CiteULike: jyuh's phosphorylation

KinasePhos: a web tool for identifying protein kinase-specific phosphorylation sites.

HD Huang — 2008-02-18T10:41:09-00:00

Nucleic Acids Res, Vol. 33, No. Web Server issue. (1 July 2005)

KinasePhos is a novel web server for computationally identifying catalytic kinase-specific phosphorylation sites. The known phosphorylation sites from public domain data sources are categorized by their annotated protein kinases. Based on the profile hidden Markov model, computational models are learned from the kinase-specific groups of the phosphorylation sites. After evaluating the learned models, the model with highest accuracy was selected from each kinase-specific group, for use in a web-based prediction tool for identifying protein phosphorylation sites. Therefore, this work developed a kinase-specific phosphorylation site prediction tool with both high sensitivity and specificity. The prediction tool is freely available at http://KinasePhos.mbc.nctu.edu.tw/.

Protein kinases associated with the yeast phosphoproteome.

RI Brinkworth — 2006-11-15T10:20:01-00:00

BMC Bioinformatics, Vol. 7 (2006)

BACKGROUND: Protein phosphorylation is an extremely important mechanism of cellular regulation. A large-scale study of phosphoproteins in a whole-cell lysate of Saccharomyces cerevisiae has previously identified 383 phosphorylation sites in 216 peptide sequences. However, the protein kinases responsible for the phosphorylation of the identified proteins have not previously been assigned. RESULTS: We used Predikin in combination with other bioinformatic tools, to predict which of 116 unique protein kinases in yeast phosphorylates each experimentally determined site in the phosphoproteome. The prediction was based on the match between the phosphorylated 7-residue sequence and the predicted substrate specificity of each kinase, with the highest weight applied to the residues or positions that contribute most to the substrate specificity. We estimated the reliability of the predictions by performing a parallel prediction on phosphopeptides for which the kinase has been experimentally determined. CONCLUSION: The results reveal that the functions of the protein kinases and their predicted phosphoprotein substrates are often correlated, for example in endocytosis, cytokinesis, transcription, replication, carbohydrate metabolism and stress response. The predictions link phosphoproteins of unknown function with protein kinases with known functions and vice versa, suggesting functions for the uncharacterized proteins. The study indicates that the phosphoproteins and the associated protein kinases represented in our dataset have housekeeping cellular roles; certain kinases are not represented because they may only be activated during specific cellular responses. Our results demonstrate the utility of our previously reported protein kinase substrate prediction approach (Predikin) as a tool for establishing links between kinases and phosphoproteins that can subsequently be tested experimentally.

Linking the kinome and phosphorylome--a comprehensive review of approaches to find kinase targets.

R Sopko — 2008-08-18T09:56:08-00:00

Molecular bioSystems, Vol. 4, No. 9. (September 2008), pp. 920-933.

Protein phosphorylation is associated with most cell signaling and developmental processes in eukaryotes. Despite the vast extent of the phosphoproteome within the cell, connecting specific kinases with relevant targets remains a significant experimental frontier. The challenge of linking kinases and their substrates reflects the complexity of kinase function. For example, kinases tend to exert their biological effects through supernumerary, redundant phosphorylation, often on multiple protein complex components. Although these types of phosphorylation events are biologically significant, those kinases responsible are often difficult to identify. Recent methods for global analysis of protein phosphorylation promise to substantially accelerate efforts to map the dynamic phosphorylome. Here, we review both conventional methods to identify kinase targets and more comprehensive genomic and proteomic approaches to connect the kinome and phosphorylome.

GPS 2.0: Prediction of kinase-specific phosphorylation sites in hierarchy.

Yu Xue — 2008-08-14T13:32:23-00:00

Molecular & cellular proteomics : MCP (6 May 2008)

Identification of protein phosphorylation sites with their cognate protein kinases (PKs) is a key step to delineate molecular dynamics and plasticity underlying a variety of cellular processes. Although nearly 10 kinase-specific prediction programs were developed, numerous PKs were casually classified into sub-groups without a standard rule. For large-scale predictions, the false positive rate (FPR) was also never addressed. In this work, we adopted a well-established rule to classify PKs into a hierarchical structure with four levels, including group, family, subfamily and single PK. In addition, we developed a simple approach to estimate the theoretically maximal FPRs. The online service and local packages of the GPS (Group-based Prediction System) 2.0 were implemented in JAVA, with the modified version of Group-based Phosphorylation Scoring algorithm. As the first stand-alone software for predicting phosphorylation, GPS2.0 can predict kinase-specific phosphorylation sites for 408 human PKs in hierarchy. A large-scale prediction of more than 13,000 mammalian phosphorylation sites by GPS2.0 was exhibited with great performance and remarkable accuracy. Using Aurora B as an example, we also conducted a proteome-wide search and provided systemic prediction of Aurora-B specific substrates including protein-protein interaction information. Thus, the GPS 2.0 is a useful tool for predicting protein phosphorylation sites and their cognate kinases, which is freely available at: http://bioinformatics.lcd-ustc.org/gps2.

Profiling Protein Tyrosine Phosphorylation : A Quantitative 45-Plex Peptide-Based Immunoassay.

Timothy K Nadler — 2008-08-14T14:29:55-00:00

Journal of biomolecular screening : the official journal of the Society for Biomolecular Screening (14 July 2008)

Cellular homeostasis and responses to stimuli are mediated by complex signaling network events dominated by changes in protein phosphorylation states. Understanding information flow in the network is essential for correlating signaling changes to cell physiology. Tyrosine phosphorylation constitutes only a small portion of all protein phosphorylation, but its importance is manifested by the significant role it plays in diseases such as cancer. A peptide-based immunoassay microarray, designed to provide site specificity, quantification, broad coverage, and accessibility, is described that profiles 45 tyrosine phosphorylation sites across 34 proteins. Epidermal growth factor-stimulated A431 cells in the absence and presence of kinase inhibitors analyzed by microarrays showed biologically validated tyrosine phosphorylation changes and unanticipated activation of other targets. The approach is scalable for increasing the breadth of content as well as for interrogating other types of protein posttranslational modifications. (Journal of Biomolecular Screening 200X:000-000).

The evolution of tools for protein phosphorylation site analysis: from discovery to clinical application.

L Bonilla — 2008-08-14T13:50:59-00:00

BioTechniques, Vol. 44, No. 5. (April 2008), pp. 671-679.

The importance of the analysis of signaling pathways has been proven for many years by the elucidation of key signaling molecules. However, in most cases these pathways tend to represent a rather narrow view of the biological state under investigation. Clearly a more detailed understanding of the complexities of cross-talk between signaling pathways is required to further our knowledge of normal and disease processes. The tools that provide the framework for this increased understanding of biology, those that enable identification, characterization, and quantitation of sites of phosphorylation in proteins, have advanced over the past 25 years. This review will present a brief overview of the history of the tools used in phosphorylation analysis and the latest technologies that are being applied in this field, such as mass spectrometry (for broad-based discovery efforts) and flow cytometry (for translation to clinical applications).

Quantitative phosphoproteomic analysis of signaling network dynamics.

Forest M White — 2008-08-14T13:50:51-00:00

Current opinion in biotechnology (23 July 2008)

Protein phosphorylation mediated cellular signaling is a highly regulated, dynamic process that controls many aspects of cellular biology. Over the past few years many methods have been developed to quantify temporal dynamics of protein phosphorylation, including mass spectrometry, which can be applied in both an unbiased, discovery mode and in a targeted mode to monitor specific phosphorylation sites. Other methods, such as kinase activity assays and antibody microarrays, have been applied to quantify central nodes in the signaling network, yielding intriguing biological insights. This review provides a concise overview of the latest advances in the quantitative analysis of signaling dynamics including a brief commentary on the future of the field.

Motif-Specific Sampling of Phosphoproteomes

Cristian Ruse — 2008-05-04T18:43:21-00:00

J. Proteome Res., Vol. 7, No. 5. (2 May 2008), pp. 2140-2150.

Abstract: Phosphoproteomics, the targeted study of a subfraction of the proteome which is modified by phosphorylation, has become an indispensable tool to study cell signaling dynamics. We described a methodology that linked phosphoproteome and proteome analysis based on Ba2+ binding properties of amino acids. This technology selected motif-specific phosphopeptides independent of the system under analysis. MudPIT (Multidimensional Identification Technology) identified 1037 precipitated phosphopeptides from as little as 250 µg of proteins. To extend coverage of the phosphoproteome, we sampled the nuclear extract of HeLa cells with three values of Ba2+ ions molarity. The presence of more than 70% of identified phosphoproteins was further substantiated by their nonmodified peptides. Upon isoproterenol stimulation of HEK cells, we identified an increasing number of phosphoproteins from MAPK cascades and AKAP signaling hubs. We quantified changes in both protein and phosphorylation levels of 197 phosphoproteins including a critical kinase, MAPK1. Integration of differential phosphorylation of MAPK1 with knowledge bases constructed modules that correlated well with its role as node in cross-talk of canonical pathways.

A multidimensional chromatography technology for in-depth phosphoproteome analysis.

Claudio P Albuquerque — 2008-06-22T21:22:40-00:00

Molecular & cellular proteomics : MCP (11 April 2008)

Protein phosphorylation is a post-translational modification widely used to regulate cellular responses. Recent studies showed that global phosphorylation analysis could be used to study signaling pathways and to identify targets of protein kinases in cells. A key objective of global phosphorylation analysis is to obtain an in-depth mapping of low abundance protein phosphorylation in cells, which necessitates the use of suitable separation techniques due to the complexity of the phosphoproteome. Here we developed a multidimensional chromatography technology, combining Immobilized Metal Affinity Chromatography (IMAC), Hydrophilic Interaction Chromatography (HILIC) and Reverse-Phase Liquid Chromatography (RP-LC), for phosphopeptide purification and fractionation. Its application to the yeast S. cerevisiae after DNA damage led to the identification of 8,764 unique phosphopeptides from 2,278 phosphoproteins using tandem mass spectrometry (MS). Analysis of two low abundance proteins, Rad9 and Mrc1, revealed that approximately 50 % of their phosphorylation was identified via this global phosphorylation analysis. Thus, this technology is suited for in-depth phosphoproteome studies.

Computational identification of ubiquitylation sites from protein sequences

Chun-Wei Tung — 2008-07-15T14:52:42-00:00

BMC Bioinformatics, Vol. 9 (15 July 2008), 310.

KinasePhos 2.0: a web server for identifying protein kinase-specific phosphorylation sites based on sequences and coupling patterns.

YH Wong — 2008-04-16T03:06:56-00:00

Nucleic acids research, Vol. 35, No. Web Server issue. (July 2007)

Due to the importance of protein phosphorylation in cellular control, many researches are undertaken to predict the kinase-specific phosphorylation sites. Referred to our previous work, KinasePhos 1.0, incorporated profile hidden Markov model (HMM) with flanking residues of the kinase-specific phosphorylation sites. Herein, a new web server, KinasePhos 2.0, incorporates support vector machines (SVM) with the protein sequence profile and protein coupling pattern, which is a novel feature used for identifying phosphorylation sites. The coupling pattern [XdZ] denotes the amino acid coupling-pattern of amino acid types X and Z that are separated by d amino acids. The differences or quotients of coupling strength C(XdZ) between the positive set of phosphorylation sites and the background set of whole protein sequences from Swiss-Prot are computed to determine the number of coupling patterns for training SVM models. After the evaluation based on k-fold cross-validation and Jackknife cross-validation, the average predictive accuracy of phosphorylated serine, threonine, tyrosine and histidine are 90, 93, 88 and 93%, respectively. KinasePhos 2.0 performs better than other tools previously developed. The proposed web server is freely available at http://KinasePhos2.mbc.nctu.edu.tw/.

Prediction of kinase-specific phosphorylation sites with sequence features by a log-odds ratio approach.

T Li — 2008-08-14T13:16:46-00:00

Proteins, Vol. 70, No. 2. (1 February 2008), pp. 404-414.

Protein phosphorylation plays important roles in a variety of cellular processes. Detecting possible phosphorylation sites and their corresponding protein kinases is crucial for studying the function of many proteins. This article presents a new prediction system, called PhoScan, to predict phosphorylation sites in a kinase-family-specific way. Common phosphorylation features and kinase-specific features are extracted from substrate sequences of different protein kinases based on the analysis of published experiments, and a scoring system is developed for evaluating the possibility that a peptide can be phosphorylated by the protein kinase at the specific site in its sequence context. PhoScan can achieve a specificity of above 90% with sensitivity around 90% at kinase-family level on the data experimented. The system is applied on a set of human proteins collected from Swiss-Prot and sets of putative phosphorylation sites are predicted for protein kinase A, cyclin-dependent kinase, and casein kinase 2 families. PhoScan is available at http://bioinfo.au.tsinghua.edu.cn/phoscan/.

NetworKIN: a resource for exploring cellular phosphorylation networks.

R Linding — 2008-03-24T13:27:42-00:00

Nucleic Acids Res, Vol. 36, No. Database issue. (January 2008)

Protein kinases control cellular responses by phosphorylating specific substrates. Recent proteome-wide mapping of protein phosphorylation sites by mass spectrometry has discovered thousands of in vivo sites. Systematically assigning all 518 human kinases to all these sites is a challenging problem. The NetworKIN database (http://networkin.info) integrates consensus substrate motifs with context modelling for improved prediction of cellular kinase-substrate relations. Based on the latest human phosphoproteome from the Phospho.ELM and PhosphoSite databases, the resource offers insight into phosphorylation-modulated interaction networks. Here, we describe how NetworKIN can be used for both global and targeted molecular studies. Via the web interface users can query the database of precomputed kinase-substrate relations or obtain predictions on novel phosphoproteins. The database currently contains a predicted phosphorylation network with 20,224 site-specific interactions involving 3978 phosphoproteins and 73 human kinases from 20 families.

Predikin and PredikinDB: a computational framework for the prediction of protein kinase peptide specificity and an associated database of phosphorylation sites

Neil Saunders — 2008-05-26T16:41:40-00:00

BMC Bioinformatics, Vol. 9 (26 May 2008), 245.

PhosphoPOINT: a comprehensive human kinase interactome and phospho-protein database.

CY Yang — 2008-08-14T12:47:34-00:00

Bioinformatics (Oxford, England), Vol. 24, No. 16. (15 August 2008)

MOTIVATION: To fully understand how a protein kinase regulates biological processes, it is imperative to first identify its substrate(s) and interacting protein(s). However, of the 518 known human serine/threonine/tyrosine kinases, 35% of these have known substrates, while 14% of the kinases have identified substrate recognition motifs. In contrast, 85% of the kinases have protein-protein interaction (PPI) datasets, raising the possibility that we might reveal potential kinase-substrate pairs from these PPIs. RESULTS: PhosphoPOINT, a comprehensive human kinase interactome and phospho-protein database, is a collection of 4195 phospho-proteins with a total of 15 738 phosphorylation sites. PhosphoPOINT annotates the interactions among kinases, with their down-stream substrates and with interacting (phospho)-proteins to modulate the kinase-substrate pairs. PhosphoPOINT implements various gene expression profiles and Gene Ontology cellular component information to evaluate each kinase and their interacting (phospho)-proteins/substrates. Integration of cSNPs that cause amino acids change with the proteins with the phosphoprotein dataset reveals that 64 phosphorylation sites result in a disease phenotypes when changed; the linked phenotypes include schizophrenia and hypertension. PhosphoPOINT also provides a search function for all phospho-peptides using about 300 known kinase/phosphatase substrate/binding motifs. Altogether, PhosphoPOINT provides robust annotation for kinases, their downstream substrates and their interaction (phospho)-proteins and this should accelerate the functional characterization of kinomemediated signaling. AVAILABILITY: PhosphoPOINT can be freely accessed in http://kinase. bioinformatics.tw/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Phospho3D: a database of three-dimensional structures of protein phosphorylation sites.

A Zanzoni — 2008-08-14T12:45:49-00:00

Nucleic acids research, Vol. 35, No. Database issue. (January 2007)

Phosphorylation is the most common protein post-translational modification. Phosphorylated residues (serine, threonine and tyrosine) play critical roles in the regulation of many cellular processes. Since the amount of data produced by screening assays is growing continuously, the development of computational tools for collecting and analysing experimental data has become a pivotal task for unravelling the complex network of interactions regulating eukaryotic cell life. Here we present Phospho3D, http://cbm.bio.uniroma2.it/phospho3d, a database of 3D structures of phosphorylation sites, which stores information retrieved from the phospho.ELM database and is enriched with structural information and annotations at the residue level. The database also collects the results of a large-scale structural comparison procedure providing clues for the identification of new putative phosphorylation sites.

Analysis of Transient Phosphorylation-Dependent Protein-Protein Interactions in Living Mammalian Cells Using Split-TEV

Michael Wehr — 2008-07-14T02:50:50-00:00

BMC Biotechnology, Vol. 8 (13 July 2008), 55.

Recent developments in mass spectrometry-based quantitative phosphoproteomics.

JC Smith — 2008-07-03T06:35:31-00:00

Biochemistry and cell biology = Biochimie et biologie cellulaire, Vol. 86, No. 2. (April 2008), pp. 137-148.

Protein phosphorylation is a reversible post-translational modification that is involved in virtually all eukaryotic cellular processes and has been studied in great detail in recent years. Many developments in mass spectrometry (MS)-based proteomics have been successfully applied to study protein phosphorylation in highly complicated samples. Furthermore, the emergence of a variety of enrichment strategies has allowed some of the challenges associated with low phosphorylation stoichiometry and phosphopeptide copy number to be overcome. The dynamic nature of protein phosphorylation complicates its analysis; however, a number of methods have been developed to successfully quantitate phosphorylation changes in a variety of cellular systems. The following review details some of the most recent breakthroughs in the study of protein phosphorylation, or phosphoproteomics, using MS-based approaches. The majority of the focus is placed on detailing strategies that are currently used to conduct MS-based quantitative phosphoproteomics.

Integrated analytical strategies for the study of phosphorylation and glycosylation in proteins.

C Temporini — 2008-05-20T04:08:10-00:00

Mass spectrometry reviews, Vol. 27, No. 3. (n 2008), pp. 207-236.

The post-translational modification (PTM) of proteins is a common biological mechanism for regulating protein localization, function, and turnover. The direct analysis of modifications is required because they are not coded by genes, and thus are not predictable. Different MS-based proteomic strategies are used for the analysis of PTMs, such as phosphorylation and glycosylation, and are composed of a structural simplification step of the protein followed by specific isolation step to extract the classes of modified peptides (also called "sub-proteomes") before mass spectrometry. This specific isolation step is necessary because PTMs occur at a sub-stoichiometric level and signal suppression of the modified fractions in the mass spectrometer occurs in the presence of the more-abundant non-modified counterpart. The request of innovative analytical strategies in PTM studies is the capability to localize the modification sites, give detailed structural information on the modification, and determine the isoform composition with increased selectivity, sensitivity, and throughput. This review focuses on the description of recent integrated analytical systems proposed for the analysis of PTMs in proteins, and their application to profile the glycoproteome and the phosphoproteome in biological samples. Comments on the difficulties and usefulness of the analytical strategies are given.

Advances in the Analysis of Protein Phosphorylation

Alberto Paradela — 2008-03-10T02:26:57-00:00

J. Proteome Res. (8 March 2008)

Abstract: Phosphorylation is one of the most relevant and ubiquitous post-translational modifications. Despite its relevance, the analysis of protein phosphorylation has been revealed as one of the most challenging tasks due to its highly dynamic nature and low stoichiometry. However, the development and introduction of new analytical methods are modifying rapidly and substantially this field. Especially important has been the introduction of more sensitive and specific methods for phosphoprotein and phosphopeptide purification as well as the use of more sensitive and accurate MS-based analytical methods. The integration of both approaches has enabled large-scale phosphoproteome studies to be performed, an unimaginable task few years ago. Additionally, methods originally developed for differential proteomics have been adapted making the study of the highly dynamic nature of protein phosphorylation feasible. This review aims at offering an overview on the most frequently used methods in phosphoprotein and phosphopeptide enrichment as well as on the most recent MS-based analysis strategies. Current strategies for quantitative phosphoproteomics and the study of the dynamics of protein phosphorylation are highlighted.

Ubiquitination, phosphorylation, and acetylation--triple threat in muscle wasting.

PO Hasselgren — 2008-02-27T05:15:53-00:00

J Cell Physiol, Vol. 213, No. 3. (December 2007), pp. 679-689.

Loss of muscle mass is commonly seen in patients with critical illness and is associated with increased expression of multiple genes controlling protein breakdown. Transcription factors that are activated during muscle wasting include NF-kB and members of the FOXO and C/EBP transcription factor families. The activity of these transcription factors is regulated by multiple posttranslational modifications, including ubiquitination, phosphorylation, and acetylation, providing for a complex and integrated network of regulatory mechanisms in muscle wasting. Targeting posttranslational modifications of transcription factors may prove important in the prevention and treatment of the debilitating consequences of muscle wasting.

Quantitative proteomic analysis of protein complexes: concurrent identification of interactors and their state of phosphorylation.

D Pflieger — 2008-02-11T09:54:50-00:00

Mol Cell Proteomics, Vol. 7, No. 2. (February 2008), pp. 326-346.

Protein complexes have largely been studied by immunoaffinity purification and (mass spectrometric) analysis. Although this approach has been widely and successfully used it is limited because it has difficulties reliably discriminating true from false protein complex components, identifying post-translational modifications, and detecting quantitative changes in complex composition or state of modification of complex components. We have developed a protocol that enables us to determine, in a single LC-MALDI-TOF/TOF analysis, the true protein constituents of a complex, to detect changes in the complex composition, and to localize phosphorylation sites and estimate their respective stoichiometry. The method is based on the combination of fourplex iTRAQ (isobaric tags for relative and absolute quantification) isobaric labeling and protein phosphatase treatment of substrates. It was evaluated on model peptides and proteins and on the complex Ccl1-Kin28-Tfb3 isolated by tandem affinity purification from yeast cells. The two known phosphosites in Kin28 and Tfb3 could be reproducibly shown to be fully modified. The protocol was then applied to the analysis of samples immunopurified from Drosophila melanogaster cells expressing an epitope-tagged form of the insulin receptor substrate homologue Chico. These experiments allowed us to identify 14-3-3epsilon, 14-3-3zeta, and the insulin receptor as specific Chico interactors. In a further experiment, we compared the immunopurified materials obtained from tagged Chico-expressing cells that were either treated with insulin or left unstimulated. This analysis showed that hormone stimulation increases the association of 14-3-3 proteins with Chico and modulates several phosphorylation sites of the bait, some of which are located within predicted recognition motives of 14-3-3 proteins.

Mechanisms of specificity in protein phosphorylation

Jeffrey Ubersax — 2007-06-23T21:02:26-00:00

Nature Reviews Molecular Cell Biology, Vol. 8, No. 7., pp. 530-541.

Processive phosphorylation: Mechanism and biological importance

Parag Patwardhan — 2008-01-12T03:15:00-00:00

Cellular Signalling, Vol. 19, No. 11. (November 2007), pp. 2218-2226.

Recent proteomic data indicate that a majority of the phosphorylated proteins in a eucaryotic cell contain multiple sites of phosphorylation. In many signaling events, a single kinase phosphorylates multiple sites on a target protein. Processive phosphorylation occurs when a protein kinase binds once to a substrate and phosphorylates all of the available sites before dissociating. In this review, we discuss examples of processive phosphorylation by serine/threonine kinases and tyrosine kinases. We describe current experimental approaches for distinguishing processive from non-processive phosphorylation. Finally, we contrast the biological situations that are suited to regulation by processive and non-processive phosphorylation.

PhosphoBlast, a Computational Tool for Comparing Phosphoprotein Signatures among Large Datasets.

Y Wang — 2008-01-08T03:55:54-00:00

Mol Cell Proteomics, Vol. 7, No. 1. (January 2008), pp. 145-162.

Identification of specific protein phosphorylation sites provides predicative signatures of cellular activity and specific disease states such as cancer, diabetes, Alzheimer disease, and rheumatoid arthritis. Recent progress in phosphopeptide isolation technology and tandem mass spectrometry has provided the means to identify thousands of phosphorylation sites from a single biological sample. These advances now make it possible to profile global changes in the phosphoproteome at an unprecedented level. However, although this technology is generating a wealth of information, there is currently no efficient means to identify phosphoprotein signatures shared among large phosphoprotein databases. Identification of common phosphoprotein signatures found in biologically relevant systems and their conservation throughout evolution would provide valuable insight into mechanisms of signal transduction and cell function. Here we describe the development of a computational program (PhosphoBlast) that can rapidly match thousands of phosphopeptides that share phosphorylation sites within and across species. PhosphoBlast analysis of several large phosphoprotein datasets from the literature revealed common phosphorylation signatures shared across diverse experimental platforms and species. Moreover PhosphoBlast is a powerful analysis tool to identify specific phosphosite mutations. Comparison of the mouse and human phosphoproteomes revealed more than 130 specific phosphoamino acid mutations, some of which are predicted to alter protein function. Further analysis revealed that known phosphorylated amino acids are more evolutionally conserved than the Ser/Thr/Tyr amino acids not known to be phosphorylated. Together our results demonstrate that PhosphoBlast is a versatile mining tool capable of identifying related phosphorylation signatures and phosphoamino acid mutations among complex proteomics datasets in a highly efficient and accurate manner. PhosphoBlast will aid in the informatics analysis of the phosphoproteome and the identification of phosphoprotein biomarkers of disease.

Phospho.ELM: a database of phosphorylation sites update 2008

Francesca Diella — 2007-10-29T09:45:04-00:00

Nucl. Acids Res. (25 October 2007), gkm772.

Phospho.ELM is a manually curated database of eukaryotic phosphorylation sites. The resource includes data collected from published literature as well as high-throughput data sets. The current release of Phospho.ELM (version 7.0, July 2007) contains 4078 phospho-protein sequences covering 12 025 phospho-serine, 2362 phospho-threonine and 2083 phospho-tyrosine sites. The entries provide information about the phosphorylated proteins and the exact position of known phosphorylated instances, the kinases responsible for the modification (where known) and links to bibliographic references. The database entries have hyperlinks to easily access further information from UniProt, PubMed, SMART, ELM, MSD as well as links to the protein interaction databases MINT and STRING. A new BLAST search tool, complementary to retrieval by keyword and UniProt accession number, allows users to submit a protein query (by sequence or UniProt accession) to search against the curated data set of phosphorylated peptides. Phospho.ELM is available on line at: http://phospho.elm.eu.org 10.1093/nar/gkm772

Kinases and protein phosphorylation as regulators of steroid hormone action.

NL Weigel — 2007-10-01T14:56:10-00:00

Nucl Recept Signal, Vol. 5 (2007)

Although the primary signal for the activation of steroid hormone receptors is binding of hormone, there is increasing evidence that the activities of cell signaling pathways and the phosphorylation status of these transcription factors and their coregulators determine the overall response to the hormone. In some cases, enhanced cell signaling is sufficient to cause activation of receptors in medium depleted of steroids. Steroid receptors are targets for multiple kinases. Many of the phosphorylation sites contain Ser/Thr-Pro motifs implicating proline-directed kinases such as the cyclin-dependent kinases and the mitogen-activated kinases (MAPK) in receptor phosphorylation. Although some sites are constitutively phosphorylated, others are phosphorylated in response to hormone. Still others are only phosphorylated in response to specific cell signaling pathways. Phosphorylation of specific sites has been implicated not only in overall transcriptional activity, but also in nuclear localization, protein stability, and DNA binding. The studies of the roles of phosphorylation in coregulator function are more limited, but it is now well established that many of them are highly phosphorylated and that phosphorylation regulates their function. There is good evidence that some of the phosphorylation sites in the receptors and coregulators are targets of multiple signaling pathways. Individual sites have been associated both with functions that enhance the activity of the receptor, as well as with functions that inhibit activity. Thus, the specific combinations of phosphorylations of the steroid receptor combined with the expression levels and phosphorylation status of coregulators will determine the genes regulated and the biological response.

Systematic discovery of in vivo phosphorylation networks.

R Linding — 2007-07-08T16:28:02-00:00

Cell, Vol. 129, No. 7. (29 June 2007), pp. 1415-1426.

Protein kinases control cellular decision processes by phosphorylating specific substrates. Thousands of in vivo phosphorylation sites have been identified, mostly by proteome-wide mapping. However, systematically matching these sites to specific kinases is presently infeasible, due to limited specificity of consensus motifs, and the influence of contextual factors, such as protein scaffolds, localization, and expression, on cellular substrate specificity. We have developed an approach (NetworKIN) that augments motif-based predictions with the network context of kinases and phosphoproteins. The latter provides 60%-80% of the computational capability to assign in vivo substrate specificity. NetworKIN pinpoints kinases responsible for specific phosphorylations and yields a 2.5-fold improvement in the accuracy with which phosphorylation networks can be constructed. Applying this approach to DNA damage signaling, we show that 53BP1 and Rad50 are phosphorylated by CDK1 and ATM, respectively. We describe a scalable strategy to evaluate predictions, which suggests that BCLAF1 is a GSK-3 substrate.