Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M503214200 on October 3, 2005

J. Biol. Chem., Vol. 280, Issue 48, 39925-39933, December 2, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/48/39925    most recent
M503214200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hirano, Y.
Right arrow Articles by Horigome, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hirano, Y.
Right arrow Articles by Horigome, T.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

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

Yasuhiro Hirano{ddagger}§, Masashi Segawa§, Fumiko S. Ouchi¶, Yoshio Yamakawa¶, Kazuhiro Furukawa§, Kunio Takeyasu{ddagger}, and Tsuneyoshi Horigome§||1

From the {ddagger}Graduate School of Biostudies, Kyoto University, Kitashirakawa-ohiwakecho, Sakyo-ku, Kyoto 606-8205, the §Course of Functional Biology, Graduate School of Science and Technology, Niigata University, Igarashi-2, Niigata 950-2181, the Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, and the ||Center for Transdisciplinary Research, Niigata University, Igarashi-2, Niigata 950-2181, Japan

Received for publication, March 23, 2005 , and in revised form, September 6, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The nuclear envelope (NE)2 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-2{beta} (LAP2{beta}), 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 (1). 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 LAP2{alpha} and 2{beta} are well understood (2–7). LBR directly binds to DNA in vitro and dissociates on phosphorylation by cdc2 kinase and other kinase(s) in a mitotic egg extract (3). LAP2{beta} binds to lamin B1 and chromatin, and cell cycle-dependent phosphorylation of LAP2{beta} cancels this binding (4). 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.

Human emerin is a serine-rich protein exhibiting an apparent mass of 34 kDa on SDS-PAGE (8) and is phosphorylated in a cell cycle-dependent manner (9). Emerin belongs to the LEM (LAP2{beta}, emerin, MAN1) protein family, whose members have approximately a 40-residue domain named the LEM (10). These proteins directly bind to barrier-to-autointegration factor (BAF) (11–13). 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 (14). 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 (15, 16). 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 (16). It is also known that emerin has many binding partners, including transcriptional repressors and intermediate filament proteins (17–25). 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 ({Delta}95–99), which causes Emery-Dreifuss muscular dystrophy and cannot bind to lamin A, exhibits aberrant cell cycle-dependent phosphorylation forms (9). The study also suggested that the phosphorylation of emerin regulates the binding of emerin to lamin A (9). 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of GST-fused Emerin Fragment Proteins and His6 Tag BAF—Cloning of the nucleoplasmic region of human emerin ({Delta}TM, 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-{Delta}TM), a GeneTailor mutagenesis kit (Invitrogen) was used according to the manufacturer's procedure. PCR was carried out with pBluescript II SK(–)-emerin {Delta}TM using the following primers: 5'-CTGTTTCGCCTCCAGGGCCTCCCTGGACC-3' and 5'-CCTGGAGGCTGAAACAGGGCGGTAGTCGT-3', followed by verification by DNA sequence analysis. The pBluescript II SK(–)-S175A-{Delta}TM 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 {Delta}LT plasmid, pBluescript II SK(–)-emerin {Delta}TM 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 [GenBank] ) 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 {Delta}TM 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 (26, 27).

Chromatin Binding Assay—Using beads bearing GST-emerin {Delta}TM or GST-emerin {Delta}LT, the chromatin binding assay was carried out as previously described (3, 28) 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 x g for 10 min. A 50-µl aliquot of the supernatant was reacted with beads bearing GST-emerin {Delta}TM 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 (28), 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 {Delta}TM or S175A-{Delta}TM 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 (29).

In Vitro Dissociation Assay of Emerin and BAF—Beads bearing ~10 µgof {Delta}TM 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 [{gamma}-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 {Delta}TM bands were excised from the gel and the phosphorylated emerin {Delta}TM was quantified by scintillation counting.

Phosphopeptide Mapping—Approximately 30 µg of emerin {Delta}TM or {Delta}LT 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.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 1.
N-terminal fragments of emerin expressed as GST-fusion proteins. A, schematic diagram of N-terminal fragments of emerin expressed as GST fusion proteins. The binding sites for emerin-binding proteins are shown as reported previously (upper, 11–13, 17–25). Slashed boxes indicate the hydrophobic amino acid-rich region, which was demonstrated by Wolff et al. (31). Two GST-fused emerin fragments were constructed and used in this study (middle and lower). B, SDS-PAGE of GST fusion proteins. GST-emerin {Delta}TM ({Delta}TM) and GST-emerin {Delta}LT ({Delta}LT) were expressed in E. coli and then purified with glutathione-Sepharose beads. Beads bearing {Delta}TM (1), {Delta}LT (2), or marker proteins (M) were analyzed by SDS-PAGE on a 10% gel and then stained with CBB. The bands with the added asterisk at the right are GST-containing degradation products of {Delta}TM and {Delta}LT. The values at the left are the relative molecular masses of the marker proteins.

 
Separation of Phosphopeptides Derived from Emerin {Delta}TM Treated with Mitotic Cytosol Using a Titansphere Column—This experiment was carried out according to the method of Kuroda et al. (30) with some modification. Beads bearing ~100 µg of GST-fused emerin {Delta}TM were treated with a Xenopus egg M-phase cytosol fraction as described above except that [{gamma}-32P]ATP was omitted. The beads thus treated were separated by 10% gel SDS-PAGE and visualized by CBB R-250 staining. Emerin {Delta}TM 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 x 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 x 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 {Delta}TM was carried out according to the method of Kuyama et al. (31). 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 x 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The Binding of Emerin {Delta}TM to Chromatin—Two kinds of emerin fragments, i.e. emerin {Delta}TM (residues 1–213) and emerin {Delta}LT (residues 37–213), fused to GST were expressed in E. coli and used in this study (Fig. 1A). Emerin {Delta}TM lacks the transmembrane domain, and emerin {Delta}LT lacks both the transmembrane domain and most of LEM domain. GST-fused emerin {Delta}TM ({Delta}TM) and GST-fused emerin {Delta}LT ({Delta}LT) were purified from E. coli extract using a GSH-Sepharose bead (Fig. 1B). Western blotting with anti-GST antibody of {Delta}TM and {Delta}LT 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 {Delta}TM and {Delta}LT (data not shown). All experiments in this study were done using beads bearing GST-emerin {Delta}TM or {Delta}LT. To determine whether {Delta}TM 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 LAP2{beta}, 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 (28). When beads bearing {Delta}TM 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 {Delta}TM to chromatin on SC treatment seemed to be independent of phosphorylation of {Delta}TM, 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 {Delta}LT (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 {Delta}TM, 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 {Delta}LT, which lacks the BAF binding domain (mentioned below in more detail), and (iii) Xenopus egg cytosol contains 12 µM BAF (14). On the other hand, suppression of the binding of SC-treated {Delta}TM 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).



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 2.
The suppression of the binding of emerin to chromatin is caused by mitotic phosphorylation of emerin. Beads bearing {Delta}TM (~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 {Delta}LT (~4 µg) treated with buffer (column 12) or SC (column 13) were reacted with chromatin in the same way as for the beads bearing {Delta}TM. The results are the means ± S.D. for three independent experiments. *, a significant difference from the respective control (p < 0.05).

 
Participation of BAF in the Binding of Emerin {Delta}TM to Chromatin— To clarify the stimulation mechanism for the binding of {Delta}TM 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 {Delta}TM 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). {Delta}TM 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 {Delta}TM 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 {Delta}TM to chromatin is mediated by BAF and also support our idea that the stimulation of the binding of beads bearing {Delta}TM to chromatin on pretreatment with SC is mediated by the binding of BAF to emerin.

M-phase-specific Phosphorylation of Emerin {Delta}TM Suppressed the Binding to BAF—We examined whether the binding of emerin {Delta}TM beads to BAF is cell cycle-dependent or not. Beads bearing {Delta}TM were pretreated with E. coli extract containing His tag BAF to bind His tag BAF to {Delta}TM. The beads thus treated were further treated with a buffer or cell cycle-dependent Xenopus egg extract (Fig. 4A). In the cases of treatments with the buffer and SC, His tag BAF bound to {Delta}TM remained on beads (Fig. 4A, buffer and SC). However, His tag BAF bound to {Delta}TM disappeared by MC treatment (Fig. 4A, MC). These results showed that MC, but not SC, treatment of {Delta}TM-BAF complex causes dissociation of BAF from {Delta}TM. On the other hand, beads bearing {Delta}TM pretreated with buffer, SC, or MC were treated with the E. coli extract containing His tag BAF, then proteins bound to the beads were separated by SDS-PAGE and transferred to a nitrocellulose membrane, and BAF was detected with an anti-His tag antibody (Fig. 4B). The binding of BAF was suppressed by the pretreatment of {Delta}TM beads with MC but not that with buffer or SC (Fig. 4B, buffer, SC, and MC). Furthermore, SC treatment followed by MC treatment (SC-MC) suppressed the binding of BAF to beads bearing {Delta}TM (Fig. 4B, SC-MC). These results were consistent with the earlier results. The phosphorylation levels of emerin shown in Fig. 4C were demonstrated by ProQ diamond staining, which is known as a means of phosphoprotein staining (compare Fig. 4C, ProQ stain with CBB stain) (29). The weak "Pro Q staining" observed for the band of {Delta}TM treated with buffer (Fig. 4C, buffer) is nonspecific staining. Therefore, staining over the background can be considered to be phosphoprotein-specific staining. The band-shift on SDS-PAGE of emerin on treatment with MC (Fig. 4C, MC and SC-MC) was consistent with in vivo phosphorylated emerin in mitoticphase lymphoblastoid cells (9). However, the four bands depending on the phosphorylation states reported for lymphoblastoid cell were not clear in this system. {Delta}TM preparations treated with MC (MC and SC-MC) were strongly phosphorylated and the binding of His-BAF was strongly suppressed. On the other hand, pretreatment of MC with apyrase or staurosporine to prevent the mitotic phosphorylation of emerin canceled this suppression activity (Fig. 4, B and C, MC+Apy. and MC+Sta.). A part of phosphorylation of {Delta}TM by MC seems to be caused by staurosporine-insensitive kinase, because 5 µM staurosporine could not cause complete inhibition (Fig. 4, ProQ stain and MC+Sta.). Therefore, we concluded that the binding of emerin to BAF is suppressed by M-phase-specific direct or indirect phosphorylation. Thereafter, we focused on analysis of the cell cycle-dependent phosphorylation of emerin, especially mitotic phosphorylation sites of emerin.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 3.
The binding of emerin to chromatin is caused by BAF. A, in vitro chromatin binding assay involving {Delta}TM treated with BAF. Beads bearing {Delta}TM (~10 µg) were treated with buffer (buffer), an E. coli-soluble fraction containing His tag BAF (BAF (+) E. coli extract), or a blank E. coli-soluble fraction expressing a His tag insoluble protein (BAF (–) E. coli extract) for 3 h. 25,000 decondensed sperm chromatin were added to the beads thus treated, followed by incubation for 10 min. Then, the "percentage of beads with bound chromatin" values were determined as described in Fig. 2. The results are the means ± S.D. for three independent experiments. *, a significant difference from the respective control (p < 0.05). B, confirmation of the binding of emerin to BAF. Proteins bound to beads treated as in A were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and then incubated with an anti-His antibody. Bound antibodies were detected as enhanced chemiluminescence. A bar at the right indicates the position of 17-kDa marker protein electrophoresed.

 



View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 4.
Phosphorylation of emerin by M-phase cytosol fraction caused dissociation of emerin from BAF. A, BAF was dissociated from emerin by MC treatment. Beads bearing {Delta}TM (~10 µg) were pretreated with E. coli extract containing His tag BAF at 4 °C for 3 h. After washing, these beads were treated with extraction buffer (buffer) or a cell cycle-dependent Xenopus egg cytosol fraction (SC or MC) at 23 °C for 1 h. Then, proteins on beads thus treated were subjected to 12% gel SDS-PAGE, transferred to a nitrocellulose membrane, and then incubated with an anti-His tag antibody. Ten percent of loading amount of His-BAF in the reaction mixture was also reacted with an anti-His tag antibody (Load). Bound antibodies were detected by enhanced chemiluminescence. B, the binding of BAF to emerin was suppressed by mitotic phosphorylation of emerin. Beads bearing {Delta}TM (~10 µg) were pretreated with SC in the case of (SC-MC), and then subsequently treated with buffer (Buffer), SC (SC), MC (MC and SC-MC), MC pretreated with apyrase (MC+Apy.), or MC pretreated with staurosporine (MC+Sta.), respectively. BAF bound to beads were detected by anti-His tag antibody as in A. C, the phosphorylation states of emerin. Beads bearing {Delta}TM treated as in B were subjected to 10% gel SDS-PAGE, and then gel was stained with ProQ diamond to confirm the phosphorylation states of emerin (ProQ stain). Total emerin protein was stained by CBB (CBB stain). Bars at the right indicate positions of 17-, 43-, and 66-kDa marker proteins electrophoresed.

 
Cell Cycle-dependent Phosphorylation States of Emerin—We next examined the cell cycle-dependent phosphorylation of emerin in a Xenopus egg cell-free system (Fig. 5). {Delta}TM on beads was treated with egg cytosol fractions containing [{gamma}-32P]ATP, washed with binding buffer, and then analyzed by SDS-PAGE. The gel was stained with CBB and then subjected to autoradiography. The {Delta}TM was strongly phosphorylated upon treatment with MC, although the phosphorylation by SC was very low (Fig. 5A, Autoradiography) (9). A mitotic phase-specific band-shift was also observed for {Delta}LT treated with MC (Fig. 5A, {Delta}LT). These results show that one or more phosphorylation sites that are outside of the LEM domain caused the main band-shift, because {Delta}LT lacking almost all of the LEM domain showed a clear band-shift on treatment with MC (Fig. 5A, {Delta}LT lanes). To compare the amounts of incorporated phosphate groups, {Delta}TM treated with SC or MC in the presence of [{gamma}-32P]ATP was electrophoresed, and {Delta}TM bands were excised from the gel and counted (Fig. 5B). The {Delta}TM phosphorylated in the M-phase was 6.6 times as strong as that in the S-phase. These results demonstrated that the phosphorylation of emerin in the S-phase is very low.

To determine the mitotic phosphorylation state of emerin, and the differences in the phosphorylation site(s) between {Delta}TM and {Delta}LT, we performed phosphopeptide mapping (Fig. 6). Beads bearing {Delta}TM and {Delta}LT were treated with SC or MC in the presence of [{gamma}-32P]ATP. After washing, the beads thus treated bearing {Delta}TM and {Delta}LT were subjected to SDS-PAGE, and proteins were transferred to a nitrocellulose membrane. Then, the full-length emerin bands were excised from the membrane and digested with trypsin (Fig. 6A) or chymotrypsin (Fig. 6B). The peptides generated were subjected to two-dimensional separation on a cellulose plate. Although many spots of phosphorylated peptides were observed for the preparations derived from both {Delta}TM and {Delta}LT treated with MC, the major spots indicated with arrows in Fig. 6 were not observed for {Delta}LT. When 32P-labeled {Delta}TM and {Delta}LT were digested with chymotrypsin, well focused patterns were obtained (Fig. 6B). In these patterns, major spots 1 and 2 and three weak spots, 8, 10, and 11, were absent in {Delta}LT map (compare Fig. 6B, {Delta}TM and {Delta}LT). These results suggested the following three possibilities: 1) at least one major phosphorylation site exists in the LEM domain, 2) deletion of the LEM domain causes obstruction of a major phosphorylation site present in other than the LEM domain, and 3) BAF binds an emerin kinase and recruits it to {Delta}TM. These three possibilities are explained under "Discussion." The phosphorylation levels of emerin fragments treated with SC were much lower than for those treated with MC (Fig. 6A), although the phosphorylation patterns of SC-treated samples were found to be very similar to those of MC-treated ones when the autoradiography films were superposed (data not shown). Therefore, we only examined the mitotic phosphorylation sites in the following experiments.



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 5.
Emerin was strongly phosphorylated in the M-phase. A, detection of phosphorylation. Beads bearing 3 µg of emerin {Delta}TM or {Delta}LT pretreated with SC (SC-MC) or not were incubated with 20 µl of a synthetic (SC) or mitotic (MC and SC-MC) phase egg cytosol fraction containing 1 µCi of [{gamma}-32P]ATP at 23 °C for 1 h. The beads thus treated were washed with binding buffer and then subjected to SDS-PAGE. The gel was stained by CBB and subjected to autoradiography. Arrows, arrowheads, and double arrowheads indicate {Delta}TM, the mitotic-specific band-shift of {Delta}TM, and emerin {Delta}LT bands, respectively. Bars at the right indicate positions of 43- and 66-kDa marker proteins electrophoresed. B, the {Delta}TM bands in A were excised from the gel, and then phosphate groups incorporated into {Delta}TM were compared by their radioactivities. Results are shown as means ± S.D. for three independent experiments.

 



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 6.
Tryptic and chymotryptic phosphopeptide maps of {Delta}TM and {Delta}LT. Beads bearing ~30 µgof {Delta}TM or {Delta}LT were incubated with 20 µl of SC or MC containing 20 µCi of [{gamma}-32P]ATP at 23 °C for 1 h. The proteins thus treated were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The {Delta}TM or {Delta}LT bands were excised and digested with trypsin (A) or chymotrypsin (B). The phosphopeptides thus obtained were separated by electrophoresis at pH 8.9 (horizontal direction; cathode to the left) and by ascending chromatography. Excised bands contained 1, 6, 0.6, 4, 6, and 4 kcpm for 6A-SC-{Delta}TM, 6A-MC-{Delta}TM, 6A-SC-{Delta}LT, 6A-MC-{Delta}LT, 6B-MC-{Delta}TM, and 6B-MC-{Delta}LT, respectively. The points of sample application can be seen as dots near the bottom-left corners. The arrows indicate major spots lacking for {Delta}LT treated with MC.

 



View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 7.
Identification of the phosphorylated peptides derived from {Delta}TM treated with MC. A, separation of phosphopeptides derived from {Delta}TM. Approximately 100 µg of {Delta}TM treated with MC as in Fig. 6 without [{gamma}-32P]ATP was chymotrypsinized or trypsinized. The peptides thus obtained were applied to a Titansphere column and eluted with 0.5 M phosphate buffer (pH 8.0). The retained chymotryptic (upper panel) or tryptic (middle panel) phosphopeptides were separated by subsequent C8 chromatography (see "Materials and Methods" for details). The collected fractions were analyzed by MALDI-TOF MS. The baseline is indicated in the lower panel. The numbered peak fractions contained phosphopeptides. B, detection of phosphopeptides of emerin on MALDI-TOF MS. One-eighth of the obtained peptides in A were analyzed by MALDI-TOF MS using CHCA as a matrix (HF(–)). For dephosphorylation, the same amount of these phosphopeptide fractions was dried, dissolved in 46% hydrofluoric acid, and incubated at room temperature for 1.5 h. The peptides thus treated were dried again and then analyzed by MALDI-TOF MS. Values shown in the figures indicate the monoisotopic peptide masses. C, identification of phosphorylation sites. A mass spectrum of the 1367.7 (((M+1H)+, left) and 685.1 (M+2H)2+, right) Da phosphopeptide (corresponding to 1367.7 Da, residues 168–179, monophosphorylated) was obtained by ESI-IT MS/MS. The prominent fragment ion series are those having b and y ions. The peptide sequence is RPVSASRSSLDL. In the spectrum, the b8 ion still has the phosphate moiety, but the b7 and y4 ones do not. This indicates that the phosphate moiety must be located at Ser175, indicated by an asterisk.

 
Identification of the Mitotic Phosphorylation Sites of Emerin—To identify the mitotic phosphorylation sites of emerin by means of mass spectrometry sequencing, we used a phosphopeptide separation system comprising a combination of a Titansphere column, which specifically binds phosphopeptides, and a reverse phase C8 column (see "Materials and Methods"). For this purpose, {Delta}TM was purified by glutathione-Sepharose and phosphorylated by MC and then digested with proteinases. We mainly used a chymotryptic digest of emerin, because a chymotryptic digest gave a clearer phosphopeptide map than a tryptic digest, as can be seen in Fig. 6. The chymotryptic or tryptic phosphopeptides derived from phosphorylated {Delta}TM were bound to the Titansphere column and then eluted with 0.5 M phosphate buffer (pH 8.0). The phosphopeptide fraction thus obtained was directly introduced onto the reverse phase C8 column, eluted with a linear gradient, and then fractionated (Fig. 7A). The molecular masses of the peptides thus obtained were determined by MALDI-TOF MS. Some of the determined masses were consistent with the calculated phosphopeptide masses (Fig. 7B, HF(–), and TABLE ONE). To confirm that these peptides contained phosphate groups, samples were pretreated with HF to hydrolyze phosphate groups and then the mass shift was analyzed by MALDI-TOF MS (Fig. 7B, HF(+)). The mass shifts of 80 or 160 Da indicate that the obtained peptides were phosphopeptides and that one or two sites in their sequences were phosphorylated (Fig. 7B and TABLE ONE). As can be seen in TABLE ONE, every phosphopeptide had more than two possible phosphorylation sites. Therefore, we performed MS/MS sequencing by means of ESI-IT MS to determine which residues are phosphorylated. The mass spectrum of the product ions of m/z 1367.7 and 685.1 ((M+1H)+ and (M+2H)2+ of the 1367.7-Da peptide, respectively) allowed localization of the phosphorylation site to residue Ser175 by b and y ion series. This was so because we could detect the b7 ion (RPVSASR) without a phosphate group on product ion m/z 1367.7 sequencing and the b8 (RPVSASRS) ion, which includes a phosphate group, and the y4 ion (SLDL) without a phosphate group on product ion m/z 685.1 sequencing (Fig. 7C). In contrast to the case of 1367.7-Da peptide, we could not determine which site, Ser175 or Ser176, was phosphorylated in 2080.0-Da peptide. However, we focused on only Ser175 because Ser175, but not 176Ser, was identified as a phosphorylation site by the analysis indicated above. Using the same approach for determination of the phosphorylated residue, the phosphorylation sites of other phosphopeptides (Ser49, Ser66, Thr67, and Ser120) were also determined (TABLE ONE). However, we could not completely exclude the possibility that another site in the 2396.8-Da peptide was phosphorylated, that is, Ser52, Ser53, or Ser54 was possibly phosphorylated. In the case of the 4109.6-Da peptide, we could not determine the phosphorylated residue, although candidates include either Ser123, Ser141, Ser142, or Ser143. In the cases of 2739.9- and 2658.2-Da peptides, moreover, sufficient information to determine the phosphorylation site could not be obtained. On the other hand, we could not detect phosphotyrosine by phosphoamino acid analysis of {Delta}TM phosphorylated by MC in the presence of [{gamma}-32P]ATP (data not shown). Finally, we identified five phosphorylation sites, Ser49, Ser66, Thr67, Ser120, and Ser175. These phosphorylation sites matched the consensus sequences of well known kinases; i.e. Ser49: protein kinase A, calmodulin-dependent kinase II, and glycogen synthetase kinase 3{beta}; Ser66: glycogen synthetase kinase 3{beta}; and Ser175: glycogen synthetase kinase 3{beta}.


View this table:
[in this window]
[in a new window]
  		TABLE ONE
Phosphopeptides detected by ESI-IT MS

This table contains data obtained in the MALDI-TOF MS and ESI-IT MS experiments shown in Fig. 7. Phosphoamino acids are given in bold and italicized letters.

 
Ser175 Phosphorylation of Emerin by MC Responsible for the Emerin-BAF Dissociation—Although five mitotic phosphorylation sites of emerin were identified and one possible phosphorylation site, i.e. Ser175, was found on MS/MS sequencing, we could not detect any phosphorylation site in the LEM domain. When these identified phosphorylation sites and phosphopeptide maps of {Delta}TM and {Delta}LT were compared, it was suggested that the phosphopeptide spots absent for the {Delta}LT digest (major spots 1 and 2 and minor spots 10 and 11 in Fig. 6B) might not be due to the LEM domain and that phosphorylation at these sites is regulated through an unknown mechanism by the LEM domain. Then, we applied the phosphopeptide separation system to {Delta}TM and {Delta}LT phosphorylated with a mitotic extract to determine the phosphorylation site(s) corresponding to the absent spots in the phosphopeptide maps in Fig. 6B. Surprisingly, a peak corresponding to a peptide containing phosphorylated Ser175 (arrow in Fig. 8A, {Delta}TM, corresponding to peak 1 in Fig. 7A) had completely disappeared for the {Delta}LT digest (Fig. 8A, {Delta}LT), although there was no clear change in other peaks. We could not examine whether the peak corresponding to peak 2 in Fig. 7A, a peak corresponding to a fragment of the peak 1 material, disappeared or not, because the peak in Fig. 8A was too small. These results indicated that phosphorylation at Ser175, which is located outside of the LEM domain, might be affected through some unknown mechanism by the LEM domain and suggested that the phosphorylation participates in emerin-BAF dissociation. Then, we generated a point mutant at Ser175 of {Delta}TM (S175A-{Delta}TM) replaced with an alanine. Using this mutant, we could confirm that the missing peak from the elution pattern of phosphopeptides derived from {Delta}LT is that of the Ser175-containing peptide (Fig. 8A, S175A). Furthermore, we performed phosphopeptide mapping to confirm that the Ser175 phosphorylation was affected by deletion of the LEM domain. As can be seen in Fig. 8B, spots 1 and 2, which disappeared from the {Delta}LT pattern, also completely disappeared from the S175A-{Delta}TM pattern. Spots 12–14 appeared irregularly (compare {Delta}TM patterns in Figs. 6 and 8). Spot 4 in Fig. 8B S175A shifted up when compared with {Delta}TM and {Delta}LT in Fig. 8B. However, the shift seems to be within a fluctuation of the spot, because the spot shifts slightly from one gel to another (compare Fig. 6B, {Delta}TM to Fig. 8B, {Delta}TM and Fig. 6B, {Delta}LT to Fig. 8B, {Delta}LT). These results suggested that the LEM domain is necessary for the Ser175 phosphorylation by MC. Then, we carried out an in vitro binding assay using S175A-{Delta}TM and BAF to determine whether the Ser175 phosphorylation regulates the dissociation of emerin and BAF or not (Fig. 8C). The S175A point mutant treated with MC retained binding activity toward BAF, although {Delta}TM treated with MC dissociated from BAF. This result showed that the Ser175 phosphorylation in the M-phase participates in the dissociation of emerin and BAF.



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 8.
Ser175 phosphorylation by MC participates in emerin-BAF dissociation. A, the separation pattern of phosphopeptides derived from {Delta}LT and S175A-{Delta}TM. Approximately 100 µg of {Delta}TM, {Delta}LT, or S175A-{Delta}TM treated with MC as in Fig. 7 was chymotrypsinized. The peptides thus obtained were applied to a phosphopeptide separation system as in Fig. 7. The peak indicated by the arrow coincides with peak 1 in Fig. 7. B, beads bearing ~30 µgof {Delta}TM, {Delta}LT, or S175A-{Delta}TM were incubated with 20 µl of SC or MC containing 10 µCi of [{gamma}-32P]ATP at 23 °C for 1 h. The proteins thus treated were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and then chymotrypsinized. The phosphopeptides thus obtained were separated by electrophoresis at pH 8.9 (horizontal direction; cathode to the left) and by ascending chromatography, as in Fig. 6. The points of sample application can be seen as dots near the bottom-left corners. C, S175A-{Delta}TM prevented the mitotic dissociation of BAF. Beads bearing {Delta}TM or S175A-{Delta}TM (~10 µg) were treated with MC at 23 °C for 1 h. The beads thus treated were incubated with an E. coli extract expressing His tag BAF, separated by 12% gel SDS-PAGE, and then transferred to a polyvinylidene difluoride membrane. BAF that bound to the beads was detected with an anti-His antibody as shown in Fig. 4.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Mitotic Phosphorylation of Emerin—Although it has been known that emerin is highly phosphorylated at the M-phase in human lymphoblastoid cells (9), the role of phosphorylation of emerin has been poorly understood. Recently, Lattanzi et al. (20) indicated that the interaction of emerin and actin is increased by dephosphorylation of emerin, suggesting that phosphorylation of emerin regulates its binding to actin. By means of a phosphopeptide separation system involving a Titansphere column, we demonstrated that emerin is phosphorylated under mitotic conditions in vitro with at least five specific residues, four serine and one threonine residues: Ser49, Ser66, Thr67, Ser120, and Ser175 (TABLE ONE). These phosphorylation sites are interestingly located at the binding region for many emerin-binding proteins: Ser49, Ser66, and Thr67 for GCL, YT521-B, and Btf (23–25) Ser120 for lamin A and actin (12, 21); and Ser175 for GCL, YT521-B, Btf, and actin (21, 23–25), respectively. Therefore, phosphorylation at some of these sites possibly regulates their interactions. In particular, dephosphorylation of Ser120 and/or Ser175 may increase the binding of emerin to actin, because the phosphorylation sites are located in the actin binding domain.

The Regulation Mechanism for the Interaction of Emerin and BAF— In this study, it was suggested that the dissociation of emerin from BAF takes place through mitotic phosphorylation of emerin in a Xenopus cell-free system. However, these results do not rule out mitotic modification of BAF, which could independently regulate its binding to emerin. Surprisingly, our point mutant study suggested that the phosphorylation at Ser175, which is located outside of the LEM domain, participates in the dissociation of emerin from BAF in the M-phase (Fig. 8C).

Bengtsson and Wilson (17) and the previous study by Lee et al. (12) indicated that when at least two emerin mutants with replacement of residues 76–83 and 207–208, which lie outside of the LEM domain, were incubated with BAF, the amount of bound BAF was decreased by these mutations. Their study suggested that not only the LEM domain but also other regions of emerin seem to participate in the binding of emerin to BAF. Our data coincide with this finding. Then, we expected that the LEM domain and another BAF binding region in emerin may comprise a "BAF binding surface," and that the phosphorylation at Ser175 on MC treatment may induce a conformational change of the BAF binding surface, because the region around Ser175 has been predicted to be a flexible region that consists of a poly-Ser cluster (32). This conformational change might cause the BAF dissociation.

Although we could not clearly show why Ser175 of {Delta}LT was not phosphorylated by MC, the following three possibilities can be pointed out. First, deletion of the LEM domain from {Delta}TM caused a conformational change around Ser175 and phosphorylation was blocked. Second, a kinase bound to the LEM domain participated in the Ser175 phosphorylation of {Delta}TM; in this case, {Delta}LT lacking the LEM domain could not be phosphorylated at Ser175, because the kinase could not be recruited. Third, BAF recruits one or more kinases that phosphorylate Ser175 of {Delta}TM; in this case, {Delta}LT lacking BAF-binding activity could not be phosphorylated. To exclude the third possibility, we performed the emerin phosphorylation assay using MC whose BAF-binding proteins were depleted. BAF-binding proteins were depleted from MC by incubation with nickel-agarose beads bearing His-BAF, and then beads bearing {Delta}TM were phosphorylated in the presence of [{gamma}-32P]ATP by thus treated MC. No difference was observed between MC- and BAF-binding protein-depleted MC in phosphorylation of {Delta}TM (data not shown). Therefore, the first and the second possibilities should be ascertained further to clarify the Ser175 phosphorylation mechanism.

Cell Cycle-dependent Phosphorylation and Dephosphorylation of Inner Nuclear Membrane Proteins—Major inner nuclear membrane proteins, i.e. LBR, LAP2{beta}, emerin, and MAN1, are known to bind directly or indirectly to chromatin and to participate in stabilization of the heterochromatin structure, regulation of transcription, and other processes (17, 33, 34). On nuclear envelope breakdown in prophase, these proteins should become dissociated from chromatin and the nuclear lamina to disperse to the ER membrane network (16, 35). In the case of LBR, we have demonstrated that phosphorylation of LBR in the RS-region by cdc2 kinase and an unknown kinase in an M-phase egg extract causes dissociation from chromatin (3). In this study, we analyzed the LEM domain protein-chromatin interaction mechanism, focusing on emerin. Our findings suggested that the binding of emerin to chromatin mediated by BAF in a synthetic egg extract was suppressed by phosphorylation of emerin by one or more kinases in a mitotic egg extract. Our suggested dissociation/association mechanism for the binding of emerin to chromatin may be applicable to other LEM proteins, including MAN1 and LAP2{beta}, because these proteins are known to bind to BAF through a common LEM domain (11, 13). Indeed, in the case of LAP2{beta}, dissociation from chromatin on treatment with a mitotic HeLa cell extract has been reported (4). Dissociation of LBR and LEM proteins from chromatin through these phosphorylation mechanisms may cause the release of the nuclear membrane from chromatin at the onset of mitosis.

It has been shown that emerin dispersed to the ER membrane in prophase accumulates in the "core" region of the chromosome in telophase in HeLa cells (16, 35). In this process, BAF is recruited to the core region faster than emerin, and this recruitment is required for the assembly of emerin (16). This observation and our results suggest that the dephosphorylation of emerin may lead to telophase recruitment of emerin to BAF around the core region of chromatin. Our observations also support this idea: the binding of emerin to chromatin, once suppressed on treatment with MC, is recovered by subsequent treatment with SC (Fig. 2, MC-SC), and the recovery of the binding activity with SC is inhibited by pretreatment of SC with okadaic acid,3 which is known as a wide spectrum serine/threonine protein phosphatase inhibitor. On the other hand, it is also important to determine what kind of mechanism regulates the BAF-chromatin interaction to understand BAF-mediated interaction of emerin and chromatin, because Haraguchi et al. (16) have indicated that emerin cannot be re-localized to the chromatin surface in late telophase without BAF re-localization.


   FOOTNOTES
 
* This work was supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan, and grants for project research from Niigata and Kyoto Universities. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 81-25-262-6160; Fax: 81-25-262-6160; E-mail: thori{at}chem.sc.niigata-u.ac.jp.

2 The abbreviations used are: NE, nuclear envelope; GST, glutathione S-transferase; {Delta}TM, GST-fused fragment comprising amino acid residues 1–213 of human emerin; {Delta}LT, GST-fused fragment comprising amino acid residues 37–213 of human emerin; BAF, barrier-to-autointegration factor; LAP, lamina-associated polypeptide; LBR, lamin B receptor; MC, M-phase Xenopus egg cytosol fraction; SC, S-phase Xenopus egg cytosol fraction; MALDI-TOF MS, matrix-assisted laser desorption ionization-time of flight mass spectrometry; ER, endoplasmic reticulum; ESI-IT MS, electrospray ionization-ion trap mass spectrometry; CHCA, {alpha}-cyano-4-hydroxycinnamic acid; HF, hydrofluoric acid; CBB, Coomassie Brilliant Blue. Back

3 Y. Hirano, M. Segawa, K. Furukawa, and T. Horigome, unpublished observation. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Gant, T. M., and Wilson, K. L. (1997) Annu. Rev. Cell. Dev. Biol. 13, 669–695[CrossRef][Medline] [Order article via Infotrieve]
  2. Nikolakaki, E., Meier, J., Simos, G., Georgatos, S. D., and Giannakouros, T. (1997) J. Biol. Chem. 272, 6208–6213[Abstract/Free Full Text]
  3. Takano, M., Koyama, Y., Ito, H., Hoshino, S., Onogi, H., Hagiwara, M., Furukawa, K., and Horigome, T. (2004) J. Biol. Chem. 279, 13265–13271[Abstract/Free Full Text]
  4. Foisner, R., and Gerace, L. (1993) Cell 73, 1267–1279[CrossRef][Medline] [Order article via Infotrieve]
  5. Dreger, M., Otto, H., Neubauer, G., Mann, M., and Hucho, F. (1999) Biochemistry 38, 9426–9434[CrossRef][Medline] [Order article via Infotrieve]
  6. Martins, S. B., Marstad, A., and Collas, P. (2003) Biochemistry 42, 10456–10461[CrossRef][Medline] [Order article via Infotrieve]
  7. Gajewski, A., Csaszar, E., and Foisner, R. (2004) J. Biol. Chem. 279, 35813–35821[Abstract/Free Full Text]
  8. Bione, S., Maestrini, E., Rivella, S., Mancini, M., Regis, S., Romeo, G., and Toniolo, D. (1994) Nat. Genet. 8, 323–327[CrossRef][Medline] [Order article via Infotrieve]
  9. Ellis, J. A., Craxton, M., Yates, J. R., and Kendrick-Jones, J. (1998) J. Cell Sci. 111, 781–792[Abstract]
  10. Lin, F., Blake, D. L., Callebaut, I., Skerjanc, I. S., Holmer, L., McBurney, M. W., Paulin-Levasseur, M., and Worman, H. J. (2000) J. Biol. Chem. 275, 4840–4847[Abstract/Free Full Text]
  11. Furukawa, K. (1999) J. Cell Sci. 112, 2485–2492[Abstract]
  12. Lee, K. K., Haraguchi, T., Lee, R. S., Koujin, T., Hiraoka, Y., and Wilson, K. L. (2001) J. Cell Sci. 114, 4567–4573[Abstract/Free Full Text]
  13. Liu, J., Lee, K. K., Segura-Totten, M., Neufeld, E., Wilson, K. L., and Gruenbaum, Y. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 4598–4603[Abstract/Free Full Text]
  14. Segura-Totten, M., Kowalski, A. K., Craigie, R., and Wilson, K. L. (2002) J. Cell Biol. 158, 475–485[Abstract/Free Full Text]
  15. Tsuchiya, Y., Hase, A., Ogawa, M., Yorifuji, H., and Arahata, K. (1999) Eur. J. Biochem. 259, 859–865[Medline] [Order article via Infotrieve]
  16. Haraguchi, T., Koujin, T., Segura-Totten, M., Lee, K. K., Matsuoka, Y., Yoneda, Y., Wilson, K. L., and Hiraoka, Y. (2001) J. Cell Sci. 114, 4575–4585[Abstract/Free Full Text]
  17. Bengtsson, L., and Wilson, K. L. (2004) Curr. Opin. Cell Biol. 16, 73–79[CrossRef][Medline] [Order article via Infotrieve]
  18. Fairley, E. A., Kendrick-Jones, J., and Ellis, J. A. (1999) J. Cell Sci. 112, 2571–2582[Abstract]
  19. Clements, L., Manilal, S., Love, D. R., and Morris, G. E. (2000) Biochem. Biophys. Res. Commun. 267, 709–714[CrossRef][Medline] [Order article via Infotrieve]
  20. Lattanzi, G., Cenni, V., Marmiroli, S., Capanni, C., Mattioli, E., Merlini, L., Squarzoni, S., and Maraldi, N. M. (2003) Biochem. Biophys. Res. Commun. 303, 764–770[CrossRef][Medline] [Order article via Infotrieve]
  21. Holaska, J. M., Kowalski, A. K., and Wilson, K. L. (2004) PLoS Biol. 2, E231[CrossRef][Medline] [Order article via Infotrieve]
  22. Mislow, J. M., Holaska, J. M., Kim, M. S., Lee, K. K., Segura-Totten, M., Wilson, K. L., and McNally, E. M. (2002) FEBS Lett. 525, 135–140[CrossRef][Medline] [Order article via Infotrieve]
  23. Holaska, J. M., Lee, K. K., Kowalski, A. K., and Wilson, K. L. (2003) J. Biol. Chem. 278, 6969–6975[Abstract/Free Full Text]
  24. Wilkinson, F. L., Holaska, J. M., Zhang, Z., Sharma, A., Manilal, S., Holt, I., Stamm, S., Wilson, K. L., and Morris, G. E. (2003) Eur. J. Biochem. 270, 2459–2466[Medline] [Order article via Infotrieve]
  25. Haraguchi, T., Holaska, J. M., Yamane, M., Koujin, T., Hashiguchi, N., Mori, C., Wilson, K. L., and Hiraoka, Y. (2004) Eur. J. Biochem. 271, 1035–1045[Medline] [Order article via Infotrieve]
  26. Kawahire, S., Takeuchi, M., Gohshi, T., Sasagawa, S., Shimada, M., Takahashi, M., Abe, T. K., Ueda, T., Kuwano, R., Hikawa, A., Ichimura, T., Omata, S., and Horigome, T. (1997) J. Biochem. (Tokyo) 121, 881–889[Abstract/Free Full Text]
  27. Nakagawa, T., Hirano, Y., Inomata, A., Yokota, S., Miyachi, K., Kaneda, M., Umeda, M., Furukawa, K., Omata, S., and Horigome, T. (2003) J. Biol. Chem. 278, 20395–20404[Abstract/Free Full Text]
  28. Takano, M., Takeuchi, M., Ito, H., Furukawa, K., Sugimoto, K., Omata, S., and Horigome, T. (2002) Eur. J. Biochem. 269, 943–953[Medline] [Order article via Infotrieve]
  29. Wang, M., Xiao, G. G., Li, N., Xie, Y., Loo, J. A., and Nel, A. E. (2005) Electrophoresis 26, 2092–2108[CrossRef][Medline] [Order article via Infotrieve]
  30. Kuroda, I., Shintani, Y., Motokawa, M., Abe, S., and Furuno, M. (2004) Anal. Sci. 20, 1313–1319[CrossRef][Medline] [Order article via Infotrieve]
  31. Kuyama, H., Toda, C., Watanabe, M., Tanaka, K., and Nishimura, O. (2003) Rapid Commun. Mass Spectrom. 17, 1493–1496[CrossRef][Medline] [Order article via Infotrieve]
  32. Wolff, N., Gilquin, B., Courchay, K., Callebaut, I., Worman, H. J., and Zinn-Justin, S. (2001) FEBS Lett. 501, 171–176[CrossRef][Medline] [Order article via Infotrieve]
  33. Cohen, M., Lee, K. K., Wilson, K. L., and Gruenbaum, Y. (2001) Trends Biochem. Sci. 26, 41–47[CrossRef][Medline] [Order article via Infotrieve]
  34. Mansharamani, M., and Wilson, K. L. (2005) J. Biol. Chem. 280, 13863–13870[Abstract/Free Full Text]
  35. Haraguchi, T., Koujin, T., Hayakawa, T., Kaneda, T., Tsutsumi, C., Imamoto, N., Akazawa, C., Sukegawa, J., Yoneda, Y., and Hiraoka, Y. (2000) J. Cell Sci. 113, 779–794[Abstract]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Cell Sci.Home page
T. Haraguchi, T. Kojidani, T. Koujin, T. Shimi, H. Osakada, C. Mori, A. Yamamoto, and Y. Hiraoka
Live cell imaging and electron microscopy reveal dynamic processes of BAF-directed nuclear envelope assembly
J. Cell Sci., August 1, 2008; 121(15): 2540 - 2554.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
J. M. Holaska
Emerin and the Nuclear Lamina in Muscle and Cardiac Disease
Circ. Res., July 3, 2008; 103(1): 16 - 23.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
N. Leach, S. L. Bjerke, D. K. Christensen, J. M. Bouchard, F. Mou, R. Park, J. Baines, T. Haraguchi, and R. J. Roller
Emerin Is Hyperphosphorylated and Redistributed in Herpes Simplex Virus Type 1-Infected Cells in a Manner Dependent on both UL34 and US3
J. Virol., October 1, 2007; 81(19): 10792 - 10803.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
J. B. Morris, H. Hofemeister, and P. O'Hare
Herpes Simplex Virus Infection Induces Phosphorylation and Delocalization of Emerin, a Key Inner Nuclear Membrane Protein
J. Virol., May 1, 2007; 81(9): 4429 - 4437.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
G. S. Wilkie and E. C. Schirmer
Guilt by Association: The Nuclear Envelope Proteome and Disease
Mol. Cell. Proteomics, October 1, 2006; 5(10): 1865 - 1875.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
R. J. Nichols, M. S. Wiebe, and P. Traktman
The Vaccinia-related Kinases Phosphorylate the N' Terminus of BAF, Regulating Its Interaction with DNA and Its Retention in the Nucleus
Mol. Biol. Cell, May 1, 2006; 17(5): 2451 - 2464.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/48/39925    most recent
M503214200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hirano, Y.
Right arrow Articles by Horigome, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hirano, Y.
Right arrow Articles by Horigome, T.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement