Dataset Information

Elucidating Fibroblast Growth Factor-induced kinome dynamics using targeted mass spectrometry and dynamic modeling

ABSTRACT: Fibroblast growth factors (FGFs) are paracrine or endocrine signaling proteins that when activated by their ligands, can elicit a wide range of healthy and disease-related processes such as cell proliferation and the epithelial-to-mesenchymal transition (EMT). The detailed molecular pathway dynamics that coordinate these responses have remained unclear. To elucidate and monitor these dynamics we stimulated MCF-7 breast cancer cells with either FGF2, 3, 4, 10, or 19. Following activation of the receptor, we quantified the kinase activity dynamics of 44 kinases using a targeted mass spectrometry assay. Our system-wide kinase activity data, supplemented with (phopsho)proteomics data, reveal ligand-dependent distinct pathway dynamics, elucidate the involvement of not earlier reported kinases such as MARK, and revise some of the pathway effects on biological outcomes. In addition, logic-based dynamic modeling of the kinome dynamics further verifies the biological goodness-of-fit of the predicted models and reveals tight regulation of the RAF kinase family.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Tim Veth

LAB HEAD: Maarten Altelaar

PROVIDER: PXD038808 | Pride | 2023-07-02

REPOSITORIES: Pride

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Publications

Elucidating Fibroblast Growth Factor-Induced Kinome Dynamics Using Targeted Mass Spectrometry and Dynamic Modeling.

Veth Tim S TS Francavilla Chiara C Heck Albert J R AJR Altelaar Maarten M

Molecular & cellular proteomics : MCP 20230615 8

Fibroblast growth factors (FGFs) are paracrine or endocrine signaling proteins that, activated by their ligands, elicit a wide range of health and disease-related processes, such as cell proliferation and the epithelial-to-mesenchymal transition. The detailed molecular pathway dynamics that coordinate these responses have remained to be determined. To elucidate these, we stimulated MCF-7 breast cancer cells with either FGF2, FGF3, FGF4, FGF10, or FGF19. Following activation of the receptor, we q ...[more]

PMID: 37328066

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Project description:Astroglia, the star-shaped cells found throughout the central nervous system are the most numerous cell type in the brain. The astroglial response to injury, generically termed reactive astrogliosis, is classically defined by cellular hypertrophy and process extension, increased glial filament production, and some degree of proliferation. Astrogliosis and its functional outcomes, glial scar formation and neuroinflammation, are often viewed as detrimental to recovery. However, increasing evidence points to neuroprotective roles of reactive astroglia in the setting of brain injury. Therapeutic modulation of this double-edged sword will require detailed knowledge of mechanisms by which specific signals induce detrimental and beneficial phenotypes. This proposal is is a logical extension of a published, peer reviewed study (Heffron DS and Mandell JW, Opposing roles of ERK and p38 MAP kinases in FGF2-induced astroglial process extension. Mol Cell Neurosci 2005, 28:779-90). The downstream gene expression changes underlying these pathway-specific responses are largely unknown. The data generated from the proposed project will delineate pathway-specific gene expression responses of astroglia to fibroblast growth factors. This information will allow a rational approach to pharmacological approaches to modulate reactive astrogliosis in brain and spinal cord injury. Determine the astroglial gene expression program elicited by fibroblast growth factor-2, and dissect specific contributions of the ERK and p38 MAP kinase pathways using highly specific pharmacological inhibitors. FGF2, acting via two distinct intracellular signaling pathways, ERK- and p38 MAPK, leads to pathway-specific gene expression changes in astrocytes which promote the morphological plasticity (ERK-dependent) and pro-inflammatory properties (p38-dependent) of reactive astroglia. Cell culture; Neonatal primary astrocyte cultures are prepared exactly as previously described (Heffron and Mandell, 2005), using slight modifications of established protocols (McCarthy and de Vellis, 1980). Astrocytes prepared with these methods comprised greater than 95% of cell cultures as determined by GFAP staining. Microglia are present as a contaminating cell type at a percentage of 3.6 Â± 0.9% as determined by CD11B staining. For process induction experiments, cells are trypsinized and replated in DMEM with 1% fetal bovine serum. This low serum concentration is necessary for good adhesion of the cells. Pharmacological inhibitors are dissolved in DMSO and added 2 h later. P38 MAP kinase inhibitors SB202190 (Calbiochem) and the MEK inhibitor U0126 (Promega) are used at 20 ÂµM. We previously determined that the inactive analog of SB 202190, SB202474 (Calbiochem) had no effects on pathway activation or astrocyte morphology. Therefore, the inactive drug analog will not be used in the array experiments. Final DMSO concentration in the drug treatments and vehicle controls is 0.2%. Human recombinant FGF2 (obtained from the National Cancer Institute Preclinical Repository) is used at 25 ng/ml, based on extensive dose/response analyses in our published work.

Dataset Information

Elucidating Fibroblast Growth Factor-induced kinome dynamics using targeted mass spectrometry and dynamic modeling

Publications

Elucidating Fibroblast Growth Factor-Induced Kinome Dynamics Using Targeted Mass Spectrometry and Dynamic Modeling.

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