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• W2052944900 abstract "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. 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. The nuclear envelope (NE) 2The abbreviations used are: NEnuclear envelopeGSTglutathione S-transferaseΔTMGST-fused fragment comprising amino acid residues 1–213 of human emerinΔLTGST-fused fragment comprising amino acid residues 37–213 of human emerinBAFbarrier-to-autointegration factorLAPlamina-associated polypeptideLBRlamin B receptorMCM-phase Xenopus egg cytosol fractionSCS-phase Xenopus egg cytosol fractionMALDI-TOF MSmatrix-assisted laser desorption ionization-time of flight mass spectrometryERendoplasmic reticulumESI-IT MSelectrospray ionization-ion trap mass spectrometryCHCAα-cyano-4-hydroxycinnamic acidHFhydrofluoric acidCBBCoomassie Brilliant Blue. is a highly dynamic structure that disassembles at the onset of mitosis and reassembles on the surface of chromatin during telophase in vertebrates. These changes of NE are crucial for cell cycle progression. The NE consists of an outer nuclear membrane, inner nuclear membrane, nuclear pore complex, and nuclear lamina. The inner nuclear membrane contains integral membrane proteins, i.e. lamin B receptor (LBR), lamina-associated polypeptide-2β (LAP2β), emerin, MAN1, and others, which interact with DNA and/or chromatin, and these proteins are proposed to participate in nuclear membrane targeting to chromatin at an early stage of nuclear assembly (1Gant T.M. Wilson K.L. Annu. Rev. Cell. Dev. Biol. 1997; 13: 669-695Crossref PubMed Scopus (200) Google Scholar). The interactions between some of the inner nuclear membrane proteins and chromatin are regulated through phosphorylation of these inner nuclear membrane proteins. The phosphorylation mechanisms for LBR and LAP2α and 2β are well understood (2Nikolakaki E. Meier J. Simos G. Georgatos S.D. Giannakouros T. J. Biol. Chem. 1997; 272: 6208-6213Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 3Takano M. Koyama Y. Ito H. Hoshino S. Onogi H. Hagiwara M. Furukawa K. Horigome T. J. Biol. Chem. 2004; 279: 13265-13271Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 4Foisner R. Gerace L. Cell. 1993; 73: 1267-1279Abstract Full Text PDF PubMed Scopus (451) Google Scholar, 5Dreger M. Otto H. Neubauer G. Mann M. Hucho F. Biochemistry. 1999; 38: 9426-9434Crossref PubMed Scopus (32) Google Scholar, 6Martins S.B. Marstad A. Collas P. Biochemistry. 2003; 42: 10456-10461Crossref PubMed Scopus (5) Google Scholar, 7Gajewski A. Csaszar E. Foisner R. J. Biol. Chem. 2004; 279: 35813-35821Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). LBR directly binds to DNA in vitro and dissociates on phosphorylation by cdc2 kinase and other kinase(s) in a mitotic egg extract (3Takano M. Koyama Y. Ito H. Hoshino S. Onogi H. Hagiwara M. Furukawa K. Horigome T. J. Biol. Chem. 2004; 279: 13265-13271Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). LAP2β binds to lamin B1 and chromatin, and cell cycle-dependent phosphorylation of LAP2β cancels this binding (4Foisner R. Gerace L. Cell. 1993; 73: 1267-1279Abstract Full Text PDF PubMed Scopus (451) Google Scholar). Phosphorylation of these inner nuclear proteins, therefore, is likely to be one of the key mechanisms that control the interactions between the inner nuclear proteins and components of the nuclear lamina as well as chromatin. In this study, we focused on the mitotic phosphorylation of emerin, one of the inner nuclear membrane proteins. nuclear envelope glutathione S-transferase GST-fused fragment comprising amino acid residues 1–213 of human emerin GST-fused fragment comprising amino acid residues 37–213 of human emerin barrier-to-autointegration factor lamina-associated polypeptide lamin B receptor M-phase Xenopus egg cytosol fraction S-phase Xenopus egg cytosol fraction matrix-assisted laser desorption ionization-time of flight mass spectrometry endoplasmic reticulum electrospray ionization-ion trap mass spectrometry α-cyano-4-hydroxycinnamic acid hydrofluoric acid Coomassie Brilliant Blue. Human emerin is a serine-rich protein exhibiting an apparent mass of 34 kDa on SDS-PAGE (8Bione S. Maestrini E. Rivella S. Mancini M. Regis S. Romeo G. Toniolo D. Nat. Genet. 1994; 8: 323-327Crossref PubMed Scopus (773) Google Scholar) and is phosphorylated in a cell cycle-dependent manner (9Ellis J.A. Craxton M. Yates J.R. Kendrick-Jones J. J. Cell Sci. 1998; 111: 781-792Crossref PubMed Google Scholar). Emerin belongs to the LEM (LAP2β, emerin, MAN1) protein family, whose members have approximately a 40-residue domain named the LEM (10Lin F. Blake D.L. Callebaut I. Skerjanc I.S. Holmer L. McBurney M.W. Paulin-Levasseur M. Worman H.J. J. Biol. Chem. 2000; 275: 4840-4847Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). These proteins directly bind to barrier-to-autointegration factor (BAF) (11Furukawa K. J. Cell Sci. 1999; 112: 2485-2492Crossref PubMed Google Scholar, 12Lee K.K. Haraguchi T. Lee R.S. Koujin T. Hiraoka Y. Wilson K.L. J. Cell Sci. 2001; 114: 4567-4573Crossref PubMed Google Scholar, 13Liu J. Lee K.K. Segura-Totten M. Neufeld E. Wilson K.L. Gruenbaum Y. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4598-4603Crossref PubMed Scopus (174) Google Scholar). BAF is a DNA-bridging protein with a dimer mass of 20 kDa and is highly conserved in metazoans, and the BAF interactions with both DNA and LEM proteins are critical for nuclear membrane targeting to chromatin and chromatin decondensation during nuclear assembly (14Segura-Totten M. Kowalski A.K. Craigie R. Wilson K.L. J. Cell Biol. 2002; 158: 475-485Crossref PubMed Scopus (154) Google Scholar). At the onset of mitosis, emerin disperses from the NE to the endoplasmic reticular (ER) network, and is re-localized to the surface of the central region of chromatin, called the “core” region, during telophase (15Tsuchiya Y. Hase A. Ogawa M. Yorifuji H. Arahata K. Eur. J. Biochem. 1999; 259: 859-865Crossref PubMed Scopus (62) Google Scholar, 16Haraguchi T. Koujin T. Segura-Totten M. Lee K.K. Matsuoka Y. Yoneda Y. Wilson K.L. Hiraoka Y. J. Cell Sci. 2001; 114: 4575-4585Crossref PubMed Google Scholar). An LEM domain deletion mutant of emerin cannot be re-localized to this region, suggesting that the binding of emerin to BAF through the LEM domain is essential for this recruitment (16Haraguchi T. Koujin T. Segura-Totten M. Lee K.K. Matsuoka Y. Yoneda Y. Wilson K.L. Hiraoka Y. J. Cell Sci. 2001; 114: 4575-4585Crossref PubMed Google Scholar). It is also known that emerin has many binding partners, including transcriptional repressors and intermediate filament proteins (17Bengtsson L. Wilson K.L. Curr. Opin. Cell Biol. 2004; 16: 73-79Crossref PubMed Scopus (111) Google Scholar, 18Fairley E.A. Kendrick-Jones J. Ellis J.A. J. Cell Sci. 1999; 112: 2571-2582Crossref PubMed Google Scholar, 19Clements L. Manilal S. Love D.R. Morris G.E. Biochem. Biophys. Res. Commun. 2000; 267: 709-714Crossref PubMed Scopus (201) Google Scholar, 20Lattanzi G. Cenni V. Marmiroli S. Capanni C. Mattioli E. Merlini L. Squarzoni S. Maraldi N.M. Biochem. Biophys. Res. Commun. 2003; 303: 764-770Crossref PubMed Scopus (75) Google Scholar, 21Holaska J.M. Kowalski A.K. Wilson K.L. PLoS Biol. 2004; 2: E231Crossref PubMed Scopus (174) Google Scholar, 22Mislow J.M. Holaska J.M. Kim M.S. Lee K.K. Segura-Totten M. Wilson K.L. McNally E.M. FEBS Lett. 2002; 525: 135-140Crossref PubMed Scopus (218) Google Scholar, 23Holaska J.M. Lee K.K. Kowalski A.K. Wilson K.L. J. Biol. Chem. 2003; 278: 6969-6975Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 24Wilkinson F.L. Holaska J.M. Zhang Z. Sharma A. Manilal S. Holt I. Stamm S. Wilson K.L. Morris G.E. Eur. J. Biochem. 2003; 270: 2459-2466Crossref PubMed Scopus (87) Google Scholar, 25Haraguchi T. Holaska J.M. Yamane M. Koujin T. Hashiguchi N. Mori C. Wilson K.L. Hiraoka Y. Eur. J. Biochem. 2004; 271: 1035-1045Crossref PubMed Scopus (119) Google Scholar). In particular, binding to A-type lamin is essential for the retention of emerin in the NE in the interphase. Furthermore, a deletion mutant of emerin residues 95–99 (Δ95–99), which causes Emery-Dreifuss muscular dystrophy and cannot bind to lamin A, exhibits aberrant cell cycle-dependent phosphorylation forms (9Ellis J.A. Craxton M. Yates J.R. Kendrick-Jones J. J. Cell Sci. 1998; 111: 781-792Crossref PubMed Google Scholar). The study also suggested that the phosphorylation of emerin regulates the binding of emerin to lamin A (9Ellis J.A. Craxton M. Yates J.R. Kendrick-Jones J. J. Cell Sci. 1998; 111: 781-792Crossref PubMed Google Scholar). Thus, we were interested in the cell cycle-dependent regulation of the binding of emerin to chromatin and BAF. We first examined the binding of emerin to chromatin by means of a binding assay involving a GST-fused N-terminal fragment of emerin and chromatin in a Xenopus egg cell-free system. We also analyzed the cell cycle-dependent phosphorylation states and sites of emerin. Phosphopeptides derived from emerin treated with a Xenopus egg mitotic cytosol were separated by means of a Titansphere column, and five phosphorylation sites were identified on mass spectrometry. Furthermore, an in vitro binding assay involving an emerin point mutant revealed that Ser175 phosphorylation is responsible for the dissociation of emerin from BAF. Construction of GST-fused Emerin Fragment Proteins and His6 Tag BAF—Cloning of the nucleoplasmic region of human emerin (ΔTM, amino acid residues 1–213) was performed by PCR. PCR was carried out with a human testis cDNA library using the following primers: 5′-CGGGATCCCCATGGACAACTAGCAGAT-3′ and 5′-CGGGATCCAGAGCACGGTTTTCAGG-3′. The PCR product was digested with BamHI and then inserted into the pBluescript II SK(–) or pGEX 3X vector (Novagen) at the BamHI site at the 3′ end of GST. To generate a point mutant with the serine at position 175 replaced with alanine (S175A-ΔTM), a GeneTailor mutagenesis kit (Invitrogen) was used according to the manufacturer's procedure. PCR was carried out with pBluescript II SK(–)-emerin ΔTM using the following primers: 5′-CTGTTTCGCCTCCAGGGCCTCCCTGGACC-3′ and 5′-CCTGGAGGCTGAAACAGGGCGGTAGTCGT-3′, followed by verification by DNA sequence analysis. The pBluescript II SK(–)-S175A-ΔTM was digested with BamHI, and the resulting fragment was inserted into the pGEX 5X-3 vector (Novagen) at the BamHI site. To construct the pGEX 3X-emerin ΔLT plasmid, pBluescript II SK(–)-emerin ΔTM was digested with BglII and BamHI, and the resulting fragment was inserted into the pGEX 3X vector using the BamHI site in the vector. The cDNA clone of human BAF (accession no. BC005942) was purchased from Invitrogen. To obtain His tag BAF, the coding region of BAF was PCR-amplified using primers 5′-CGGGATCCCGATGACAACCTCCCAAAAGCA-3′ and 5′-CGGAATTCATGCAAGAGCGAGAATCC-3′. The PCR product was digested with BamHI and EcoRI, and then inserted into the pET28c vector at the BamHI and EcoRI sites. The DNA sequences of the inserts in plasmid pGEX 3X-emerin ΔTM and pET28c-BAF were confirmed using an ALF DNA sequencer (Amersham Biosciences). Preparation of Xenopus Egg Cytosol Fractions—Xenopus eggs were collected, dejelled, and then lysed to prepare S-phase and M-phase cytosol fractions as described previously (26Kawahire S. Takeuchi M. Gohshi T. Sasagawa S. Shimada M. Takahashi M. Abe T.K. Ueda T. Kuwano R. Hikawa A. Ichimura T. Omata S. Horigome T. J. Biochem. (Tokyo). 1997; 121: 881-889Crossref PubMed Scopus (27) Google Scholar, 27Nakagawa T. Hirano Y. Inomata A. Yokota S. Miyachi K. Kaneda M. Umeda M. Furukawa K. Omata S. Horigome T. J. Biol. Chem. 2003; 278: 20395-20404Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Chromatin Binding Assay—Using beads bearing GST-emerin ΔTM or GST-emerin ΔLT, the chromatin binding assay was carried out as previously described (3Takano M. Koyama Y. Ito H. Hoshino S. Onogi H. Hagiwara M. Furukawa K. Horigome T. J. Biol. Chem. 2004; 279: 13265-13271Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 28Takano M. Takeuchi M. Ito H. Furukawa K. Sugimoto K. Omata S. Horigome T. Eur. J. Biochem. 2002; 269: 943-953Crossref PubMed Scopus (37) Google Scholar) except for the use of 20,000 Xenopus sperm chromatin per assay. When pretreatment of the beads bearing GST-emerin with an Escherichia coli extract containing BAF was necessary, it was carried out as follows. E. coli cells expressing His tag BAF were sonicated vigorously and then centrifuged at 12,000 × g for 10 min. A 50-μl aliquot of the supernatant was reacted with beads bearing GST-emerin ΔTM in binding buffer (20 mm Tris-HCl (pH 7.6), 134 mm NaCl, and 0.1% Tween-20 either containing 125 μg/ml DNase or not) at 4 °C for 3 h. The beads thus treated were washed three times with binding buffer and then used for the chromatin binding assay. Typically, Xenopus sperm chromatin, which was demembranated with lysolecithin and subsequently decondensed with heated Xenopus egg cytosol, in 20 μl of extraction buffer (10,000 per μl in 50 mm HEPES-KOH (pH 7.7), 250 mm sucrose, 50 mm KCl, 2.5 mm MgCl2, 2 mm 2-mercaptoethanol), was added to the beads, which were thus treated suspended in 10 μl of extraction buffer. After incubation at 4 °C for 10 min, the binding reaction was stopped by pipetting 15-μl samples onto glass slides spotted with 12 μl of a fixing solution (extraction buffer containing 3% formaldehyde and 6 μg/ml Hoechst dye 33342). The fixed samples were observed by fluorescence microscopy. 100–200 beads were observed for every sample, and “the percentage of beads with bound chromatin” was determined. This value was used as an index of the affinity of beads bearing emerin fragments and chromatin. The values in the figures are indicated after subtraction of a blank value. The blank value (<10%) was determined in each experiment using Sepharose beads with GST-bound instead of GST-emerin fragments. The significance of the pretreatment of beads bearing emerin fragments with various reagents in the chromatin binding assay was evaluated by means of Student's t test (n = 3). In a previous study (28Takano M. Takeuchi M. Ito H. Furukawa K. Sugimoto K. Omata S. Horigome T. Eur. J. Biochem. 2002; 269: 943-953Crossref PubMed Scopus (37) Google Scholar), we compared this method and an established in vitro binding method involving soluble proteins and chromatin, showing that this bead method gives the same results as the established method. Therefore, we used this method to determine the affinity of protein fragments to chromatin in this study. In Vitro Binding Assay of Emerin and BAF—A supernatant containing His tag BAF was prepared as described above. Beads bearing ∼10 μg of ΔTM or S175A-ΔTM preincubated with Xenopus cytosol or extraction buffer were washed twice with binding buffer (20 mm Tris-HCl (pH 7.6), 134 mm NaCl and 0.1% Tween 20), and then incubated with BAF-expressed E. coli extract at 4 °C for 3 h. The beads were washed three times with binding buffer. The sample thus obtained was subjected to SDS-PAGE and then transferred to a nitrocellulose membrane. BAF bound to beads was detected with an anti-His tag antibody and chemical luminescence. Emerin phosphorylated with a Xenopus egg cytosol fraction was separated by 10% gel SDS-PAGE, and the emerin band was excised. Phosphorylation of the protein was detected with ProQ diamond stain (Molecular Probes) according to the manufacturer's instructions and a reference (29Wang M. Xiao G.G. Li N. Xie Y. Loo J.A. Nel A.E. Electrophoresis. 2005; 26: 2092-2108Crossref PubMed Scopus (40) Google Scholar). In Vitro Dissociation Assay of Emerin and BAF—Beads bearing ∼10 μgof ΔTM were pretreated with a supernatant containing His tag BAF as described above. After washing three times, the beads thus treated were again treated with buffer or cell cycle-dependent Xenopus egg cytosol fraction at 23 °C for 1 h. The beads were washed, subjected to SDS-PAGE, and transferred to a nitrocellulose membrane. BAF bound to the beads was detected as described above. Phosphorylation Assay of Emerin Fragments with a Xenopus Egg Cytosol Fraction—Approximately 3 μg of GST-fused emerin bound to glutathione-Sepharose beads was incubated with S-phase and M-phase egg cytosol fractions containing 1 μCi of [γ-32P]ATP at 23 °C for 1 h. After washing twice with phosphate-buffered saline containing 0.05% Tween 20 (PBS-T), the beads were supplemented with 2 mm ATP and then incubated at 4 °C for 10 min. The proteins thus treated were separated by SDS-PAGE and visualized by CBB R-250 staining. After drying the gel, phosphorylation was detected with Fuji x-ray film. The emerin ΔTM bands were excised from the gel and the phosphorylated emerin ΔTM was quantified by scintillation counting. Phosphopeptide Mapping—Approximately 30 μg of emerin ΔTM or ΔLT was phosphorylated as described above, separated by SDS-PAGE, and then transferred to a nitrocellulose sheet. The full-length emerin band was excised, soaked in 0.5% poly(vinyl pyrrolidone) K-30 in 100 mm acetic acid for 30 min at 37 °C, and then washed extensively with water. The protein was digested with trypsin or chymotrypsin in 50 mm NH4HCO3 for 16 h at 37 °C. The released peptides were dried, dissolved in water, and then loaded onto a cellulose TLC plate (Funacell, Funakoshi Co., Tokyo). Electrophoresis, in the first dimension, was performed at pH 8.9 (1% ammonium carbonate) for 20 min at 1000 V, and ascending chromatography, in the second dimension, was performed with a solvent system of 37.5% 1-butanol, 25% pyridine, and 7.5% acetic acid in water (v/v). The dried plate was exposed to Fuji x-ray film. Separation of Phosphopeptides Derived from Emerin ΔTM Treated with Mitotic Cytosol Using a Titansphere Column—This experiment was carried out according to the method of Kuroda et al. (30Kuroda I. Shintani Y. Motokawa M. Abe S. Furuno M. Anal. Sci. 2004; 20: 1313-1319Crossref PubMed Scopus (97) Google Scholar) with some modification. Beads bearing ∼100 μg of GST-fused emerin ΔTM were treated with a Xenopus egg M-phase cytosol fraction as described above except that [γ-32P]ATP was omitted. The beads thus treated were separated by 10% gel SDS-PAGE and visualized by CBB R-250 staining. Emerin ΔTM bands were excised from the gel and then in-gel digested with chymotrypsin or trypsin in 50 mm NH4HCO3 at 37 °C for 16 h. The peptides thus obtained were dried, dissolved in solvent A (Milli Q grade water containing 0.1% (v/v) trifluoroacetic acid), and then applied to a Titansphere column (4.0-mm inner diameter × 10-mm column, GL Science Co., Japan) equilibrated with solvent A at the flow rate of 0.1 ml/min for 30 min. Phosphopeptides trapped on the Titansphere column were eluted with solvent C (0.5 m H3PO4·NaOH (pH 8.0)) at the flow rate of 0.5 ml/min for 15 min. The thus eluted phosphopeptides were directly applied to a reversed-phase silica-base CAPCELL PACK C8 column (4.6-mm inner diameter × 150-mm column, Shiseido, Japan), briefly washed with solvent A, and then eluted with a 90-min linear gradient, 0–45%, of solvent B (acetonitrile containing 0.0886% (v/v) trifluoroacetic acid) at a flow rate of 1 ml/min. The phosphopeptides thus isolated were analyzed with an AXIMA-CFR MALDI-TOF MS (Shimadzu Co., Japan) using CHCA as a matrix. Dephosphorylation of Phosphopeptides—Dephosphorylation of phosphopeptides derived from M-phase cytosol-treated emerin ΔTM was carried out according to the method of Kuyama et al. (31Kuyama H. Toda C. Watanabe M. Tanaka K. Nishimura O. Rapid Commun. Mass Spectrom. 2003; 17: 1493-1496Crossref PubMed Scopus (33) Google Scholar). The phosphopeptides were dried, dissolved in 40 μl of 46% hydrofluoric acid (HF, Wako, Japan), and then incubated at room temperature for 1.5 h. The peptides thus treated were dried, dissolved in 1 μl of 40% acetonitrile containing 0.1% (v/v) trifluoroacetic acid, and then analyzed with an AXIMA-CFR MALDI-TOF MS. MS/MS Sequencing—Phosphopeptides, which were separated with the Titansphere and C8 columns, were applied to an Inertsil ODS column (0.2-mm inner diameter × 50 mm, GL Science Co., Japan) equilibrated with solvent D (2% (v/v) acetonitrile containing 0.1% (v/v) formic acid) and then eluted with a 20-min linear gradient, from 5 to 55% of solvent E (98% (v/v) acetonitrile containing 0.1% (v/v) formic acid) at the flow rate of 1.5 μl/min using a MAGIC 2002 system (Michrome BioResources, Inc.). The phosphopeptides thus eluted were directly introduced to ESI-IT MS, LCQ Deca XP (Thermo Electron, San Jose, CA), equipped with a nanospray interface (AMR Inc., Japan) and a metal nanosprayer (GL Science Co.). To obtain sequence information on the eluted phosphopeptides, the mass spectrometer was operated in the ion select mode, where the MS scan was followed by the MS/MS scans of the calculated mass of the phosphopeptide as parent mass. The Binding of Emerin ΔTM to Chromatin—Two kinds of emerin fragments, i.e. emerin ΔTM (residues 1–213) and emerin ΔLT (residues 37–213), fused to GST were expressed in E. coli and used in this study (Fig. 1A). Emerin ΔTM lacks the transmembrane domain, and emerin ΔLT lacks both the transmembrane domain and most of LEM domain. GST-fused emerin ΔTM (ΔTM) and GST-fused emerin ΔLT (ΔLT) were purified from E. coli extract using a GSH-Sepharose bead (Fig. 1B). Western blotting with anti-GST antibody of ΔTM and ΔLT preparations, which were purified by GSH-Sepharose showed that smaller protein bands observed in Fig. 1B (lanes 1 and 2) indicated by asterisk were GST-containing degradation products of ΔTM and ΔLT (data not shown). All experiments in this study were done using beads bearing GST-emerin ΔTM or ΔLT. To determine whether ΔTM interacts with chromatin and its interaction is regulated in a cell cycle-dependent manner, like for some other inner nuclear membrane proteins, i.e. LBR and LAP2β, or not, we performed an in vitro chromatin binding assay. We previously developed this assay method to analyze the binding of inner nuclear membrane proteins to chromatin (28Takano M. Takeuchi M. Ito H. Furukawa K. Sugimoto K. Omata S. Horigome T. Eur. J. Biochem. 2002; 269: 943-953Crossref PubMed Scopus (37) Google Scholar). When beads bearing ΔTM were preincubated with buffer in the absence of the Xenopus egg cytosol fraction, they bound to chromatin slightly (column 1 in Fig. 2). However, when they were preincubated with a synthetic phase cytosol fraction (SC), the binding of chromatin to beads was stimulated (compare columns 1 and 2 in Fig. 2). Preincubation with a mitotic phase cytosol fraction (MC) did not stimulate the binding (column 3 in Fig. 2). Moreover, the once-stimulated chromatin binding activity of SC-treated beads was suppressed on subsequent incubation with MC (compare columns 2 and 4 in Fig. 2). On the other hand, the once-suppressed chromatin binding activity of MC-treated beads was activated on subsequent incubation with SC (compare columns 3 and 5 in Fig. 2). These results demonstrated that the emerin fragment thus expressed can bind to chromatin, and that the chromatin binding assay method can be used to analyze the cell cycle-dependent binding of emerin to chromatin in vitro. The stimulation of the binding of ΔTM to chromatin on SC treatment seemed to be independent of phosphorylation of ΔTM, because the stimulation was not suppressed on pretreatment of SC with apyrase for ATP depletion or a wide-spectrum protein kinase inhibitor, i.e. staurosporine (compare columns 2, 6, and 7 in Fig. 2). Furthermore, the stimulation of the binding did not occur on SC treatment of the beads bearing GST-emerin ΔLT (compare columns 12 and 13 in Fig. 2). These results indicated that the stimulation might be caused by the binding of the BAF in SC to ΔTM, which is known to mediate the binding of emerin to chromatin, because (i) the stimulation of the binding was not suppressed by kinase inhibitors, (ii) the stimulation was not observed for ΔLT, which lacks the BAF binding domain (mentioned below in more detail), and (iii) Xenopus egg cytosol contains 12 μm BAF (14Segura-Totten M. Kowalski A.K. Craigie R. Wilson K.L. J. Cell Biol. 2002; 158: 475-485Crossref PubMed Scopus (154) Google Scholar). On the other hand, suppression of the binding of SC-treated ΔTM to chromatin on subsequent treatment with MC should be caused by the phosphorylation of emerin, because the suppression was prevented by apyrase or a kinase inhibitor, i.e. staurosporine (columns 8–11 in Fig. 2).FIGURE 2The suppression of the binding of emerin to chromatin is caused by mitotic phosphorylation of emerin. Beads bearing ΔTM (∼6 μg) were used, and the beads for columns 4, 10, and 11 were pretreated with a synthetic-phase cytosol (columns 4, 10, and 11), a mitotic phase cytosol (column 5) or buffer (columns 1–3 and 6–9) at 23°C for 20 min. The beads were subsequently treated with buffer (column 1; Buffer), SC (columns 2 and 5; SC and MC-SC, respectively), MC (columns 3 and 4; MC and SC-MC, respectively), SC pretreated with 8 milliunits of apyrase or 5 μm staurosporine (columns 6 and 7; SC+Apy. and SC+Sta., respectively) or MC pretreated with 8 milliunits of apyrase or 5 μm staurosporine (columns 8–11; MC+Apy., MC+Sta., SC-MC+Apy., and SC-MC+Sta., respectively) at 23 °C for 20 min. The beads thus treated were incubated with 20,000 decondensed sperm chromatin at 4 °C for 10 min, and then observed by fluorescence microscopy after staining of DNA with Hoechst 33342. The “percentage of beads with bound chromatin” values were determined as described under “Materials and Methods” after subtraction of the value for blank GST beads. Beads bearing ΔLT (∼4 μg) treated with buffer (column 12) or SC (column 13) were reacted with chromatin in the same way as for the beads bearing ΔTM. The results are the means ± S.D. for three independent experiments. *, a significant difference from the respective control (p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Participation of BAF in the Binding of Emerin ΔTM to Chromatin— To clarify the stimulation mechanism for the binding of ΔTM to chromatin on SC treatment, we examined whether the binding of emerin to chromatin is mediated or not by BAF in our assay system. Beads bearing ΔTM were treated with an E. coli extract containing expressed His tag BAF. The beads thus treated were used for the chromatin-binding assay (Fig. 3). ΔTM treated with the E. coli extract containing His tag BAF (Fig. 3A, lane 2) bound to chromatin, although beads treated with buffer or the blank E. coli extract (Fig. 3A, lane 1 or 3, respectively) could not bind to chromatin. The binding of His tag BAF to the beads bearing ΔTM in this assay system was confirmed by Western blotting with anti-His tag antibody (Fig. 3B). These results clearly show that the binding of beads bearing ΔTM to chromatin is mediated by BAF and also support our idea that the stimulation of the binding of beads bearing ΔTM to chromatin on pretreatment with SC is mediated by the binding of BAF to emerin. M-phase-specific Phosphorylation of Emerin ΔTM Suppressed the Binding to BAF—We examined whether the binding of emerin ΔTM beads to BAF is cell cycle-dependent or not. Beads bearing ΔTM were pretreated with E. coli extract containing His tag BAF to bind His tag BAF to ΔTM. The beads thus treated were further treated with a buffer or cell cycle-dependent Xenopus egg extrac" @default.
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