A mass spectrometric study on the in vitro methylation of HMGA1a and HMGA1b proteins by PRMTs: methylation specificity, the effect of binding to AT-rich duplex DNA, and the effect of C-terminal phosphorylation

Biochemistry. 2007 Jul 3;46(26):7896-906. doi: 10.1021/bi6024897. Epub 2007 Jun 6.

Abstract

HMGA1a and HMGA1b are members of one subfamily of non-histone chromosomal high-mobility group (HMG) proteins. They bind to various DNA-related substrates, including the minor groove of AT-rich duplex DNA sequences, and have been postulated to be architectural transcription factors functioning in a wide variety of cellular processes. Post-translational modifications of HMGA1 proteins, such as phosphorylation, acetylation, and methylation, are widely observed in tumor cells in vivo and correlated with the modulation of protein function. Here, we investigated the in vitro methylation of recombinant human HMGA1a and HMGA1b proteins by three members of the protein arginine methyltransferase (PRMT) family: PRMT1, PRMT3, and PRMT6. PRMT1 and PRMT3 showed a preference for methylating arginine residues in the first AT-hook of HMGA1 proteins, whereas PRMT6 methylated mainly residues in the second AT-hook. The initial sites of methylation catalyzed by PRMT1 and PRMT3 were mapped by tandem mass spectrometry to be Arg25 and Arg23, respectively, while we confirmed that the initial sites of methylation catalyzed by PRMT6 were at Arg57 and Arg59. Our results also revealed that binding of HMGA1 proteins to AT-rich duplex DNA, but not GC-rich duplex DNA, significantly inhibited the methylation efficiency of all of the PRMTs toward HMGA1 proteins. Moreover, C-terminal constitutive phosphorylation of HMGA1 proteins induced by protein kinase CK2 did not have any appreciable effect on the in vitro methylation of HMGA1. Our results suggest that PRMT1 might be involved in the previously reported methylation of Arg25 in HMGA1a in vivo.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • DNA / metabolism*
  • HMGA1a Protein / metabolism*
  • HMGA1b Protein / metabolism*
  • Humans
  • Methylation
  • Nuclear Proteins / metabolism
  • Phosphorylation
  • Protein-Arginine N-Methyltransferases / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • Repressor Proteins / metabolism*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Nuclear Proteins
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • HMGA1b Protein
  • HMGA1a Protein
  • DNA
  • PRMT1 protein, human
  • PRMT3 protein, human
  • PRMT6 protein, human
  • Protein-Arginine N-Methyltransferases