title: 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.
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.
authors.affiliation: Faculty of Pharmacy, Iwaki Meisei University, Iwaki 970-8551, Japan siwast@iwakimu.ac.jp.
authors.forename: Shintaro
authors.initials: S
authors.lastname: Iwashita
authors.affiliation: Center for Sustainable Resource Science, RIKEN, Wako 351-0198, Japan.
authors.forename: Takehiro
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Research Center for Radiation Emergency Medicine, National Institute of Radiological Sciences, Chiba 263-8555, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yasuda
authors.affiliation: Institute of Neuroscience, Tokushima Bunri University, Sanuki 769-2193, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Nakashima
authors.affiliation: Mitsubishi Kagaku Institute of Life Sciences, Machida 194-0031, Japan.
authors.forename: Taiichi
authors.initials: T
authors.lastname: Sakamoto
authors.affiliation: Mitsubishi Kagaku Institute of Life Sciences, Machida 194-0031, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Kohno
authors.affiliation: Tsukuba Primate Research Center, National Institute of Biomedical Innovation, Tsukuba 305-0843, Japan.
authors.forename: Ichiro
authors.initials: I
authors.lastname: Takahashi
authors.affiliation: *Department of Project Program, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan.
authors.forename: Takayasu
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Faculty of Pharmacy, Iwaki Meisei University, Iwaki 970-8551, Japan.
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Ohno-Iwashita
authors.affiliation: †Laboratory Center for Proteomics Research, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.
authors.forename: Shinobu
authors.initials: S
authors.lastname: Imajoh-Ohmi
authors.affiliation: Institute of Neuroscience, Tokushima Bunri University, Sanuki 769-2193, Japan.
authors.forename: Si-Young
authors.initials: SY
authors.lastname: Song
authors.affiliation: Center for Sustainable Resource Science, RIKEN, Wako 351-0198, Japan.
authors.forename: Naoshi
authors.initials: N
authors.lastname: Dohmae
mesh_terms: D000107:Acetylation; D019943:Amino Acid Substitution; D050882:CREB-Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044127:Epigenesis, Genetic; D057809:HEK293 Cells; D006801:Humans; D020125:Mutation, Missense; D009687:Nuclear Proteins; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D017434:Protein Structure, Tertiary
chemical_list: C104422:CFDP1 protein, human; D009687:Nuclear Proteins; D010750:Phosphoproteins; D050882:CREB-Binding Protein; C494363:CREBBP protein, human
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