Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.

Cellular responses to DNA damage are mediated by a number of protein kinases, including ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). The outlines of the signal transduction portion of this pathway are known, but little is known about the physiological scope of the DNA damage response (DDR). We performed a large-scale proteomic analysis of proteins phosphorylated in response to DNA damage on consensus sites recognized by ATM and ATR and identified more than 900 regulated phosphorylation sites encompassing over 700 proteins. Functional analysis of a subset of this data set indicated that this list is highly enriched for proteins involved in the DDR. This set of proteins is highly interconnected, and we identified a large number of protein modules and networks not previously linked to the DDR. This database paints a much broader landscape for the DDR than was previously appreciated and opens new avenues of investigation into the responses to DNA damage in mammals.

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ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage

Global Survey of Phosphotyrosine Signaling Identifies Oncogenic Kinases in Lung Cancer

Data analysis and interpretation remain major logistical challenges when attempting to identify large numbers of protein phosphorylation sites by nanoscale reverse-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Supplementary Figure 1 online). In this report we address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. We provide a large-scale phosphorylation data set with a measured error rate as determined by the target-decoy approach, we demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and we present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra. We applied our methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where we identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-positive rate of 1.3%.

A probability-based approach for high-throughput protein phosphorylation analysis and site localization.

PhosphoSitePlus (http://www.phosphosite.org) is an open, comprehensive, manually curated and interactive resource for studying experimentally observed post-translational modifications, primarily of human and mouse proteins. It encompasses 1,30,000 non-redundant modification sites, primarily phosphorylation, ubiquitinylation and acetylation. The interface is designed for clarity and ease of navigation. From the home page, users can launch simple or complex searches and browse high-throughput data sets by disease, tissue or cell line. Searches can be restricted by specific treatments, protein types, domains, cellular components, disease, cell types, cell lines, tissue and sequences or motifs. A few clicks of the mouse will take users to substrate pages or protein pages with sites, sequences, domain diagrams and molecular visualization of side-chains known to be modified; to site pages with information about how the modified site relates to the functions of specific proteins and cellular processes and to curated information pages summarizing the details from one record. PyMOL and Chimera scripts that colorize reactive groups on residues that are modified can be downloaded. Features designed to facilitate proteomic analyses include downloads of modification sites, kinase-substrate data sets, sequence logo generators, a Cytoscape plugin and BioPAX download to enable pathway visualization of the kinase-substrate interactions in PhosphoSitePlus®.

PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse

Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.

Immunoaffinity profiling of tyrosine phosphorylation in cancer cells

A common phosphotyrosine signature for the Bcr-Abl kinase

The invention discloses 318 novel phosphorylation sites identified in carcinoma, peptides (including AQUA peptides) comprising a phosphorylation site of the invention, antibodies specifically bind to a novel phosphorylation site of the invention, and diagnostic and therapeutic uses of the above.

Tyrosine phosphorylation sites

The invention discloses nearly 474 novel phosphorylation sites identified in signal transduction proteins and pathways underlying human carcinoma, and provides phosphorylation-site specific antibodies and heavy-isotope labeled peptides (AQUA peptides) for the selective detection and quantification of these phosphorylated sites/proteins, as well as methods of using the reagents for such purpose. Among the phosphorylation sites identified are sites occurring in the following protein types: Kinase, Adaptor/Scaffold proteins, Phosphatase, G protein Regulator/Guanine Nucleotide Exchange Factors/GTPase Activating Proteins, Cytoskeleton Proteins, DNA Binding Proteins, Phospholipase, Receptor Proteins, Enzymes, DNA Repair/Replication Proteins, Adhesion Proteins, and Proteases , as well as other protein types.

Reagents for the detection of protein phosphorylation in carcinoma signaling pathways

The invention discloses 482 novel phosphorylation sites identified in carcinoma and/or leukemia, peptides (including AQUA peptides) comprising a phosphorylation site of the invention, antibodies specifically bind to a novel phosphorylation site of the invention, and diagnostic and therapeutic uses of the above.

Erik Spek

Papers

Immunoaffinity profiling of tyrosine phosphorylation in cancer cells

A common phosphotyrosine signature for the Bcr-Abl kinase

Tyrosine phosphorylation sites

Reagents for the detection of protein phosphorylation in carcinoma signaling pathways

Tyrosine phosphorylation sites and antibodies specific for them