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

Shintaro Iwashita, Takehiro Suzuki, Takeshi Yasuda, Kentaro Nakashima, Taiichi Sakamoto, Toshiyuki Kohno, Ichiro Takahashi, Takayasu Kobayashi, Yoshiko Ohno-Iwashita, Shinobu Imajoh-Ohmi, Si Young Song, Naoshi Dohmae

Research output: Contribution to journalArticlepeer-review

6 Citations (Scopus)

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, Ser250, 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 Ser250 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 Ser250 phosphorylation in BCNT-C. Furthermore, Bcnt/Cfdp1 is acetylated in vitro by CREB-binding protein (CBP) and four lysine residues including Lys268 in BCNT-C are also acetylated in vivo, revealing a protein regulated at multiple levels.

Original languageEnglish
Article numbere00228
JournalBioscience Reports
Volume35
Issue number4
DOIs
Publication statusPublished - 2015

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

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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