SimPhospho: a software tool enabling confident phosphosite assignment

Motivation: Mass spectrometry combined with enrichment strategies for phosphorylated peptides has been successfully employed for two decades to identify sites of phosphorylation. However, unambiguous phosphosite assignment is considered challenging. Given that site-specific phosphorylation events function as different molecular switches, validation of phosphorylation sites is of utmost importance. In our earlier study we developed a method based on simulated phospho-peptide spectral libraries, which enables highly sensitive and accurate phosphosite assignments. To promote more widespread use of this method, we here introduce a software implementation with improved usability and performance.Results: We present SimPhospho, a fast and user-friendly tool for accurate simulation of phospho-peptide tandem mass spectra. Simulated phosphopeptide spectral libraries are used to validate and supplement database search results, with a goal to improve reliable phosphoproteome identification and reporting. The presented program can be easily used together with the TransProteomic Pipeline and integrated in a phosphoproteomics data analysis workflow.Availability and implementation: SimPhospho is open source and it is available for Windows, Linux and Mac operating systems. The software and its user's manual with detailed description of data analysis as well as test data can be found at https://sourceforge.net/projects/simphospho/.Contact: [email protected] or G.L. [email protected] information: Supplementary data are available at Bioinformatics online

Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling

Hyperactivated RAS drives progression of many human malignancies. However, oncogenic activity of RAS is dependent on simultaneous inactivation of protein phosphatase 2A (PP2A) activity. Although PP2A is known to regulate some of the RAS effector pathways, it has not been systematically assessed how these proteins functionally interact. Here we have analyzed phosphoproteomes regulated by either RAS or PP2A, by phosphopeptide enrichment followed by mass-spectrometry-based label-free quantification. To allow data normalization in situations where depletion of RAS or PP2A inhibitor CIP2A causes a large uni-directional change in the phosphopeptide abundance, we developed a novel normalization strategy, named pairwise normalization. This normalization is based on adjusting phosphopeptide abundances measured before and after the enrichment. The superior performance of the pairwise normalization was verified by various independent methods. Additionally, we demonstrate how the selected normalization method influences the downstream analyses and interpretation of pathway activities. Consequently, bioinformatics analysis of RAS and CIP2A regulated phosphoproteomes revealed a significant overlap in their functional pathways. This is most likely biologically meaningful as we observed a synergistic survival effect between CIP2A and RAS expression as well as KRAS activating mutations in TCGA pan-cancer data set, and synergistic relationship between CIP2A and KRAS depletion in colony growth assays.Peer reviewe

Phosphorylation of Notch1 by Pim kinases promotes oncogenic signaling in breast and prostate cancer cells

Tumorigenesis is a multistep process involving co-operation between several deregulated oncoproteins. In this study, we unravel previously unrecognized interactions and crosstalk between Pim kinases and the Notch signaling pathway, with implications for both breast and prostate cancer. We identify Notch1 and Notch3, but not Notch2, as novel Pim substrates and demonstrate that for Notch1, the serine residue 2152 is phosphorylated by all three Pim family kinases. This target site is located in the second nuclear localization sequence (NLS) of the Notch1 intracellular domain (N1ICD), and is shown to be important for both nuclear localization and transcriptional activity of N1ICD. Phosphorylation-dependent stimulation of Notch1 signaling promotes migration of prostate cancer cells, balances glucose metabolism in breast cancer cells, and supports in vivo growth of both types of cancer cells on chick embryo chorioallantoic membranes. Furthermore, Pim-induced growth of orthotopic prostate xenografts in mice is associated with enhanced nuclear Notch1 activity. Finally, simultaneous inhibition of Pim and Notch abrogates the cellular responses more efficiently than individual treatments, opening up new vistas for combinatorial cancer therapy.</p

Optimization of TripleTOF spectral simulation and library searching for confident localization of phosphorylation sites

Tandem mass spectrometry (MS/MS) has been used in analysis of proteins and their post-translational modifications. A recently developed data analysis method, which simulates MS/MS spectra of phosphopeptides and performs spectral library searching using SpectraST, facilitates confident localization of phosphorylation sites. However, its performance has been evaluated only on MS/MS spectra acquired using Orbitrap HCD mass spectrometers so far. In this study, we have investigated whether this approach would be applicable to another type of mass spectrometers, and optimized the simulation and search conditions to achieve sensitive and confident site localization. Synthetic phosphopeptides and enriched K562 cell phosphopeptides were analyzed using a TripleTOF 6600 mass spectrometer before and after enzymatic dephosphorylation. Dephosphorylated peptides identified by X!Tandem database searching were subjected to spectral simulation of all possible single phosphorylations using SimPhospho software. Phosphopeptides were identified and localized by SpectraST searching against a library of the simulated spectra. Although no synthetic phosphopeptide was localized at 1% false localization rate under the previous conditions, optimization of the spectral simulation and search conditions for the TripleTOF datasets achieved the localization and improved the sensitivity. Furthermore, the optimized conditions enabled sensitive localization of K562 phosphopeptides at 1% false discovery and localization rates. These results suggest that accurate phosphopeptide simulation of TripleTOF MS/MS spectra is possible and the simulated spectral libraries can be used in SpectraST searching for confident localization of phosphorylation sites

Extracellular signal-regulated kinase and glycogen synthase kinase 3β regulate gephyrin postsynaptic aggregation and GABAergic synaptic function in a calpain-dependent mechanism

Molecular mechanisms of plasticity at GABAergic synapses are currently poorly understood. To identify signaling cascades that converge onto GABAergic postsynaptic density proteins, we performed MS analysis using gephyrin isolated from rat brain and identified multiple novel phosphorylation and acetylation residues on gephyrin. Here, we report the characterization of one of these phosphoresidues, Ser-268, which when dephosphorylated leads to the formation of larger postsynaptic scaffolds. Using a combination of mutagenesis, pharmacological treatment, and biochemical assays, we identify ERK as the kinase phosphorylating Ser-268 and describe a functional interaction between residues Ser-268 and Ser-270. We further demonstrate that alterations in gephyrin clustering via ERK modulation are reflected by amplitude and frequency changes in miniature GABAergic postsynaptic currents. We unravel novel mechanisms for activity- and ERK-dependent calpain action on gephyrin, which are likely relevant in the context of cellular signaling affecting GABAergic transmission and homeostatic synaptic plasticity in pathology

Quantitative analysis of the erythrocyte membrane proteins in polycythemia vera patients treated with hydroxycarbamide

More than 90% of polycythemia vera (PV) patients have a mutation in the protein JAK2, which is closely associated with the erythrocyte membrane. With the comparison of 1-D gels of erythrocyte membranes obtained from PV patients treated with hydroxycarbamide and those of untreated controls we observed significant differences in the region of 40–55 kDa. On the basis of the LC–MS/MS analysis of this region we report up-regulation of four protein disulfide isomerases, which was subsequently confirmed by targeted mass spectrometric analysis. In further studies it will be prudent to compare this in patients both treated and not treated with hydroxycarbamide

Quantitative Site-Specific Phosphoproteomics of <i>Trichoderma reesei</i> Signaling Pathways upon Induction of Hydrolytic Enzyme Production

The filamentous fungus <i>Trichoderma reesei</i> is used for industrial production of secreted enzymes including carbohydrate active enzymes, such as cellulases and hemicellulases. The production of many of these enzymes by <i>T. reesei</i> is influenced by the carbon source it grows on, where the regulation system controlling hydrolase genes involves various signaling pathways. <i>T. reesei</i> was cultivated in the presence of sorbitol, a carbon source that does not induce the production of cellulases and hemicellulases, and then exposed to either sophorose or spent-grain extract, which are efficient inducers of the enzyme production. Specific changes at phosphorylation sites were investigated in relation to the production of cellulases and hemicellulases using an MS-based framework. Proteome-wide phosphorylation following carbon source exchange was investigated in the early stages of induction: 0, 2, 5, and 10 min. The workflow involved sequential trypsin digestion, TiO₂ enrichment, and MS analysis using a Q Exactive mass spectrometer. We report on the identification and quantitation of 1721 phosphorylation sites. Investigation of the data revealed a complex signaling network activated upon induction involving components related to light-mediated cellulase induction, osmoregulation, and carbon sensing. Changes in protein phosphorylation were detected in the glycolytic pathway, suggesting an inhibition of glucose catabolism at 10 min after the addition of sophorose and as early as 2 min after the addition of spent-grain extract. Differential phosphorylation of factors related to carbon storage, intracellular trafficking, cytoskeleton, and cellulase gene regulation were also observed

FVIIa-sTF and Thrombin Inhibitory Activities of Compounds Isolated from Microcystis aeruginosa K-139

The rise of bleeding and bleeding complications caused by oral anticoagulant use are serious problems nowadays. Strategies that block the initiation step in blood coagulation involving activated factor VII-tissue factor (fVIIa-TF) have been considered. This study explores toxic Microcystis aeruginosa K-139, from Lake Kasumigaura, Ibaraki, Japan, as a promising cyanobacterium for isolation of fVIIa-sTF inhibitors. M. aeruginosa K-139 underwent reversed-phase solid-phase extraction (ODS-SPE) from 20% MeOH to MeOH elution with 40%-MeOH increments, which afforded aeruginosin K-139 in the 60% MeOH fraction; micropeptin K-139 and microviridin B in the MeOH fraction. Aeruginosin K-139 displayed an fVIIa-sTF inhibitory activity of ~166 µM, within a 95% confidence interval. Micropeptin K-139 inhibited fVIIa-sTF with EC50 10.62 µM, which was more efficient than thrombin inhibition of EC50 26.94 µM. The thrombin/fVIIa-sTF ratio of 2.54 in micropeptin K-139 is higher than those in 4-amidinophenylmethane sulfonyl fluoride (APMSF) and leupeptin, when used as positive controls. This study proves that M. aeruginosa K-139 is a new source of fVIIa-sTF inhibitors. It also opens a new avenue for micropeptin K-139 and related depsipeptides as fVIIa-sTF inhibitors

Phosphorylation of Notch1 by Pim kinases promotes oncogenic signaling in breast and prostate cancer cells

Tumorigenesis is a multistep process involving co-operation between several deregulated oncoproteins. In this study, we unravel previously unrecognized interactions and crosstalk between Pim kinases and the Notch signaling pathway, with implications for both breast and prostate cancer. We identify Notch1 and Notch3, but not Notch2, as novel Pim substrates and demonstrate that for Notch1, the serine residue 2152 is phosphorylated by all three Pim family kinases. This target site is located in the second nuclear localization sequence (NLS) of the Notch1 intracellular domain (N1ICD), and is shown to be important for both nuclear localization and transcriptional activity of N1ICD. Phosphorylation-dependent stimulation of Notch1 signaling promotes migration of prostate cancer cells, balances glucose metabolism in breast cancer cells, and supports in vivo growth of both types of cancer cells on chick embryo chorioallantoic membranes. Furthermore, Pim-induced growth of orthotopic prostate xenografts in mice is associated with enhanced nuclear Notch1 activity. Finally, simultaneous inhibition of Pim and Notch abrogates the cellular responses more efficiently than individual treatments, opening up new vistas for combinatorial cancer therapy