(Received for publication, October 19, 1995; and in revised form, December 20, 1995)
From the
Previous studies have identified a subassembly of nuclear
envelope proteins, termed ``the LBR complex.'' This complex
includes the lamin B receptor protein (LBR or p58), a kinase which
phosphorylates LBR in a constitutive fashion (LBR kinase), the nuclear
lamins A and B, an 18-kDa polypeptide (p18), and a 34-kDa protein
(p34/p32). The latter polypeptide has been shown to interact with the
HIV-1 proteins Rev and Tat and with the splicing factor 2 (SF2). Using
recombinant proteins produced in bacteria and synthetic peptides
representing different regions of LBR, we now show that the LBR kinase
modifies specifically arginine-serine (RS) dipeptide motifs located at
the nucleoplasmic, NH-terminal domain of LBR and in members
of the SR family of splicing factors. Furthermore, we show that the
NH
-terminal domain of LBR binds to p34/p32, whereas a
mutated domain lacking the RS region does not. Phosphorylation of LBR
by the RS kinase completely abolishes binding of p34/p32, suggesting
that this enzyme regulates interactions among the components of the LBR
complex.
Several integral proteins of the inner nuclear membrane have
been characterized recently (for recent reviews, see Gerace and Foisner
(1994) and Georgatos et al.(1994)). One such protein,
originally identified in nucleated avian erythrocytes is the
``lamin B receptor'' (LBR) or ``p58.'' cDNA
sequencing of chicken and human LBR has revealed that the protein
possesses a long, hydrophilic NH-terminal domain protruding
into the nucleoplasm, eight hyrdophobic segments which are predicted to
span the membrane, and a hydrophilic COOH-terminal domain (Worman et al., 1990; Ye and Worman, 1994). The
NH
-terminal domain of LBR contains distinct sites for
protein kinase A and Cdc2 kinase phosphorylation (Simos and Georgatos,
1992; Courvalin et al., 1992), as well as a stretch rich in
arginine-serine (RS) motifs (Simos and Georgatos, 1994). Such dipeptide
motifs have been identified in a variety of splicing factors and have
been shown to mediate protein-protein interactions between components
of the splicing machinery (Wu and Maniatis, 1993; Kohtz et
al., 1994).
LBR is widely expressed in cells of higher
eukaryotes, and the human gene has been recently characterized (Schuler et al., 1994). In addition, three yeast proteins have been
identified that are homologous to the hydrophobic regions and the
COOH-terminal domain of LBR, but lack most of the
NH-terminal domain (Chen et al., 1991; Lorenz and
Parks, 1992; Shimanuki et al., 1992). One of these LBR-related
polypeptides (ERG24) is involved in sterol metabolism, and its function
in yeast is not complemented by higher eukaryotic LBR (Smith and
Blobel, 1994). On the basis of this evidence, it has been previously
proposed that ``full-length'' and
``NH
-truncated'' forms of LBR may represent
distinct members of a multigene family which includes nuclear envelope
and ER (
)proteins (Georgatos et al., 1994).
LBR
associates with B-type lamins both in vitro and in vivo (Worman et al., 1988; Simos and Georgatos, 1992; Ye and
Worman, 1994; Smith and Blobel, 1994), consistent with its presumed
function as a lamin receptor. Although the NH-terminal
domain of LBR is probably responsible for lamin B binding (Ye and
Worman, 1994), interactions between the farnesyl group of lamin B and
the transmembrane regions of LBR also seem likely (Georgatos et
al., 1994; Smith and Blobel, 1994). The association of B-type
lamins with LBR is not disrupted during mitosis, when the nuclear
envelope is fragmented and the nuclear lamina depolymerized (Meier and
Georgatos, 1994).
Recent work has shown that during interphase LBR
forms a multimeric complex which includes the nuclear lamins A and B, a
specific LBR kinase, and three other polypeptides with molecular masses
of 18 (p18), 34 (p34), and 150 (p150) kDa, respectively (Simos and
Georgatos, 1992). p18 has been characterized recently as a new integral
membrane protein of the bird erythrocyte nuclear envelope. ()Furthermore, p34 has been identified as the avian
equivalent of a human nuclear protein known as p32 (Simos and
Georgatos, 1994). p32 has been characterized previously and found to
co-isolate with splicing factor 2 (SF2) (Krainer et al.,
1991). Recently, Luo et al.(1994) have shown that p32 also
interacts with the viral trans-activator Rev, which is
required for the replication of human immunodeficiency virus type 1
(HIV-1). Another interaction of p32 seems to involve the HIV-1 protein
Tat (Fridell et al., 1995).
The LBR kinase was previously
shown to cofractionate with LBR and to phosphorylate LBR in vivo and in vitro, exclusively at serine residues. The enzyme
is clearly distinct from protein kinase A and Cdc2 kinase, for both of
which LBR is a substrate (Simos and Georgatos, 1992). Reasoning that
the LBR kinase may regulate interactions between LBR and its partners,
we decided to characterize this activity in detail. Results presented
below show that the LBR kinase belongs to a novel class of protein
kinases which modify specifically RS motifs (Woppmann et al.,
1993; Gui et al., 1994). The LBR kinase regulates, through
phosphorylation of the RS region, the binding of p34/p32 to the
NH-terminal domain of LBR.
Peptides R (
KQRKSQSSSSSPSRRSRSRS
), R
(
SRSRSRSPGRPAKG
), R
(
SSPSRRSRSRSRSRSPGRPAKG
), R
(
KGRRRSSSHSRE
), R
(
KIFEAIKTPEKPSSKT
), R
(*C
ANSQKNNFRRNPADPK
), R
(*C
KPSENNTRRYNGEPDSTERND
), R
(*C
TERNDTSSKLLEQQKLKPDVE
), and
R
(
DEHHCKKKYGLAWERY
*C),
representing different regions of chicken LBR (Worman et al.,
1990), were made at the Protein Sequencing and Peptide Synthesis
Facility of EMBL. A cysteine residue (*C) was added to the sequences
for coupling purposes. R
peptide was coupled to Affi-Gel 10
by incubating 30 mg of the peptide with 3 ml of the column as described
previously (Georgatos and Blobel, 1987b). The anti-LBR antibody
aR
, raised against the peptide R
, as well as an
anti-p34 antibody (ap34-C), raised against the COOH-terminal residues
(CGG
TGESEWKDTNYTLNTDS
) of HeLa p32 (Honore et al., 1993), were prepared and affinity-purified as
described previously (Simos and Georgatos, 1992, 1994). The tripeptide
CGG was added to the sequences for coupling purposes. All other
chemicals were purchased from Sigma (Sigma, Deisenhofen, Germany).
To isolate the LBR
kinase, turkey erythrocyte nuclear envelopes were extracted either with
50 mM Tris-HCl, pH 7.5, 1 M NaCl, 1 mM dithiothreitol and 1 mM PMSF, or with 20 mM Tris-HCl, pH 7.5, 2 mM MgCl, 150 mM NaCl, 1 mM dithiothreitol, 1 mM PMSF, and 1%
Triton X-114. The aqueous phase of the Triton X-114 extract (Bordier,
1981) or the 1 M NaCl extract (following dilution to 0.3 M and clarification by centrifugation at 15,000
g for 20 min) were applied to a phosphocellulose column previously
equilibrated with 20 mM Tris-HCl, pH 7.5, 0.3 M NaCl,
1 mM dithiothreitol, and 1 mM PMSF. The bound
proteins were eluted by a linear (0.3-1 M) NaCl
gradient. Kinase-containing fractions were pooled, the salt
concentration was adjusted to 0.35 M, and the material was
further chromatographed through an Affi-Gel 10 column containing the
R
peptide. Analysis of the flow-through fractions showed
that all the kinase activity was bound to the column. The column was
subsequently washed with 0.9 M NaCl, and elution of the kinase
activity was accomplished by a linear (0.9-2.2 M) NaCl
gradient. The active fractions were pooled, concentrated with an Amicon
device, and used in subsequent experiments. RS kinase activity was
determined by measuring the incorporation of
PO
from
[
-
P]ATP to electroeluted LBR. Routine
assays were carried out at 30 °C in a total volume of 25 µl
containing 25 mM Tris-HCl, pH 7.5, 10 mM MgCl
, 200 mM NaCl, 50 µM [
-
P]ATP (6,000 Ci/mmol), 0.3% Triton
X-100, 1.5 µg of LBR, and an aliquot of the enzyme. After 30 min,
the reaction was terminated by the addition of 5 µl of 5
electrophoresis sample buffer (Laemmli, 1970) and analyzed by SDS-PAGE.
SDS-PAGE was performed according to Laemmli(1970) using 12%
polyacrylamide gels. Protein concentration was determined by the method
of Bradford(1976). For the determination of K
, the
amount of substrate in the reaction mixture was varied between 0.1 and
5 µg, and incorporation of radioactivity was measured by excising
the radioactive bands from an SDS-PAGE gel and scintillation counting.
The K
values were calculated using the MicroCal
Origin (version 2.94) program. For determination of stoichiometry,
phosphorylation was carried out using a concentration of substrate at
the end of the linear range of the reaction, and the incorporation of
radioactivity was measured by scintillation counting of excised
radioactive bands from an SDS-PAGE gel.
In situ kinase
assays were performed according to Kameshita and Fujisawa(1989). LBR
was added to the separating gel, at a concentration of 0.1 mg/ml, prior
to polymerization. For control experiments, LBR was omitted or replaced
with 0.1 mg/ml bovine serum albumin. Electrophoresis was carried out at
25 mA for approximately 1 h, using 12% acrylamide minigels. SDS was
removed from the gel by equilibration in 20% 2-propanol, 50 mM Tris-HCl, pH 8.0. The kinase was then fully denatured by
incubating in 6 M guanidinium hydrochloride for 1 h at room
temperature and allowed to renature overnight at 4 °C in 50 mM Tris-HCl, 14 mM 2-mercaptoethanol, and 0.04% Tween 40, pH
8.0. Gels were incubated in 4 ml of assay buffer (10 mM MgCl, 200 mM NaCl, 25 mM Tris-HCl)
for 20 min, at room temperature. The kinase assay was then initiated by
the addition of 50 µM cold ATP and 50 µCi of
[
-
P]ATP (6,000 Ci/mmol). Incubation was
carried out for an additional 60 min. The reaction was terminated by
extensive washing with 5% trichloroacetic acid containing 10 mM sodium pyrophosphate. The gels were dried and exposed to Kodak
X-Omat film.
Proteolytic peptide mapping was performed essentially
as described by Luo et al.(1991). Briefly, phosphorylated LBR
was run on a SDS-PAGE gel and then transferred to a nitrocellulose
sheet. The radioactive LBR bands were excised, soaked in 0.5% PVP 360
(polyvinylpyrrolidone) in 100 mM acetic acid, for 1 h, at 37
°C and washed extensively with water. Samples were then digested to
completion with tosylphenylalanyl chloromethyl ketone-treated trypsin
in 50 mM NHHCO
, at 37 °C,
overnight. Residual salt was removed by repeated lyophilization, and
the digests were subsequently applied to thin layer cellulose plates
(Kodak) for two-dimensional peptide mapping. Electrophoresis (in the
first dimension) was run at pH 8.9 (1% ammonium carbonate) for 1 h at
500 V. Ascending chromatography (in the second dimension) was performed
using as a solvent a mixture of 1-butanol:pyridine:acetic acid:water in
ratios of 750:500:150:600.
An oligonucleotide-directed in vitro mutagenesis system (Altered SitesII, In Vitro Mutagenesis System, Promega) was used to delete five serine and
arginine repeats in the NH
-terminal domain of LBR. Using
the oligonucleotide 5`-TGCTGGCCGACCAGGTCTGGAAGAGAACT-3`, the codons for
amino acids 75 to 84 were deleted (
RSwtNt). In addition, using the
oligonucleotides 5`-TCTGCTTCTACCTCTTCTGGA-3`,
5`-TCGAGATCTGCCTCTACTTCT-3`, 5`-TCTGGATCGAGCTCTGCTTCT-3`,
5`-AGGAGATCTGGCTCGAGATCT-3`, and 5`-GACCAGGAGCTCTGGATCG-3`,
Ser
, Ser
, Ser
, Ser
,
and Ser
were mutated to Gly (GST-NtG
), Gly
(GST-NtG
), Ala (GST-NtA
), Ala
(GST-NtA
), and Ala (GST-NtA
), respectively.
The mutated cDNAs were sequenced and subcloned into the pGEX-2T
expression vector as described previously for wtNt.
A fragment containing all five arginine-serine repeats was generated using the following two complementary oligonucleotides 5`-GATCCAGAAGTAGAAGCAGATCTCGATCCAGATCTAG-3` and 5`-AATTCTAGATCTGGATCGAGATCTGCTTCTACTTCTG-3` and inserted into the BamHI/EcoRI site of pGEX-2T expression vector.
GST fusion proteins were produced in bacteria and purified using glutathione-Sepharose (Pharmacia), as described by Smith and Johnson (1988). When used in phosphorylation experiments, the fusion proteins were dialyzed previously against 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM PMSF, and 1 mM 2-mercaptoethanol.
Figure 1:
Subcellular distribution of LBR kinase.
Fractions of turkey erythrocyte cytosol (CYT), nuclear content (NC) released after DNase I digestion of nuclei, high salt
extract (SE) of nuclear envelopes, salt-washed nuclear
envelopes (SNE), and plasma membranes (PM) were
incubated with 1.5 µg of purified LBR and 50 µM [-
P]ATP. Samples were analyzed by
SDS-PAGE and autoradiographed. The radioactive bands corresponding to
LBR were excised, and the radioactivity was determined by Cerenkov
counting. LBR kinase activity of the different fractions is expressed
either as total units (%) or as specific activity (cpm/mg of protein).
The protein concentration of the cytosol is much higher than the other
fractions due to the abundance of hemoglobin, and, therefore, the
specific activity in this fraction appears close to
zero.
To selectively extract the LBR kinase we used two methods. First, we treated nuclear envelopes at 4 °C with the detergent Triton X-114 and then induced phase separation by raising the temperature of the extracted material to 37 °C. Under these conditions, the bulk of solubilized LBR partitioned with the detergent phase, whereas a significant portion of the kinase activity was released in the aqueous phase (Fig. 2A). Alternatively, we treated the nuclear envelopes with 1 M NaCl which extracted considerable amounts of the enzyme, but did not release significant amounts of LBR or other integral membrane proteins (Fig. 2B). The detergent extract of nuclear envelopes was practically devoid of histone kinase activity and contained low levels of casein kinase; however, the salt extract of nuclear envelopes contained significant amounts of both histone and casein kinase activity (Fig. 2, A and B, and data not shown).
Figure 2:
Extraction of LBR kinase activity from
nuclear envelopes by Triton X-114 or 1 M NaCl. A,
equivalent fractions of whole turkey erythrocyte nuclear envelopes (NE), extracted nuclear envelopes with 1% Triton X-114 (TX-Pel), the Triton X-114 extract of nuclear envelopes (TX-Sup), the aqueous phase of the Triton X-114 extract (TX-Aq), and the detergent phase of the Triton X-114 extract (TX-Det) were incubated with
[-
P]ATP in the absence(-) or presence
(+) of exogenous LBR. Samples were analyzed by SDS-PAGE and
stained with Coomassie Blue (left panel) or autoradiographed (right panel). B, equivalent fractions of turkey
erythrocyte nuclear envelopes and 1 M NaCl extracts of nuclear
envelopes were incubated with [
-
P]ATP in
the absence (-) or presence (+) of exogenous LBR. Samples
were analyzed by SDS-PAGE and stained with Coomassie Blue (left
panel) or autoradiographed (right panel). LBR is
indicated by an arrow. Bars on the left indicate
molecular masses (in kDa).
As a first step to characterize the enzyme we were
interested in, we used salt or Triton X-114 extracts which contained
LBR-free kinase to phosphorylate a set of peptides representing
different regions of the NH- and the COOH-terminal domains
of LBR (Fig. 3B, for details see ``Materials and
Methods''). Two NH
-terminal peptides (R
and R
) which contained three or five arginine-serine
(RS) motifs, respectively, could be phosphorylated by the LBR kinase (Fig. 3A). However, a third NH
-terminal
peptide (R
) which contained three RS motifs but no
downstream flanking sequence could not serve as a substrate and
affected LBR phosphorylation marginally (Fig. 3A).
Interestingly, the synthetic derivative R
(which contained
the highest number of RS motifs) inhibited completely the
phosphorylation of purified LBR by the corresponding kinase, while the
other two peptides inhibited LBR phosphorylation to a lower extent (Fig. 3A). These data strongly suggested that the RS
dipeptide motifs represent the major phosphorylation sites of LBR
modified by the LBR kinase.
Figure 3:
Phosphorylation of purified LBR by protein
kinases present in the aqueous phase of the Triton X-114 extract of
nuclear envelopes in the presence of various synthetic peptides. A, 1.5 µg of electroeluted LBR were incubated with 0.5
mM each peptide and the aqueous phase of the Triton X-114
extract in the presence of 50 µM [-
P]ATP in a total reaction volume of
25 µl. Samples were subsequently analyzed by SDS-PAGE on 15% gels
and autoradiographed. An autoradiogram of the SDS-gel is shown. B, amino acid sequences of the peptides used. The relative
position of the peptides in the LBR molecule is schematically
indicated. Black boxes along the LBR sequence numbered with Roman numerals, represent potential transmembrane
domains.
To explore this point further, we
expressed in E. coli a fusion protein consisting of GST and
the NH-terminal domain of LBR (residues 1-205;
construct termed GST-wtNt). For control purposes, we also expressed in
bacteria a similar fusion protein missing the RS motifs (deletion of
residues 75-84; construct termed GST-
RSNt), as well as a
protein consisting of GST and the RS region of LBR (residues
75-84; construct termed GST-RS). The three recombinant proteins
were used as substrates for in vitro phosphorylation assays.
Data depicted in Fig. 4reveal that LBR kinase present in
salt or Triton X-114 extracts could efficiently phosphorylate GST-wtNt,
whereas GST-RSNt was not phosphorylated. This was not due to a
global misfolding of the polypeptide chain induced by the deletion of
the RS region, because both GST-
RSNt and GST-wtNt (both of which
contain a consensus protein kinase A site) were efficiently modified by
protein kinase A and exhibited the same solubility and ligand-binding
properties. (
)Finally, GST-RS could serve as a substrate for
the LBR kinase, but was phosphorylated at a lower stoichiometry than
GST-wtNt. The relatively lower extent of phosphorylation in the latter
case might be due to the lack of ``context'' information
normally provided by sequences flanking the RS region. This idea is
further supported by the fact that the synthetic peptide R
,
which includes the RS region but lacks long neighboring sequences, was
also phosphorylated substoichiometrically in comparison to intact LBR
or GST-wtNt (data not shown).
Figure 4:
Phosphorylation of GST, GST fusion protein
containing the NH-terminal domain of LBR (GST-wtNt, amino acids 1-205), GST fusion protein
containing the NH
-terminal domain of LBR, but missing the
RS motifs (GST-
RSNt, amino acids deleted 75-84),
and GST fusion protein containing five RS dipeptide repeat (GST-RS) by LBR kinase present in the aqueous phase of the
Triton X-114 extract of turkey erythrocyte nuclear envelopes. The
full-length fusion protein migrates with an apparent molecular mass of
approximately 51 kDa. The lower bands represent degradation products
(see also Ye and Worman(1994)). A, SDS-PAGE analysis and
Coomassie Blue staining of GST, GST-wtNt, GST-
RSNt, and GST-RS. B, immunoblotting of bacterially expressed proteins using an
affinity-purified anti-LBR antibody, raised against peptide R
(aR
). The blots were stained using an alkaline
phosphatase-conjugated rabbit goat anti-rabbit antibody. Note that in
addition to full-length fusion protein, aR
also reacts with
degradation products. C, in vitro phosphorylation of
bacterially expressed proteins by the LBR kinase. The samples were
analyzed by SDS-PAGE on 12% gels and autoradiographed. Molecular mass
standards are shown at left (in
kDa).
Exploiting this information, we
proceeded with the purification of the LBR kinase from nuclear envelope
extracts. To this end, we first chromatographed the salt extract or the
aqueous phase of the Triton X-114 extract through phosphocellulose and
loaded the pool of the fractions possessing LBR kinase activity onto an
agarose column containing immobilized R peptide (for
details see ``Materials and Methods''). Analysis of the
eluted fractions by SDS-PAGE and staining of the corresponding gels
with silver nitrate revealed the presence of two bands, a major one at
54 kDa and a minor one at 110 kDa (Fig. 5A). In
situ phosphorylation assays in polyacrylamide gels to which 0.1
mg/ml purified LBR were incorporated revealed that a protein with a
molecular mass of 110 kDa could modify LBR (Fig. 5B, lane 2). This was specific because no labeling was detected
when LBR was omitted from the gel or replaced by bovine serum albumin
(data not shown). In addition, the same 110-kDa polypeptide appeared to
phosphorylate LBR when, instead of the column-purified preparation, LBR
kinase co-immunoprecipitated with LBR from a Triton X-100 lysate of
nuclear envelopes was used in the in situ gel assay (Fig. 5B, lane 1). These data suggest that the
110-kDa band corresponds to the catalytic subunit of the LBR kinase.
The nature of the 54-kDa protein which copurifies with the kinase but
contains no LBR-phosphorylating activity is presently unknown.
Figure 5:
Partial purification and characterization
of the LBR kinase. A, SDS-PAGE analysis on 8% gel and silver
staining of the material eluted from the R-agarose affinity
column (for details see text). Bars on the left indicate molecular masses (in kDa). B, in situ kinase assay of column-purified LBR kinase (lane 2) and
of the kinase co-immunoprecipitated with LBR by affinity-purified
aR
antibody from a Triton X-100 extract of nuclear
envelopes (lane 1). Samples were electrophoresed on a 12%
SDS-polyacrylamide gel containing 0.1 mg/ml electroeluted LBR,
renatured in situ, incubated with
[
-
P]ATP, and subjected to autoradiography. Bars on the left indicate the same molecular masses
as in A. C, binding of column-purified LBR kinase to
the NH
-terminal domain of LBR. Purified LBR kinase was
incubated with Protein A-Sepharose beads (lane 1),
electroeluted LBR bound to aR
antibody/Protein A-Sepharose
beads (lane 2), glutathione-Sepharose beads (lane 3),
GST-wtNt immobilized on glutathione-Sepharose beads (lane 4),
and GST-
RSNt immobilized on glutathione-Sepharose beads (lane
5). The co-sedimenting material was incubated with
[
-
P]ATP and analyzed by SDS-PAGE and
autoradiographed. In control assays (lanes 1 and 3)
and in the case of GST-
RSNt-glutathione-Sepharose beads (lane
5), 2.5 µg of GST-wtNt were added in the reaction mixtures to
provide a substrate for the LBR kinase. Bars on the left indicate molecular masses (in kDa).
The
column-purified enzyme was also able to bind to LBR in solution and
could be co-immunoprecipitated with LBR using affinity-purified
antibodies (Fig. 5C, lanes 1 and 2).
Binding involved the RS dipeptide motifs of the LBR, since the kinase
was able to associate with the GST-wtNt immobilized on
glutathione-Sepharose beads, whereas no interaction with GST-RSNt
was observed (Fig. 5C, lanes 3-5).
The partially purified LBR kinase did not modify histones, casein,
and myelin basic protein, but did phosphorylate intact LBR and GST-wtNt (Fig. 6B). Interestingly, when a well-characterized
subcellular fraction containing SR proteins (Zhaler et al.,
1992) was incubated with column-purified kinase, we found that the
enzyme could efficiently phosphorylate the 30-kDa major component (Fig. 6B). The other proteins present in the SR
fraction were not phosphorylated to a significant extent suggesting
that the LBR kinase may show substrate selectivity. The 30-kDa band
contains two distinct polypeptides SRp30a and SRp30b, which have also
been described as SF2 and SC35, respectively. The phosphorylation of
LBR and SRp30 was inhibited by an excess of the synthetic peptide
R as well as by a peptide containing six arginine-serine
(RS) repeats (R
, Fig. 6C). From the sum of
these observations it can be inferred that the LBR kinase belongs to a
novel class of enzymes which can also modify SR proteins (Gui et
al. 1994).
Figure 6:
Substrate specificity of LBR kinase.
Phosphorylation of H, H
, H
,
H
, H
, myelin basic protein, casein,
electroeluted LBR, GST-wtNt, and RS proteins (RSP), previously
heated to 70 °C for 10 min, by purified LBR kinase. RSP* shows the
phosphorylation of heated RS proteins in the absence of LBR kinase. All
substrates were added to the assay mixture at a final concentration of
0.15 mg/ml except for LBR, the final concentration of which was 0.08
mg/ml. The samples were analyzed by SDS-PAGE on 12% gels and stained
with Coomassie Blue (A) or autoradiographed (B). Bars on the left side of A and B indicate molecular masses (in kDa). C, inhibition of
phosphorylation of LBR and SR proteins by 0.5 mM R
and R
peptide. Autoradiograms of the gels are shown.
The five top bars on the left indicate the same
molecular masses as in A, and the sixth bar corresponds to 14 kDa.
To determine more specifically the serine residues
of LBR that are phosphorylated by the LBR kinase, we expressed in E. coli fusion proteins identical with GST-wtNt except that in
each case one of Ser, Ser
, Ser
,
Ser
, and Ser
of the RS motif was mutated to
glycine or alanine (Table 1). Mutation of Ser
to Gly
resulted in a construct that could not be expressed in E.
coli, even though the sequence and the proper subcloning of the
mutated cDNA into the pGEX-2T expression vector were confirmed.
However, the other four recombinant proteins were appropriately
expressed, purified, and used as substrates for in vitro phosphorylation assays with the partially purified LBR kinase.
Results presented in Table 1and Fig. 7reveal that all
four fusion proteins could be phosphorylated similarly to wtNt. The
apparent K
of the kinase for the recombinant
proteins was in the range of 1.7-2.4 µM. Taking into
consideration that the stoichiometry of the phosphorylation reaction
for both wtNt and the mutants was close to 1, any one of the serines of
the RS motif, but only one per molecule, should be phosphorylated at
steady state. That several spots have been observed previously in
two-dimensional phosphopeptide maps of in vivo or in vitro phosphorylated LBR (for relevant information, see Simos and
Georgatos(1992)) is consistent with this interpretation. Similar
phosphopeptide mapping confirm that the peptides phosphorylated by the
partially purified LBR kinase correspond to the peptides phosphorylated in vivo. (
)
Figure 7:
Inhibition of LBR phosphorylation by
GST-wtNt and serine mutants of GST-Nt (for nomenclature see text and Table 1). One µg of purified LBR was incubated with
radiolabeled ATP and the LBR kinase in the presence of buffer(-),
GST-wtNt, GST-RSNt, or mutated forms of GST-Nt (10 µg), as
indicated. The reaction products were run on SDS-polyacrylamide gels
and autoradiographed. Note that LBR phosphorylation is competed off by
GST-wtNt and all serine mutants, but not by GST-
RS. Also notice
that GST-Nt and the serine mutants are all phosphorylated by the
enzyme, whereas GST-
RS is not.
Figure 8:
The NH-terminal domain of LBR
binds to p34/p32 in vitro. A, immunoblot showing
binding of p34 when a fraction highly enriched in p34 was incubated
with GST (lane 2), GST-wtNt (lane 3), or
GST-
RSNt (lane 4) immobilized on glutathione-Sepharose
beads. Lane 1 shows a reference sample containing purified
p34. B, GST-wtNt (lane 1) or GST-
RSNt (lane
3) immobilized on glutathione-Sepharose beads were incubated with
a Triton X-100 lysate of nuclear envelopes, and the material bound to
the beads was analyzed by immunoblotting. In lane 2, binding
to GST-wtNt was assessed in the presence of 0.25 mM R
peptide. The blots were probed with affinity-purified ap34-C
antibodies and stained using an alkaline phosphatase-conjugated rabbit
goat anti-rabbit antibody.
The
specificity of this binding was assessed by performing the same type of
experiment with the deletion mutant GST-RSNt. Under these
conditions, the binding of p34/p32 to the glutathione beads carrying
the mutant protein was greatly inhibited (Fig. 8A, lane 4) or completely abolished (Fig. 8B, lane 3). Finally, repetition of the assay in the presence of
an excess of R
peptide also abolished binding between
GST-wtNt and p34/p32 (Fig. 8B, lane 2). From
these results it can be concluded that p34/p32 binds to LBR by
interacting with the RS-containing region.
Figure 9:
Phosphorylation by the LBR kinase inhibits
binding of p34 to the NH-terminal domain of LBR. Immunoblot
showing binding of p34 when a Triton X-100 lysate of turkey erythrocyte
nuclear envelopes was incubated with GST-wtNt immobilized on
glutathione-Sepharose beads (lane 1). In lanes 2, 3, and 4, immobilized GST-wtNt was incubated with
buffer and 100 µM ATP (lane 2), purified LBR
kinase and ATP (lane 3), or purified LBR kinase, in the
absence of ATP (lane 4), prior to incubation with the Triton
X-100 lysate. The blots were processed as described in Fig. 8.
To distinguish whether the binding is indeed inhibited by phosphorylation, or whether the kinase and p34/p32 compete for the same binding site on LBR, we repeated the same type of experiment in the presence of the LBR kinase but adding or omitting ATP in the course of the assay. Fig. 9, lane 4, shows that binding of p34/p32 to the fusion protein was inhibited only when ATP was present in the reaction mixture. Thus, binding of p34/p32 to LBR seems to be inhibited after phosphorylation of the RS motifs.
The family of SR proteins
includes essential splicing factors that commit precursor mRNA to
splicing and mediate spliceosome assembly (Fu, 1993). Although their
role in the splicing mechanism is not yet clear, mutational studies
have shown that the RS domains in U2AF and in ASF/SF2 are
required for splicing activity (Zamore et al., 1992; Caceres
and Krainer, 1993; Zuo and Manley, 1993). Recent data suggest that
phosphorylation promotes spliceosome assembly but blocks the catalytic
steps of splicing, and the prime candidates for the targets of
phosphorylation are the RS domain-containing splicing factors (Mermoud et al., 1994; Gui et al., 1994).
There have been two reports on protein kinases which phosphorylate specifically RS motifs (Woppmann et al., 1993; Gui et al., 1994). One such activity is associated with snRNP particles and phosphorylates the U1 snRNP 70-kDa protein at a subset of the sites phosphorylated in vivo (Woppmann et al., 1993). This activity also phosphorylates the COOH-terminal, RS-rich domain of ASF/SF2. In addition, Gui et al.(1994) identified a cell cycle-regulated serine kinase (SRPK1 = SR Protein Kinase 1), with an apparent molecular mass of 92 kDa, which can phosphorylate splicing factors of the RS family. Purified SRPK1 can induce disassembly of speckled intranuclear snRNP structures in interphase nuclei. The exact location of the kinase in the nucleus is not known.
At present, we do not
know if the LBR kinase is the same with the enzyme purified by Gui et al.(1994). SDS-PAGE analysis together with in situ gel assays predict a molecular mass of 110 kDa for the catalytic
subunit of the LBR kinase, which is significantly higher than that of
SRPK1. This observation indicates that the two kinases are distinct;
however, species-specific differences between birds and mammals may
also be the reason for this difference in apparent M.
Because p34 does not co-isolate with LBR when extraction and purification are performed under conditions that favor phosphorylation (e.g. in the presence of ATP and phosphatase inhibitors; Simos and Georgatos(1992, 1994)), we suspected that p34 may interact with the RS domain of LBR in a phosphorylation-dependent manner. Results obtained by in vitro binding assays clearly show that p34 binds tightly to the RS motifs of LBR when the latter is unphosphorylated, but dissociates from it upon phosphorylation mediated by the LBR kinase.
Recent studies have shown that the RS domains mediate protein-protein interactions between components of the splicing machinery (Wu and Maniatis, 1993; Kohtz et al., 1994), probably in a phosphorylation-dependent manner (Woppmann et al., 1993; Mermoud et al., 1994). The existence of RS motifs in the LBR molecule and the occurrence of a splicing factor-associated protein among the constituents of the LBR complex raise the possibility that LBR, alone or in combination with p34, may interact with components of the splicing machinery. Taking into account earlier observations (Spector et al., 1991), it can be speculated that LBR and its partners act as transient docking sites for nuclear ``speckles,'' in the nuclear envelope. Such a possibility is further supported by the fact that snRNPs migrate to the nuclear periphery when murine erythroleukemia cells (MEL) are induced to differentiate in vitro (Antoniou et al., 1993).
Based on the fact that the lamins are peripheral membrane proteins,
whereas LBR traverses the inner nuclear membrane, LBR was considered to
function as a lamin receptor. On the other hand, LBR, together with the
integral membrane protein LAP2 (Foisner and Gerace, 1993), are the most
obvious candidates to mediate the association of interphase nuclear
membranes to chromatin (Maison et al., 1995). ()The
data presented here expand the possible functions of LBR, raising the
possibility that the LBR complex is a molecular device that may couple
the karyoskeleton (nuclear lamina) to regulatory factors involved in
different aspects of gene expression.
It is also noteworthy that protein kinase A and Cdc2, which can be induced by hormones and mitotic factors, modify sites which are close, but distinct, from those phosphorylated by the RS kinase. Given that this segment of the molecule is exposed to the nucleoplasm and is charged (Worman et al., 1990), these modifications may as well participate in the regulation of LBR-protein and/or LBR-DNA interactions during interphase or mitosis. Such potential interactions remain to be addressed in future studies.
This article is dedicated to Stavros and Adamantia Politis.