The Subcellular Localization of SF2/ASF Is Regulated by
Direct Interaction with SR Protein Kinases (SRPKs)*
Jun
Koizumi
,
Yoshichika
Okamoto§,
Hiroshi
Onogi
,
Akila
Mayeda¶,
Adrian R.
Krainer¶, and
Masatoshi
Hagiwara
From the
Department of Functional Genomics, Medical
Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, the § Department of Surgery,
Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya
466, Japan, and the ¶ Cold Spring Harbor Laboratory,
Cold Spring Harbor, New York 11724-2208
 |
ABSTRACT |
Serine/arginine-rich (SR) proteins play an
important role in constitutive and alternative pre-mRNA splicing.
The C-terminal arginine-serine domain of these proteins, such as
SF2/ASF, mediates protein-protein interactions and is phosphorylated
in vivo. Using glutathione S-transferase
(GST)-SF2/ASF-affinity chromatography, the SF2/ASF kinase activity was
co-purified from HeLa cells with a 95-kDa protein, which was recognized
by an anti-SR protein kinase (SRPK) 1 monoclonal antibody. Recombinant
SRPK1 and SRPK2 bound to and phosphorylated GST-SF2/ASF in
vitro. Phosphopeptide mapping showed that identical sites were
phosphorylated in the pull-down kinase reaction with HeLa extracts and
by recombinant SRPKs. Epitope-tagged SF2/ASF transiently expressed in
COS7 cells co-immunoprecipitated with SRPKs. Deletion analysis mapped
the phosphorylation sites to a region containing an
(Arg-Ser)8 repeat beginning at residue 204, and far-Western
analysis showed that the region is required for binding of SRPKs to
SF2/ASF. Further binding studies showed that SRPKs bound
unphosphorylated SF2/ASF but did not bind phosphorylated SF2/ASF.
Expression of an SRPK2 kinase-inactive mutant caused accumulation of
SF2/ASF in the cytoplasm. These results suggest that the formation of
complexes between SF2/ASF and SRPKs, which is influenced by the
phosphorylation state of SF2/ASF, may have regulatory roles in the
assembly and localization of this splicing factor.
 |
INTRODUCTION |
Pre-mRNA splicing is an essential process required for the
expression of most eukaryotic protein-coding genes. Splicing catalysis occurs in a spliceosome complex (1). Components of the spliceosome include the U1, U2, and U4/U6.U5 small nuclear ribonucleoprotein particles (snRNPs)1 (2) and
numerous non-snRNP protein factors (3). The latter include all members
of the SR protein family, which play important roles during mammalian
spliceosome assembly. All SR proteins have one or two N-terminal
RNA-recognition motifs (RRMs) and a C-terminal domain rich in
arginine-serine dipeptide repeats (RS domain). The RS domain is
involved in protein-protein interactions with related domains of other
splicing factors, and these interactions are thought to be important
for splice site selection (4, 5).
The SR proteins are phosphorylated at multiple serines located
predominantly within the RS domain (6, 7). At least eight members of
the SR family contain phosphopeptides that are recognized by the
monoclonal antibody mAb104 (8). Analysis of tryptic phosphopeptides
derived from SF2/ASF showed that the RS domain of this protein is
phosphorylated at multiple sites both in vivo and in
vitro (7). Although the physiological role of SR protein phosphorylation is unknown, recent studies suggested that
phosphorylation of the RS domain of SF2/ASF enhances the interactions
between this domain and the U1-70K polypeptide and that
phosphorylation or dephosphorylation cycles may be required for
splicing (9, 10).
Several protein kinases can phosphorylate SR proteins, such as SF2/ASF,
within its RS domain in vitro. A U1 snRNP-associated kinase
was the first kinase activity reported to phosphorylate RS domains
although cDNAs encoding this kinase have not been isolated (11). A
second kinase, SRPK1, was purified and cloned on the basis of its
ability to phosphorylate SC35 or other SR proteins in vitro
(6, 12). The Clk/Sty kinase has an RS domain at its N terminus and was
found to interact with several members of the SR protein family in a
yeast two-hybrid screen (7). SRPK2, for which we isolated a mouse brain
cDNA, also gave the same pattern of serine phosphorylation of
SF2/ASF in vitro as SRPK1 (13) and is probably identical to
WBP, a cDNA fragment isolated as encoding a WW-domain-binding
protein in a two-hybrid screen (14). A human cDNA for SRPK2 was
also recently isolated from a fetal brain library (15). In addition to
these recently identified kinases, protein kinases C and A can also
phosphorylate SF2/ASF in vitro (7), as can the
p34cdc2 kinase (32). Moreover, DNA topoisomerase
I has been reported to phosphorylate SR proteins although it has no
obvious kinase domain (16). Finally, a nuclear envelope-bound kinase
activity phosphorylates the RS motif at the N-terminal domain of the
lamin B receptor (17).
To identify kinases that bind specifically to SF2/ASF in mammalian
cells, we performed pull-down kinase assays using a GST-SF2/ASF fusion
protein immobilized on glutathione-Sepharose beads. We found that SRPK1
and SRPK2 interact specifically with the RS domain of SF2/ASF in a
manner that depends on the phosphorylation state of SF2/ASF. This
interaction appears to modulate the subcellular localization of
SF2/ASF.
 |
EXPERIMENTAL PROCEDURES |
Preparation of HeLa Cell Extracts--
HeLa cells were grown in
suspension culture. The cells were harvested at the logarithmic growth
stage (4-6 × 105 cells/ml), suspended in lysis
buffer (20 mM HEPES (pH 7.8), 50 mM KCl, 1 mM EDTA, 10 µg/ml leupeptin, 1 mM PMSF, 1%
Triton X-100), and incubated on ice for 20 min. The lysates were
centrifuged at 12,000 × g for 30 min at 4 °C. Nuclear
and S100 extracts were prepared as described (20).
Preparation of Recombinant Proteins--
His-tagged SRPK1 and
SRPK2 proteins were expressed in Escherichia coli and
purified as described (13) using Ni-NTA matrix (QIAGEN). GST-SF2/ASF
was expressed in E. coli DH5 and induced with 0.1 mM IPTG for 2 h. The cells were lysed in GST binding buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM benzamidine, 2 mM dithiothreitol, 1 mM PMSF, 1% Triton X-100) and passed through a
glutathione-Sepharose 4B column (Amersham Pharmacia Biotech). GST-SF2/ASF and its mutant derivatives were eluted with 30 mM glutathione and dialyzed twice against TSE buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1 mM EDTA, 2 mM benzamidine, 2 mM
dithiothreitol, 0.5 mM PMSF, 20% glycerol) for 6 h
each at 4 °C.
Pull-down Kinase Assay--
HeLa cell nuclear extract or
recombinant kinases were incubated with GST-SF2/ASF in 1 ml of PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM
Na2HPO4·7H2O, 1.4 mM
KH2PO4) for 1 h at 4 °C.
Glutathione-Sepharose 4B was added and rotated for 30 min at 4 °C.
The beads were washed with cold PBS five times for 5 min each,
resuspended in 50 µl of 40 mM HEPES (pH 7.8), 10 mM MgCl2, 2 mM dithiothreitol, and 1 µCi of [
-32P]ATP and incubated for 30 min at
30 °C. The beads were then boiled in SDS-PAGE buffer (1% SDS, 1%
mercaptoethanol, 10 mM Tris-HCl (pH 8.0), 20% glycerol,
0.05% bromphenol blue), and the proteins were analyzed on a 10%
SDS-polyacrylamide gel.
Phosphopeptide Mapping--
Phosphorylation sites of SF2/ASF
were analyzed by phosphopeptide mapping as described before (31) after
the pull-down kinase assay. The phosphorylated SF2/ASF bands were
excised from SDS-polyacrylamide gels, and the proteins were eluted and
digested with trypsin in 50 mM
NH4HCO3 (pH 8.4) for 16 h at 37 °C.
After digestion, the samples were lyophilized, spotted onto thin layer
silica gel plates, and subjected to electrophoresis for 70 min at 1000 V in 15% acetic acid, 5% formic acid. This was followed by
chromatography in 37.5% n-butyl alcohol, 25% pyridine,
7.5% acetic acid.
Affinity Chromatography--
A 1-ml column of GST-SF2/ASF
immobilized on glutathione-Sepharose was equilibrated with buffer A (20 mM HEPES (pH 7.8), 50 mM KCl, 1 mM
EDTA, 10 mg/ml leupeptin, 1 mM PMSF, 1% Triton X-100). A
5-ml HeLa cell extract was loaded, and the column was washed with ten
volumes of buffer A. The column was developed with a 10-ml gradient of
NaCl from 50 to 500 mM.
Western and Far-Western Blotting--
GST-SF2/ASF was incubated
with HeLa cell nuclear extract, and complexes were captured on
glutathione-Sepharose 4B beads. The beads were boiled in SDS sample
buffer, and the supernatant was loaded on a 10% SDS-polyacrylamide
gel. After electrophoresis, the proteins were transferred to a
nitrocellulose membrane. The blots were blocked with 5% skim milk for
16 h at 4 °C and rinsed with PBS. For Western blotting, then
the membranes were incubated with anti-SRPK1 antibody (PharMingen) or
anti-hemagglutinin (HA) antibody (Medical & Biological Laboratories
Co., Ltd.) for 1 h at room temperature and rinsed with PBS. Bound
antibodies were detected using AP-conjugated goat anti-mouse IgG
(Zymed Laboratories Inc.) or anti-rabbit IgG
(Zymed Laboratories Inc.). For far-Western blotting,
the membrane was incubated with recombinant HA-SRPK1 protein (1 µg/ml) for 1 h at room temperature and rinse with PBS. Then it
was incubated with anti-SRPK1 antibody for 1 h at room temperature. Antibody was detected using AP-conjugated goat anti-mouse IgG.
Immunoprecipitations--
COS7 cells were cultured at 37 °C
in 10-cm dishes in Dulbecco's modified Eagle's medium (Nissui
Pharmaceutical Co., Ltd.) supplemented with 10% fetal bovine serum and
transfected with 20 µg of plasmid DNA in DEAE-dextran. After 48 h, the cells were washed twice with PBS and harvested. The cells were
resuspended in 200 µl of lysis buffer (20 mM HEPES (pH
7.8), 150 mM NaCl, 1 mM EDTA, 10 µg/ml
leupeptin, 1 mM PMSF, 1% Triton X-100) and incubated on
ice for 20 min. The solution was cleared by centrifugation at 15,000 rpm for 30 min at 4 °C. Anti-HA antibody or anti-c-Myc antibody was
added to the supernatant, followed by incubation for 30 min at 4 °C.
The solution was then incubated with protein G-Sepharose for 30 min at
4 °C. The beads were washed with cold PBS five times for 5 min each.
The immunoprecipitates were analyzed by further incubation under
phosphorylation conditions, followed by SDS-PAGE and autoradiography,
or by Western blotting.
Immunostaining--
HeLa cells were grown on coverslips and
cultivated in a CO2 incubator for 24 h after
transfection. The coverslips were washed twice with PBS. Subsequently,
the cells were fixed in 4% paraformaldehyde in PBS for 15 min, at room
temperature, permeabilized, and blocked in PBS containing 0.4% Triton
X-100, 1.5% bovine serum albumin, and 5% normal goat serum. For the
double immunostaining of the cells expressing HA-SRPKs and
myc-SF2, anti-HA polyclonal antibody (MBL), and anti-c-Myc
monoclonal antibody (Santa-Cruz) were used, followed by incubation with
secondary fluorescein isothiocyanate-conjugated goat anti-rabbit IgG
(Zymed Laboratories Inc. Laboratories) and Texas
red-labeled goat anti-mouse IgG (Southern Biotechnology Associates,
Inc.).
 |
RESULTS |
Recombinant human GST-SF2/ASF was expressed in and purified from
E. coli. The protein was incubated with HeLa cell extract and recovered by pull-down with glutathione-Sepharose beads. The beads
were then used for kinase reactions in the presence of radiolabeled ATP
(Fig. 1A). Phosphorylation of
the 60-kDa GST-SF2/ASF protein, detected by autoradiography (lane
1), suggested the existence of an SF2/ASF-associated kinase in
HeLa cell extracts.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 1.
Purification of SF2/ASF-bound kinases from
HeLa cell extracts. A, GST-SF2/ASF was incubated in
HeLa cell nuclear extract, recovered by pull-down with
glutathione-Sepharose beads, and incubated under in vitro
phosphorylation conditions in the presence of
[ -32P]ATP. Phosphorylation was detected by SDS-PAGE
and autoradiography. Lane 1, phosphorylation of GST-SF2/ASF;
lane 2, negative control with unfused GST. B,
elution profile of proteins bound to immobilized GST-SF2/ASF. HeLa cell
extract was applied to a GST-SF2/ASF-glutathione-Sepharose column. A
linear gradient from 50 to 500 mM NaCl was applied from
fractions 16 to 25. 25-µl aliquots were assayed for kinase activity
using 2 µg of GST-SF2/ASF as a substrate (13). Fractions of 1 ml were
collected, and the protein concentration of each fraction was
determined with a DC protein assay kit (Bio-Rad). Open
circles indicate kinase activity, as indicated on the
right y-axis. Closed circles indicate
total protein concentration, as indicated on the left axis.
C, protein composition of selected fractions. 10 µl of
fractions from the flow-through and wash (fractions 2 and
10, respectively), and 10 µl of 10-fold concentrated
fractions at or near the SF2/ASF kinase peak (fractions
17-24) were analyzed by SDS-PAGE and silver staining. The
molecular masses of standards are indicated on the left.
D, detection of SRPK1 in the GST-SF2/ASF column eluate.
Aliquots of the same column fractions as in panel C were
analyzed by Western blotting with a monoclonal antibody against
SRPK1.
|
|
To purify the kinase that binds to and phosphorylates SF2/ASF in HeLa
cells, we used affinity chromatography. The extracts were loaded on a
GST-SF2/ASF-glutathione-Sepharose column, and bound material was eluted
with a salt gradient. Individual fractions were assayed and SF2/ASF
kinase activity eluted between 300-500 mM salt, whereas
the bulk of bound protein eluted between 100-200 mM salt
(Fig. 1B). SDS-PAGE and silver staining analysis of the active fractions revealed a prominent band with a relative molecular mass of 95 kDa (Fig. 1C). The 95-kDa protein reacted with a
monoclonal antibody against SRPK1 (Fig. 1D).
The association of SRPK1 in HeLa extracts with GST-SF2/ASF was
confirmed by Western blotting of the complexes with anti-SRPK1 antibody
(Fig. 2). GST-SF2/ASF or GST were
incubated in HeLa cell nuclear (NE, lanes 1 and
3) or cytosolic S100 (lanes 2 and 4) extract and recovered by pull-down with glutathione-Sepharose beads.
The complexes were recovered and analyzed by Western blotting with a
monoclonal antibody against SRPK1.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 2.
Western blotting of GST-SF2/ASF-bound SRPK1
of HeLa cells. GST or GST-SF2/ASF were incubated in HeLa cell
nuclear (NE, lanes 1 and 3) or
cytosolic S100 (lanes 2 and 4) extracts and
recovered by pull-down with glutathione-Sepharose beads. The complexes
were recovered and analyzed by Western blotting with a monoclonal
antibody against SRPK1. The mobility of SRPK1 is indicated on the
right.
|
|
The pull-down kinase assays were also carried out using recombinant
SRPK1 and SRPK2, instead of the HeLa extract (Fig.
3A). Both SRPK1 and SRPK2
bound stably to and subsequently phosphorylated GST-SF2/ASF under these
conditions (lanes 2 and 3), whereas Clk/Sty did
not (lane 4).

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 3.
Pull-down assay of recombinant SF2/ASF
kinases. A, GST-SF2/ASF was incubated with HeLa cell
extract (lane 1), recombinant HA-SRPK1 (lane 2),
HA-SRPK2 (lane 3), or GST-Clk/Sty kinase (lane
4). As a negative control, GST protein was incubated with SRPK1
(lane 5), SRPK2 (lane 6), or Clk/Sty (lane
7). After incubation, the GST-SF2/ASF and GST proteins were
recovered by binding to glutathione-Sepharose beads, and the
immobilized complexes were phosphorylated in vitro.
Phosphorylated proteins were detected by SDS-PAGE and autoradiography.
The mobility of GST-SF2/ASF is indicated on the right. B,
tryptic phosphopeptide mapping of phosphorylated GST-SF2/ASF. After the
pull-down kinase assay, the phosphorylated SF2/ASF bands were excised
from SDS-polyacrylamide gels, digested with trypsin, and the fragments
were analyzed by two-dimensional thin layer electrophoresis and
chromatography as described under "Experimental Procedures."
a, HeL, HeLa.
|
|
We compared the phosphorylation sites of SF2/ASF phosphorylated by the
GST-SF2/ASF-bound kinase in HeLa extracts (Fig. 3B, panel a), recombinant SRPK1 (Fig. 3B, panel
b), or recombinant SRPK2 (Fig. 3B, panel c)
using two-dimensional thin layer electrophoresis and chromatography.
The resulting patterns are indistinguishable. These results suggest
that the SRPKs are the major kinases capable of associating with
GST-SF2/ASF in HeLa cell extracts.
To determine whether SRPKs interact with SF2/ASF in vivo, we
carried out immunoprecipitation experiments. HA-tagged SRPK1 or SRPK2
and myc-tagged SF2/ASF expression plasmids were transiently transfected into COS7 cells, and the cells were harvested and lysed at
48 h post-transfection. Immunoprecipitations were carried out with
either anti-c-Myc (Fig. 4A) or
anti-HA monoclonal antibodies (Fig. 4B), and the
immunoprecipitates were then incubated in kinase buffer in the presence
of radiolabeled ATP. Phosphorylated SF2/ASF was detected only when both
SRPK and SF2/ASF expression plasmids were co-transfected (Fig.
4A, lanes 2 and 3, Fig. 4B,
lanes 2 and 3), although the endogenous kinase
gave a faint band (Fig. 4A, lane 1). These
results indicate that SF2/ASF and the SRPKs can form stable complexes
in vivo. These results were confirmed by
immunoprecipitation/Western analysis (Fig. 4C). Complexes
between SF2/ASF and SRPKs were immunoprecipitated with anti-HA
polyclonal antibodies and separated by SDS-PAGE. The proteins were then
transferred to nitrocellulose, and epitope-tagged SF2/ASF was detected
with anti-c-Myc monoclonal antibody. When myc-tagged SF2/ASF
was co-transfected with HA-tagged SRPK1 or SRPK2, the 35-kDa myc-tagged
SF2/ASF polypeptide was detected in the anti-HA immunoprecipitate (Fig.
4C, lanes 2 and 3), again
demonstrating a stable interaction between SF2/ASF and the SRPKs.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
Binding of SRPK1 or SRPK2 to SF2/ASF in COS7
cells. A, COS7 cells were co-transfected with plasmids
expressing myc-tagged SF2/ASF and a HA-tagged kinase: SRPK1 (lane
2) or SRPK2 (lane 3). Extracts were prepared at 48 h post-transfection, and myc-SF2/HA-kinase complexes were
immunoprecipitated with anti-c-Myc monoclonal antibody. The
immunoprecipitates were incubated under in vitro
phosphorylation conditions, and labeled proteins were detected by
SDS-PAGE and autoradiography. The mobility of epitope-tagged SF2/ASF is
indicated on the right. B, same as panel
A, but the complexes were immunoprecipitated with anti-HA
polyclonal antibody. C, same as panel B, but the
immunoprecipitates were analyzed by Western blotting with anti-c-Myc
monoclonal antibody.
|
|
Because the RS domain of SR proteins is thought to mediate
protein-protein interactions (4, 5), we constructed several deletion
mutants of the RS domain of SF2/ASF to determine what regions are
required for stable interaction with the SRPKs (Fig. 5A). The deleted proteins,
RS (aa 1-197),
204 (aa 1-203),
227 (aa 1-226),
234 (aa
1-233), and
238 (aa 1-237), as well as the wild-type parent
protein (aa 1-248) were prepared as GST-fusion proteins in E. coli (Fig. 5B, top). The ability of these
proteins to interact with SRPKs was examined by a far-Western assay.
The recombinant proteins were separated by SDS-PAGE and blotted onto nitrocellulose. The filters were incubated with recombinant HA-SRPK1, and the bound kinase was detected using anti-SRPK1 antibody (Fig. 5B, middle). SRPK1 bound to
227,
234, and
238 but not to
RS or
204. The latter two mutants lack a
segment of eight consecutive RS dipeptide repeats. In agreement with
these results,
227,
234, and
238 were phosphorylated by SRPK1
in vitro, whereas neither
RS nor
204 proteins could be
phosphorylated (Fig. 5B, bottom). Identical
results were obtained with SRPK2 (data not shown). Together, these
results show that the SRPK phosphorylation sites in SF2/ASF are located
within a region containing the eight RS repeats (aa 204-226) and that
the same site is required for stable interaction between these kinases
and SF2/ASF.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 5.
Deletion analysis of SF2/ASF domains required
for SRPK1 binding. A, top, schematic diagram
of wild-type and deletion mutants of SF2/ASF. The C-terminal RS domain
is shown in black. Bottom, sequence of the RS
domain of SF2/ASF. RS and SR dipeptides are shown in bold,
and the deletion end point for each mutant is indicated by the
arrows. B, top, Coomassie Brilliant
Blue (CBB) staining; middle, interaction between
SRPK1 and SF2/ASF mutants. 0.5 µg of wild-type (lane 1) or
deletion mutants of GST-SF2/ASF (lanes 2-6) or GST only
(lane 7) were blotted onto nitrocellulose after SDS-PAGE.
The blot was incubated with SRPK1 protein, and SF2/ASF-bound SRPK1 was
visualized with anti-SRPK1 antibody. Bottom, in vitro
phosphorylation of SF2/ASF mutants by SRPK1. 0.3 µg of the indicated
proteins was phosphorylated in vitro with recombinant SRPK1.
The samples were analyzed by SDS-PAGE and autoradiography.
|
|
Next, we examined the effect of SF2/ASF phosphorylation on
protein-protein interactions between SF2/ASF and SRPK1. The
phosphorylation state of SF2/ASF was checked by the mobility shift on
SDS-PAGE (Fig. 6, left panel).
The interaction of HA-tagged SRPK1 with either phosphorylated (ATP+) or
unphosphorylated (ATP
) SF2/ASF was examined by the far-Western assay
(Fig. 6, right panel), and the interaction efficiencies were
quantitated by the relative intensity of the staining for HA-SRPK1. The
SRPK1 bound to unphosphorylated SF2/ASF was 5 times more than that
bound to phosphorylated SF2/ASF, indicating that the interaction
between these proteins depends on the phosphorylation state of
SF2/ASF.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of SF2/ASF phosphorylation state on
interaction with SRPK1. Left, Coomassie Brilliant Blue
(CBB) staining; right, SF2/ASF was incubated with
SRPK1 in the presence (+) or absence ( ) of ATP. Phosphorylated (+) or
unphosphorylated ( ) SF2/ASF were resolved by SDS-PAGE and transferred
to a nitrocellulose membrane. The membrane was incubated with SRPK1 and
probed with anti-SRPK1 antibody as in Fig. 5B.
|
|
As addition of SRPK1 kinase to permeabilized cells or overexpression of
Clk/Sty kinase results in a diffuse distribution of SC35, it has been
suggested that hyperphosphorylation of the RS domains may control
the subcellular distributions of SR proteins (6, 7, 18). We likewise
observed that expression of SRPK2 induced changes in the subcellular
localization of transfected SF2/ASF (Fig.
7, g and h) and
endogenous SC35 (13). Overexpression of SRPK1 also changed the
subcellular localization of both endogenous and transiently expressed
SF2/ASF (Fig. 7, c-f). To examine the effect of direct
interaction of SF2/ASF with SRPKs on the subcellular localization of
SF2/ASF, we analyzed the distribution of SF2/ASF in cells expressing a
catalytically inactive kinase mutant, SRPK2K108R, and
observed that SF2/ASF accumulated in the cytoplasm of cells expressing
the inactive SRPK2 (Fig. 7, i and j). In
contrast, a mutant that lacks the presumed SF2/ASF-interaction domain,
had no effect on the distribution of SF2/ASF (data not shown).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of SRPKs expression on SF2/ASF
distribution. HeLa cells were co-transfected with plasmids
expressing myc-tagged SF2/ASF and HA-tagged SRPKs and then fixed
24 h after transfection. Localization of endogenous SF2/ASF
(a and d) and myc-SF2/ASF (b,
f, h, and j) were visualized by
indirect immunofluorescence staining of transfected HeLa cells using an
anti-SF2/ASF monoclonal antibody or an anti-c-Myc monoclonal antibody
followed by Texas red-conjugated secondary antibody. HA-SRPKs were
detected using an anti-HA polyclonal antibody followed by fluorescein
isothiocyanate-conjugated secondary antibody (c, e, g, and
i). Bar, 10 µm, endogenous SF2/ASF (a),
myc-SF2/ASF only (b), SRPK1 wild type only (c and
d), myc-SF2/ASF with SRPK1 wild type (e and
f), SRPK2 wild type (g and h), and
SRPK2K108R (i and j),
respectively.
|
|
 |
DISCUSSION |
SR proteins are essential splicing factors that promote
splice-site recognition at an early stage of spliceosome assembly and
also influence the selection of alternative splice sites (19, 20, 21,
22). The phosphorylation state of SR proteins appears to influence
their activities in general, and alternative splicing, as well as their
subnuclear localization and nuclear-cytoplasmic transport properties.
Protein phosphatase 1 (PP1) can block an early step of spliceosome
assembly (23) and can affect alternative 5' splice-site selection
in vitro (24). These effects are thought to occur via
dephosphorylation of SR proteins, because addition of phosphorylated SR
proteins relieved the block to spliceosome assembly (23). Nuclear
speckles are subnuclear domains where SR proteins and other splicing
components are concentrated, and they are thought to represent sites of
storage or assembly for splicing factors (25). SRPK1 was originally
identified as a kinase of SC35 in extracts from HeLa cells (6, 12).
Addition of purified SRPK1 to permeabilized cells or
overexpression of SRPK1 or Clk/Sty in transfected cells results in an
apparent disassembly of the nuclear speckles (6, 7). These results
suggest that phosphorylation, or hyperphosphorylation, causes
release of these factors from the speckles, or perhaps that the
integrity of these structures is compromised.
Information about kinases that can phosphorylate SR proteins has
recently been obtained. Although the kinase domains of SRPK1 and
Clk/Sty are approximately 30% identical, including key amino acids
that are expected to be involved in substrate specificity (6), SRPK1
has a more restricted substrate specificity in vitro (26).
Clk/Sty phosphorylates the basic proteins histone H1 and myelin basic
protein, albeit to a lower specific activity than it phosphorylates
SF2/ASF. It does not phosphorylate more acidic substrates, such as
-casein, enolase, or an N-terminal region of c-Jun (26). SRPK1 was
calculated to be 150-fold more active with SF2/ASF than Clk/Sty with
SF2/ASF as the substrate (26). The Km values for
SF2/ASF were similar (0.28 µM for SRPK1, 0.40 µM for Clk/Sty), indicating that SRPK1 turns over SF2/ASF much faster than Clk/Sty does. Our present findings are consistent with
these published data. We found that SRPK1 and SRPK2 interact more
strongly with SF2/ASF than Clk/Sty does but that this interaction is
weakened upon phosphorylation, allowing product release.
Clk/Sty has an RS domain at its N-terminus, through which it was shown
to interact with five RNA-binding proteins, including three SR
proteins, in a two-hybrid screen (7). Our pull-down kinase assay failed
to detect an interaction between SF2/ASF and Clk/Sty. The discrepancy
may be attributable to the higher sensitivity of the two-hybrid screen
for what may be weak or transient interactions, or perhaps the
phosphorylation states of the SF2/ASF and Clk/Sty RS domains are
different in vitro and in yeast, with the latter resulting
in more stable interactions. Alternatively, the interaction of the
Clk/Sty RS domain with SF2/ASF may be weak but long lived compared with
the interaction between the SRPKs and SF2/ASF, resulting in the slower
release of SF2/ASF from Clk/Sty.
SRPK1 was reported to be closely related in sequence to a hypothetical
Caenorhabditis elegans kinase (CEHK) and to the fission yeast kinase Dsk1 (6, 27, 28). We recently cloned a mouse brain
cDNA encoding a novel SR protein-specific kinase, SRPK2 (13). The
amino acid sequence of SRPK2 is 58% identical to that of human SRPK1
and 32% similar to that of yeast Dsk1. Immunolocalization experiments
showed that both SRPK1 and SRPK2 are primarily localized in the
cytoplasm, despite having putative nuclear localization signals (13).
In contrast, SF2/ASF accumulates in the nucleus, concentrating in the
speckle domains (29). The RS domain of SF2/ASF is a nuclear targeting
signal, although it is not sufficient to direct accumulation in the
speckle domains. The RS domain of SF2/ASF is also involved in shuttling
of the protein between the nucleus and the cytoplasm, and co-expression
of Clk/Sty, presumably resulting in hyperphosphorylation of SF2/ASF,
interferes with shuttling, resulting in accumulation of SF2/ASF in the
cytoplasm (30). Expression of a catalytically inactive kinase mutant, Clk/StyK190R had a much more limited effect on the
distribution of SR proteins, suggesting that the phosphorylation state
of SR proteins strongly influences their cellular distribution. The
present study, however, shows that SF2/ASF accumulated in the cytoplasm
even in cells expressing the catalytically inactive kinase
SRPK2K108R. The discrepancy in the effects of
kinase-inactive mutants Clk/StyK190R and
SRPK2K108R may reflect the differential roles of Clk/Sty
and SRPK in vivo. The stable interactions between SF2/ASF
and SPRKs may be a key determinant of the subcellular distribution of
SF2/ASF. In support of this notion, expression of a deletion mutant of
SRPK1, which lacks the presumed SF2/ASF-interaction domain, had no
effect on the localization of SF2/ASF (data not shown).
SRPK1 activity was shown to be highest in metaphase cells, suggesting
that SRPK1 expression or activity is subject to cell cycle control (6).
The fact that recombinant SRPK1 expressed in E. coli binds
to and phosphorylates SF2/ASF suggests the existence of a cell-cycle
regulated kinase inhibitor or inhibitory modification of the kinase in
mammalian cells. An inactive form of SRPKs would still be expected to
form a stable complex with SF2/ASF in the cytoplasm. In this study, we
obtained approximately 6 mg of SRPK1 from 0.9 g of HeLa cells by
GST-SF2/ASF affinity chromatography. The amount of endogenous SRPKs
seems to be relatively high in comparison with the endogenous kinase
activity measured by the pull-down kinase assay, suggesting that the
majority of SRPKs may be free from SF2/ASF in living cells. Considering
that SRPK1 is inactive through G1/S phase (6), our results
suggest the existence of a potential regulator that works as a
pseudosubstrate, although other regulatory mechanisms cannot be
eliminated. Thus, the activation state of SRPKs may determine the
assembly of SR proteins into kinase-substrate complexes, as well as
their subcellular localization.
 |
FOOTNOTES |
*
This work was supported by grants from the Ministry of
Education, Science, and Culture in Japan (to M. H.) and in part by Grant CA13106 from the NCI, National Institutes of Health (to A. M.,
and A. R. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
+81-3-5803-5836; Fax: +81-3-5803-5853; E-mail:
m.hagiwara.end{at}mri.tmd.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
snRNP, small nuclear
ribonucleoprotein particle;
SR protein, serine/arginine-rich protein;
SRPK, SR protein-specific kinase;
HA, hemagglutinin;
GST, glutathione
S-transferase;
PBS, phosphate-buffered saline;
RS domain, domain rich in arginine-serine dipeptide repeat;
PMSF, phenylmethylsulfonyl fluoride;
IPTG, isopropyl-1-thio-
-D-galactopyranoside;
PAGE, polyacrylamide gel electrophoresis;
aa, amino acid.
 |
REFERENCES |
-
Staley, J. P.,
and Guthrie, C.
(1998)
Cell
92,
315-326[Medline]
[Order article via Infotrieve]
-
Will, C. L.,
and Lührmann, R.
(1997)
Curr. Opin. Cell Biol.
9,
320-328[CrossRef][Medline]
[Order article via Infotrieve]
-
Krämer, A.
(1996)
Annu. Rev. Biochem.
65,
367-409[CrossRef][Medline]
[Order article via Infotrieve]
-
Wu, J. Y.,
and Maniatis, T.
(1993)
Cell
75,
1061-1070[Medline]
[Order article via Infotrieve]
-
Kohtz, J. D.,
Jamison, S. F.,
Will, C. L.,
Zuo, P.,
Luhrmann, R.,
Gercia-Blanco, M. A.,
and Manley, J. L.
(1994)
Nature
368,
119-124[CrossRef][Medline]
[Order article via Infotrieve]
-
Gui, J. F.,
Lane, W. S.,
and Fu, X. D.
(1994)
Nature
369,
678-682[CrossRef][Medline]
[Order article via Infotrieve]
-
Colwill, K.,
Pawson, T.,
Andrews, B.,
Prasad, J.,
Manley, J. L.,
Bell, J. C.,
and Duncan, P. I.
(1996)
EMBO J.
15,
265-275[Abstract]
-
Zahler, A. M.,
Lane, W. S.,
Stolk, J. A.,
and Roth, M. B.
(1992)
Genes Dev.
6,
837-847[Abstract]
-
Xiao, S. H.,
and Manley, J. L.
(1997)
Genes Dev.
11,
334-344[Abstract]
-
Cao, W.,
Jamison, S. F.,
and Garcia-Blanco, M. A.
(1997)
RNA
3,
1456-1467[Abstract]
-
Woppmann, A.,
Will, C. L.,
Kornstadt, U.,
Zuo, P.,
Manley, J. L.,
and Luhrmann, R.
(1993)
Nucleic Acids Res.
21,
2815-2275[Abstract]
-
Gui, J. F.,
Tronchere, H.,
Chandler, S. D.,
and Fu, X. D.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
10824-10828[Abstract/Free Full Text]
-
Kuroyanagi, N.,
Onogi, H.,
Wakabayashi, T.,
and Hagiwara, M.
(1997)
Biochem. Biophys. Res. Commun.
242,
357-364[CrossRef]
-
Bedford, M. T.,
Chan, D. C.,
and Leder, P.
(1997)
EMBO J.
16,
2376-2383[Abstract/Free Full Text]
-
Wang, H.-Y.,
Lin, W.,
Dyck, J. A.,
Yeakley, J., M.,
Songyang, Z.,
Cantley, L. C.,
and Fu, X.-D.
(1998)
J. Cell Biol.
140,
737-750[Abstract/Free Full Text]
-
Rossi, F.,
Labourier, E.,
Fornr, T.,
Divita, G.,
Derancourt, J.,
Riou, J. F.,
Antone, E.,
Cathala, G.,
Brunel, C.,
and Tazi, J.
(1996)
Nature
381,
80-82[CrossRef][Medline]
[Order article via Infotrieve]
-
Nikolakaki, E.,
Meier, J.,
Simos, G.,
Georgatos, S. D.,
and Giannakouros, T.
(1997)
J. Biol. Chem.
272,
6208-6213[Abstract/Free Full Text]
-
Misteli, T.,
and Spector, D. L.
(1996)
Trends Cell Biol.
7,
135-138[CrossRef]
-
Ge, H.,
and Manley, J. L.
(1990)
Cell
62,
25-34[Medline]
[Order article via Infotrieve]
-
Krainer, A. R.,
Conway, G. C.,
and Kozak, D.
(1990)
Genes Dev.
4,
1158-1171[Abstract]
-
Krainer, A. R.,
Conway, G. C.,
and Kozak, D.
(1990)
Cell
62,
35-42[Medline]
[Order article via Infotrieve]
-
Fu, X.-D.,
Mayeda, A.,
Maniatis, T.,
and Krainer, A. R.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
11224-11228[Abstract]
-
Mermoud, J. E.,
Cohen, P. T. W.,
and Lamond, A. I.
(1994)
EMBO J.
13,
5679-5688[Abstract]
-
Cardinali, B.,
Cohen, P. T. W.,
and Lamond, A. I.
(1994)
FEBS Lett.
352,
276-280[CrossRef][Medline]
[Order article via Infotrieve]
-
Fu, X.-D.,
and Maniatis, T.
(1990)
Nature
343,
437-441[CrossRef][Medline]
[Order article via Infotrieve]
-
Colwill, K.,
Feng, L. L.,
Yeakley, J. M.,
Gish, G. D.,
Cáceres, J. F.,
Pawson, T.,
and Fu, X.-D.
(1996)
J. Biol. Chem.
271,
24569-24575[Abstract/Free Full Text]
-
Takeuchi, M.,
and Yanagida, M.
(1993)
Mol. Biol. Cell
4,
247-260[Abstract]
-
Wilson, R.,
Ainscough, R.,
Anderson, K.,
Baynes, C.,
Berks, M.,
Bonfield, J.,
Burton, J.,
Connell, M.,
Copsey, T.,
Cooper, J.,
et al..
(1994)
Nature
368,
32-38[CrossRef][Medline]
[Order article via Infotrieve]
-
Cáceres, J. F.,
Misteli, T.,
Screaton, G., R.,
Spector, D., L.,
and Krainer, A., R.
(1997)
J. Cell Biol.
138,
225-238[Abstract/Free Full Text]
-
Cáceres, J. F.,
Screaton, G. R.,
and Krainer, A. R.
(1998)
Genes Dev.
12,
55-66[Abstract/Free Full Text]
-
Hagiwara, M.,
Alberts, A.,
Brindle, P.,
Meinkoth, J.,
Feramisco, J.,
Deng, T.,
Karin, M.,
Shenolikar, S.,
and Montminy, M.
(1992)
Cell
70,
105-113[Medline]
[Order article via Infotrieve]
-
Okamoto, Y.,
Onogi, H.,
Honda, R.,
Yasuda, H.,
Wakabayashi, T.,
Nimura, Y.,
and Hagiwara, M.
(1998)
Biochem. Biophys. Res. Commun.
248,
872-878[CrossRef]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.