Phosphoproteomic analysis of endothelial cells treated with VEGF and Ang-1
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Velos
ORGANISM(S): Bos Taurus (bovine)
TISSUE(S): Aortic Endothelial Cell
SUBMITTER: 
Rony Chidiac
LAB HEAD: Jean-Philippe Gratton
PROVIDER: PXD002456 | Pride | 2016-02-09
REPOSITORIES: Pride
Publications
Comparative Phosphoproteomics Analysis of VEGF and Angiopoietin-1 Signaling Reveals ZO-1 as a Critical Regulator of Endothelial Cell Proliferation.
Chidiac Rony R Zhang Ying Y Tessier Sylvain S Faubert Denis D Delisle Chantal C Gratton Jean-Philippe JP
Molecular & cellular proteomics : MCP 20160204 5
VEGF and angiopoietin-1 (Ang-1) are essential factors to promote angiogenesis through regulation of a plethora of signaling events in endothelial cells (ECs). Although pathways activated by VEGF and Ang-1 are being established, the unique signaling nodes conferring specific responses to each factor remain poorly defined. Thus, we conducted a large-scale comparative phosphoproteomic analysis of signaling pathways activated by VEGF and Ang-1 in ECs using mass spectrometry. Analysis of VEGF and Ang ...[more]
Similar Datasets
Project description:Skeletal muscle insulin resistance is the earliest defect in type 2 diabetes (T2D), preceding and predicting disease development. Whether this represents the underlying primary defect in T2D or effects of changes in hormones or circulating metabolites is unknown. To address this question, we have developed a “disease-in-a-dish” model by differentiating iPS cells from T2D patients and controls into myoblasts (iMyo) and studied their function in vitro. We find that T2D iMyos exhibit multiple defects mirroring human disease including altered insulin signaling through the IRS/AKT pathway, decreased insulin-stimulated glucose uptake, and reduced mitochondrial oxidation. In addition, using global phosphoproteomics we find that T2D alters phosphorylation of a large network of targets of mTOR, S6K, PKC and other kinases including proteins involved in regulation of Rho-GTPases, mRNA splicing/processing, vesicular trafficking, gene transcription and chromatin remodeling. This cell-autonomous dysregulated phosphorylation network reveals a new dimension in the mechanism underlying insulin resistance in T2D.
2020-08-27 | PXD015430 | Pride
Project description:Bacterial cell wall components provide various unique molecular structures that are detected by pattern recognition receptors (PRRs) of the innate immune system as non-self. Most bacterial species form a cell wall that consists of peptidoglycan, a polymeric structure comprising of alternating amino sugars. Muramyl dipeptide (MDP) is the minimal, biologically active molecular structure derived from peptidoglycan, and its immunogenicity is well documented in numerous studies. MDP is sensed by the cytosolic nucleotide-binding and nucleotide oligomerization domain-like receptor 2 (NOD2), which triggers a pro-inflammatory response upon engagement 1,2. Conducting a forward genetic screen to identify factors required for MDP detection, we discovered that N-acetylglucosamine kinase (NAGK) is essential for the immunostimulatory activity of MDP. NAGK, which has previously been identified to contribute to the hexosamine salvage pathway in humans, is broadly expressed and inducible in innate immune cells. Mechanistically, NAGK functions upstream of NOD2 in that it directly phosphorylates the N-acetylmuramic moiety of MDP at the hydroxyl group of its C6 position, yielding 6-O-phospho-MDP. NAGK-phosphorylated MDP constitutes an essential component of NOD2 dependent signal transduction. Altogether, these results unveil a previously unknown interconnection of amino sugar metabolism and innate immunity to bacterial cell walls.
2022-06-13 | PXD022384 | Pride
Project description:Tumor necrosis factor (TNF) is one of the few cytokines successfully targeted by therapies against inflammatory diseases. However, blocking this well studied and pleiotropic ligand can cause dramatic side-effects. We reasoned that a systems-level proteomic analysis of TNF signaling could dissect its diverse functions and offer a base for developing more targeted therapies. Combining phosphoproteomics time course experiments with spatial and kinase inhibitor analysis clusters phosphorylations into functional modules. The majority of regulated phosphorylations could be assigned to an upstream kinase by inhibiting master kinases and spatial proteomics revealed phosphorylation-dependent translocations of hundreds of proteins upon TNF stimulation. Phosphoproteome analysis of TNF-induced apoptosis and necroptosis revealed a key role for transcriptional cyclin-dependent kinase (CDK) activity to promote cytokine production and prevent excessive cell death downstream of the TNF signaling receptor. Our comprehensive interrogation of TNF induced pathways and sites can be explored at http://tnfviewer.biochem.mpg.de/.
2021-10-11 | PXD021366 | Pride
Project description:Insulin acts through the insulin receptor (IR) tyrosine kinase to exert its classical metabolic and mitogenic actions. Here, using receptors with either short or long deletion of the β-subunit or mutation of the kinase active site (K1030R, we uncover a second novel IR signaling pathway that is intracellular domain dependent, but ligand and tyrosine kinase-independent (LYK-I). These LYK-I actions of the IR are linked to changes in phosphorylation of a network of proteins involved in the regulation of extracellular matrix organization, cell cycle, ATM signaling and cellular senescence; and result in upregulation of expression of multiple extracellular matrix-related genes and proteins, down-regulation of immune/interferon-related genes and proteins, and increased sensitivity to apoptosis. Thus, in addition to classical ligand and tyrosine kinase-dependent (LYK-D) signaling, the IR regulates a second, novel ligand and tyrosine kinase-independent (LYK-I) pathway which regulates the cellular machinery involved in senescence, matrix interaction and response to extrinsic challenges.
2023-01-02 | PXD035587 | Pride
Project description:Insulin and IGF-1 receptors (IR/IGF1R) are highly homologous and share similar signaling systems, but each has a unique physiological role, with IR primarily regulating metabolic homeostasis and IGF1R regulating mitogenic control and growth. Here, we show that replacement of a single amino acid at position 973, just distal to the NPEY motif in the intracellular juxtamembrane region, from leucine, which is highly-conserved in IRs, to phenylalanine, the highly-conserved homologous residue in IGF1Rs, results in decreased IRS-1-PI3K-Akt-mTORC1 signaling and increased of Shc-Gab1-MAPK-cell cycle signaling. As a result, cells expressing L973F-IR exhibit decreased insulin-induced glucose uptake, increased cell growth and impaired receptor internalization. Mice with knockin of the L973F-IR show similar alterations in signaling in vivo, and this leads to decreased insulin sensitivity, a modest increase in growth and decreased weight gain when challenged with high-fat diet. Thus, leucine973 in the juxtamembrane region of the IR acts is a crucial residue differentiating IR signaling from IGF1R signaling.
2022-12-21 | PXD038649 | Pride
Project description:Background Annexin-1 (ANXA1) plays pivotal roles in regulating various physiological processes including inflammation, proliferation and apoptosis, and deregulations of ANXA1 functions have been associated with tumorigenesis and metastasis events in several cancers. Though ANXA1 levels correlate with breast cancer disease status and outcome, its distinct functional involvement in breast cancer initiation and progression remains unclear. We hypothesized ANXA1-responsive kinase signaling alteration and associated phosphorylation signaling underlie early events in breast cancer initiation events and hence profiled ANXA1-dependent phosphorylation changes in mammary gland epithelial cells. Methods Quantitative phosphoproteomics analysis of mammary gland epithelial cells derived from ANXA1-heterozygous and ANXA1-deficient mice was carried out using SILAC-based mass spectrometry. Kinase and signaling changes underlying ANXA1 perturbations were derived by upstream kinase prediction and integrated network analysis of altered proteins and phosphoproteins. Results We identified a total of 8,110 unique phosphorylation sites, of which 582 phosphorylation sites on 372 proteins showed ANXA1-responsive changes. A majority of these phosphorylation changes occurred on proteins associated with cytoskeletal reorganization spanning the focal adhesion, stress fibers, and also the microtubule network pressing on new roles for ANXA1 in regulating microtubule dynamics. Comparative analysis of regulated global proteome and phosphoproteome highlighted key differences in translational and post-translational effects of ANXA1, and stressed on a close-coordinated rewiring of the cell adhesion network. Kinase prediction analysis suggested activity modulation of CAMK2, PAK, ERK, and IKK kinases upon loss of ANXA1. Integrative analysis revealed regulation of WNT and also Hippo signaling pathways in ANXA1-deficient mammary epithelial cells, wherein there is a downregulation of transcriptional effects of TEAD suggestive of ANXA1-responsive transcriptional rewiring. Conclusions The phosphoproteome landscape uncovered several novel perspectives for ANXA1 in mammary gland biology and stressed on its involvement in key signaling pathways modulating cell adhesion and migration that could contribute to breast cancer initiation.
2017-12-13 | PXD007051 | Pride
Project description:Protein phosphorylation has a major role in controlling the life-cycle and infection stages of bacteria. Proteome-wide occurrence of S/T/Y phosphorylation has been reported for many prokaryotic systems. Previously, we reported the phosphoproteome of Pseudomonas aeruginosa and Pseudomonas putida. In this study, we show the role of S/T phosphorylation of one motility protein, FliC, in regulating multiple surface-associated phenomena of Pseudomonas aeruginosa PAO1. The absence of phosphorylation in the conserved T27 and S28 residues of flagellin FliC, interestingly, did not affect swimming motility, but affected the secretome of type 2 secretion system (T2SS) and biofilm formation of PAO1. Flagellin phosphomutants had increased levels and activities of type 2 secretome proteins. This occurs by a possible mechanism affecting the secretion efficiency of T2SS machinery. Flagellin phosphomutants also formed reduced biofilm at 24h and had delayed biofilm dispersal under static and dynamic flow conditions, respectively. The levels of type 2 secretome and biofilm formation under static conditions had an inverse correlation. Hence, increase in the levels of type 2 secretome was accompanied by reduced biofilm formation in the flagellin phosphomutants. Altogether, we found a system of phosphorylation that co-ordinately regulates surface related processes such as proteases secretion by T2SS, biofilm formation and dispersal.
2022-03-02 | PXD003645 | Pride
Project description:Although many affinity adsorbents have been developed for phosphopeptides enrichment, high-specifically capturing the multi-phosphopeptides is still a big challenge. Here, we investigated the mechanism of phosphate ions coordination and substitution on affinity adsorbents surfaces and modulated the selectivity of affinity adsorbents to multi-phosphopeptides based on the different capability of mono- and multi-phosphopeptides in competitively substituting the pre-coordinated phosphate ions at strong acidic condition. We demonstrated both the species of pre-coordinated phosphate ions and the substituting conditions played crucial roles in modulating the enrichment selectivity to multi-phosphopeptides, and the pre-coordinated affinity materials with relative more surfaces positive charges exhibited better enrichment efficiency due to the cooperative effect of electrostatic interaction and competitive substitution. Finally, an enrichment selectivity of 85% to multi-phosphopeptides was feasibly achieved with 66% improvement in identification numbers for complex protein sample extracted from HepG2 cells.
2016-08-03 | PXD004252 | Pride
Project description:Adenosine triphosphate (ATP) synthase is located on the inner membrane of mitochondria which generate ATP for various cellular processes. Our previous studies showed that ATP synthases existed on plasma membrane of several types of cancer cells, which was defined as ectopic ATP synthase. However, the trafficking pathway of ectopic ATP synthase is still unclear. To investigate how ATP synthase subunits are involved in the trafficking process, we performed shot-gun proteomic analysis to reveal the plasma membrane proteins of A549 lung cancer cells and identified four subunits of ATP synthases, ATP5A1, ATP5B, ATP5H, and MT-ATP6. With the results of gene set enrichment analysis, we inferred that ectopic ATP synthase subunits are assembled as complex consistently and then translocated to cell surface through the trafficking of mitochondria. On the other hand, microtubules are involved in many intracellular transport. During protein trafficking, mitochondria and vesicles are transported to their final destination along microtubules by motor proteins such as kinesins and dyneins. To explore the function of microtubules in the trafficking of ectopic ATP synthase, we treated MCF-7 breast and A549 lung cancer cells with nocodazole, a microtubule depolymerization agent, and found that the expression level of ectopic ATP synthase decreased using both immunocytochemistry and flow cytometry. Besides, since kinesin family member 5B (KIF5B) is an important motor proteins involved in the transport of mitochondria, we further performed the immunoprecipitation of KIF5B followed by liquid chromatography−tandem mass spectrometry (LC−MS/MS). The result revealed that one of the mitochondrial outer membrane proteins, dynamin-1 like proteins (Drp1), was able to associate to KIF5B. From our recent research results and other reports show that Drp1 is a mediator of mitochondrial dynamic through regulation of mitochondrial fission. Taken together, our findings suggested that Drp1-KIF5B complex-mediated mitochondrial trafficking along microtubules may play a critical role in the transport of ectopic ATP synthase
2023-05-10 | PXD006791 | Pride
Project description:Philadelphia-like (Ph-like) acute lymphoblastic leukaemia (ALL) is a high-risk subtype of B-cell ALL characterised by a gene expression profile resembling Philadelphia Chromosome positive ALL (Ph+ ALL) in the absence of BCR-ABL1. Tyrosine kinase activating fusions, some involving ABL1, are recurrent drivers of Ph-like ALL and are targetable with tyrosine kinase inhibitors (TKIs). We identified a rare instance of SFPQ-ABL1 in a child with Ph-like ALL. SFPQ-ABL1 expressed in cytokine-dependent cell lines was sufficient to transform cells which were sensitive to ABL1-targeting TKIs. In contrast to BCR-ABL1, SFPQ-ABL1 localised to the nuclear compartment and was a weaker driver of cellular proliferation. Phosphoproteomics analysis showed upregulation of cell cycle, DNA replication and spliceosome pathways, and downregulation of signal transduction pathways, including ErbB, NF-kappa B, VEGF, and MAPK signalling in SFPQ-ABL1-, compared to BCR-ABL1-expressing cells. SFPQ-ABL1 expression did not activate PI3K/AKT signalling and was associated with phosphorylation of G2/M cell cycle proteins. SFPQ-ABL1 was sensitive to navitoclax and S-63845 and promotes cell survival through upregulation of Mcl-1 and Bcl-xL. SFPQ-ABL1 has functionally distinct mechanisms by which it drives ALL, including subcellular localisation, proliferative capacity, and activation of cellular pathways, highlighting the role that fusion partners have in mediating the function of ABL1 fusions.
2022-05-20 | PXD028925 | Pride