The binding of lamin B receptor to chromatin is regulated by phosphorylation in the RS region Makoto Takano 1 , Masaki Takeuchi 1 , Hiromi Ito 2 , Kazuhiro Furukawa 1,2,3 , Kenji Sugimoto 4 , Saburo Omata 1,2,3 and Tsuneyoshi Horigome 1,5 1 Courses of Biosphere Science and 2 Functional Biology, Graduate School of Science and Technology, Niigata University, Japan; 3 Department of Biochemistry, Faculty of Science, Niigata University, Japan; 4 Laboratory of Applied Molecular Biology, Department of Applied Biochemistry, University of Osaka Prefecture, Osaka, Japan; 5 Center for Instrumental Analysis, Niigata University, Japan Binding of lamin B receptor (LBR) to chromatin was studie d by means o f an in vitro assay system involving recombinant fragments of human LBR and Xenopus sperm chromatin. Glutathione-S-transferase (GST)-fused proteins including LBR fragments containing the N -terminal region (residues 1–53) and arginine-serine repeat-containing region (residues 54–89) bound to chromatin. The b inding of GST-fusion proteins incorporating t he N-terminal and arginine-serine repeat-containing regions to chromatin was suppressed by mild trypsinization of the chromatin and by pretreatment with a DNA solution. A new cell-free system for analyzing the c ell cycle-dependent binding of a protein to chromatin was developed from recombinant proteins, a Xenopus egg cytosol fraction and sperm chromatin. The system was applied to a nalyse the binding of LBR to chromatin. It was shown that the binding of LBR fragments to chromatin was stimulated by phosphorylation in the arginine-serine repeat- containing region by a protein kinase(s) in a synthetic phase egg cytosol. However, the binding of LBR fragments was suppressed by phosphorylation at different residues in the same region by a kinase(s) in a m itotic phase cytosol. These results s uggested that the cell cycle-dependent binding of LBR to chromatin is regulated by phosphorylation in the arginine-serine repeat-containing region b y multiple kinases. Keywords: chromatin binding; lamin B receptor; LBR; Xenopus egg extract. The mature eggs of m ost vertebrates stay at the m etaphase of the second meiotic division until they meet sperm. In that phase, the nuclear envelope is dispersed in the cytop lasm as nuclear envelope precursor vesicles. Cell cycle progression is triggered by fertilization, the nuclear envelope of the pronucleus being formed first. Then, the formation and disruption of nuclear envelopes occurs repeatedly during cleavage and in further differentiated somatic cell divisions. Thus, the structure of nuclear envelopes changes very dynamically d epending on the stage o f the cell cycle. T o ensure the p recise assembly/disassembly of nuclear envel- opes in t he cell c ycle, the binding of proteins on nuclear envelope precursor vesicles/inner nuclear membranes to chromatin should be precisely regulated. Major nuclear envelope proteins known to bind to chromatin are lamins [1–4], lamin B receptor (LBR) [5,6], and L AP2b [7,8]. A peripheral nuclear membrane protein, Ya, is also known as a chromatin binding protein in e arly embryos of Drosophila melanogaster [9]. LAP2 was found as lamina-associated polypeptides in r at liver nuclear envelopes andshowntobindtochromatinattheN-terminalregion [7,8]. It was shown recently that when a recombinant fragment of the p rotein was added to cell-free Xenopus egg nuclear assembly reactions at high concentrations, mem- brane binding to chromatin is inhibited [10]. LBR was found first as an avian e rythrocyte- a nd liver-nuclear membrane protein [11,12]. Then, LBR was shown to b e a chromatin-binding protein [5,6,13]. The segment two-thirds from the C -terminal of t he LBR molecule contains eight transmembrane-segments [6,14,15] and exhibits sterol C14 reductase activity [16,17]. The segment one-third from the N-terminal (1–208) of human LBR is located in the nucleoplasm [14], and this portion is responsible for the binding of chromatin, DNA and most other proteins reported previously. In chicken erythrocytes, an 18-kDa membrane protein [18] and an LBR kinase were found to be associated with LBR [19]. LBR also bound a nuclear localization signal peptide [6,20], nucleoplasmin [6,20], and DNA [15] in vitro . It was shown by means of a t wo hybrid method that heterochromatin protein 1 ( HP1) binds to LBR [21], a nd the binding site was localized to a region (residues 97–174) of the N-terminal portion of human LBR [22]. Importantly, it was shown that LBR, but not LAP2, i s essential for the vesicle binding to chromatin using vesicles selectively depleted of these proteins by means of specific antibodies [5]. There have been some reports on regulation of the binding of LBR to other proteins. Phosphorylation of the arginine-serine repeat-region in the N-terminal portion of LBR by an LBR kinase inhibits the binding of p34 protein Correspondence to T. Horigome, Department of Biochemistry, Faculty of Science, Niigata University, 2-Igarashi, Niigata 950-2181, Japan, Fax/Tel.: + 81 25 262 6160; E-mail: thor i@chem.sc.niigata-u.ac.jp Abbreviations: CBB, Coomassie Brilliant Blue R-250; GST, glutathi- one-S-transferase; LBR, lamin B receptor; HP1, heterochromatin- associated protein 1; LAPs, lamina-associated polypeptides; PKA, protein kinase A; PKI, protein kinase inhibitor, a proteinous inhibitor specific for protein kinase A; PKII, calmodulin-dependent protein kinase II; SRPK, SR protein-specific kinase. (Received 9 October 2001, revised 4 December 2001, accepted 7 December 2001) Eur. J. Biochem. 269, 943–953 (2002) Ó FEBS 2002 [23]. LBR was phosphorylated in the mitotic phase in vivo by an SR protein-specific kinase (SRPK) and cdc2 kinase [24]. However, phosphorylation of LBR by cdc2 kinase in vitro has no effect on the binding to lamin B [24]. There has been no report about the effect of phosphorylation on the interaction of LBR and chromatin. T herefore, a study on the cell cycle-dependent regulation of the interaction of LBR and chromatin is important for elucidation of the nuclear envelope assembly/disassembly mechanism. In this stud y, we first determined the chromatin binding region of LBR by using beads bearing recombinant fragments of LBR and Xenopus sperm chromatin. Then, a system for analyzing the regulation m echanism for the binding of LBR to chromatin was developed through the combination of the above binding method and Xenopus egg cytosol fractions. It was suggested, with this method, that the c ell cycle-dependent binding of LBR to chromatin is regulated by phosphorylation in the arginine-serine repeat- containing region (RS-region) by multiple kinases. The potential function of LBR and LAP2 in vesicle targeting to chromatin is discussed. MATERIALS AND METHODS Materials Protein kinase inhibitors: A3 and K-252b were purchased from Calbiochem. Apyrase, the catalytic s ubunit of protein kinase A, and protein kinase inhibitor (PKI) were obtained from Sigma Chemicals Co. Calmodulin-dependent protein kinase II (PKII) was purified from bovine brain [25]. Buffers NaCl/P i :10m M sodium phosphate (pH 7.4), 140 m M NaCl and 2.7 m M KCl; extraction buffer: 50 m M Hepes-KOH (pH 7 .7), 250 m M sucrose, 50 m M KCl and 2.5 m M MgCl 2 ; elution buffer: 25 m M Tris/HCl (pH 7.5), 150 m M NaCl and 50 m M glutathione (reduced form); buffer X: 15 m M Pipes-KOH (pH 7.4), 200 m M sucrose, 7 m M MgCl 2 , 80 m M KCl, 15 m M NaCl and 5 m M EDTA; SRPK reaction buffer: 25 m M Tris/HCl (pH 7.5), 10 m M MgCl 2 and 200 m M NaCl; and buffer M: 20 m M Hepes-KOH (pH 7 .5), 60 m M b-glycerophosphate, 20 m M EGTA and 15 m M MgCl 2 . Preparation of demembranated Xenopus sperm chromatin Xenopus spermwastreatedwithlysolecithintoremovethe plasma and nuclear membranes without the highly co n- densed chromatin being affected, according to t he method of Smythe & Newport [ 26]. The chromatin concentration is expressed as the number o f chromatin com plexes in the binding reaction mixture. The nu mber was determined by counting with a hemacytometer. Preparation of a synthetic phase Xenopus egg cytosol Xenopus eggs were collected, dejelled, and then lysed to prepare a synthetic phase (interphase) extract, essentially as described previously [27]. The extraction buffer was supple- mented with 2 m M 2-mercaptoethanol, 10 lgÆmL )1 aproti- nin and leupeptin immediately before use. Eggs were packed into tubes by brief centrifugation for several seconds at 6000 g. Excess buffer a bove the packed eggs was removed and the eggs were then crushed by centrifugation at 15 000 g for 10 min. The crude extract, i.e. the supernatant between the lipid cap and pellet, was collected and mixed with 10 lgÆmL )1 cytochalasin B. The crude extract was further sep arated into cytosol, membrane and gelatinous pellet fractions by ultracentrifugation a t 200 000 g for 4 h in an RP55S rotor (Hitachi, Tokyo). The cytosol fraction was then re-centrifuged at 200 000 g for 30 min to remove residual m embr anes a nd st ored a t )80 °C until use. Preparation of a mitotic phase Xenopus egg extract Eggs were dejelled with 2% cysteine/NaOH (pH 8.0) at 23 °C. After washing three times with 100 m M NaCl and twice with buffer M at 23 °C,theeggswerewashedtwice with cold buffer M containing 100 m M NaCl and 250 m M sucrose at 4 °C. Then the eggs were supplemented with 10 lgÆmL )1 aprotinin and leupeptin, and packed into tubes by brief centrifugation f or several seconds at 6000 g. Excess buffer above t he packed eggs was removed and the eggs were then crushed by centrifugation a t 1 5 000 g for 1 0 min The crude extract w as collected, a nd further s eparated into cytosol, membrane and gelatinous pellet fractions as for the preparation of the synthetic phase extract, except that cytochalasin B was not added and buffer M was u sed instead of extraction buffe r. Chromatin binding assay (I): a method involving soluble proteins This method was used in the experiments for Fig. 2. A cytosol fraction o f Xenopus eggs was boiled for 10 min, cooled in ice-water for 5 min, and then centrifuged at 10 000 g for 10 min to remove denatured proteins. The resulting supernatant, i.e. heated cytosol, containing nucleo- plasmin was stored at )80 °C until use. To determine chromatin b inding of GST-fused proteins, 5 lL o f demem- branated sperm chromatin (40 000 per lL) in buffer X was incubated with 50 lL of heated c ytosol at 23 °Cfor30min for decondensation o f the chromatin. Then the c hromatin was precipitated by centrifugation at 2000 g for 10 min The pellet was suspended in 1 0 lL of extraction buffer c ontain- ing 0 .1% T riton X-100 and 0 .5 lg o f GST, GST–NK, G ST– NM, GST–RS or GST–SK, and then incubated at 4 °Cfor 20 min. Chromatin was reprecipitated by centrifugation at 7000 g for 10 min The resulting supernatant was designated as th e Ôunbound fractionÕ. The precipitated chromatin w as washed with 200 lL of extraction buffer, and then dissolved in 25 lL of 1% SDS. The resulting solution was centrifuged at 100 000 g for 1 h and the supernatant was designated as the Ôbound fractionÕ. The obtained ÔboundÕ (20 lL) and ÔunboundÕ (8 lL) fractions were separated by SDS/PAGE, transferred t o a nitrocellulose filter, and immunoblotted w ith affinity purified anti-GST Ig as described previously [28]. Chromatin binding assay (II): a method involving immobilized proteins This method was used for all chromatin-binding experi- ments other than those in Fig. 2. Demembranated sperm 944 M. Takano et al. (Eur. J. Biochem. 269) Ó FEBS 2002 chromatin (40 000 per lL) in 0.5 lL of buffer X was incubated with 10 lLofXenopus egg heated cytosol at 23 °C for 30 min for decondensation of the chromatin. Then the reaction mixture was centrifuged at 300 g for 10 min and the p recipitated chromatin was suspended i n 20 lL of extraction buffer. After centrifugation, the preci- pitated chromatin was resuspended in 10 lLofextraction buffer, and then added to 2 lg o f GST-fused proteins attached to 2 lL of glutathione–Sepharose 4B beads suspended in 10 lL of extraction buffer. After incubation at 4 °C f or 20 min, the binding reaction was s topped by pipetting 10-lL samples onto glass slides spotted with 8 lL of fixing solution (3% formaldehyde, 2 lLÆmL )1 Hoechst dye 33342, 80 m M KCl, 15 m M NaCl, 50% glycerol and 15 m M Pipes, pH 7.2). The fixed samples w ere o bserved by phase-contrast and fluorescence microscopy. O ne hundred beads were observed for every sample and ‘the percentage of beads w ith bound chromatin’ was c alculated. This value was used as an index of the affinity of beads bearing LBR fragments and chromatin. The values shown in the figures are the averages of three or more experiments, and are shown as values after subtraction of a blank value, except in Fig. 4. The blank value was determined in every experiment using GST–Sepharose beads instead of GST–LBR frag- ment-Sepharose beads, as shown in Fig. 4. The bars in the figure show the standard error. Assay for cell cycle dependency of the binding of LBR fragments to sperm chromatin GST-fused proteins attached to glutathione–Sepharose beads were preincubated with either a synthetic or mitotic phase egg cytosol fraction at 23 °C for one hour. After washing twice with extraction buffer, the binding to chromatin w as examined by chromatin binding assay II, as shown above. The addition of 1 M NaCl to the washing buffer to remove possible bound proteins from gel beads had n o e ffect on the binding of chromatin t o beads. Expression of LBR fragments and preparation of beads bearing these fragments Cloning of various fragm ents of human LBR fused with GST was carried out as previously described for Escher- ichia coli [6]. Expression of fusion proteins was induced by the addition of 0.1 m M isopropyl thio-b- D -galactoside, followed by incubation for 6 h at 30 °C. The bacterial cells were collected by centrifugation a nd resuspended in a buffered saline solution. The cell s uspension was sonicated vigorously and then centrifuged at 15 000 g for 10 min. An aliquot of the prepared supernatant was reacted with glutathione–Sepharose beads at 4 °Cfor2h.After washing twice with the buffered saline solution, the beads were stored at 4 °C until use. The amount of protein immobilized on beads was estimated b y the Lowry method after elution with glutathione followed by acetone-precipitation. Phosphopeptide mapping GST–NK phosphorylated with [c- 32 P]ATP in vitro was separated by SDS/PAGE and then transferred to a nitrocellulose sheet. The GST–NK band was excised, soaked in 0.5% poly(vinyl pyrrolidone) K)30 (Wako, Tokyo) in 100 m M acetic acid for 3 0 m in at 37 °Candthen washed exten sively w ith water. The protein was digested with trypsin in 50 m M NH 4 HCO 3 at 37 °C for 24 h. 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; ascending chromatography in the second dimension was performed using 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 with intensifying screens. Preparation of a heterochromatin-associated protein 1 (HP1) fragment Recombinant GST-fused HC1 (83–191 amino acids), a n human HP1 HSa fragment containing the LBR binding domain (104–191 amino acids) [22], was expressed as a GST-fusion protein i n E. coli, as previously described [29], and then bound to glutathione–Sepharose beads. The beads were treated with Factor Xa to hydrolyze the hinge region of GST and HC1 at 37 °C for 3 h. Then, the cleaved HC1 portion, which was recovered i n the supernatant, was concentrated and used for the binding assay. RESULTS Identification of chromatin binding regions of LBR To analyze t he binding of LBR to chromatin, we used the N-terminal portion of LBR, because this portion is respon- sible for the binding to chromatin [ 5,6]. A fragment containing the whole N-terminal portion, NK, and its subfragments, shown in Fig. 1A, were expressed in E. coli as GST fusion proteins (Fig. 1B), and then bound to glutathione–Sepharose beads. These beads were incubated with demembranated a nd decondensed sperm c hromatin. After fixation and staining of DNA with Hoechst 33342, the beads were observed b y phase contrast and fluorescence microscopy (Fig. 1C). Most GST–NK (Fig. 1C) and GST– RS (not shown) beads bound chromatin, however, G ST beads only bound a little (Fig. 1C). Then, we introduced Ôpercentage of beads with boun d chromatinÕ as an index for estimating the affinity of protein fragment-bearing beads with chromatin. One hundred beads were counted and the percentage of beads with bound chromatin was calculated. GST–NK, GST–RS and G ST bearing beads gave values of 65 ± 7, 60 ± 5 a nd 18 ± 5%, respectively. These values clearly show that the RS moiety w ithin the NK region of LBR exhibits affinity with chromatin. Then, to confirm these results, we tried an established in vitro chromatin- binding assay involving soluble proteins. GST–NK, GST– NM, GST–RS, GST–SK and G ST in a soluble state were incubated with chromatin. The chromatin bound and unbound fractions were analyzed by immunoblotting (Fig. 2 ). GST–NK and GST–RS were bound to chromatin, although GST–NM, GST–SK and the GST moiety alone were not bound (Fig. 2). Furthermore, bindings of GST– NK and GST–RS to chromatin were inhibited in the Ó FEBS 2002 Regulation of the binding of LBR to chromatin (Eur. J. Biochem. 269) 945 presence of free DNA (Fig. 2). The inhibition with DNA is consistent with that observed with an assay involving GST- fusion protein b earing beads, as shown below. From these results, we concluded t hat Ôthe percentage of beads with bound chromatinÕ obtained with GST-fusion protein-bear- ing beads can be u sed as a n index of t he affinity of protein fragments to chromatin. We then applied t his b ead method to characterize the binding of LBR to chromatin because it is faster and needs only a one-tenth amount of chromatin compared to t he established chromatin binding assay. GST–NK, GST–NM, GST–RS and GST–SK beads were prepared, and chro- matin binding was examined (Fig. 3, empty columns). It is Fig. 2. Chromatin binding assay involving soluble GST-fusion proteins. Approximately 0 .5 lg o f G S T–NK, GST–NM, GST–RS , GST –S K or GST w as incubated w ith various am ounts of dec onde nsed Xenopus sperm chromatin, as shown in the figure, and then centrifuged to separate the unbound f raction (supernatant) f rom chromatin. T he pellet was washed with extraction buff er, dissolved in 1% SDS and then ultracentrifuged to remo ve DNA. The t hus obt ained supe rnatant was designated the Ôbound fractionÕ.TheÔboundÕ and ÔunboundÕ frac- tions were separated by SDS/PAG E a nd th en analyzed by immu no- blotting with anti-GST Ig. In the case of Ô(+ DNA)Õ, GST-fusion proteins were preincubated with 0.5 mgÆmL )1 of porcine liver DNA and then used for the binding assay. ÔUÕ and ÔBÕ in the figure den ote the Ôunbou ndÕ and Ôbo undÕ fractions, respectively. For other details, see Materials and methods. Fig. 1. N-Terminal fragments of LBR expressed as GST fusion pro- teins, and the binding of beads bearing these fragments to chromatin. (A) Schematic diagrams of N-terminal fragments of L BR exp ressed as GST fusion proteins. The line numbered 1 and 211 shows the N-terminal portion of the LBR molecule, and these numbers are those of amino-acid residues from the N-terminal of LBR. ÔRSÕ is the site of arginine-serine repeats. (B) SDS/PAGE of GST fusion proteins. Samples were expressed in E. coli, purified with glutathione–Sepha- rose,analyzedbySDS/PAGEona10%gel,andthenstainedwith Coomassie Brilliant Blue R-250 (CBB). The lines at the left show the positions of marker pro teins having relative molecular masses of 66, 43 and 29 kDa, from top t o bottom. (C) Binding of GST–NK bearing beads to chromatin. GST and GST–NK bearing glutathione-Sepha- rose beads were incubated with decondensed Xenopus sperm chro- matin at 4 °C for 20 min, and then observed by phase contrast and fluorescence microscopy after staining of DNA with Hoec hst 33342. Arrows, a rrowhead s and d ouble-arrow heads indica te b eads, unbound chromatin and bound chromatin, respectively. Bar ¼ 10 lm. 946 M. Takano et al. (Eur. J. Biochem. 269) Ó FEBS 2002 known that GST–NM, GST–RS and GST–SK carry binding sites f or chromatin [ 6], naked DNA [15], and a heterochromatin specific protein, HP1 [22], respectively. Beads bearing GST–NK and GST–RS bound chromatin. GST–NM beads also showed apparent but lower affinity to chromatin. The lower affinity could not be detected in an assay involving soluble proteins (Fig. 2). These results showed that the NM and RS regions have affinity for chromatin. However, the binding of chromatin to GST–SK beads was very low, although the fragment in question carries a binding site for chromatin protein HP1. This point is discussed below. To characterize the mode of binding of LBR to chromatin, beads bearing LBR fragments were preincu- bated with a DNA solution and then the binding to chromatin was examined (Fig. 3, h atched c olumn s). T he binding of RS- a nd NK-fragm ents to chromatin w as strongly suppressed, but the binding of the NM-fragment was little a ffected. T hese results suggested that the RS region of LBR binds to the DNA region of chromatin. On the other hand, with the pretreatment of the chromatin with a low c oncentration of trypsin, the binding was suppressed strongly in the c ase of the NM fragment but not the NK and RS fragments (Fig. 3, filled columns). These results suggested that the NM region binds to a protein(s) on chromatin. These results also suggested that the binding of the RS region to chromatin DNA is superior to the binding of the NM region to the chromatin p rotein because the binding mode of NK, which contains the NM and RS regions, is similar to that of RS. An assay system for the cell cycle-dependent binding of LBR to chromatin We wondered whether this binding system can be applied to the analysis o f the cell cycle-dependent interaction of LBR and chromatin. Th erefore, we p retreated beads bearing LBR fragments w ith a Xenopus egg cytosol fraction at the synthetic phase of the cell cycle, and then chromatin binding was e xamined. T he binding was stimulated (data not shown). W hen NK b eads were pretre ated with a m itotic phase cytosol fraction, however, the binding was strongly suppressed (data not shown). Changes in the affinity of chromatin to NK on pretreatment with the two phases egg extracts were the same as the predicted c hanges in living cells. T hese preliminary results s uggested t hat an in vitro assay system f or the a nalysis of t he cell c ycle-dependent interaction of LBR and chromatin can be developed using this binding assay system. Then, we optimized the assay conditions for a nalysis o f the cell cycle-dependent interaction of LBR and chro- matin (Fig. 4). The preincubation time for GST–NK beads at 23 °C. with a synthetic phase cytosol fraction was examined a nd it was found that 60 min is necessary to reach a plateau of increased binding affinity (Fig. 4A). The same preincubation time was applicable to experi- ments involving a mitotic phase cytosol fraction (data not shown). The binding of chromatin to GST–NK beads almost linearly increased with increasing chromatin con- centration up to 70–80% (Fig. 4B). V arious concentra- tions of GST–NK on beads, 1–10 lgÆlL )1 , had no effect on the percentage of beads with bound chromatin (data not shown). The binding of chromatin to GST–NK beads was very fast, being completed with in one minute at 4 °C (Fig. 4 C). Then, as standard conditions, we chose 60 min as the preincubation time, 20 000 chromatin per assay, and 20 min for t he time o f binding of chromatin to beads, as shown under Materials and methods. The chromatin concentration can be varied, depending on the experimental purpose, i.e. lower and higher chromatin concentrations can be used to analyze increases or decreases in binding activity (for example, Fig. 5A,B). Then, we applied this method to analyze the regulation mechanism for the b inding of LBR to chromatin, as described below. Cell cycle-dependent binding of LBR fragments to chromatin When beads were pretreated with a synthetic phase cytosol fraction, the numbers of NK- and RS-beads with bound chromatin were significantly increased, but not that of NM-beads (Fig. 5A). SK-beads showed no significant binding of chromatin regardless of treatment with a synthetic phase cytosol fraction (Fig. 5A). On the other hand, when beads were pretreated with a mitotic phase cytosol fraction, the numbers of NK- and RS-beads with bound chromatin were significantly decreased, but not that of NM-beads (Fig. 5B). SK-beads again showed no significant b inding regardless o f treatment with a m itotic phase cytosol fraction (Fig. 5B). These r esults show that the affinity of the N-terminal portion of LBR and chromatin increases in a synthetic phase extract and decreases in a m itotic phase one in vitro.Moreover,the Fig. 3. Identification of chromatin binding domains in the N-terminal region of LBR and analysis of the binding mode. Empty columns: four kinds o f GST fusion proteins inclu ding N-terminal domains of LBR attached to glutathione–Sepharose beads were incubated with decon- densed sperm chromatin at 4 °C for 20 min, and then o bserved by fluorescence microscopy after staining of DNA with Hoechst 33342. The Ôpercentage of beads with bound chromatinÕ values were deter- mined as described under Materials a nd methods after subtraction of the value for blank GST-beads. (Hatched columns) Four GST fusion proteins including LBR fragments a ttached to glutathione–Sepharose beads were preincubated with a 0.5-m gÆmL )1 DNA solution at 4 °C for 1 h, and t hen the binding to chrom atin was examined as above. (Filled columns) Decondensed chromatin was pretreated with 10 lgÆmL )1 trypsin at 23 °C for 10 min, and then aft er the a ddit ion o f leupeptin and aprotinin (final, 0.5 mgÆmL )1 ), the binding to beads bearing GST–LBR fr agments was examined a s above. Ó FEBS 2002 Regulation of the binding of LBR to chromatin (Eur. J. Biochem. 269) 947 binding of the N -terminal portion of LBR to chromatin is regulated through the RS region, not the NM- and SK-regions. The directions of the changes in the affinity of NK and chromatin on treatment with the two cytosol fractions in vitro, i.e., increasing with a synthetic phase cytosol f raction d ecreasing with a mitotic one, w ere strikingly the same as those of the changes in the affinity of nuclear envelope precursor vesicles and chromatin in vivo. The results obtained with this in vitro system seemed to reflect this phenomenon in vivo. Stimulation of the binding of LBR to chromatin by phosphorylation by a kinase(s) in a synthetic phase egg cytosol Chromatin binding of beads bearing GST–NK was incr- eased by pretreatment w ith a synthetic phase egg cytosol fraction (compare columns 1 an d 2 in Fig. 6A). The increase could be suppressed by apyrase and protein kinase-inhib- itors having broad specificities: staurosporine, A3 [30], and K252b (compare columns 3–6 with column 2 in Fig. 6A). Fifty percent suppression with staurosporine was achieved with as little as  4n M (data not shown). These results indicate that the increase in the affinity of NK to chromatin is an ATP-dependent reaction, and is caused by a kinase(s) in the c ytosol. Then, authentic p rotein kinase A (PKA) and calmodulin-dependent protein kinase II (CaMKII) were applied instead of the c ytosol fractio n, as we p reviously observed that LBR is phosphorylated by these kinases [28]. PKA but not PKII caused a similar increase in the affinity of NK to chromatin (Fig. 6A, column 7). However, protein kinase inhibitor (PKI), a PKA-specific inhibitor, could not suppress the stimulation of the binding of NK to chromatin Fig. 5 . Effects of pretreatment of LBR fragments with Xenopus egg cytosol fractions on the binding to chromatin. (A) Beads, with bound GST and GST-fused proteins including LBR fragments, were pre- treated with extraction buffer (empty bars) or a synthetic phase egg cytosol fraction (filled bars), an d then the b inding to chrom atin was determined as in Fig. 3. (B) The same as (A) except that a mitotic phase egg cytosol fraction was u sed i nstead of th e synthetic phase one. Instead of 20 000 chromatin per assay as a standard condition, 10 000 and 25 000 chromatin p er assa y w ere u sed in ( A ) and ( B) , r espectively, to clearly s ho w t he cha n ges i n Ôpercentage of beads with bo und chromatinÕ. Fig. 4. Assay conditions for the binding of chromatin to GST–NK beads pretreated with a synthetic pha se Xenopus egg cytosol fraction. GST– NK (filled circles) and b lank GST ( open circles) beads, 2 lL, were preincubated with 20 lL of a synthetic phase Xenopus egg cytosol fraction at 23 °C f or 1 h. Thus treated b eads wer e then i ncubated w ith 25 000 chromatin per assay at 4 °C for 20 min. Then, the p ercentage of beadswithboundchromatinwasdetermined.Ineachfigure,thetime of preincu bation o f beads with a cytosol fraction (A), the chromatin concentration (B), or the time of incubation of beads with chromatin (C) was varied. 948 M. Takano et al. (Eur. J. Biochem. 269) Ó FEBS 2002 by a s ynthetic phase cytosol (Fig. 6A, column 9 ). T hese results suggested that a kinase(s) in the synthetic phase egg cytosol fraction phosphorylates NK at a functionally similar site(s) to i n the case of PKA, and thereby increases t he affinity of LBR and chromatin. The b inding of chromatin to GST– RS beads was also stimulated by a synthetic phase cytosol and PKA (Fig. 6A, columns 10–13). These data suggested that phosphorylation at a site(s) within the RS region is responsible for the stimulation. To confirm t he phosphorylation in the RS region, beads bearing GST, GST–NK and GST–RS w ere i ncubated with a synthetic phase cytosol in the presence of [c- 32 P]ATP. Then, the proteins were eluted with SDS and analyzed by SDS/ PAGE. The gel was stained with CBB and then subjected to autoradiography (Fig. 6 B). Radioactivity was detected for the GST–NK and GST–RS beads, but not for GST itself (Fig. 6 B, autoradiography, lanes 1–3). Incorporation of radioactivity into the GST–N K and GST–RS bands was completely suppressed by the addition of staurosporine (Fig. 6 B, autoradiography, lanes 5 and 6). These results indicate that LBR is indeed phosphorylated in the RS region by a synthetic phase cytosol, which stimulated the binding to chromatin. Suppression of the binding of LBR to chromatin by phosphorylation with a kinase(s) in a mitotic phase egg cytosol The affinity of NK-bead s and chromatin was decreased by preincubation of the beads with a mitotic phase egg cytosol fraction ( compare columns 1 and 2 in Fig. 7A). The decrease could be suppressed by apyrase and protein kinase inhibitors having broad specificities: staurosporine, A3 and K252b (Fig. 7A). Fifty percent suppression with stauro- sporine was achieved with  6n M (data not shown). These results suggest that the decrease in the affinity of NK and chromatin is an ATP-dependent reaction, and is caused by a kinase(s) in the cytosol. When GST–RS was used instead of GST–NK, similar suppression of the b inding to chromatin was observed with preincubation with a mitotic cytosol (Fig. 7 A, columns 7–9). These results suggested that phos- phorylation in the RS region is responsible for the suppression. To confirm the phosphorylation in the RS region, beads bearing GST–NK, GST–RS and only GST wereincubatedwithamitoticphasecytosolfractioninthe presence of [c- 32 P]ATP. Then, the proteins were eluted with SDS and analyzed by SDS/PAGE, followed by C BB staining and autoradiography (Fig. 7B). Radioactivity was detected for the GST–NK and GST–RS beads, but not for GST (Fig. 7B, Autoradiography, lanes 1–3). Incorporation of the radioactivity into the GST–NK and GST–RS bands was completely suppressed by the addition of staurosporine (Fig. 7 B, autoradiography, lanes 5 and 6). These results indicate that LBR is phosphorylated in the RS region by a mitotic phase cytosol, which suppressed the binding to chromatin. Phosphopeptide mapping Synthetic phase and mitotic phase egg extracts both phosphorylated GST–NK and had opposite effects on chromatin b inding affinity (Figs 6 and 7). Therefore, the phosphorylation sites for the two extracts w ere exp ected to be different. Then, to confirm this difference, tryptic phosphopeptides of GST–NK treated with synthetic phase and mitotic phase egg e xtracts were c ompared with each other by means of two-dimensional separation (Fig. 8). A s can be s een in Fig. 8, several phosphop eptide spots were different, although some were the same. These results c learly showed that the NK fragment is phosphorylated with synthetic phase and mitotic phase egg extracts at common multiple sites, however, as expected, several sites are Fig. 6. Stimulation of the binding of LBR fragments to chromatin by phosphorylation with a synthetic phase e gg cytosol fraction. (A) Effects of apyrase, protein kinases, a nd protein kinase inhibitors on stimula- tion of the binding of LBR fragments to chromatin by pretreatment with a synthetic phase egg cytosol fraction. GST–NK-beads (columns 1–9) and G ST–RS-beads (columns 10– 13) were preincubated w ith extraction buffer (Buffer), a synthetic phase egg cytosol fraction (SC), 1 lgÆmL )1 protein kinase A (PKA), 1 lgÆmL )1 calmodulin-dependent protein kinase II (CaMKII), and SC containing either 8 mU apyrase, 10 n M stauros porin e (Sta.), 1 m M A3, 1 l M K252b or 50 lgÆmL )1 protein kinase inhibitor (PKI), and then the binding to chromatin was examined as in Fig. 3. (B) Detection of phosphorylation. Beads bearing 1–2 lg GST, GST–NK, or GST–RS were incubated with 20 lL of a synthetic phase egg cytosol fraction supplemented with 0.1 lLof3.3l M [c- 32 P]ATP ( 110 TBqÆmmol )1 ) in the presence (SC/Sta.) o r absence (SC) of 10 l M staurosporine at 23 °Cfor1h. Thus treated proteins w ere extracted with SDS and then analyzed by SDS/PAGE, followed by CBB staining and autoradiography. Lanes 1 and 4 , GST; lanes 2 and 5, GST–NK; l anes 3 and 6, GST– RS. The arrowhead and double arrowhead indicate the GST–NK and GST–RS bands, respectively. Ó FEBS 2002 Regulation of the binding of LBR to chromatin (Eur. J. Biochem. 269) 949 phosphorylated specifically with a synthetic phase or mitotic phase extract. DISCUSSION Binding sites on the N-terminal portion of LBR for chromatin Ye et al. reported that free-DNA [15] and a chromatin protein, H P1 [22], bind with LBR in regions corresponding to the RS and SK regions, respectively. On the other hand, we previously reported t hat the NM region of LBR, w hich is different from the RS and SK regions, binds with chromatin [6]. Therefore, we analyzed the binding of LBR to chromatin in more detail using a n assay method involving GST-fusion fragments of LBR and Xeno pus sperm c hromatin in this study. It was shown that the RS region of LBR binds with chromatin and that the b inding is inhibited by the addition of free DNA (Figs 2 and 3). These results suggested that LBR binds to a DNA region on chromatin in the RS region. This idea was consistent with a report by Ye & Worman [15], i.e. that a region corresponding to RS binds free DNA. Duband-Goulet & Courvalin recently show ed that LBR binds linker DNA but not the nucleosome core using in vitro reconstituted nucleosomes and short DNA fragments [31]. Therefore, the b inding site on chromatin for the RS r egion of LBR seems to be linker DNA. On the o ther hand, it was suggested that the NM-region, not the SK-region, binds to a protein(s) on s perm chromatin ([6]; Fig. 3). HP1, the only known chromatin protein which binds to LBR, was reported by Ye et al.tobindtoaregion of SK [22]. Then, we examined which region of LBR binds to HP1 i n our binding assay system. A HP1 fragment (83–191 amino acids) containing the LBR binding region was expressed in E. coli, and then the binding to beads bearing GST–NK, GST–NM, GST–RS and GST–SK was examined. The HP1 fragment bound to beads bearing GST–NK and GST–SK, but not to ones bearing GST–NM (data not shown). T hese results are consis tent with those reported by Ye et al. [22]. On the o ther hand, James et al. reported t hat HP1 is not observed in the nuclei o f e arly syncytial e mbryos, but becomes concentrated in the nuclei at the syncytial blastoderm stage (about nuclear division cycle 10) in Drosophila melanogaster [32]. Therefore, HP1 may not participate in the binding of LBR to sperm chromatin in eggs. In t he case of the binding of LBR to Fig. 7 . Suppression of the b inding of LBR fragments to chromatin by phosphorylation with a mitotic phase egg cytosol fraction. (A) Effects of apyrase and protein kinase inhibitors on sup pression of the bind ing of LBR fragme nts to chromatin b y pretreatment with a mitotic phase egg cytosol fraction. GST-NK-beads (columns 1–6) and GST–RS-beads (columns 7–9) were preincubated with extraction buffer (Buffer), a mitotic phase egg cytosol fraction (MC), and MC containing either 8mUapyrase,10n M staurosporine (Sta.), 1 m M A3 or 1 l M K252b, and then the binding to chromatin was examined as in Fig. 3. (B) Beads bearing 1–2 lg GST, G ST–N K, o r GST–RS were treated and analyzed as in Fig. 6B, except that a mitotic phase egg cytosol fraction was u sed instead of the synthetic phase o ne. Lanes 1 a nd 4, GST; lanes 2 and 5, GST –NK; lanes 3 an d 6, G ST–RS. The arrowhead and double arrowhead indicate the GST–NK and GST–RS bands, respectivel y. Fig. 8. Tryptic phosphopeptide analysis of GST-NK. Beads bearing 20 lg GST–NK were i ncubated with 20 lL of a synthetic phase (SC) or a mitotic phase (MC) egg cytosol fraction supplemented with 2 lLof3.3l M [c- 32 P]ATP (110 TBqÆmmol )1 )at23°C for 1 h. The thus treated proteins were separated b y S DS/PAG E and the n transfe rred to a nitrocellulose sheet. The G ST–NK b ands we re e xcised a nd dige sted with trypsin. The eluted phosphopeptides were separated by electrophoresis at pH 8.9 (horizontal direction; cathode to the left) and by a scending chromato- graphy. T he points o f sample application can be seen as dots near the bottom-left corners. 950 M. Takano et al. (Eur. J. Biochem. 269) Ó FEBS 2002 sperm chromatin in eggs, binding through the RS region of LBR to linker DNA of chromatin s eems to b e predominant. Then, the binding is supported by the interaction of the NM region and a protein(s ) other t han HP1 on sperm chromatin. An assay system for the cell cycle-dependent binding of LBR to chromatin To analyze the regulatory mechanism for the cell cycle- dependent binding of chromatin and nuclear membranes, we developed a new in vitro assay system comprising a Xenopus egg extract and a binding assay involving sperm chromatin and beads bearing LBR fragments. The binding was stimulated by preincubation of beads bearing LBR fragments w ith a synthetic p hase extr act, whereas it w as suppressed by that with a mitotic phase extract (Fig. 5). The binding of chromatin t o LBR fragments on beads could be estimated semiquantitatively by t his method. The effects of enzymes, inhibitors and other reagents on the cell cycle– dependent interaction could also be examined very easily by means of t his method. This method is applicable to the analysis of phosphorylated residues o n LBR fragments and protein kinases responsible for the phosphorylation. This method should a lso be a pplicable to the analysis of cell cycle-dependent regulation of the binding of other proteins to chromatin, such as LAP2, emerin, MAN1, lamins and nuclear matrix proteins. Kinases responsible for cell cycle-dependent regulation of the binding of LBR to chromatin It was suggested that the binding of LBR to sperm chromatin is stimulated by phosphorylation in the RS region of LBR by a kinase(s) in a s ynthetic phase egg c ytosol (Fig. 6). In interphase somatic c ell nuclei, the b inding of LBR to lamin B may be stimulated by in vivo phosphory- lation by PKA [33]. However, PKA seems not to participate in s timulation o f the bind ing o f LBR to chromatin in a Xenopus egg system, because PKI, a specific inhibitor for PKA, could not suppress the stimulation (Fig. 6A). On the other hand, an SR-repeat specific kinase 1 (SRPK1), which is expressed ubiquitously [34] and phosphorylates LBR in a constitutive manner, is known to phosphorylate serine/ threonine residues within the RS-repeat ( Fig. 1) in the R S region at multiple sites [23,35]. This phosphorylation inhibits the binding of LBR to p34/p32 [23], whereas there has been no report about the effect on the interaction of LBR an d chromatin. Identification of the kinase(s) respon- sible for the stimulation of the binding of LBR to chromatin is important for c larifying the p hysiological function of the phosphorylation, and such work is currently underway. It was suggested that the binding of LBR to sperm chromatin is strongly suppressed by phosphorylation in the RS region of LBR b y a kinase(s) in a mitotic phase egg cytosol (Fig. 7). Results of phosphopeptide mapping of GST–NK treated with synthetic phase and mitotic phase egg extracts showed different patterns (Fig. 8). It is known that recombinant cdc2 kinase and a mitotic extract of cultured chicken cells phosphorylate serine 71 within the RS region [24]. The binding of LBR to lamin B is not affected by such phosphorylation, whereas the effect on the binding to chromatin is not known [24]. In a zebrafish egg system, it was found that PKC and cdc2-kinase mediate phosphory- lation events that elicit nuclear envelope disassembly [36]. In a sea urchin egg system, an LBR-like protein mediates targeting of nuclear envelope vesicles to sperm chromatin [37]. These observations are c onsistent with the idea that phosphorylation of serine 71 of LBR by cdc2 kinase in a mitotic egg cytosol participates in the dissociation of LBR and chromatin. Therefore, identification of the kinase(s) and phosphorylation site(s) responsible for the suppression is important, and such work is currently underway. Cell cycle-dependent regulation of the interaction of nuclear membranes and chromatin The dissociation/association of membranes with chromatin in pronuclei formation, and the beginning and end of mitosis a re critical for control of the nuclear dynamics during these stages of the cell c ycle. Inner nuclear membran e proteins, LBR [5,6,13], LAP2 [7], and emerin (M. S egawa, K. Furakawa, S. Omata & T. Horigone, unpublished observations) have been shown to bind directly to chrom- atin. Therefore, precise regulation of the cell-cycle depen- dent dissociation/association of these proteins and chromatin is i mportant for t he cell cycle. In the case of LBR, binding to chromatin was shown by sperm chromatin in vitro ([6,13]; this study), and by mitotic phase chromo- somes f rom C HO cells [5]. Regulation of the binding of LBR to chromatin by phosphorylation was shown in this study using s perm chromatin and a Xenopu s egg extract. In the case of LAP2, phosphorylation in the interphase [38] and mitotic phase [7] has been shown in somatic cells. It has also been shown that t he phosphorylation of LAP2 with a mitotic H eLa cell extract in hibits its b inding to chromo- somes [ 7]. In the cases of emerin a nd MAN1, which share the LEM domain with LAP2 [39,40], the regulatory mechanism for the binding to chromatin remains to be elucidated. The function of LBR in the targeting of nuclear membranes or nuclear envelope precursor vesicles to chromatin remains elusive. In rat liver and tu rkey erythro- cyte in vitro systems, Pyrpasopoulou et al. [5] showed that the binding of vesicles to chromatin was suppressed when LBR, but not LAP2, was immuno-depleted from the vesicles. In a Xenopus e gg cell-free system, however, it was found that ves icles containing NEP-B78 bind first to chromatin a nd then to vesicles containing an LBR-like protein [41]. LBR-containing vesicles alone can no t bind to chromatin [41]. These observations may suggest that LBR does not participate i n the binding of vesicles. However, surface remodeling of chromatin through initial interactions between NEP-B78 containing vesicles and chromatin may permit LBR-chromatin binding activity [41]. Therefore, the possibility of direct participation of LBR in cell cycle- dependent vesicle targeting to chrom atin still remains f or the Xenopus egg system, too. In the case of the sea urchin egg system, it was suggested that a 56-kDa LBR-like protein, which reacts w ith anti-(human LBR) Ig, participates in the targeting [37]. Therefore, the participation o f LBR in the targeting of nuclear membranes to chromatin may vary a little from system to system a nd/or LBR acts together with other proteins. Indeed, LBR and a LEM domain protein, emerin, are targeted to different regions on the surface of chromatin in the telophase very early, suggesting that the two proteins may participate in t he targeting of nuclear Ó FEBS 2002 Regulation of the binding of LBR to chromatin (Eur. J. Biochem. 269) 951 membranes to different regions on the surface of chromatin [42]. We also observed the binding of a truncated emerin protein directly to sperm chromatin in vitro (M. S egawa, K. Furakawa, S. Omata & T. Horigone, unpublished observation). LAP2 s eems to participate in the targeting of nuclear envelope precursor vesicles in the Xenopus egg extract system because membrane binding to chromatin is inhibited on the addition of a high concentration of a truncated recombinant LAP2 protein to the cell-free Xenopus egg extract system [10]. 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