| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2005: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2004: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
Participation of multiple kinases in regulation of the binding of lamin B receptor (LBR) to chromatin was previously suggested (Takano et al. 2002. Eur.J.Biochem. 269, 943-953). To identify these kinases, regulation of the binding of LBR to sperm chromatin was studied using a cell-cycle dependent Xenopus egg extract in vitro. The binding of LBR to chromatin pretreated with an S-phase extract was suppressed by incubation with an M-phase extract. Enzyme inhibitor experiments revealed that multiple kinases participate in the suppression. One of these kinases was shown to be cdc2 kinase using a specific inhibitor and protein depletion with beads bearing p13, which specifically binds cdc2 kinase. Experiments involving a mutant LBR showed that the phosphorylation of serine 71 by cdc2 kinase is responsible for the suppression.
Emerin is the gene product of STA whose mutations cause Emery-Dreifuss muscular dystrophy. 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, ^<49>Ser, ^<66>Ser, ^<67>Thr, ^<120>Ser and ^<175>Ser, 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 MS/MS sequencing. An in vitro binding assay involving an emerin S175A point mutant protein suggested that phosphorylation at ^<175>Ser regulates the dissociation of emerin from BAF.
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