Journal Article 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

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

35 ( 4 )  , p.e00228 , 2015-06
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 DrosophilaYeti homologues play crucial roles in chromatin remodelling organization, mammalian Bcnt/Cfdp1 (craniofacialdevelopmental protein 1) remains poorly understood. The protein, which lacks cysteine, is largely disordered andcomprises an acidic N-terminal region, a lysine/glutamic acid/proline-rich 40 aa sequence and BCNT-C. It showscomplex mobility on SDS/PAGE at ∼50 kDa, whereas its calculated molecular mass is ∼33 kDa. To characterize thismobility 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 thanthe protein expressed in Escherichia coli but faster than the endogenous protein on SDS/PAGE. Among seven in vivophosphorylation sites, Ser250, which resides in a region between disordered and ordered regions in BCNT-C, is heavilyphosphorylated and detected predominantly in the 49 kDa band. Together with experiments involving treatment withphosphatases and Ser250 substitutions, the results indicate that the complex behaviour of Bcnt/Cfdp1 on SDS/PAGEis 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-Care also acetylated in vivo, revealing a protein regulated at multiple levels.

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