Mammalian Bcnt/Cfdp1, a potential epigenetic factor characterized by an acidic stretch in the disordered N-terminal and Ser250 phosphorylation in the conserved C-terminal regions

Biosci Rep. 2015 Jun 12;35(4):e00228. doi: 10.1042/BSR20150111.

Abstract

The BCNT (Bucentaur) superfamily is classified by an uncharacteristic conserved sequence of ∼80 amino acids (aa) at the C-terminus, BCNT-C (the conserved C-terminal region of Bcnt/Cfdp1). Whereas the yeast Swc5 and Drosophila Yeti homologues play crucial roles in chromatin remodelling organization, mammalian Bcnt/Cfdp1 (craniofacial developmental protein 1) remains poorly understood. The protein, which lacks cysteine, is largely disordered and comprises an acidic N-terminal region, a lysine/glutamic acid/proline-rich 40 aa sequence and BCNT-C. It shows complex mobility on SDS/PAGE at ∼50 kDa, whereas its calculated molecular mass is ∼33 kDa. To characterize this mobility discrepancy and the effects of post-translational modifications (PTMs), we expressed various deleted His-Bcnt in E. coli and HEK cells and found that an acidic stretch in the N-terminal region is a main cause of the gel shift. Exogenous BCNT/CFDP1 constitutively expressed in HEK clones appears as a doublet at 49 and 47 kDa, slower than the protein expressed in Escherichia coli but faster than the endogenous protein on SDS/PAGE. Among seven in vivo phosphorylation sites, Ser(250), which resides in a region between disordered and ordered regions in BCNT-C, is heavily phosphorylated and detected predominantly in the 49 kDa band. Together with experiments involving treatment with phosphatases and Ser(250) substitutions, the results indicate that the complex behaviour of Bcnt/Cfdp1 on SDS/PAGE is caused mainly by an acidic stretch in the N-terminal region and Ser(250) phosphorylation in BCNT-C. Furthermore, Bcnt/Cfdp1 is acetylated in vitro by CREB-binding protein (CBP) and four lysine residues including Lys(268) in BCNT-C are also acetylated in vivo, revealing a protein regulated at multiple levels.

Keywords: Bucentaur (BCNT) superfamily; disordered protein; mass spectroscopy; post-translational modification (PTM); sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) mobility; the conserved C-terminal region of Bcnt/Cfdp1 (BCNT-C) domain.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylation
  • Amino Acid Substitution
  • CREB-Binding Protein / chemistry
  • CREB-Binding Protein / genetics
  • CREB-Binding Protein / metabolism
  • Epigenesis, Genetic
  • HEK293 Cells
  • Humans
  • Mutation, Missense
  • Nuclear Proteins
  • Phosphoproteins / chemistry*
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • Phosphorylation / physiology
  • Protein Structure, Tertiary

Substances

  • CFDP1 protein, human
  • Nuclear Proteins
  • Phosphoproteins
  • CREB-Binding Protein
  • CREBBP protein, human