Tandem Mass Spectrometry for the Examination of the Posttranslational Modifications of High-Mobility Group Al Proteins:Symmetric and Asymmetric imethylation of Arg-25 in HMGAla Protein

摘要

High-mobility group (HMG) Ala and Alb 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 memylation of HMGA1 proteins that were isolated from the PC-3 human prostate cancer cells.We showed by LC-MS/MS that SerlOl and Serl02 were completely phosphorylated in HMGAla protein,whereas only a portion of the protein was phosphorylated at Ser98.We also found that the HMGAlb 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 HMGAla,was both mono- and dimethylated.Moreover,both symmetric and asymmetric dimethylations were observed.The closely related HMGAlb 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.