title: Mass spectrometric analysis of the phosphorylation levels of the SWI/SNF chromatin remodeling/tumor suppressor proteins ARID1A and Brg1 in ovarian clear cell adenocarcinoma cell lines.
abstract: Protein phosphorylation is one of the major factors involved in tumor progression and malignancy. We performed exploratory studies aimed at identifying phosphoproteins characteristic to cell lines derived from ovarian clear cell adenocarcinoma (CCA), a highly malignant type of ovarian cancer. Comparative phosphoproteome analysis revealed that the phosphopeptides of five SWI/SNF chromatin remodeling/tumor suppressor components, including ARID1A and BRG1, were significantly down-regulated in CCA cells. We then quantitatively determined the phosphorylation levels of ARID1A and BRG1 by immunoprecipitation-multiple reaction monitoring (IP-MRM) that we used for analysis of the cognate phospho- and nonphosphopeptides of low-abundance proteins. The phosphorylation level of Brg1 at Ser1452 was down-regulated in CCA cells, whereas the phosphorylation level of ARID1A at Ser696 did not significantly differ between CCA and non-CCA cells. These results were consistent with the results of immunoblotting showing that Brg1 levels were comparable, but ARID1A levels were lower, in CCA cells relative to non-CCA cells. This is the first report to demonstrate reduced phosphorylation of Brg1 in CCA-derived cells. Our data also indicated that the IP-MRM/MS method we used is a powerful tool for validation of the phosphoproteins detected by shotgun analysis of phosphopeptides.
authors.affiliation: Advanced Medical Research Center and Graduate School of Medical Life Science, Yokohama City University , Fukuura 3-9, Kanazawa, Yokohama 236-0004, Japan.
authors.forename: Ayuko
authors.initials: A
authors.lastname: Kimura
authors.forename: Noriaki
authors.initials: N
authors.lastname: Arakawa
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hirano
mesh_terms: D018262:Adenocarcinoma, Clear Cell / Q000378:metabolism* / Q000473:pathology; D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D042002:Chromatin Assembly and Disassembly; D004265:DNA Helicases / Q000032:analysis / Q000378:metabolism*; D004268:DNA-Binding Proteins; D015536:Down-Regulation; D005260:Female; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000032:analysis / Q000378:metabolism*; D010051:Ovarian Neoplasms / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation; D020543:Proteome / Q000032:analysis; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014157:Transcription Factors / Q000032:analysis / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism
chemical_list: C112476:ARID1A protein, human; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D020543:Proteome; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C084108:SMARCA4 protein, human; D004265:DNA Helicases
keywords: ARID1A; Brg1; clear cell adenocarcinoma; multiple reaction monitoring; ovarian cancer; phosphorylation