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

Author:

Iwashita Shintaro1,Suzuki Takehiro2,Yasuda Takeshi3,Nakashima Kentaro4,Sakamoto Taiichi5,Kohno Toshiyuki5,Takahashi Ichiro6,Kobayashi Takayasu7,Ohno-Iwashita Yoshiko1,Imajoh-Ohmi Shinobu8,Song Si-Young4,Dohmae Naoshi2

Affiliation:

1. Faculty of Pharmacy, Iwaki Meisei University, Iwaki 970-8551, Japan

2. Center for Sustainable Resource Science, RIKEN, Wako 351-0198, Japan

3. Research Center for Radiation Emergency Medicine, National Institute of Radiological Sciences, Chiba 263-8555, Japan

4. Institute of Neuroscience, Tokushima Bunri University, Sanuki 769-2193, Japan

5. Mitsubishi Kagaku Institute of Life Sciences, Machida 194-0031, Japan

6. Tsukuba Primate Research Center, National Institute of Biomedical Innovation, Tsukuba 305-0843, Japan

7. *Department of Project Program, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan

8. †Laboratory Center for Proteomics Research, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan

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.

Publisher

Portland Press Ltd.

Subject

Cell Biology,Molecular Biology,Biochemistry,Biophysics

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