Tài liệu Báo cáo Y học: The binding of lamin B receptor to chromatin is regulated by phosphorylation

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

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 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

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; lamin B receptor; LBR;

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.

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

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].

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: [email protected]

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