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Unlabelled: We here present a neural network-based method for the prediction of protein phosphorylation sites in yeast--an important model organism for basic research. Existing protein phosphorylation site predictors are primarily based on mammalian data and show reduced sensitivity on yeast phosphorylation sites compared to those in humans, suggesting the need for an yeast-specific phosphorylation site predictor. NetPhosYeast achieves a correlation coefficient close to 0.75 with a sensitivity of 0.84 and specificity of 0.90 and outperforms existing predictors in the identification of phosphorylation sites in yeast., Availability: The NetPhosYeast prediction service is available as a public web server at http://www.cbs.dtu.dk/services/NetPhosYeast/.

Symbiosis receptor-like kinase SYMRK is required for root nodule symbiosis between legume plants and nitrogen-fixing bacteria. To understand symbiotic signaling from SYMRK, we determined the crystal structure to 1.95 Å and mapped the phosphorylation sites onto the intracellular domain. We identified four serine residues in a conserved "alpha-I" motif, located on the border between the kinase core domain and the flexible C-terminal tail, that, when phosphorylated, drives organogenesis. Substituting the four serines with alanines abolished symbiotic signaling, while substituting them with phosphorylation-mimicking aspartates induced the formation of spontaneous nodules in the absence of bacteria. These findings show that the signaling pathway controlling root nodule organogenesis is mediated by SYMRK phosphorylation, which may help when engineering this trait into non-legume plants. [ABSTRACT FROM AUTHOR]

Protein phosphorylation tightly regulates specific binding of effector proteins that control many diverse biological functions of cells (e. g. signaling, migration and proliferation). p140Cap is an adaptor protein, specifically expressed in brain, testis and epithelial cells, that undergoes phosphorylation and tunes its interactions with other regulatory molecules via post- translation modification. In this work, using mass spectrometry, we found that p140Cap is in vivo phosphorylated on tyrosine (Y) within the peptide GEGLpYADPYGLLHEGR (from now on referred to as EGLYA) as well as on three serine residues. Consistently, EGLYA has the highest score of in silico prediction of p140Cap phosphorylation. To further investigate the p140Cap function, we performed site specific mutagenesis on tyrosines inserted in EGLYA and EPLYA, a second sequence with the same highest score of phosphorylation. The mutant protein, in which both EPLYA/EGLYA tyrosines were converted to phenylalanine, was no longer tyrosine phosphorylated, despite the presence of other tyrosine residues in p140Cap sequence. Moreover, this mutant lost its ability to bind the C-terminal Src kinase (Csk), previously shown to interact with p140Cap by Far Western analysis. In addition, we found that in vitro and in HEK-293 cells, the Abelson kinase is the major kinase involved in p140Cap tyrosine phosphorylation on the EPLYA and EGLYA sequences. Overall, these data represent an original attempt to in vivo characterise phosphorylated residues of p140Cap. Elucidating the function of p140Cap will provide novel insights into its biological activity not only in normal cells, but also in tumors. [ABSTRACT FROM AUTHOR]

Human JAK2 tyrosine kinase mediates signaling through numerous cytokine receptors. The JAK2 JH2 domain functions as a negative regulator and is presumed to be a catalytically inactive pseudokinase, but the mechanism(s) for its inhibition of JAK2 remains unknown. Mutations in JH2 lead to increased JAK2 activity, contributing to myeloproliferative neoplasms (MPNs). Here we show that JH2 is a dual-specificity protein kinase that phosphorylates two negative regulatory sites in JAK2: Ser523 and Tyr570. Inactivation of JH2 catalytic activity increased JAK2 basal activity and downstream signaling. Notably, different MPN mutations abrogated JH2 activity in cells, and in MPN (V617F) patient cells phosphorylation of Tyr570 was reduced, suggesting that loss of JH2 activity contributes to the pathogenesis of MPNs. These results identify the catalytic activity of JH2 as a previously unrecognized mechanism to control basal activity and signaling of JAK2. [ABSTRACT FROM AUTHOR]

Soil-living rhizobia secrete lipochitin oligosaccharides known as Nod factors, which in Lotus japonicus are perceived by at least two Nod-factor receptors, NFR1 and NFR5. Despite progress in identifying molecular components critical for initial legume host recognition of the microsymbiont and cloning of downstream components, little is known about the activation and signalling mechanisms of the Nod-factor receptors themselves. Here we show that both receptor proteins localize to the plasma membrane, and present evidence for heterocomplex formation initiating downstream signalling. Expression of NFR1 and NFR5 in Nicotiana benthamiana and Allium ampeloprasum (leek) cells caused a rapid cell-death response. The signalling leading to cell death was abrogated using a kinase-inactive variant of NFR1. In these surviving cells, a clear interaction between NFR1 and NFR5 was detected in vivo through bimolecular fluorescence complementation (BiFC). To analyse the inter- and intramolecular phosphorylation events of the kinase complex, the cytoplasmic part of NFR1 was assayed for in vitro kinase activity, and autophosphorylation on 24 amino acid residues, including three tyrosine residues, was found by mass spectrometry. Substitution of the phosphorylated amino acids of NFR1 identified a single phosphorylation site to be essential for NFR1 Nod-factor signalling in vivo and kinase activity in vitro. In contrast to NFR1, no in vitro kinase activity of the cytoplasmic domain of NFR5 was detected. This is further supported by the fact that a mutagenized NFR5 construct, substituting an amino acid essential for ATP binding, restored nodulation of nfr5 mutant roots. [ABSTRACT FROM AUTHOR]

The molecular basis for the interaction of insulin granules with the cortical cytoskeleton of pancreatic β-cells remains unknown. We have proposed that binding of the granule protein ICA512 to the PDZ domain of β2-syntrophin anchors granules to actin filaments and that the phosphorylation/dephosphorylation of β2-syntrophin regulates this association. Here we tested this hypothesis by analyzing INS-1 cells expressing GFP-β2-syntrophin through the combined use of biochemical approaches, imaging studies by confocal and total internal reflection fluorescence microscopy as well as electron microscopy. Our results support the notion that β2-syntrophin restrains the mobility of cortical granules in insulinoma INS-1 cells, thereby reducing insulin secretion and increasing insulin stores in resting cells, while increasing insulin release upon stimulation. Using mass spectrometry, in vitro phosphorylation assays and β2-syntrophin phosphomutants we found that phosphorylation of β2-syntrophin on S75 near the PDZ domain decreases its binding to ICA512 and correlates with increased granule motility, while phosphorylation of S90 has opposite effects. We further show that Cdk5, which regulates insulin secretion, phosphorylates S75. These findings provide mechanistic insight into how stimulation displaces insulin granules from cortical actin, thus promoting their motility and exocytosis. [ABSTRACT FROM AUTHOR]

Abstract: Serine racemase (SR) catalyses the synthesis of the transmitter/neuromodulator d-serine, which plays a major role in synaptic plasticity and N-methyl d-aspartate receptor neurotoxicity. We now report that SR is phosphorylated at Thr71 and Thr227 as revealed by mass spectrometric analysis and in vivo phosphorylation assays. Thr71 phosphorylation was observed in the cytosolic and membrane-bound SR while Thr227 phosphorylation was restricted to the membrane fraction. The Thr71 site has a motif for proline-directed kinases and is the main phosphorylation site of SR. Experiments with a phosphorylation-deficient SR mutant indicate that Thr71 phosphorylation increases SR activity, suggesting a novel mechanism for regulating d-serine production. [Copyright &y& Elsevier]

Abstract: At yeast vacuoles, phosphorylation of the HOPS subunit Vps41 depends on the Yck3 kinase. In a screen for mutants that mimic the yck3Δ phenotype, in which Vps41 accumulates in vacuolar dots, we observed that mutants in the V0-part of the V0/V1-ATPase, in particular in vma16Δ, also accumulate Vps41. This accumulation is not due to a phosphorylation defect, but to reduced release of Vps41 from vma16Δ vacuoles. One reason could be a connection to vacuole fission, which is blocked in V-ATPase mutants. Vacuole fusion is not impaired between vacuoles lacking the V0-subunits Vma16 or Vma6 and wild-type vacuoles, whereas fusion between mutant vacuoles is reduced. Our data suggest a connection between vacuole biogenesis and membrane fusion. [Copyright &y& Elsevier]

Human UNG2 is a multifunctional glycosylase that removes uracil near replication forks and in non-replicating DNA, and is important for affinity maturation of antibodies in B cells. How these diverse functions are regulated remains obscure. Here, we report three new phosphoforms of the non-catalytic domain that confer distinct functional properties to UNG2. These are apparently generated by cyclin-dependent kinases through stepwise phosphorylation of S23, T60 and S64 in the cell cycle. Phosphorylation of S23 in late G1/early S confers increased association with replication protein A (RPA) and replicating chromatin and markedly increases the catalytic turnover of UNG2. Conversely, progressive phosphorylation of T60 and S64 throughout S phase mediates reduced binding to RPA and flag UNG2 for breakdown in G2 by forming a cyclin E/c-myc-like phosphodegron. The enhanced catalytic turnover of UNG2 p-S23 likely optimises the protein to excise uracil along with rapidly moving replication forks. Our findings may aid further studies of how UNG2 initiates mutagenic rather than repair processing of activation-induced deaminase-generated uracil at Ig loci in B cells. [ABSTRACT FROM AUTHOR]

The growth factor/insulin-stimulated AGC kinases share an activation mechanism based on three phosphorylation sites. Of these, only the role of the activation loop phosphate in the kinase domain and the hydrophobic motif (HM) phosphate in a C-terminal tail region are well characterized. We investigated the role of the third, so-called turn motif phosphate, also located in the tail, in the AGC kinases PKB, S6K, RSK, MSK, PRK and PKC. We report cooperative action of the HM phosphate and the turn motif phosphate, because it binds a phosphoSer/Thr-binding site above the glycine-rich loop within the kinase domain, promoting zipper-like association of the tail with the kinase domain, serving to stabilize the HM in its kinase-activating binding site. We present a molecular model for allosteric activation of AGC kinases by the turn motif phosphate via HM-mediated stabilization of the αC helix. In S6K and MSK, the turn motif phosphate thereby also protects the HM from dephosphorylation. Our results suggest that the mechanism described is a key feature in activation of upto 26 human AGC kinases. [ABSTRACT FROM AUTHOR]

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling by insulin receptor and several G-protein-coupled receptors. However, there is only limited knowledge of the physical structure of JAK2 or which of the 49 tyrosines in JAK2 are autophosphorylated. In this study, mass spectrometry and two-dimensional peptide mapping were used to determine that tyrosines 221, 570, and 1007 in JAK2 are autophosphorylated. Phosphorylation of tyrosine 570 is particularly robust. In response to growth hormone, JAK2 was rapidly and transiently phosphorylated at tyrosines 221 and 570, returning to basal levels by 60 min. Analysis of the sequences surrounding tyrosines 221 and 570 in JAK2 and tyrosines in other proteins that are phosphorylated in response to ligands that activate JAK2 suggests that the YXX[L/I/V] motif is one of the motifs recognized by JAK2. Experiments using JAK2 with tyrosines 221 and 570 mutated to phenylalanine suggest that tyrosines 221 and 570 in JAK2 may serve as regulatory sites in JAK2, with phosphorylation of tyrosine 221 increasing kinase activity and phosphorylation of tyrosine 570 decreasing kinase activity and thereby contributing to rapid termination of ligand activation of JAK2. [ABSTRACT FROM AUTHOR]

Analyzes the phosphorylation and oligomerization state of native pig brain HSP90. Performance of the mass spectrometry technique; Determination of the relative abundance of the isoforms in solution; Description of heat shock proteins.

The article presents the author's comments on the research conducted by S. A. Beausoleil and colleagues on improved computational techniques that facilitate for automated phosphorylation site assignment in large-scale proteomic experiments. Protein phosphorylation is arguably the most important post-translational modification as it plays the most decisive roles during cellular development. In his view, their methods promise to alleviate an important bottleneck in phosphoproteomics.

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling for multiple receptor tyrosine kinases and several G-protein-coupled receptors. In this study, phosphopeptide affinity enrichment and mass spectrometry identified serine 523 (Ser523) in JAK2 as a site of phosphorylation. A phosphoserine 523 antibody revealed that Ser523 is rapidly but transiently phosphorylated in response to growth hormone (GH). MEK1 inhibitor UO126 suppresses GH-dependent phosphorylation of Ser523, suggesting that extracellular signal-regulated kinases (ERKs) 1 and/or 2 or another kinase downstream of MEK1 phosphorylate Ser523 in response to GH. Other ERK activators, phorbol 12-myristate 13-acetate and epidermal growth factor, also stimulate phosphorylation of Ser523. When Ser523 in JAK2 was mutated, JAK2 kinase activity as well as GH-dependent tyrosyl phosphorylation of JAK2 and Stat5 was enhanced, suggesting that phosphorylation of Ser523 inhibits JAK2 kinase activity. We hypothesize that phosphorylation of Ser523 in JAK2 by ERKs 1 and/or 2 or other as-yet-unidentified kinases acts in a negative feedback manner to dampen activation of JAK2 in response to GH and provides a mechanism by which prior exposure to environmental factors that regulate Ser523 phosphorylation might modulate the cell's response to GH. [ABSTRACT FROM AUTHOR]

Reversible protein phosphorylation plays a critical role in cell signaling and is responsible for the regulation of many biological processes in most living organisms. The low stoichiometry of protein phosphorylation requires sensitive analysis by tandem mass spectrometry. However, incomplete peptide fragmentation and the loss of labile phosphate groups complicate identification of the site of the phosphate motif. Here, we have implemented and evaluated a novel approach for phospho-site localization by the combined use of peptide tandem mass spectrometry data obtained using both collision-activated dissociation and electron transfer dissociation, an approach termed the Cscore. The scoring algorithm used in the Cscore was adapted from the widely used Ascore method. The analytical benefit of integrating the product ion information of both ETD and CAD data are evident by increased confidence in phospho-site localization and the number of assigned phospho-sites at a fixed false-localization rate. The average calculated Cscore from a large data set (>7000 phosphopeptide MS/MS spectra) was ~32 compared to ~23 and ~17 for the Ascore using collision-activated dissociation (CAD) or electron transfer dissociation (ETD), respectively. Compared with the Ascore using either CAD or ETD, the Cscore identified up to 88% more phosphorylation sites. Using a phosphopeptide library revealed that the score threshold for obtaining a false-localization rate of 0.5% was lower for the Cscore than either the Ascore (CAD) or the Ascore (ETD). [ABSTRACT FROM AUTHOR]

Phosphorylation is an important posttranslational modification of proteins in living cells and primarily serves regulatory purposes. Several methods were employed for isolating phosphopeptides from proteolytically digested plasma membranes of Arabidopsis thaliana. After a mass spectrometric analysis of the resulting peptides we could identify 10 different phosphorylation sites in plasma membrane H+-ATPases AHA1, AHA2, AHA3, and AHA4/11, five of which have not been reported before, bringing the total number of phosphosites up to 11, which is substantially higher than reported so far for any other P-type ATPase. Phosphosites were almost exclusively (9 of 10) in the terminal regulatory domains of the pumps. The AHA2 isoform was subsequently expressed in the yeast Saccharomyces cerevisiae. The plant protein was phosphorylated at multiple sites in yeast, and surprisingly, seven of nine of the phosphosites identified in AHA2 were identical in the plant and fungal systems even though none of the target sequences in AHA2 show homology to proteins of the fungal host. These findings suggest an unexpected accessibility of the terminal regulatory domain of plasma membrane H+-ATPase to protein kinase action. [ABSTRACT FROM AUTHOR]

Eisosomes are recently described fungal structures that play roles in the organization of the plasma membrane and endocytosis. Their major protein components are Pil1 and Lsp1, and previous studies showed that these proteins are phosphorylated by the sphingolipid long-chain base-activated Pkh1 and Pkh2 protein kinases in vitro. We show that Pkh1 and Pkh2 phosphorylate Pil1 and Lsp1 in vivo to produce species B, and that heat stress, which activates Pkh1 and Pkh2, generates a more highly phosphorylated species, C. Cells with low Pkh activity lack species B and C and contain abnormally organized eisosomes. To verify that Pil1 phosphorylation is essential for correct eisosome organization, phosphorylated serine and threonine residues were identified and changed to alanines, A variant Pil1 protein lacking five phosphorylation sites did not form eisosomes during log phase growth, indicating that phosphorylation is critical for eisosome organization. We also found that eisosomes are dynamic structures and disassemble when the Ypk protein kinases, which are activated by the sphingolipid-Pkh signaling pathway, are inactivated or when the sphingolipid signal is pharmacologically blocked with myriocin. We conclude that eisosome formation and turnover are regulated by the sphingolipid-Pkh1/2-Ypk1/2 signaling pathway. These data and previous data showing that endocytosis is regulated by the sphingolipid-Pkh1/2-Ypk1/2 signaling pathway suggest that Pkh1 and -2 respond to changes in membrane sphingolipids and transmit this information to eisosomes via Pil1 phosphorylation. Eisosomes then control endocytosis to align the composition and function of the plasma membrane to match demand. [ABSTRACT FROM AUTHOR]

Phosphorylation of proteins is essential in intracellular signal transduction pathways in eukaryotic and prokaryotic cells. Histidine phosphorylation plays an important role in two-component signal transduction in bacteria. In this study, we describe the characterization of a synthetic histidine-phosphorylated peptide with four different mass spectrometric (MS) fragmentation techniques: Collision-induced dissociation (CID), electron capture dissociation, electron-transfer dissociation, and electron detachment dissociation. Furthermore, LC-MS methods were developed to detect histidine-phosphorylated peptides, which are acid-labile, in more complex samples. From these results, we concluded that nonacidic solvent systems or fast LC methods provide the best conditions for separation of histidine-phosphorylated peptides prior to electrospray ionization mass spectrometry analysis. Electron-based fragmentation methods should be used for determination of histidine phosphorylation sites, since CID results in very facile phosphate-related neutral losses. The developed LC—MS/MS methods were successfully applied to a tryptic digest of the cytoplasmic part of the histidine kinase EnvZ, which was in vitro autophosphorylated. Finally, a new method is described for nonretentive solid-phase extraction of histidine-phosphorylated peptides using polymeric Strata-X microcolumns. [ABSTRACT FROM AUTHOR]

Nucleoplasmin (NP) is a histone chaperone involved in nucleosome assembly, chromatin decondensation at fertilization, and apoptosis. To carry out these activities NP has to interact with different types of histones, an interaction that is regulated by phosphorylation. Here we have identified a number of phosphorylated residues by mass spectrometry and generated mutants in which these amino acids are replaced by Asp to mimic the effect of phosphorylation. Our results show that, among the eight phosphoryl groups experimentally detected, four are located at the flexible N terminus, and the rest are found at the tail domain, flanking the nuclear localization signal. Phosphorylation-mimicking mutations render a recombinant protein as active in chromatin decondensation as hyperphosphorylated NP isolated from Xenopus laevis eggs. Comparison of mutants in which the core and tail domains of the protein were independently or simultaneously "activated" indicates that activation or phosphorylation of both protein domains is required for NP to efficiently extract linker-type histones from chromatin. [ABSTRACT FROM AUTHOR]

Neutrophil NADPH oxidase plays a key role in host defense and in inflammation by releasing large amounts of superoxide and other ROSs. Proinflammatory cytokines such as GM-CSF and TNF-α prime ROS production by neutrophils through unknown mechanisms. Here we used peptide sequencing by tandem mass spectrometry to show that GM-CSF and TNF-α induce phosphorylation of Ser345 on p47phox, a cytosolic component of NADPH oxidase, in human neutrophils. As Ser345 is located in the MAPK consensus sequence, we tested the effects of MAPK inhibitors. Inhibitors of the ERK1/2 pathway abrogated GM-CSF-induced phosphorylation of Ser345, while p38 MAPK inhibitor abrogated TNF-α-induced phosphorylation of Ser345. Transfection of HL 60 cells with a mutated p47phox (S345A) inhibited GM-CSF- and TNF-α-induced priming of ROS production. This event was also inhibited in neutrophils by a cell-permeable peptide containing a TAT-p47phox-Ser345 sequence. Furthermore, ROS generation, p47phox-Ser345 phosphorylation, and ERK1/2 and p38 MAPK phosphorylation were increased in synovial neutrophils from rheumatoid arthritis (RA) patients, and TAT Ser345 peptide inhibited ROS production by these primed neutrophils. This study therefore identifies convergent MAPK pathways on Ser345 that are involved in GM-CSF- and TNF-a-induced priming of neutrophils and are activated in RA. Inhibition of the point of convergence of these pathways might serve as a novel antiinflammatory strategy. [ABSTRACT FROM AUTHOR]

Abstract: Previous studies suggest that dephosphorylation of connexin43 (Cx43) is related to uncoupling of gap junction communication, which plays an important role in the genesis of ischemia-induced ventricular tachycardia. We studied changes in Cx43 phosphorylation during global ischemia in the absence and presence of the antiarrhythmic peptide analogue rotigaptide (formerly known as ZP123). Phosphorylation analysis was performed on Cx43 purified from isolated perfused rat hearts using matrix-assisted laser desorption/ionization mass spectrometry and liquid chromatography electrospray ionization tandem mass spectrometry. Thirteen different serine phosphorylation sites were identified in Cx43 during non-ischemic conditions, three of which had not previously been described. Within the first 7 min of ischemia, Ser306 became fully dephosphorylated whereas Ser330 became phosphorylated. Between 15 and 30 min of ischemia, the critical time interval where gap junction uncoupling occurs, Ser297 and Ser368 also became fully dephosphorylated. During the same time period, all untreated hearts developed asystole. Treatment with rotigaptide significantly increased the time to ischemia-induced asystole and suppressed dephosphorylation of Ser297 and Ser368 at 30 min of ischemia. Our results suggest that phosphorylation of Ser297 and Ser368 may be involved in functional gating of Cx43 during ischemia and may be possible downstream targets for rotigaptide signaling. [Copyright &y& Elsevier]

Focuses on the detection of phosphorylated proteins after two-dimensional gel electrophoretic separation of the matrix fraction isolated from potato tuber mitochondria. Identification of the phosphoproteins by mass spectrometry; Measurement of the extent of phosphorylation; Increase of formate dehydrogenase activity at low oxygen concentrations inside the potato tubers.

In signal transduction in eukaryotes, protein phosphorylation is a key event. To understand signaling processes, we must first acquire an inventory of phosphoproteins and their phosphorylation sites under different conditions. Because phosphorylation is a dynamic process, elucidation of signaling networks also requires quantitation of these phosphorylation events. In this article, we outline several methods for enrichment of phosphorylated proteins and peptides and discuss various options for their identification and quantitation with special emphasis on mass spectrometry-based techniques. [Copyright &y& Elsevier]