Project description:Aberrant signaling pathway activity is a hallmark of tumorigenesis and progression, which has guided targeted inhibitor design for over 30 years. Yet, adaptive resistance mechanisms, induced by rapid, context-specific signaling network rewiring, continue to challenge therapeutic efficacy. By leveraging progress in proteomic technologies and network-based methodologies over the past decade we developed VESPA—an algorithm designed to elucidate mechanisms of cell response and adaptation to drug perturbations—and used it to analyze 7-point phosphoproteomic time series from colorectal cancer cells treated with clinically-relevant inhibitors and control media. Interrogation of tumor-specific enzyme/substrate interactions accurately inferred kinase and phosphatase activity, based on their inferred substrate phosphorylation state, effectively accounting for signal cross-talk and sparse phosphoproteome coverage. The analysis elucidated time-dependent signaling pathway response to each drug perturbation and, more importantly, cell adaptive response and rewiring that was experimentally confirmed by CRISPRko assays, suggesting broad applicability to cancer and other diseases.
Project description:AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis. Although AMPK has been studied extensively in cellular processes, understanding of its substrates and downstream functional network, and their contributions to cell fate and disease development, remains incomplete. To elucidate the AMPK-dependent signaling pathways, we performed global quantitative phosphoproteomic analysis using wild-type and AMPK1/2-double knockout cells and discovered 160 AMPK-dependent phosphorylation sites.
Project description:Tumor heterogeneity attributes substantial challenges in determining the treatment regimen. Along with the conventional treatment, such as chemotherapy and radiotherapy, targeted therapy has greater impact in cancer management. Owing to the recent advancements in proteomics, we aimed to mine and re-interrogate the Clinical Proteomic Tumor Analysis Consortium (CPTAC) data sets which contain deep scale, mass spectrometry (MS)-based proteomic and phosphoproteomic data sets conducted on human tumor samples. Quantitative proteomic and phosphoproteomic data sets of tumor samples were explored and downloaded from the CPTAC database for six different cancers types (breast cancer, clear cell renal cell carcinoma (CCRCC), colon cancer, lung adenocarcinoma (LUAD), ovarian cancer, and uterine corpus endometrial carcinoma (UCEC)). We identified 880 phosphopeptide signatures for differentially regulated phosphorylation sites across five cancer types (breast cancer, colon cancer, LUAD, ovarian cancer, and UCEC). We identified the cell cycle to be aberrantly activated across these cancers. The correlation of proteomic and phosphoproteomic data sets identified changes in the phosphorylation of 12 kinases with unchanged expression levels. We further investigated phosphopeptide signature across five cancer types which led to the prediction of aurora kinase A (AURKA) and kinases-serine/threonine-protein kinase Nek2 (NEK2) as the most activated kinases targets. The drug designed for these kinases could be repurposed for treatment across cancer types.
Project description:To elucidate the mechanism of neuronal activation, we conducted a global quantitative phosphoproteomic analysis of hypothalamic neurons treated with Raptin or PBS. By COG analysis of the differentially expressed phosphorylation protein, we found obvious alterations of transport and catabolism, as well as cell motility. By conducting a KEGG analysis using phosphoproteomic data, we identified the PI3K-AKT pathway.
Project description:During aging, senescent cells accumulate in bone marrow and secrete the dysfunctional factors, termed senescence associated secretory phenotype (SASP), which is implied to regulate bone metabolism. To identify the key SASP factors in bone marrow that influence skeletal aging, we analyzed the dysregulated factors in the bone marrow supernatant from young and aged rat through mass spectrometry. In another hand, BMSCs treated with rGCA, transfection of siRNA-Plxnb2 or controls were subjected to global quantitative phosphoproteomic analysis.
Project description:BackgroundThe non-receptor tyrosine kinase, SRMS (Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites) is a member of the BRK family kinases (BFKs) which represents an evolutionarily conserved relative of the Src family kinases (SFKs). Tyrosine kinases are known to regulate a number of cellular processes and pathways via phosphorylating substrate proteins directly and/or by partaking in signaling cross-talks leading to the indirect modulation of various signaling intermediates. In a previous study, we profiled the tyrosine-phosphoproteome of SRMS and identified multiple candidate substrates of the kinase. The broader cellular signaling intermediates of SRMS are unknown.MethodsIn order to uncover the broader SRMS-regulated phosphoproteome and identify the SRMS-regulated indirect signaling intermediates, we performed label-free global phosphoproteomics analysis on cells expressing wild-type SRMS. Using computational database searching and bioinformatics analyses we characterized the dataset.ResultsOur analyses identified 60 hyperphosphorylated (phosphoserine/phosphothreonine) proteins mapped from 140 hyperphosphorylated peptides. Bioinfomatics analyses identified a number of significantly enriched biological and cellular processes among which DNA repair pathways were found to be upregulated while apoptotic pathways were found to be downregulated. Analyses of motifs derived from the upregulated phosphosites identified Casein kinase 2 alpha (CK2α) as one of the major potential kinases contributing to the SRMS-dependent indirect regulation of signaling intermediates.ConclusionsOverall, our phosphoproteomics analyses identified serine/threonine phosphorylation dynamics as important secondary events of the SRMS-regulated phosphoproteome with implications in the regulation of cellular and biological processes.
Project description:Ebola virus (EBOV) causes severe human disease with a high case fatality rate. The balance of evidence implies that the virus circulates in bats. The molecular basis for host-viral interactions, including the role for phosphorylation during infections, is largely undescribed. To address this, and to better understand the biology of EBOV, the phosphorylation of EBOV proteins was analyzed in virions purified from infected monkey Vero-E6 cells and bat EpoNi/22.1 cells using high-resolution mass spectrometry. All EBOV structural proteins were detected with high coverage, along with phosphopeptides. Phosphorylation sites were identified in all viral structural proteins. Comparison of EBOV protein phosphorylation in monkey and bat cells showed only partial overlap of phosphorylation sites, with shared sites found in NP, VP35, and VP24 proteins, and no common sites in the other proteins. Three-dimensional structural models were built for NP, VP35, VP40, GP, VP30 and VP24 proteins using available crystal structures or by de novo structure prediction to elucidate the potential role of the phosphorylation sites. Phosphorylation of one of the identified sites in VP35, Thr-210, was demonstrated to govern the transcriptional activity of the EBOV polymerase complex. Thr-210 phosphorylation was also shown to be important for VP35 interaction with NP. This is the first study to compare phosphorylation of all EBOV virion proteins produced in primate versus bat cells, and to demonstrate the role of VP35 phosphorylation in the viral life cycle. The results uncover a novel mechanism of EBOV transcription and identify novel targets for antiviral drug development.