title: Establishment of an ectopically expressed and functional PRMT1 for proteomic analysis of arginine-methylated proteins.
abstract: Protein arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), plays crucial roles in a variety of cellular processes. Mammalian PRMT1 exists in a large protein complex in cells, which has been implied in modulating the regulatory and catalytic properties of this enzyme. Establishment of a mammalian comparative approach will help to identify putative substrates of PRMT1 in an authentic condition. Here, we showed that ectopically expressed PRMT1 in mammalian HEK293 cells not only exhibited catalytic properties comparable to the endogenous enzyme but also existed in a functional complex together with endogenous PRMT1 and thus functioned as an endogenous counterpart. In addition, the measured methylation level of cellular proteins using a tritium-labeled methyl donor was accordingly enhanced upon ectopic expression of PRMT1. Subsequent proteomic analysis with such PRMT1-expressing cells allowed us to identify several known and putative methylated proteins. In vitro methylation of selected proteins, eukaryotic translation initiation factor 4A-I and vimentin, by cellular PRMT1 was shown. Together, we have demonstrated the functional equivalence of ectopically expressed PRMT1 in HEK293 cells and its application to systematically identify the substrate proteins in a mammalian cell context.
authors.affiliation: Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Yuan-I
authors.initials: YI
authors.lastname: Chang
authors.forename: Sheng-Wei
authors.initials: SW
authors.lastname: Lin
authors.forename: Yi-Ying
authors.initials: YY
authors.lastname: Chiou
authors.forename: Jung-Sung
authors.initials: JS
authors.lastname: Sung
authors.forename: Lee-Chun
authors.initials: LC
authors.lastname: Cheng
authors.forename: Yu-Ling
authors.initials: YL
authors.lastname: Lu
authors.forename: Kuang-Hui
authors.initials: KH
authors.lastname: Sun
authors.forename: Keejong
authors.initials: K
authors.lastname: Chang
authors.forename: Chao-Hsiung
authors.initials: CH
authors.lastname: Lin
authors.forename: Wey-Jinq
authors.initials: WJ
authors.lastname: Lin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional; D039601:Eukaryotic Initiation Factor-4A / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006388:Hemagglutinins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D008745:Methylation; D008969:Molecular Sequence Data; D011484:Protein-Arginine N-Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D014746:Vimentin / Q000737:chemistry / Q000378:metabolism
chemical_list: D006388:Hemagglutinins; D011993:Recombinant Fusion Proteins; D014746:Vimentin; D001120:Arginine; C541817:PRMT1 protein, rat; D011484:Protein-Arginine N-Methyltransferases; D039601:Eukaryotic Initiation Factor-4A