Báo cáo Y học: The binding of lamin B receptor to chromatin is regulated by phosphorylation in the RS region

Eur. J. Biochem. 269, 943–953 (2002) (cid:211) FEBS 2002

The binding of lamin B receptor to chromatin is regulated by phosphorylation in the RS region

Makoto Takano1, Masaki Takeuchi1, Hiromi Ito2, Kazuhiro Furukawa1,2,3, Kenji Sugimoto4, Saburo Omata1,2,3 and Tsuneyoshi Horigome1,5 1Courses of Biosphere Science and 2Functional Biology, Graduate School of Science and Technology, Niigata University, Japan; 3Department of Biochemistry, Faculty of Science, Niigata University, Japan; 4Laboratory of Applied Molecular Biology, Department of Applied Biochemistry, University of Osaka Prefecture, Osaka, Japan; 5Center for Instrumental Analysis, Niigata University, Japan

lamin B receptor; LBR;

applied to analyse 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 mitotic phase cytosol. These results suggested that the cell cycle-dependent binding of LBR to chromatin is regulated by phosphorylation in the arginine-serine repeat-containing region by multiple kinases.

Keywords: chromatin binding; Xenopus egg extract.

The mature eggs of most vertebrates stay at the metaphase of the second meiotic division until they meet sperm. In that phase, the nuclear envelope is dispersed in the cytoplasm 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 depending on the stage of the cell cycle. To ensure the precise assembly/disassembly of nuclear envel- opes in the cell cycle, the binding of proteins on nuclear envelope precursor vesicles/inner nuclear membranes to chromatin should be precisely regulated.

Binding of lamin B receptor (LBR) to chromatin was studied by means of 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 binding of GST-fusion proteins incorporating the 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 cell cycle-dependent binding of a protein to chromatin was developed from recombinant proteins, a Xenopus egg cytosol fraction and sperm chromatin. The system was

embryos of Drosophila melanogaster [9]. LAP2 was found as lamina-associated polypeptides in rat liver nuclear envelopes and shown to bind to chromatin at the N-terminal region [7,8]. It was shown recently that when a recombinant fragment of the protein 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 erythrocyte- and liver-nuclear membrane protein [11,12]. Then, LBR was shown to be a chromatin-binding protein [5,6,13]. The segment two-thirds from the C-terminal of the 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 two hybrid method that heterochromatin protein 1 (HP1) binds to LBR [21], and 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, is essential for the vesicle binding to chromatin using vesicles selectively depleted of these proteins by means of specific antibodies [5].

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: thori@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)

Major nuclear envelope proteins known to bind to chromatin are lamins [1–4], lamin B receptor (LBR) [5,6], and LAP2b [7,8]. A peripheral nuclear membrane protein, Ya, is also known as a chromatin binding protein in early

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

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[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. Therefore, 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.

nin and leupeptin immediately before use. Eggs were packed into tubes by brief centrifugation for several seconds at 6000 g. Excess buffer above 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 separated into cytosol, membrane and gelatinous pellet fractions by ultracentrifugation at 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 membranes and stored at )80 (cid:176)C until use.

Preparation of a mitotic phase Xenopus egg extract

In this study, 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 mechanism 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 cell 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.

M A T E R I A L S A N D M E T H O D S

Materials

Protein kinase inhibitors: A3 and K-252b were purchased from Calbiochem. Apyrase, the catalytic subunit 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].

Eggs were dejelled with 2% cysteine/NaOH (pH 8.0) at 23 (cid:176)C. After washing three times with 100 mM NaCl and twice with buffer M at 23 (cid:176)C, the eggs were washed twice with cold buffer M containing 100 mM NaCl and 250 mM sucrose at 4 (cid:176)C. Then the eggs were supplemented with 10 lgÆmL)1 aprotinin and leupeptin, and packed into tubes by brief centrifugation for several seconds at 6000 g. Excess buffer above the packed eggs was removed and the eggs were then crushed by centrifugation at 15 000 g for 10 min The crude extract was collected, and further separated 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 used instead of extraction buffer.

NaCl/Pi: 10 mM sodium phosphate (pH 7.4), 140 mM NaCl and 2.7 mM KCl; extraction buffer: 50 mM Hepes-KOH (pH 7.7), 250 mM sucrose, 50 mM KCl and 2.5 mM MgCl2; elution buffer: 25 mM Tris/HCl (pH 7.5), 150 mM NaCl and 50 mM glutathione (reduced form); buffer X: 15 mM Pipes-KOH (pH 7.4), 200 mM sucrose, 7 mM MgCl2, 80 mM KCl, 15 mM NaCl and 5 mM EDTA; SRPK reaction buffer: 25 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 200 mM NaCl; and buffer M: 20 mM Hepes-KOH (pH 7.5), 60 mM b-glycerophosphate, 20 mM EGTA and 15 mM MgCl2.

Chromatin binding assay (I): a method involving soluble proteins Buffers

Preparation of demembranated Xenopus sperm chromatin

This method was used in the experiments for Fig. 2. A cytosol fraction of 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 (cid:176)C until use. To determine chromatin binding of GST-fused proteins, 5 lL of demem- branated sperm chromatin (40 000 per lL) in buffer X was incubated with 50 lL of heated cytosol at 23 (cid:176)C for 30 min for decondensation of the chromatin. Then the chromatin was precipitated by centrifugation at 2000 g for 10 min The pellet was suspended in 10 lL of extraction buffer contain- ing 0.1% Triton X-100 and 0.5 lg of GST, GST–NK, GST– NM, GST–RS or GST–SK, and then incubated at 4 (cid:176)C for 20 min. Chromatin was reprecipitated by centrifugation at 7000 g for 10 min The resulting supernatant was designated as the (cid:212)unbound fraction(cid:213). The precipitated chromatin was 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 (cid:212)bound fraction(cid:213). The obtained (cid:212)bound(cid:213) (20 lL) and (cid:212)unbound(cid:213) (8 lL) fractions were separated by SDS/PAGE, transferred to a nitrocellulose filter, and immunoblotted with affinity purified anti-GST Ig as described previously [28].

Preparation of a synthetic phase Xenopus egg cytosol

Chromatin binding assay (II): a method involving immobilized proteins

Xenopus sperm was treated with lysolecithin to remove the plasma and nuclear membranes without the highly con- densed chromatin being affected, according to the method of Smythe & Newport [26]. The chromatin concentration is expressed as the number of chromatin complexes in the binding reaction mixture. The number was determined by counting with a hemacytometer.

This method was used for all chromatin-binding experi- ments other than those in Fig. 2. Demembranated sperm 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 mM 2-mercaptoethanol, 10 lgÆmL)1 aproti-

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nitrocellulose sheet. The GST–NK band was excised, soaked in 0.5% poly(vinyl pyrrolidone) K)30 (Wako, Tokyo) in 100 mM acetic acid for 30 min at 37 (cid:176)C and then washed extensively with water. The protein was digested with trypsin in 50 mM NH4HCO3 at 37 (cid:176)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

chromatin (40 000 per lL) in 0.5 lL of buffer X was incubated with 10 lL of Xenopus egg heated cytosol at 23 (cid:176)C for 30 min for decondensation of the chromatin. Then the reaction mixture was centrifuged at 300 g for 10 min and the precipitated chromatin was suspended in 20 lL of extraction buffer. After centrifugation, the preci- pitated chromatin was resuspended in 10 lL of extraction buffer, and then added to 2 lg of GST-fused proteins attached to 2 lL of glutathione–Sepharose 4B beads suspended in 10 lL of extraction buffer. After incubation at 4 (cid:176)C for 20 min, the binding reaction was stopped by pipetting 10-lL samples onto glass slides spotted with 8 lL of fixing solution (3% formaldehyde, 2 lLÆmL)1 Hoechst dye 33342, 80 mM KCl, 15 mM NaCl, 50% glycerol and 15 mM Pipes, pH 7.2). The fixed samples were observed by phase-contrast and fluorescence microscopy. One hundred beads were observed for every sample and ‘the percentage of beads with bound chromatin’ was calculated. 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.

Recombinant GST-fused HC1 (83–191 amino acids), an human HP1HSa fragment containing the LBR binding domain (104–191 amino acids) [22], was expressed as a GST-fusion protein in 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 (cid:176)C for 3 h. Then, the cleaved HC1 portion, which was recovered in the supernatant, was concentrated and used for the binding assay.

Assay for cell cycle dependency of the binding of LBR fragments to sperm chromatin

R E S U L T S

GST-fused proteins attached to glutathione–Sepharose beads were preincubated with either a synthetic or mitotic phase egg cytosol fraction at 23 (cid:176)C for one hour. After washing twice with extraction buffer, the binding to chromatin was 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 no effect on the binding of chromatin to beads.

Identification of chromatin binding regions of LBR

Cloning of various fragments 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 mM isopropyl thio-b-D-galactoside, followed by incubation for 6 h at 30 (cid:176)C. The bacterial cells were collected by centrifugation and resuspended in a buffered saline solution. The cell suspension 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 (cid:176)C for 2 h. After washing twice with the buffered saline solution, the beads were stored at 4 (cid:176)C until use. The amount of protein immobilized on beads was estimated by the Lowry method after elution with glutathione followed by acetone-precipitation.

To analyze the 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 and decondensed sperm chromatin. After fixation and staining of DNA with Hoechst 33342, the beads were observed by phase contrast and fluorescence microscopy (Fig. 1C). Most GST–NK (Fig. 1C) and GST– RS (not shown) beads bound chromatin, however, GST beads only bound a little (Fig. 1C). Then, we introduced (cid:212)percentage of beads with bound chromatin(cid:213) 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 GST bearing beads gave values of 65 (cid:139) 7, 60 (cid:139) 5 and 18 (cid:139) 5%, respectively. These values clearly show that the RS moiety within 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 GST 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

Phosphopeptide mapping GST–NK phosphorylated with [c-32P]ATP in vitro was separated by SDS/PAGE and then transferred to a

Expression of LBR fragments and preparation of beads bearing these fragments

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Fig. 2. Chromatin binding assay involving soluble GST-fusion proteins. Approximately 0.5 lg of GST–NK, GST–NM, GST–RS, GST–SK or GST was incubated with various amounts of decondensed Xenopus sperm chromatin, as shown in the figure, and then centrifuged to separate the unbound fraction (supernatant) from chromatin. The pellet was washed with extraction buffer, dissolved in 1% SDS and then ultracentrifuged to remove DNA. The thus obtained supernatant was designated the (cid:212)bound fraction(cid:213). The (cid:212)bound(cid:213) and (cid:212)unbound(cid:213) frac- tions were separated by SDS/PAGE and then analyzed by immuno- blotting with anti-GST Ig. In the case of (cid:212)(+ DNA)(cid:213), GST-fusion proteins were preincubated with 0.5 mgÆmL)1 of porcine liver DNA and then used for the binding assay. (cid:212)U(cid:213) and (cid:212)B(cid:213) in the figure denote the (cid:212)unbound(cid:213) and (cid:212)bound(cid:213) 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 LBR expressed 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. (cid:212)RS(cid:213) 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, analyzed by SDS/PAGE on a 10% gel, and then stained with Coomassie Brilliant Blue R-250 (CBB). The lines at the left show the positions of marker proteins having relative molecular masses of 66, 43 and 29 kDa, from top to 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 (cid:176)C for 20 min, and then observed by phase contrast and fluorescence microscopy after staining of DNA with Hoechst 33342. Arrows, arrowheads and double-arrow heads indicate beads, unbound chromatin and bound chromatin, respectively. Bar (cid:136) 10 lm.

We then applied this bead method to characterize the binding of LBR to chromatin because it is faster and needs only a one-tenth amount of chromatin compared to the 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 presence of free DNA (Fig. 2). The inhibition with DNA is consistent with that observed with an assay involving GST- fusion protein bearing beads, as shown below. From these results, we concluded that (cid:212)the percentage of beads with bound chromatin(cid:213) obtained with GST-fusion protein-bear- ing beads can be used as an index of the affinity of protein fragments to chromatin.

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An assay system for the cell cycle-dependent binding of LBR to chromatin

Fig. 3. Identification of chromatin binding domains in the N-terminal region of LBR and analysis of the binding mode. Empty columns: four kinds of GST fusion proteins including N-terminal domains of LBR attached to glutathione–Sepharose beads were incubated with decon- densed sperm chromatin at 4 (cid:176)C for 20 min, and then observed by fluorescence microscopy after staining of DNA with Hoechst 33342. The (cid:212)percentage of beads with bound chromatin(cid:213) values were deter- mined as described under Materials and methods after subtraction of the value for blank GST-beads. (Hatched columns) Four GST fusion proteins including LBR fragments attached to glutathione–Sepharose beads were preincubated with a 0.5-mgÆmL)1 DNA solution at 4 (cid:176)C for 1 h, and then the binding to chromatin was examined as above. (Filled columns) Decondensed chromatin was pretreated with 10 lgÆmL)1 trypsin at 23 (cid:176)C for 10 min, and then after the addition of leupeptin and aprotinin (final, 0.5 mgÆmL)1), the binding to beads bearing GST–LBR fragments was examined as above.

i.e.

known that GST–NM, GST–RS and GST–SK carry binding sites for 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.

We wondered whether this binding system can be applied to the analysis of the cell cycle-dependent interaction of LBR and chromatin. Therefore, we pretreated beads bearing LBR fragments with a Xenopus egg cytosol fraction at the synthetic phase of the cell cycle, and then chromatin binding was examined. The binding was stimulated (data not shown). When NK beads were pretreated with a mitotic 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 changes in living cells. These preliminary results suggested that an in vitro assay system for the analysis of the cell cycle-dependent interaction of LBR and chromatin can be developed using this binding assay system.

Then, we optimized the assay conditions for analysis of the cell cycle-dependent interaction of LBR and chro- matin (Fig. 4). The preincubation time for GST–NK beads at 23 (cid:176)C. with a synthetic phase cytosol fraction was examined and 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). Various 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 within one minute at 4 (cid:176)C (Fig. 4C). Then, as standard conditions, we chose 60 min as the preincubation time, 20 000 chromatin per assay, and 20 min for the time of binding of chromatin to beads, as shown under Materials and methods. The chromatin concentration can be varied, depending on the lower and higher chromatin experimental purpose, 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 binding of LBR to chromatin, as described below.

Cell cycle-dependent binding of LBR fragments to chromatin

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, hatched columns). The binding of RS- and NK-fragments to chromatin was strongly suppressed, but the binding of the NM-fragment was the suggested that RS region of LBR binds to the DNA region of chromatin.

little affected. These results

On the other hand, with the pretreatment of trypsin,

the chromatin with a low concentration of the binding was suppressed strongly in the case 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 protein because the binding mode of NK, which contains the NM and RS regions, is similar to that of RS. 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 treatment with a binding of chromatin regardless of 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 binding regardless of treatment with a mitotic phase cytosol fraction (Fig. 5B). These results show that the affinity of the N-terminal portion of LBR and chromatin increases in a synthetic phase extract and decreases in a mitotic phase one in vitro. Moreover, the

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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), and then the binding to chromatin was determined as in Fig. 3. (B) The same as (A) except that a mitotic phase egg cytosol fraction was used instead of the synthetic phase one. Instead of 20 000 chromatin per assay as a standard condition, 10 000 and 25 000 chromatin per assay were used in (A) and (B), respectively, to clearly show the changes in (cid:212)percentage of beads with bound chromatin(cid:213).

Fig. 4. Assay conditions for the binding of chromatin to GST–NK beads pretreated with a synthetic phase Xenopus egg cytosol fraction. GST– NK (filled circles) and blank GST (open circles) beads, 2 lL, were preincubated with 20 lL of a synthetic phase Xenopus egg cytosol fraction at 23 (cid:176)C for 1 h. Thus treated beads were then incubated with 25 000 chromatin per assay at 4 (cid:176)C for 20 min. Then, the percentage of beads with bound chromatin was determined. In each figure, the time of preincubation of beads with a cytosol fraction (A), the chromatin concentration (B), or the time of incubation of beads with chromatin (C) was varied.

Stimulation of the binding of LBR to chromatin by phosphorylation by a kinase(s) in a synthetic phase egg cytosol

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 fraction decreasing with a mitotic one, were 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. Chromatin binding of beads bearing GST–NK was incr- eased by pretreatment with a synthetic phase egg cytosol fraction (compare columns 1 and 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 (cid:25) 4 nM (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 cytosol. Then, authentic protein kinase A (PKA) and calmodulin-dependent protein kinase II (CaMKII) were applied instead of the cytosol fraction, as we previously 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

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phosphorylation in the RS region, beads bearing GST, GST–NK and GST–RS were incubated with a synthetic phase cytosol in the presence of [c-32P]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. 6B). Radioactivity was detected for the GST–NK and GST–RS beads, but not for GST itself (Fig. 6B, autoradiography, lanes 1–3). Incorporation of radioactivity into the GST–NK and GST–RS bands was completely suppressed by the addition of staurosporine (Fig. 6B, 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-beads 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 (cid:25) 6 nM (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 binding to chromatin was observed with preincubation with a mitotic cytosol (Fig. 7A, 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 were incubated with a mitotic phase cytosol fraction in the presence of [c-32P]ATP. Then, the proteins were eluted with SDS and analyzed by SDS/PAGE, followed by CBB 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. 7B, 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.

Fig. 6. Stimulation of the binding of LBR fragments to chromatin by phosphorylation with a synthetic phase egg cytosol fraction. (A) Effects of apyrase, protein kinases, and 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 GST–RS-beads (columns 10–13) were preincubated with 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 nM staurosporine (Sta.), 1 mM A3, 1 lM 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 lL of 3.3 lM [c-32P]ATP (110 TBqÆmmol)1) in the presence (SC/Sta.) or absence (SC) of 10 lM staurosporine at 23 (cid:176)C for 1 h. Thus treated proteins were 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; lanes 3 and 6, GST–RS. The arrowhead and double arrowhead indicate the GST–NK and GST–RS bands, respectively.

Phosphopeptide mapping

by a synthetic phase cytosol (Fig. 6A, column 9). These results suggested that a kinase(s) in the synthetic phase egg cytosol fraction phosphorylates NK at a functionally similar site(s) to in the case of PKA, and thereby increases the affinity of LBR and chromatin. The binding 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 the Synthetic phase and mitotic phase egg extracts both phosphorylated GST–NK and had opposite effects on chromatin binding affinity (Figs 6 and 7). Therefore, the phosphorylation sites for the two extracts were expected to be different. Then, to confirm this difference, tryptic phosphopeptides of GST–NK treated with synthetic phase and mitotic phase egg extracts were compared with each other by means of two-dimensional separation (Fig. 8). As can be seen in Fig. 8, several phosphopeptide spots were different, although some were the same. These results clearly 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

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phosphorylated specifically with a synthetic phase or mitotic phase extract.

D I S C U S S I O N

Binding sites on the N-terminal portion of LBR for chromatin

Fig. 7. Suppression of the binding of LBR fragments to chromatin by phosphorylation with a mitotic phase egg cytosol fraction. (A) Effects of apyrase and protein kinase inhibitors on suppression of the binding of LBR fragments to chromatin by 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 8 mU apyrase, 10 nM staurosporine (Sta.), 1 mM A3 or 1 lM K252b, and then the binding to chromatin was examined as in Fig. 3. (B) Beads bearing 1–2 lg GST, GST–NK, or GST–RS were treated and analyzed as in Fig. 6B, except that a mitotic phase egg cytosol fraction was used instead of the synthetic phase one. Lanes 1 and 4, GST; lanes 2 and 5, GST–NK; lanes 3 and 6, GST–RS. The arrowhead and double arrowhead indicate the GST–NK and GST–RS bands, respectively.

On the other hand, it was suggested that the NM-region, not the SK-region, binds to a protein(s) on sperm chromatin ([6]; Fig. 3). HP1, the only known chromatin protein which binds to LBR, was reported by Ye et al. to bind to a region of SK [22]. Then, we examined which region of LBR binds to HP1 in 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). These results are consistent with those reported by Ye et al. [22]. On the other hand, James et al. reported that HP1 is not observed in the nuclei of early syncytial embryos, 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 the case of the binding of LBR to

Fig. 8. Tryptic phosphopeptide analysis of GST-NK. Beads bearing 20 lg GST–NK were incubated with 20 lL of a synthetic phase (SC) or a mitotic phase (MC) egg cytosol fraction supplemented with 2 lL of 3.3 lM [c-32P]ATP (110 TBqÆmmol)1) at 23 (cid:176)C for 1 h. The thus treated proteins were separated by SDS/PAGE and then transferred to a nitrocellulose sheet. The GST–NK bands were excised and digested with trypsin. The eluted phosphopeptides were separated by electrophoresis at pH 8.9 (horizontal direction; cathode to the left) and by ascending chromato- graphy. The points of sample application can be seen as dots near the bottom-left corners.

Ye et al. reported that free-DNA [15] and a chromatin protein, HP1 [22], bind with LBR in regions corresponding to the RS and SK regions, respectively. On the other hand, we previously reported that the NM region of LBR, which 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 an assay method involving GST-fusion fragments of LBR and Xenopus sperm chromatin in this study. It was shown that the RS region of LBR binds with chromatin and that the binding 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 showed that LBR binds linker DNA but not the nucleosome core using in vitro reconstituted nucleosomes and short DNA fragments [31]. Therefore, the binding site on chromatin for the RS region of LBR seems to be linker DNA.

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sperm chromatin in eggs, binding through the RS region of LBR to linker DNA of chromatin seems to be predominant. Then, the binding is supported by the interaction of the NM region and a protein(s) other than HP1 on sperm chromatin.

An assay system for the cell cycle-dependent binding of LBR to chromatin

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 consistent 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

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 with a synthetic phase extract, whereas it was suppressed by that with a mitotic phase extract (Fig. 5). The binding of chromatin to LBR fragments on beads could be estimated semiquantitatively by this method. The effects of enzymes, inhibitors and other reagents on the cell cycle– dependent interaction could also be examined very easily by means of this method. This method is applicable to the analysis of phosphorylated residues on LBR fragments and protein kinases responsible for the phosphorylation. This method should also be applicable 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 synthetic phase egg cytosol (Fig. 6). In interphase somatic cell nuclei, the binding of LBR to lamin B may be stimulated by in vivo phosphory- lation by PKA [33]. However, PKA seems not to participate in stimulation of the binding of 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 RS 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 and chromatin. Identification of the kinase(s) respon- sible for the stimulation of the binding of LBR to chromatin is important for clarifying the physiological function of the phosphorylation, and such work is currently underway.

The function of LBR in the targeting of nuclear membranes or nuclear envelope precursor vesicles to chromatin remains elusive. In rat liver and turkey 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 egg cell-free system, however, it was found that vesicles containing NEP-B78 bind first to chromatin and then to vesicles containing an LBR-like protein [41]. LBR-containing vesicles alone can not bind to chromatin [41]. These observations may suggest that LBR does not participate in 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 chromatin still remains for 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 with anti-(human LBR) Ig, participates in the targeting [37]. Therefore, the participation of LBR in the targeting of nuclear membranes to chromatin may vary a little from system to system and/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 the targeting of nuclear

The dissociation/association of membranes with chromatin in pronuclei formation, and the beginning and end of mitosis are critical for control of the nuclear dynamics during these stages of the cell cycle. Inner nuclear membrane proteins, LBR [5,6,13], LAP2 [7], and emerin (M. Segawa, 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 important for the 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 from CHO cells [5]. Regulation of the binding of LBR to chromatin by phosphorylation was shown in this study using sperm chromatin and a Xenopus 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 the phosphorylation of LAP2 with a mitotic HeLa cell extract inhibits its binding to chromo- somes [7]. In the cases of emerin and MAN1, which share the LEM domain with LAP2 [39,40], the regulatory mechanism for the binding to chromatin remains to be elucidated.

It was suggested that the binding of LBR to sperm chromatin is strongly suppressed by phosphorylation in the RS region of LBR by 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-

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LAP210 proteins alter lamina assembly, envelope formation, nuclear size, and DNA replication e(cid:129)ciency in Xenopus laevis extracts. J. Cell Biol. 144, 1083–1096.

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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. Segawa, K. Furakawa, S. Omata & T. Horigone, unpublished observation). LAP2 seems 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]. Further analysis of the regulation mechanism for the binding of a set of inner nuclear membrane proteins to chromatin is necessary for understanding the molecular mechanism of dissociation/ association of membranes with chromatin in pronucleus formation and the mitotic phase of somatic cells.

A C K N O W L E D G E M E N T S

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We wish to thank Dr Masatoshi Hagiwara for the helpful discussion. We also wish to thank Hitomi Susa and Satomi Hoshino for their help in the construction of the plasmids encoding GST–RS and the phosphopeptide mapping, respectively.

This work was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan, and grants from the Biodesign Research Project and for Project Research of Niigata University.

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