Dissociation of Emerin from Barrier-to-autointegration Factor Is Regulated through Mitotic Phosphorylation of Emerin in a Xenopus Egg Cell-free System

• Published in: The Journal of biological chemistry Vol. 280; no. 48; pp. 39925 - 39933
• Main Authors: Hirano, Yasuhiro, Segawa, Masashi, Ouchi, Fumiko S., Yamakawa, Yoshio, Furukawa, Kazuhiro, Takeyasu, Kunio, Horigome, Tsuneyoshi
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02.12.2005; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Cell Cycle; Cell-Free System; Chromatin - chemistry; Chromatin - metabolism; Chymotrypsin - chemistry; Cytosol - metabolism; DNA-Binding Proteins - metabolism; Glutathione Transferase - metabolism; Humans; Mass Spectrometry; Membrane Proteins - metabolism; Mitosis; Muscular Dystrophy, Emery-Dreifuss - genetics; Muscular Dystrophy, Emery-Dreifuss - metabolism; Mutation; Nuclear Proteins - metabolism; Oocytes - metabolism; Peptides - chemistry; Phosphopeptides - chemistry; Phosphorylation; Point Mutation; Protein Binding; Recombinant Fusion Proteins - chemistry; Serine - chemistry; Spectrometry, Mass, Electrospray Ionization; Thymopoietins - metabolism; Trypsin - chemistry; Xenopus
• DOI: 10.1074/jbc.M503214200
• ISSN: 0021-9258

Emerin is the gene product of STA whose mutations cause Emery-Dreifuss muscular dystrophy. It is an inner nuclear membrane protein and phosphorylated in a cell cycle-dependent manner. However, the means of phosphorylation of emerin are poorly understood. We investigated the regulation mechanism for the binding of emerin to chromatin, focusing on its cell cycle-dependent phosphorylation in a Xenopus egg cell-free system. It was shown that emerin dissociates from chromatin depending on mitotic phosphorylation of the former, and this plays a critical role in the dissociation of emerin from barrier-to-autointegration factor (BAF). Then, we analyzed the mitotic phosphorylation sites of emerin. Emerin was strongly phosphorylated in an M-phase Xenopus egg cell-free system, and five phosphorylated sites, Ser49, Ser66, Thr67, Ser120, and Ser175, were identified on analysis of chymotryptic and tryptic emerin peptides using a phosphopeptide-concentrating system coupled with a Titansphere column, which specifically binds phosphopeptides, and tandem mass spectrometry sequencing. An in vitro binding assay involving an emerin S175A point mutant protein suggested that phosphorylation at Ser175 regulates the dissociation of emerin from BAF.