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

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
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]. Further analysis of the
regulation mechanism for the binding of a set of inner
nuclear membrane proteins to chromatin i s necessary for
understanding the molecular mechanism of dissociation/
association of membranes with chromatin in pronucleus
formation and the mitotic phase o f somatic cells.

ACKNOWLEDGEMENTS
We wish to thank D r Masatoshi H agiwara for the helpful d iscussion.
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 s upported b y a G rant-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.
REFERENCES
1. Glass, J.R. & Gerace, L. (199 0) Lami ns A and C bind a nd
assemble at the surface of mitotic chromosomes. J. Cell Biol. 111,
1047–1057.
2. Taniura, H., Glass, C. & Gerace, L. (1995) A chromatin binding
site in the tail domain of nuclear lamins that interacts with core
histones. J. Cell Biol. 131, 33–44.
3. Lourim, D. & Krohne, G. (1998) Chromatin binding and poly-
merization of the endogenous Xenopus egg lamin s: the opposing
effects of glycogen and ATP. J. Cell Sci. 111, 3675 –3686.
4. Stuurman, N ., Heins, S. & Aebi, U. (1998) N uclear lamins: their
structure, assembly, and interactions. J. Struct. Biol. 12 2 , 42–66.
5. Pyrpasopoulou, A., Meier, J., Maison, C ., Simos, G . & G eorgatos,
S.D. (1996) The lamin B receptor (LBR) provides essential
chromatin docking sites at the nuclear e nvelope. EMBO J. 15,
7108–7119.
6. Kawahire, S., Takeuchi, M., Gohshi, T., Sasagawa, S., Shimada,
M., Takahashi, M ., Abe, T.K., Ueda, T., Kuwano, R., Hikawa,
A., Ichimura, T. , Omata, S . & Horigome, T. (1997) cDNA cloning
of nuclear l ocalizatio n signal binding protein NBP60, a rat
homologue of lam in B rec eptor, a nd id entification o f b inding site s

of human lamin B receptor for nuclear localization s ignals and
chromatin. J. Biochem. 121, 881–889.
7. Foisner, R. & Gerace, L. (1993) Integral membrane prote ins of the
nuclear e nvelope interact with lamins and c h romosomes, and
binding is modulated by mitotic pho sphorylation. Cell 73, 1267–
1279.
8. Furukawa, K., Fritze, C.E. & Gerace, L . (1998) The major nuclear
envelope targetin g d omain o f L AP2 co incid es with its lamin
binding region but is distinct from its chromatin interaction
domain. J. Biol. C hem. 273, 4213–4219.
9. Lopez, J.M. & W olfner, M.F. (1997) The developmentally regu-
lated Drosophila em bryonic nuclear lamina protein ÔYoung A rre stÕ
(fs(1)Ya) is capable of associating with chromatin. J. Cell Sci . 11 0 ,
643–651.
10. Gant, T.M., Harris, C.A. & Wilson, K.L. ( 1999) Roles of LAP2
proteins in nuclear assembly and DNA replication: truncated
LAP210 proteins alter lamina assembly, envelope formation,
nuclear size, and DNA replication efficiency in Xenopus laevis
extracts. J. Cell Biol. 144, 1083–1096.
11. Worman, H.J., Yuan, J., Blobel, G. & Georgatos, S.D. (1988)
A lamin B receptor in the nuclear envelope. Proc. Natl Acad. Sci.
USA 85, 8531–8534.
12. Bailer, S.M., Eppenbe rger, H .M., G riffiths, G . & Nigg, E.A.
(1991) Characterization of a 54-kDa p rotein of the inner nuclear
membrane: evidence for cell cycle–dependent interaction with the
nuclear lamina. J. Cell Biol. 114, 389–400.
13. Gajewski, A. & Krohne, G. (1999) Subcellular distribution of the
Xenopus p58/lam in B receptor in oocytes and eggs. J. Cell Sci. 112,
2583–2596.
14. Worman, H.J., Evans, C.D. & Blobel, G. (1990) The lamin B

receptor of the nuclear envelope inner membrane: a polytopic
protein with eight potential transmembrane domains. J. Cell Biol.
111, 1535–1542.
15. Ye, Q. & Worman, H.J. (1994) Primary structure analysis, and
lamin B and DNA binding of human LBR, an integral protein of
the nuclear envelope inner membrane. J. Biol. Chem. 269, 11306–
11311.
16. Silve, S., Dupuy, P H., Ferrara, P. & Loison, G. (1998)
Human lamin B receptor exhibits sterol C14-reductase activity in
Saccharomyces cerevisiae. Biochim. Biophys. Acta 1392, 233–244.
17. Prakash, A., Sengupta, K., Aparna, K. & Kasbekar, D.P. (1999)
The erg-3 (stero l D
14,15
-reductase) gene o f N eurospora crassa:
generation of null mutants by repeat-induced point mutation and
complementation by proteins chimeric for human lamin B recep-
tor sequences. Microbiology 145, 1443–1451.
18. Simos, G., Maison, C. & G eorgatos, S.D. (1996) Characterization
of p18, a component of the lamin B receptor complex and a new
integral me mbrane protein of the avian erythrocyte nuclear
envelope. J. Biol. C hem. 271, 12617–12625.
19. Simos, G. & Georgatos, S.D. (1992) The inner nuclear membrane
protein p58 associates in vivo with a p58 kinase and the nuclear
lamin s. EMBO J. 11, 4027–4036.
20. Haino, M., Kawahire, S., Omata, S . & Horigome, T . (1993)
Purification o f a 60 kDa nuclear localization signal binding protein
in rat liver nuclear e nvelopes and characterization of its properties.
J. Biochem. 113, 308–313.
21. Ye, Q. & Worman , H.J. (1996) Interaction between an integral
protein of the nuclear envelope inner membrane and hu man

chromodomain proteins ho mologous to Drosophila HP1. J. B io l.
Chem. 271, 14653–14656.
22. Ye, Q., Callebaut, I., Pezhman, A., Courvalin, J.C. & Worman,
H.J. (1997) Domain–sp ecific inte ractions of human HP1-type
chromodomain p roteins a n d inn er nuc lear membrane protein
LBR. J. Biol. Chem. 272, 14983–14989.
23. Nikolakaki, E., Simos, G., Georgatos, S.D . & G iannakouros, T.
(1996) A nuclear envelope-associated kinase phosphoryla tes
arginine-serine m otifs and m odulates interactions betwee n the
lamin B receptor an d o ther nuclear p roteins. J. Biol. Chem. 271,
8365–8372.
24. Nikolakaki, E., Meier, J., Simos, G., Georgatos, S.D. & Gian-
nakouros, T. ( 1997) M itotic p hosphorylation o f th e l am in B
receptor by a serine/arginine kinase and p34
cdc2
. J. Biol. Chem.
272, 6208–6213.
25. Yamauchi, T. & Fujisawa, H. (1983) Purification and character-
ization of th e brain c almodu lin-de pendent protein kinase (kinase
II), which is involved in the activation of tryptophan 5-mono-
oxygenase. Eur. J. Bioc hem. 132, 15–21.
26. Smythe, C. & Newport, J.W. (1991) Systems for the study of
nuclear assembly, DNA replication, and nuclear breakdown in
Xenopus laevis egg extracts. Methods Cell Biol. 35, 449–468.
27. Kubota, Y. & Takisawa, H. (1993) Determination of initiation of
DNA replication before and after n uclear formation in Xenopus
egg cell free e xtracts. J. Cell Biol. 123, 1 321–1331.
952 M. Takano et al. (Eur. J. Biochem. 269) Ó FEBS 2002
28. Kawahire, S., Tachibana, T., Umemoto, M., Yoneda, Y., Imai, N.,
Saito,M.,Ichimura,T.,Omata,S.&Horigome,T.(1996)Sub-

cellular distribution and phosphorylation of the nuclear localiza-
tion signal binding protein, NBP60. Exp. Cell Res. 222, 385–394.
29. Sugimoto, K., Yamada, T., Muro, Y. & Himeno, M. (1996)
Human hom olog of Drosophila heterochromatin-associated pro-
tein 1 (HP1) is a DNA-binding protein which possesses a DNA-
binding motif with weak similarity to that of human centromere
protein C (CENP-C). J. Biochem. 12 0, 153–159.
30. Inagaki, M., Kawamoto, S., I toh, H., S aitoh, M., Hagiwara, M.,
Takahashi, J. & Hidaka, H. (1986) Naphthalenesulfonamides as
calmodulin antagonists and protein kinase inhibitors. Mol. Phar-
macol. 29, 577–581.
31. Duband-Goulet, I. & Courvalin, J.C. (2000) Inner nuclear me m-
brane protein LBR preferentially interacts with D NA secondary
structures and nucleosomal linkers. Biochemistry 39, 6483–6488.
32. James, T.C., Eissenberg, J.C., Craig, C., Dietrich, V., Hobson, A.
& E lgin, S.C. (1989) Distribution patterns of HP1, a
heterochromatin- associated nonhistone chromosomal p rotein of
Drosophila. Eur. J. Cell Biol. 50, 1 70–180.
33. Appelbaum, J ., Blobel, G. & Georgatos, S.D . (1990) In vivo
phosphorylation o f the lamin B receptor. Bindin g of lamin B to its
nuclear membrane r eceptor i s affected by phosphorylation. J. Biol.
Chem. 265, 4181–4184.
34. Kuroyanagi, N., Onogi, H., Wakabayashi, T. & Hagiwara, M.
(1998) Novel SR-protein-specific kinase, SRPK2, disassembles
nuclear s peckles. Biochem. Bio phys. Res. Commun. 242, 357–364.
35. Papoutsopoulou, S., N ikolakaki, E. & Giannakouros, T. (1999)
SRPK1 and LBR protein kinase show identical s ubstrate speci-
ficities. Biochem. Biophys. Res. Commun. 255, 602–607.
36. Collas, P. (1999) Sequential PKC- and C dc2-mediated phospho-
rylation ev ents elicit zebrafish nuclear envelope disassembly.

J. Cell Sci. 112, 977–987.
37. Collas, P., Courvalin, J .C. & Poccia, D. (1996) Targ eting o f
membranes to sea urchin chromatin is mediated by a lamin B
receptor-like integral membrane protein. J. Cell Biol. 135, 1715–
1725.
38. Dreger, M., Otto, H., Neubauer, G., Mann, M. & Hucho, F.
(1999) Identification of phosphorylation sites in native lamina-
associated po lypeptide 2 beta. Biochemistry 38, 9426–9434.
39. Lin, F., Blake, D.L., Callebaut, I., Skerjanc, I.S., Holmer, L.,
MacBurney, M.W., P aulin-Levasseur, M. & Worman, H.J. ( 2000)
MAN1, an inner nuclear membrane protein that shares the LEM
domain with lamina-associated polypeptide 2 and emerin. J. Biol.
Chem. 275, 4840–4857.
40. Shumaker, D.K., Lee, K.K ., Tanhe hco, Y.C., C raigie, R. &
Wilson, K.L. ( 2001) LAP2 binds to BAF40D NA c omplexes:
requirement for the LEM domain and modulation by variable
regions. EMBO J. 20, 1754–1764.
41. Drummond, S., F errign o, P., L yon, C., Murphy, J., Goldb erg, M.,
Allen, T., Smythe, C. & Hutchison, C.J. ( 1999) Temp oral differ-
ences in the appearance of NEP-B78 and an LBR-like protein
during Xenopus nuclear envelope reassembly reflect the ordered
recruitment of functionally discrete vesicle types. J. Cell Biol. 144,
225–240.
42. Haraguchi, T., Koujin, T., Hayakawa, T., Kaneda, T., Tsutsumi,
C., Imamoto, N., Akazawa, C., Sukegawa, J., Yoneda, Y.
& Hiraoka, Y . (2000) Live fluorescence i maging reveals e arly
recruitment of emerin, LBR, RanBP2, and Nup153 to ref orming
functional nuclear envelopes. J. Cell Sci. 113, 7 79–794.
Ó FEBS 2002 Regulation of the binding of LBR to chromatin (Eur. J. Biochem. 269) 953