of about 12, 000 mol wt. In addition to this, the Kirsten strain of murine sarcoma virus contained a second major phosphoprotein of about 10, 000 mol wt, and mouse erythroblastosis virus contained a second major phosphoprotein that was either identical to or comigrated with the virion glycoprotein of about 74, 000 mol wt. The major phosphoprotein of RD-114 virus was found to be of about 16, 000 mol wt. The major phosphoamino acid of the 12, 000-mol wt polypeptide of the mouse erythroblastosis virus was identified as phosphoserine, and that of the 16, 000-mol wt polypeptide of the RD-114 virus was identified as phosphothreonine.
Topics: Electrophoresis, Paper; Electrophoresis, Polyacrylamide Gel; Gammaretrovirus; Leukemia Virus, Feline; Molecular Weight; Oncogenic Viruses; Peptides; Phosphoproteins; Phosphorus Radioisotopes; RNA Viruses; Rauscher Virus; Retroviridae; Tritium
PubMed: 163371
DOI: 10.1128/JVI.15.3.540-549.1975