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.

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

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

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
.

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.

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

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

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.

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

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

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

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

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,

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

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

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

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.

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