title: Phosphoproteomic analysis of mouse thymoma cells treated with tributyltin oxide: TBTO affects proliferation and energy sensing pathways.
abstract: We report the results of phosphoproteomic analysis of mouse thymoma cells treated with tributyltin oxide (TBTO), an immunotoxic compound. After cell lysis, phosphoproteins were isolated using Phosphoprotein Purification Kit, separated by SDS-PAGE and subsequently digested with trypsin. Phosphopeptides were enriched employing titanium dioxide, and the obtained fractions were analyzed by nano-LC-MS/MS. A total of 160 phosphoproteins and 328 phosphorylation sites were identified in thymoma cells. Among the differentially phosphorylated proteins identified in TBTO-treated cells were key enzymes, which catalyze rate-limiting steps in pathways that are sensitive to cellular energy status. These proteins included acetyl-CoA carboxylase isoform 1, which catalyzes the rate-limiting step of fatty acid synthesis. Another enzyme was glutamine: fructose-6-phosphate amidotransferase, GFAT1, the first and rate-limiting enzyme for the hexoamine synthesis pathway. Pyruvate dehydrogenase (PDH), a multicomplex enzyme that catalyzes the rate-limiting step of aerobic oxidation of fuel carbohydrates, was identified in both TBTO-treated and control cells; however, phosphorylation at residue S293, known to inhibit PDH activity, was identified only in control cells. A lower expression level of ribosomal protein S6 kinase 1, a downstream kinase of the mammalian target of rapamycin signaling pathway implicated in protein synthesis through phosphorylation of 40 ribosomal S6, was observed in the treated cells. Giant kinases like AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKAR1A), which are known to mediate the phosphorylation of these enzymes, were identified in TBTO-treated cells. Downregulation of proteins, such as MAPK, matrin-3 and ribonucleotide reductase, subunit RRM2, which are implicated in cell proliferation, was also observed in TBTO-treated cells. Together, the results show that TBTO affects proliferation and energy sensor pathways.
authors.affiliation: National Institute for Public Health and the Environment (RIVM), NL-3720 BA Bilthoven, The Netherlands. ahmed.osman@rivm.nl
authors.forename: Ahmed M
authors.initials: AM
authors.lastname: Osman
authors.forename: Henk
authors.initials: H
authors.lastname: van Loveren
mesh_terms: D000103:Acetyl-CoA Carboxylase / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D002851:Chromatography, High Pressure Liquid; D004734:Energy Metabolism / Q000187:drug effects*; D004785:Environmental Pollutants / Q000633:toxicity*; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects; D051379:Mice; D055616:Microtechnology; D019884:Nitrogenous Group Transferases / Q000737:chemistry / Q000378:metabolism; D034521:Nuclear Matrix-Associated Proteins / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D040901:Proteomics / Q000379:methods; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D013950:Thymus Gland / Q000187:drug effects* / Q000378:metabolism; D014220:Trialkyltin Compounds / Q000633:toxicity*
chemical_list: D004785:Environmental Pollutants; D007527:Isoenzymes; D034521:Nuclear Matrix-Associated Proteins; D010750:Phosphoproteins; D011768:Pyruvate Dehydrogenase Complex; D016601:RNA-Binding Proteins; D014220:Trialkyltin Compounds; C573692:matrin-3 protein, mouse; C005961:bis(tri-n-butyltin)oxide; D019884:Nitrogenous Group Transferases; C488523:Gfpt1 protein, mouse; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D000103:Acetyl-CoA Carboxylase