Tandem mass spectrometry for the examination of the posttranslational modifications of high-mobility group A1 proteins: symmetric and asymmetric dimethylation of Arg25 in HMGA1a protein.
High-mobility group (HMG) A1a and A1b proteins are among a family of HMGA proteins that bind to the minor groove of AT-rich regions of DNA. Here we employed tandem mass spectrometry and determined without ambiguity the sites of phosphorylation and the nature of methylation of HMGA1 proteins that were isolated from the PC-3 human prostate cancer cells. We showed by LC-MS/MS that Ser101 and Ser102 were completely phosphorylated in HMGA1a protein, whereas only a portion of the protein was phosphorylated at Ser98. We also found that the HMGA1b protein was phosphorylated at the corresponding sites, that is, Ser90, Ser91 and Ser87. In addition, Arg25, which is within the first DNA-binding AT-hook domain of HMGA1a, was both mono- and dimethylated. Moreover, both symmetric and asymmetric dimethylations were observed. The closely related HMGA1b protein, however, was not methylated. The unambiguous identification of the sites of phosphorylation and the nature of methylation facilitates the future examination of the biological implications of the HMGA1 proteins.
Zou
Yan
Y
Department of Chemistry-027, University of California at Riverside, Riverside, California 92521-0403, USA.
Wang
Yinsheng
Y
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D025741:HMGA1a Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D025742:HMGA1b Protein / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008745:Methylation; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012694:Serine / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D025742:HMGA1b Protein; D025741:HMGA1a Protein; D012694:Serine; D004247:DNA; D001120:Arginine