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PXD039689 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleIdentification of phosphorylation sites regulated by lysophosphatidic acid (LPA) in OCMI91s ovarian cancer cells
DescriptionElevated levels of lysophosphatidic acid (LPA) species accumulate in the ascites of ovarian high grade serous cancer (HGSC) and are associated with a short relapse-free survival. LPA is known to support the metastatic spread of cancer cells by activating a multitude of signaling pathways through G-protein-coupled receptors of the LPAR family. Systematic unbiased analyses of the LPA-regulated signal transduction network in ovarian cancer cells have, however, not been reported to date. Methods: LPA-induced signaling pathways were identified by phosphoproteomics of both patient-derived cells and the HGSC cell line OVCAR8, RNA sequencing, pharmacological inhibition, measurements of intracellular Ca2+ and cAMP levels as well as cell imaging. The function of LPARs and downstream signaling components in migration and the formation of entotic cell-in-cell structures was analyzed by applying selective pharmacological inhibitors and RNA interference. Results: Transcriptional signaling by LPA is mainly mediated by LPAR1 to target genes promoting cell motility and migration via multiple cooperating pathways, including PKC, PKD1 and ERK1/2, as demonstrated for IL6 and THBS1 genes. Likewise, cytoplasmic signaling targeting actomyosin is mediated by interconnected phosphorylation-driven pathways triggered prominently by LPAR1. A central component of these signaling pathways is the protein phosphatase 1 regulatory subunit MYPT1, which is a negative regulator of myosin light chain 2 (MYL2). MYPT1 is negatively regulated by PKC- and ERK-mediated phosphorylation in response to LPA, and is indispensable for LPA-induced actomyosin-dependent cell migration and entosis (cell-in-cell invasion). We further show a novel LPAR2-DOCK7 pathway to be essential for the induction of entosis. Conclusion: The LPAR1-ERK/PKC-MYPT1 axis is a critical pathway of LPA-triggered cytoskeletal changes, cell migration and entotic cell-in-cell invasion, pointing to MYPT1 as a promising candidate for therapeutic intervention. For phosphoproteomic analyses cells were treated with LPA, antagonists or solvent at least in triplicate as described above and lysed in 100 mM Tris pH 7.6, 4% SDS, PhosSTOP (Roche, #4906845001), Protease Inhibitor Cocktail (Sigma, P8340). Analysis of OCMI91s lysates were performed as described [PMID: 19029910]. The protocol was modified to use 12 ug of peptide per channel for TMT labeling, resulting in 500 ug multiplexed samples. LC-MS2 analysis was performed on 50% of the corresponding sample material rehydrated in 0.1% formic acid as published [PMID: 19029910]. Detailed information on instrumentation parametrization was extracted and summarized using MARMoSET [PMID: 310976732] and is included in the repository-deposited data set. Peptide/spectrum matching as well as TMT quantitation was performed using the MaxQuant suit of algorithms (versions, [PMID: 19029910]) against the human canonical and isoforms Uniprot database (downloaded 20190819, 173199 entries). Mass spectrometric raw data along with documentation of instrumentation parameters governing its acquisition as well as MaxQuant parameters employed are deposited. Data were filtered for rows containing no zero values, and analyzed by paired Student's t-test (blocking on replicate set / phosphoproteome analysis chip). Thresholds were set according to the dynamic range of the assay to FC >1.2x or <0.83x and p <0.1.
ReviewLevelNon peer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportUnsupported dataset by repository
PrimarySubmitterJohannes Graumann
SpeciesList scientific name: Homo sapiens; common name: human; NCBI TaxID: 9606;
ModificationListPhospho; Acetyl; Carbamidomethyl
InstrumentQ Exactive HF
Dataset History
RevisionDatetimeStatusChangeLog Entry
02023-01-26 08:33:00ID requested
12023-03-18 16:08:58announced
Publication List
no publication
Keyword List
submitter keyword: lysophosphatidic acid, phosphoproteome, signal transduction, protein kinases, ovarian cancer metastasis, actomyosin dynamics, entosis, protein phosphatase 1 regulatory subunit MYPT1, DOCK7
Contact List
Rolf Mueller
contact affiliationCenter for Tumor Biology and Immunology (ZTI), Philipps University
contact emailrolf.mueller@uni-marburg.de
lab head
Johannes Graumann
contact affiliationPhilipps-University Marburg
contact emailjohannes.graumann@uni-marburg.de
dataset submitter
Full Dataset Link List
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Dataset FTP location
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