* Division of Cellular and Molecular Medicine, Abstract. Reversible phosphorylation plays an important role in pre-mRNA splicing in mammalian cells.
Two kinases, SR protein-specific kinase (SRPK1) and
Clk/Sty, have been shown to phosphorylate the SR
family of splicing factors. We report here the cloning
and characterization of SRPK2, which is highly related
to SRPK1 in sequence, kinase activity, and substrate
specificity. Random peptide selection for preferred
phosphorylation sites revealed a stringent preference of
SRPK2 for SR dipeptides, and the consensus derived may be used to predict potential phosphorylation sites
in candidate arginine and serine-rich (RS) domain-containing proteins. Phosphorylation of an SR protein
(ASF/SF2) by either SRPK1 or 2 enhanced its interaction with another RS domain-containing protein (U1
70K), and overexpression of either kinase induced specific redistribution of splicing factors in the nucleus.
These observations likely reflect the function of the
SRPK family of kinases in spliceosome assembly and in
mediating the trafficking of splicing factors in mammalian cells. The biochemical and functional similarities
between SRPK1 and 2, however, are in contrast to their
differences in expression. SRPK1 is highly expressed in
pancreas, whereas SRPK2 is highly expressed in brain, although both are coexpressed in other human tissues
and in many experimental cell lines. Interestingly,
SRPK2 also contains a proline-rich sequence at its NH2
terminus, and a recent study showed that this NH2-terminal sequence has the capacity to interact with a WW
domain protein in vitro. Together, our studies suggest that different SRPK family members may be uniquely
regulated and targeted, thereby contributing to splicing
regulation in different tissues, during development, or
in response to signaling.
PRE-MRNA splicing in mammalian cells takes place in
a multi-component complex known as the spliceosome, which contains two classes of splicing factors:
U1, U2, U5, and U4/6 small nuclear ribonucleoprotein
particles (snRNPs)1 and non-snRNP splicing factors (for
review see Krämer, 1996 All RS domain-containing proteins are probably posttranslationally modified by phosphorylation, and reversible phosphorylation has been shown to play an important
role in splicing. For example, no splicing complexes could
be detected in nuclear extract treated with the phosphatase PP1, indicating that phosphorylation is essential for early steps of spliceosome assembly, and SR proteins
appear to be the major targets of the PP1 treatment (Mermoud et al., 1994 Two families of kinases, SR protein-specific kinase
(SRPK) and Clk/Sty, have been identified that phosphorylate RS domain-containing splicing factors. Our lab identified and cloned human SRPK1 in the pursuit of an activity
that mediates splicing factor redistribution in the cell cycle
(Gui et al., 1994a In this paper, we report the cloning and characterization
of a new SRPK family member named SRPK2 that was
discovered based on its sequence similarity to SRPK1.
A series of biochemical experiments demonstrate that
SRPK1 and 2 are very similar with respect to their enzymatic activity and substrate specificity. Both kinases promoted specific protein-protein interactions between RS
domain-containing splicing factors and their overexpression induced the redistribution of splicing factors from nuclear speckles to nucleoplasm, indicating that both kinases
may be involved in the regulation of spliceosome assembly
in vivo. However, these two kinases are differentially expressed in various human tissues and each kinase contains
unique sequence features that may contribute to their specific function and/or regulation in vivo. Therefore, mammalian cells may have evolved multiple kinases to regulate
RNA splicing, and these SR protein kinases may themselves be targets for regulation.
cDNA Cloning of SRPK2
A database search revealed multiple expression seqence tag (EST) clones
that are homologous but not identical to SRPK1. One such cDNA clone
(Genbank/EMBL/DDBJ accession number H00135) was obtained from
Research Genetics, Inc. (Huntsville, AL), and sequencing analysis showed
that the clone encodes a serine/threonine kinase and displays 78% identity
to SRPK1 in their kinase domains. DNA probes derived from the clone
were used to screen a human fetal brain cDNA library in the Lambda
ZAP II vector (Stratagene, La Jolla, CA). From 5 × 105 plaques, 31 positive clones were obtained. Restriction analysis showed that all the clones
are derived from different regions of a single cDNA, and thus, the longest
clone was sequenced in both strands.
Expression of SRPK2 and Its Substrates
The HpaI-NheI fragment encoding the full-length SRPK2 was cloned into
the pAcG2T vector (PharMingen, San Diego, CA) to express the kinase
as a glutathione-S-transferase (GST) fusion protein by baculovirus according to manufacturer's instructions. GST-SRPK2 was purified on glutathione-Sepharose 4B beads (Pharmacia Biotechnology Inc., Piscataway,
NJ). ASF/SF2 and its mutant derivatives were expressed and purified
from bacteria (Gui et al., 1994b Northern Blotting Analysis
A human multiple tissue Northern blot was purchased from Clontech
Laboratories Inc. (Palo Alto, CA). Soas2, HBL100, Bosc23, SF763,
MG63, and Hela cells were cultured in DME (Life Technologies, Gaithersburg, MD) plus 10% heat-inactivated FCS (Hyclone, Logan, UT).
Lan5 cells were grown in DME-F12 plus 10% FCS. THP1, U937, Weri1,
and Lnz308 cells were cultured in RPMI 1640 (Irvine Scientific, Santa
Ana, CA) plus 10% FCS. Total RNA from 6 × 105 cultured cells was extracted using RNAexol (BioChain, San Leandro, CA); 15-20 µg of total
RNA was separated in 1% agarose gel containing 2.2 M formaldehyde
and then blotted onto Hybond-N+ nylon membrane (Amersham Corp.,
Arlington Heights, IL). After baking the membrane at 80°C for 2 h, the
membrane was stained with methylene blue to visualize RNA molecular
markers (Life Technologies) and destained in H2O. The blots were prehybridized at 68°C for 1 h in 10 ml ExpressHyb Hybridization solution
(Clontech Laboratories Inc.), and then hybridized with random primed
probes as specified in Fig. 8. After hybridization for 1-2 h, the blots were
washed at 50°C for 15 min sequentially in 0.1% SDS plus 0.5, 0.25, and 0.1× SSC before exposure to X-ray film. For normalization, each blot was
stripped by boiling in 0.5% SDS for 5 min and reprobed for glyceraldehyde-3-phosphate dehydrogenase.
Molecular Pathology Program,
Department of Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139; and ¶ Division of Signal Transduction, Beth Israel Hospital and Department of
Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
). A series of interactions between pre-mRNA and snRNPs during spliceosome assembly are critical for splice-site selection, and most importantly, for establishing a catalytic core for the splicing
reaction to occur in the spliceosome. However, most of
these interactions are mediated by non-snRNP factors.
Among the best characterized non-snRNP factors are the
superfamily of arginine/serine-rich (RS) domain-containing splicing factors (for review see Fu, 1995
). The family is
composed of "classic" SR proteins that are characterized by one or two RNA recognition motifs at the NH2 terminus, an RS domain at the COOH terminus, and other RS
domain-containing polypeptides. RNA recognition motifs
are responsible for binding to RNA, whereas RS domains
appear to function as protein-protein interaction modules to mediate various spliceosome assembly steps. Thus,
some RS domain-containing proteins may mediate complex assembly on RNA, whereas others may function as
adaptor molecules to bring complexes together in spliceosome assembly. Because RS domain-containing proteins
play a critical role in selecting and pairing functional splice
sites, many splicing factors in this family are not only essential for constitutive splicing, but can also affect alternative splicing (for review see Fu, 1995
; Manley and Tacke,
1996
; Valcárcel and Green, 1996
).
). Consistent with these findings, phosphorylation was recently shown to promote specific protein-protein interactions between U1 70K and ASF/SF2,
both of which contain an RS domain (Xiao and Manley,
1997
). On the other hand, dephosphorylation is critical for
splicing after spliceosome assembly. When nuclear extracts were treated with phosphatase inhibitors, spliceosome assembly was not affected, but splicing was blocked.
Splicing in the stalled spliceosome could then be rescued
by phosphatase PP1 or PP2A (Mermoud et al., 1992
).
Moreover, incorporation of nonhydrolyzable
-S-ATP in
the RS domain-containing U1 70K blocked splicing after
spliceosome assembly (Tazi et al., 1993
). Together, these
studies strongly indicate that a phosphorylation-dephosphorylation cycle is important for RNA splicing. Additionally, PP1 was also shown to affect splice-site selection in
vitro (Cardinal et al., 1994). Because SR proteins are
known to affect splice-site selection both in vitro and in vivo, and PP1 clearly caused dephosphorylation of these
splicing factors in nuclear extract, it is likely that the PP1
effect on alternative splicing is mediated through changes
in the phosphorylation state of SR proteins (Mermoud et al.,
1994
).
). SRPK1 is a kinase highly specific for
RS domain-containing splicing factors because it recognizes only arginine (not lysine) and phosphorylates only serine (not threonine) in its substrates (Gui et al., 1994b
). Clk/Sty was initially cloned as a cdk-like kinase by PCR
(Johnson and Smith, 1991
; Ben-David et al., 1991
) as well
as a dual specificity kinase in an expression screening
(Howell et al., 1991
). In fact, three highly related Clk/Sty
kinases are expressed in mammalian cells (Hanes et al.,
1994
). Clk/Sty was later found to interact with RS domain-
containing splicing factors in the yeast two-hybrid system,
and to efficiently phosphorylate these splicing factors in
vitro (Colwill et al., 1996a
). Interestingly, Clk/Sty itself contains several SR or RS repeats at the NH2 terminus,
which appear to contribute to its high affinity binding with
RS domain-containing substrates. SRPK1 and Clk/Sty
share 32% identity in their kinase domains and both contain a signature amino acid sequence referred to as the
LAMMER motif (Colwill et al., 1996a
). A systematic comparison between the two kinases revealed that SRPK1 displays high specific activity towards RS domain-containing
splicing factors, whereas Clk/Sty has broader substrate
specificity (Colwill et al., 1996b
). These observations suggest that these two kinases may have distinct functions in
pre-mRNA splicing. Clearly, splicing is likely to be regulated by multiple kinases and phosphatases, given the
complexity of the splicing reaction and the diversity of
splicing factors involved.
Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
; Colwill et al., 1996b
). GST-ASF/SF2 and
U2AF65 were also made in bacteria. Other RS domain-containing proteins (SRp20, GST-SRp40, GST-SC35, SRp55) were expressed by baculovirus as described (Gui et al., 1994a
).
View larger version (68K):
[in a new window]
Fig. 8.
Northern blotting
analysis of SR protein kinases (indicated in the right
of each panel) in multiple human tissues (A and B) and
cell lines (C-E).
In Vitro Kinase Assay
The kinase activity of purified SRPK1 and 2 was normalized using bacterial ASF/SF2 as a substrate and then used to phosphorylate other substrates as described (Gui et al., 1994b). Each purified substrate was normalized (using BSA as a standard) to 0.1 µg for testing phosphorylation in
Fig. 2 B or to 1 µg in Fig. 2 C. To determine whether bacterial ASF/SF2
could gain the mAb104 phosphoepitope upon phosphorylation by
SRPK2, 0.1 µg of GST-ASF/SF2 was incubated with purified SRPK2 in
the presence of 1 mM ATP under kinase assay conditions followed by
Western blotting with mAb104 (culture supernatant). The blot was developed by enhanced chemiluminescence (ECL; Pierce, Rockford, IL). To
compare the relative specific activities of SRPK1 and 2, full-length SRPK1
and 2 cDNAs were fused with a FLAG-tag sequence at their NH2 termini,
and cloned into the pSP73 vector for in vitro transcription/translation in
the TNT system (Promega Corp., Madison, WI). The translated products
were immunoprecipitated using the M2 anti-FLAG monoclonal antibody
followed by a kinase assay on beads as previously described (Colwill et al.,
1996b
). After SDS-PAGE, 35S-labeled kinases and 32P-labeled ASF/SF2
were quantitated by phosphoimaging (Molecular Dynamics Inc., Sunnyvale, CA).
|
Determination of Phosphorylation Consensus by Peptide Selection
Rationale for peptide library design and synthesis were as previously described (Songyang et al., 1994). In the current study, a serine-oriented and
arginine-directed peptide library (see Fig. 4 A) was used. For the kinase
reaction, purified SRPK2 (100 U, as defined in Gui et al., 1994a
) was incubated at 25°C for 1 h with 1 mg of degenerate peptide mixture in a 300-µl
reaction volume in the presence of 100 mM ATP, trace labeled with [
-
32P]ATP (~6 × 105 cpm), 1 mM DTT, 10 mM MgCl2, and 50 mM Tris, pH
7.5. After the reaction, free ATP was removed by DEAE-Sephacel chromatography, and phosphopeptides were quantitatively separated from the
unphosphorylated mixture on a ferric chelation column as previously described (Songyang et al., 1994
). Peptide sequencing and data analysis were
also as described (Songyang et al., 1994
).
|
|
|
Protein-Protein Interaction Determined by the GST Pulldown Assay
The assay was performed essentially as described (Xiao and Manley,
1997). Bacterially expressed GST-ASF/SF2 was kinased in vitro by baculovirus-expressed GST-SRPK1 or 2 as described (Gui et al., 1994b
), except that the ATP concentration was raised to 1 mM, and the reaction was
incubated at 30°C for 4 h. Unphosphorylated GST-ASF/SF2 was prepared
in a parallel sample lacking ATP. Treated proteins were desalted on G25
columns equilibrated in NETN (20 mM Tris-Cl, pH 8.0, 100 mM NaCl,
0.5 mM EDTA, 0.5% NP-40; Xiao and Manley, 1997
), and rebound to glutathione-Sepharose. Beads were washed twice with NETN containing 1 M
NaCl, and then three times with NETN. Bead aliquots containing 1-2 µg
GST or GST-ASF/SF2 were incubated in 200 µl NETN with 3 µl in vitro
translated [35S]methionine U1 70K for 30 min at 4°C. Beads were washed
three times in NETN, treated with RNase and washed once, and then
boiled in SDS-PAGE sample buffer. After SDS-PAGE, U1 70K was detected by autoradiography.
Indirect Immunofluorescence Microscopy
Full-length SRPK2 (the HpaI-NheI fragment) was subcloned into the
NcoI-XbaI sites in the pUHD10-3 vector to create pUHD-SRPK2, in
which a FLAG tag was fused with SRPK2 at its NH2 terminus. This vector
system is used in cells expressing a tetracycline-controlled transactivator
(tTA), and thus, gene expression is constitutive in the absence of tetracycline (Gossen and Bujard, 1992). 1 d before transfection, 5 × 104 tTA
HeLa cells were seeded in 6-cm plates containing two or three 1.8-cm
round coverslips coated with 0.1% gelatin. 10 µg of pUHD-SRPK2 plasmid DNA was transfected per plate by the calcium-phosphate method
and the transfected cells were washed three times with PBS 16-24 h later.
After culturing in fresh medium for another 16-24 h, cells were processed
for immunostaining at room temperature as described (Fu and Maniatis,
1990
). Briefly, cells were washed twice in PBS, and then fixed in 2% formaldehyde plus 0.2% Triton X-100 for 10 min. Fixed cells were permeablized in 1% Triton X-100 for 10 min, washed three times in PBS, and
then blocked for 30 min in 20% FBS plus 0.5% Tween 20. Cells were
stained for 1 h with the primary monoclonal M2 anti-FLAG (IgG1; 1:1,200
dilution from the 2 mg/ml stock purchased from Eastman Kodak Co.
(Rochester, NY) and B1C8 (IgM; 1:50 dilution from culture supernatant; a
gift from B. Blencowe and P. Sharp, Massachusetts Institute of Technology, Cambridge, MA) or mAb104 (IgM; undiluted culture supernatant), washed three times with PBS plus 0.1% NP-40, and developed for 1 h with
secondary rhodamine-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-mouse IgM (Southern Biotechnology, Birmingham, AL).
Finally, the coverslips were mounted in an antiquenching solution (25 mg/
ml 1,4-diazabicyclo [2.2.2] octane and 70% glycerol in PBS) for examination under a Zeiss Axiophot fluorescence microscope.
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Results |
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Identification and Cloning of SRPK2
We previously described the cloning and characterization
of SRPK1, a serine kinase highly specific for the RS domain present in many splicing factors (Gui et al., 1994a,b;
Colwill et al., 1996b
). To gain further understanding of its
biological functions, we conducted chromosomal mapping.
Unexpectedly, probes derived from SRPK1 hit multiple
loci in both mice and humans (our unpublished results),
suggesting the possibility of multiple related kinases. These observations prompted us to search for SRPK1-
related expression sequence tags in databases. We found
eight clones displaying extensive homology to various regions within the kinase domain of SRPK1. Sequencing
analysis revealed two kinases, one of which is SRPK1 and
the other (represented by the clone H00135) is highly related, but not identical to SRPK1. We then screened a human fetal brain cDNA library using probes derived from
H00135 and isolated a full-length clone, which contains an
open reading frame encoding a novel kinase of 686 amino
acids (Fig. 1 A).
Sequence comparison with SRPK1 revealed several features of this new kinase: first, the new kinase displays 77%
identity and 90% similarity to SRPK1 over their entire kinase domains (kinase domains are boxed in Fig. 1 A, and
individual domains are underlined and marked in Fig. 1 B).
Second, like SRPK1, the kinase domains of this new kinase are divided into two halves by a large spacer sequence (Fig. 1), a structural feature rare among serine/
threonine kinases, but common among tyrosine kinases
(Hanks and Quinn, 1991). Interestingly, one-third of the
spacer region is also highly conserved between the two kinases (Fig. 1 B). Comparison with cAMP-dependent protein kinase A (PKA; Fig. 1 B) indicates that the spacers
are inserted between two
sheets (
8 and
9; Taylor and
Radzio-Andzelm, 1994), suggesting that the spacers may
function as an autonomous domain in these kinases (see
below). Third, both SRPK1 and the new kinase have a
small deletion between kinase domain II and III, and a
small insertion in the linker loop within kinase domain V
(Fig. 1 B). These differences may contribute significantly
to the activity and specificity of these kinases (see Discussion for further details). Based on these structural similarities as well as on many shared biochemical properties with
SRPK1 described below, we named the new kinase SRPK2.
SRPK2 also contains unique features that may be indicative of its function and/or regulation in vivo. Most notably, SRPK2 contains a proline-rich sequence APLVPPPPPPPPPPPPPLPDPTPPEP at the NH2 terminus (underlined
in Fig. 1 A). This motif contains the binding consensus
core for Src Homology 3 (SH3) domain-containing proteins (PXXP, X-any amino acid, P-proline; Ren et al., 1993), and for a subclass of WW domain-containing proteins that
bind to proteins containing PPLP cores (underlined above)
usually in the context of polyprolines (Chan et al., 1996
;
Sudol, 1996a
; Bedford et al., 1997
). Indeed, a recent two-hybrid screen using a WW domain-containing protein as
bait identified a mouse cDNA fragment, WBP6, which is
highly homologous to SRPK2 (Bedford et al., 1997
; see
Discussion). Accordingly, this NH2-terminal proline-rich motif of SRPK2 may function as a targeting signal to interact with substrates and/or regulators.
SRPK2 Is Specific for RS Domain-containing Splicing Factors
The extensive sequence homology between SRPK1 and 2 suggests that they may have similar enzymatic activity and substrate specificity. To test this possibility, we prepared both kinases by in vitro transcription/translation followed by immunoprecipitation, and SR protein-kinase activity was assayed on beads using bacterially expressed ASF/SF2 as substrate. As shown in Fig. 2 A, both SRPK1 and 2 phosphorylated ASF/SF2 with a similar specific activity, demonstrating that SRPK2 is an active kinase for this SR protein. We then tested phosphorylation of other RS domain-containing splicing factors using purified baculovirus-expressed SRPK2. As shown in Fig. 2 B, SRPK2 efficiently phosphorylated SRp20, ASF/SF2, SC35, SRp40, and SRp55, as well as U2AF65. SRPK2 could not phosphorylate other common kinase substrates such as histone H1 or myelin basic protein (data not shown). Therefore, like SRPK1, SRPK2 is specific for RS domain-containing splicing factors.
To determine whether SRPK2-mediated phosphorylation took place in the RS domain and to further characterize the sequence requirement for SRPK2-mediated phosphorylation, we tested phosphorylation of a panel of ASF/
SF2 mutants. Previous studies demonstrate that, although
both SRPK1 and Clk/Sty phosphorylate this splicing factor in its RS domain, they have different sequence requirements (Colwill et al., 1996b). For example, changing
serines to threonines in all RS dipeptides in ASF/SF2 abolished phosphorylation by both kinases, indicating that
both proteins are serine-specific kinases. However, changing arginines to lysines abolished phosphorylation by
SRPK1, but had little effect on phosphorylation by Clk/
Sty, indicating that SRPK1, but not Clk/Sty, requires arginine for substrate recognition and phosphorylation. As
shown in Fig. 2 C, SRPK2-mediated phosphorylation of
ASF/SF2 was abolished when the RS domain was deleted
(
RS), showing that phosphorylation occurs in this domain. Replacing serines by threonines (RT) or glycines
(RG) in all RS dipeptides in ASF/SF2 diminished phosphorylation (some residual phosphorylation likely takes
place at unmutagenized serines outside the RS repeats; Gui
et al., 1994b
), and mutation of arginines in the RS dipeptides, KS and GS, resulted in a complete loss of phosphorylation of ASF/SF2 by SRPK2. Therefore, SRPK2 has the
same substrate specificity as SRPK1, displaying a stringent requirement for both arginine and serine in phosphorylating RS domain-containing splicing factors.
Previous studies also showed that a specific phosphorylated epitope is present in all standard SR proteins purified
from mammalian cells and is recognized by the monoclonal antibody mAb104 (Zahler et al., 1992). This phosphoepitope can be restored to bacterially expressed ASF/
SF2 by both SRPK1- and Clk/Sty-mediated phosphorylation (Gui et al., 1994b
; Colwill et al., 1996b
). To verify if
this is also true for SRPK2, bacterially expressed GST-ASF/SF2 was phosphorylated by SRPK2 and analyzed by
Western blotting using mAb104. Fig. 2 D shows that ASF/
SF2 did regain the mAb104 specific phosphoepitope upon
phosphorylation by SRPK2, and the phosphorylation was
also accompanied by a mobility shift in SDS-PAGE as expected (data not shown, see Fig. 5 A). Taken together, these
data demonstrate that SRPK1 and 2 have identical biochemical properties in phosphorylating RS domain-containing splicing factors in vitro, and support the idea that
SRPK2 is one of the kinases responsible for phosphorylating those splicing factors in vivo.
|
Selection of Preferred Substrates
The above biochemical characterization demonstrates that
the SRPK family members are highly specific for RS domain-containing splicing factors. To further define the
substrate specificity and understand the structural basis
for phosphorylation-site selection by the SRPK family of
kinases, we took a peptide selection approach that has
been developed to determine the substrate specificity of
protein kinases (Songyang et al., 1994, 1996
). The method
involves phosphorylating a random peptide library using a
purified kinase followed by quantitative separation and
isolation of phosphopeptides, and then sequencing of the
selected phosphopeptide pool. Amino acid(s) enriched in
a given position will indicate a preference for this amino
acid at that position in substrate recognition and phosphorylation. Because the SRPK family members phosphorylate RS domain-containing splicing factors, they are likely to
be arginine-directed kinases (Songyang et al., 1996
). Therefore, we chose a biased library in which serine was placed
at position 0, and arginine at position
3 (Fig. 3 A). Purified SRPK2 was used to phosphorylate the peptide library.
After quantitative removal of unphosphorylated peptides
by iron-chelating chromatography, SRPK2-phosphorylated peptides were sequenced. Preferred amino acids at each
position are presented in Fig. 3 B. Although the experiment was done using SRPK2, it is reasonable to assume
that the data are representive of the SRPK family of kinases because both SRPK1 and 2 are highly related, displaying very similar kinase activity and substrate specificity as described above.
As shown in Fig. 3 B, SRPK2 generally prefers arginine,
but not lysine, near the phosphorylation site, which is consistent with the fact that both SRPK1 and 2 phosphorylate
SR proteins, but not the KS mutant of ASF/SF2 (Fig. 2).
Besides arginine, SRPK2 also selected histidine NH2-terminal of the serine. These observations suggest that the kinase may require positive charges on both sides of the
serine, indicating that arginine-serine-arginine (RSR) is
the best substrate for the kinase. Based on these selection data in combination with the mutagenesis results, it appears that phosphorylation mediated by the SRPK family
of kinases requires three criteria: (a) SR and RS dipeptides are preferred; (b) A basic environment around the
SR or RS dipeptide is critical; and (c) Lysine is not allowed, especially at the 2 position (Fig. 2 C and Fig. 3 B).
These criteria explain why the SRPK family of kinases
could not phosphorylate most common kinase substrates including histone H1 and myelin basic proteins (many contain lysines and hydrophobic amino acids around phosphorylation sites). Further, one may use these criteria to
predict potential SRPK phosphorylation sites in a candidate protein substrate (see Discussion).
There are two notable exceptions to RSR as the best
substrate in the selection. First, proline was also preferentially selected at the +1 position (compared to other positions). This finding explains the previous observation that
SRPK1 could phosphorylate the SPRY peptide in ASF/
SF2 in vitro (Colwill et al., 1996b). However, a synthetic
SPRY peptide was not a good substrate for SRPK1 (Colwill et al., 1996b
), likely due to the lack of surrounding basic amino acids. Second, both polar (N and Q) and acidic
(D and E) amino acids are significantly selected at the
2
position. This observation implies that the kinase may
phosphorylate an SRSR-containing peptide, regardless of
whether the first serine is unmodified (polar) or phosphorylated (acidic; note that serine was not included in randomized positions in the library so that its selection was
not detected). Therefore, the SRPK family of kinases may
be capable of phosphorylating both unphosphorylated and
partially phosphorylated SR proteins.
Structural Basis for Substrate Specificity
Perhaps the most striking feature of the SRPK family of
kinases is the strong selection of arginine at the P+1 position, which is in contrast to most protein kinases characterized so far (Songyang et al., 1994, 1996
). Previous structural studies have revealed a pocket (called the P+1
pocket) in the kinase catalytic core that is specific for selecting the amino acid at the P+1 position in the substrate.
Previous studies have established that the composition of
the P+1 pocket is highly predictive of substrate specificity (Songyang, Z., unpublished results). To understand the
mechanism for the P+1 selection by the SRPK family
members, we aligned SRPK sequences with a number of
kinases whose tertiary structures have been determined,
and therefore the composition of their P+1 pockets is
known (the sequence alignment of SRPK1 and 2 with
PKA is shown in Fig. 1 B). The sequence comparison revealed identical P+1 pockets for SRPK1 and 2, which is
consistent with identical substrate specificity between the
two kinases, and provides a rationale for the selection of
arginine at the P+1 position, as described below.
At positions marked in Fig. 1 B and summarized in Fig.
4, the P+1 pockets for PKA and many other kinases (their
sequences not shown, see Hanks and Quinn, 1991) are
composed of hydrophobic residues, explaining the preference of hydrophobic residues at the P+1 position in both
selected and natural substrates (Songyang et al., 1994
). In
contrast, previous modeling predicted that the arginine in
mitogen-activated protein kinase (MAPK) at the PKA 205 equivalent position sits in the bottom of the P+1 pocket
and directly interacts with a secondary amine group in the
substrate (Songyang et al., 1994
). Proline was strongly selected by MAPK at the P+1 position because it is the only
natural amino acid that has a secondary amine. For a similar reason, perhaps, proline was selected (although to a
lesser extent) by SRPK2 as the kinase also has an arginine
at this position (Arg550 in SRPK2, see Fig. 1 B and Fig. 4).
Casein kinase II
(CKII
) has a highly basic P+1 pocket
including an arginine at the PKA equivalent position 198, which is consistent with its selection for an acidic residue at the P+1 position in the substrate as explained earlier
(Songyang et al., 1996
). In contrast, the P+1 pocket of
both SRPK1 and 2 contains conserved polar (Asn532 in
SRPK2, which corresponds to Phe187 in PKA) and acidic
(Asp543, which corresponds to Leu198 in PKA) residues.
This acidic amino acid in the P+1 pocket of these kinases
may be responsible for strong selection of arginine in the
P+1 position in the substrate.
As described in the Introduction, Clk/Sty is capable of
phosphorylating SR proteins; however it can also phosphorylate many other substrates which do not contain any
RS motif (Colwill et al., 1996a,b). Sequence alignment revealed that the P+1 pocket of Clk/Sty (Fig. 4) is similar to
that of MAPK, explaining why Clk/Sty can phosphorylate
the SPRY peptide more efficiently than SRPK1 (Colwill
et al., 1996b
). Further, a polar serine (Ser327 in Clk/Sty) at
the PKA equivalent position 187 may be partially responsible for the selection of arginine at the P+1 position. This
may be functionally significant as this serine residue is conserved among all three Clk/Sty family members from
both mouse and human (Hanes et al., 1994
). One may further speculate that the phosphorylation specificity of Clk/
Sty for RS domain-containing splicing factors may be regulated by its own phosphorylation, thereby converting this
polar amino acid (Ser327) to a negatively charged residue
(phosphoserine). Clk/Sty may be autophosphorylated (Johnson and Smith, 1991
; Ben-David et al., 1991
) or phosphorylated by other kinases, but the phosphorylation
site(s) remains to be determined and the postulated function and regulation of the serine residue in the P+1 pocket
in the determination of substrate specificity will have to be
proven by mutagenesis.
The SRPK Family of Kinases Mediates In Vitro Interactions between SR Domain-containing Splicing Factors
The biochemical data strongly suggest a role of SRPK1
and 2 in splicing. Recently, it was demonstrated that Clk/
Sty-mediated phosphorylation of ASF/SF2 facilitates its
interaction with another RS domain-containing splicing
factor, U1 70K (Xiao and Manley, 1997). To determine
whether the SRPK family of kinases has a similar function,
we tested the effect of SRPK1- and 2-mediated phosphorylation on protein-protein interactions by the GST-pulldown assay. Bacterially produced ASF/SF2 was efficiently
phosphorylated by either kinase, resulting in a marked
mobility shift in SDS-PAGE (Fig. 5 A), and phosphorylated ASF/SF2 interacted with in vitro translated U1 70K
much more efficently than the mock-phosphorylated protein (Fig. 5 B). GST alone did not bring down any U1 70K,
demonstrating the specificity of the assay. These data support a role for the SRPK family of kinases in spliceosome
assembly by facilitating specific protein-protein interactions among spliceosomal components.
Localization of SRPK1 and SRPK2 and Evidence for Their Interaction with Splicing Factors In Vivo
It is known that splicing factors are concentrated in a
speckled pattern in the nucleus, yet splicing takes place
cotranscriptionally on nascent transcripts (for review see
Spector, 1993). Thus, splicing factors must be recruited to
sites of transcription and splicing (Jimenez-Garcia and
Spector, 1993
; Huang and Spector, 1996
; Misteli and Spector, 1997). Previous studies showed that kinases, such as
SRPK1 (Gui et al., 1994a
) and Clk/Sty (Colwill et al.,
1996a
), were able to induce the redistribution of splicing
factors from nuclear speckles to the nucleoplasm, indicating that these kinases may play an active role in the recruitment process. To provide further evidence that the
SRPK family members interact with their substrates in
vivo and mediate the localization of splicing factors in the
nucleus, we determined the localization of both SRPK1
and 2 by peptide tagging or fusing to green fluorescent
protein (GFP), and examined the effect of overexpression
of these kinases on the localization of endogenous splicing
factors.
Previous studies showed that Clk/Sty is localized in the
nucleus, consistent with its function in splicing (Colwill et al., 1996a). As shown in Fig. 6, we also detected both SRPK1
and 2 in the nucleus. Surprisingly, both these kinases are
also found in the cytoplasm. Among transfected cells,
some displayed predominant cytoplasmic (Fig. 6, a and d),
whereas others displayed nuclear (Fig. 6, b and e) localization. This heterogeneous pattern could be attributed to the
level of the expressed kinases, the physiological state of
the cell, or fixation and permeabilization conditions during
immunolocalization. To examine the localization of the expressed kinases without fixation and permeabilization,
we directly visualized GFP-SRPK1 (Fig. 6 c) and GFP-SRPK2 (Fig. 6 f) in living cells. The cytoplasmic signal appears to be predominant (Fig. 6, c and f), although the expressed kinases are also clearly visible in the nucleus (see
below for further details). Therefore, it is likely that the
endogenous kinases are present in both cellular compartments.
|
The localization of the SRPK family of kinases in the cytoplasm is interesting because many kinases with a nuclear
function are regulated by nuclear translocation. In fact,
Dsk1, an SRPK family member from fission yeast, was
also found to localize predominantly in the cytoplasm in
interphase, but in the nucleus during mitosis (note that the
yeast nuclear envelope does not break down in mitosis; Takeuchi and Yanagida, 1993). Thus, only a fraction of
these kinases may be required for splicing in the nucleus in
interphase, but a maximal level of such kinases may be
necessary for the reorganization of the splicing machinery
during mitosis, as suggested previously (Gui et al., 1994a
).
Our studies, however, do not preclude the possibility that
the SRPK family of kinases has a function in the cytoplasm. We further observed that SRPK1 and more clearly SRPK2 are not diffusely localized in the cytoplasm (Fig. 6,
a, d, and g). Instead, they appear to be attached to some
structure, which may function by anchoring these kinases
in the cytoplasm. We are currently determining the sequence requirements for the localization of both kinases in
the cytoplasm as well as in the nucleus.
Upon a close inspection of transfected cells, a punctate
nuclear population of both kinases was occasionally seen
in both fixed and living cells (see Fig. 6 c, for an example).
When focused on different focal planes (compare Fig. 6, g
and h), SRPK2 was clearly colocalized with B1C8 (Fig. 6,
h-j), a novel RS domain-containing splicing factor (Blencowe and Sharp, personal communication) previously characterized as one of the markers for nuclear speckles
(Blencowe et al., 1994). Similarly, we also detected colocalization of GFP-SRPK1 with SC35 in nuclear speckles
(data not shown). These observations suggest that at least
some fraction of SRPK1 and 2 interacts with their substrates in the nucleus.
In most SRPK-transfected cells, images of nuclear
speckles appear diffused, a situation reminiscent of overexpression of wild-type Clk/Sty (Colwill et al., 1996a). In
fact, this effect may reflect the movement of splicing factors mediated by the SRPK and Clk/Sty families of kinases
in the nucleus (for review see Fu, 1995
). As shown in Fig.
7, overexpression of both SRPK1 (Fig. 7, a and b) and
SRPK2 (Fig. 7, e-h) effectively induced the redistribution of endogenous splicing factors from nuclear speckles to
nucleoplasm. Splicing factors, such as the B1C8 antigen
(Fig. 7 b) and SR proteins detected by mAb104 (data not
shown), are concentrated in nuclear speckles in untransfected cells, but became diffusely localized in cells overexpressing SRPK1. Similar results were also obtained with
SRPK2 (Fig. 7, e-h). In contrast, overexpression of a mutant SRPK1, which contains a point mutation (K109M) at
the ATP-binding site and was previously shown to be an
inactive kinase (Colwill et al., 1996b
), had little effect on
the distribution of endogenous SR proteins (Fig. 7, c and d).
These data are consistent with our published results that
application of purified SRPK1 onto permeabilized cells in
vitro induced the disassembly of the speckled nuclear domains (Gui et al., 1994a
), and strongly suggest that SRPK
family members are directly involved in releasing SR proteins from nuclear speckles to the nucleoplasm. This may
represent an essential step in recruiting splicing factors to
nascent transcripts for cotranscriptional splicing suggested
by Spector and his colleagues (Jimenez-Garcia and Spector, 1993
; Huang and Spector, 1996
; Misteli and Spector,
1997).
|
Finally, other nuclear structures, such as the PML oncogenic domains (Dyck et al., 1994) detected by the anti-PML mAb5E10 (Stuurman et al., 1992
) and nuclear envelope detected by anti-lamine A/C antibodies (Gui et al.,
1994a
), were not affected by overexpression of either
SRPK1 or 2 (Fig. 7, i-l). Therefore, the SRPK family of kinases appears to specifically mediate the redistribution of
splicing factors in the nucleus.
SR Protein Kinases Are Differentially Expressed
Current data suggest that both the Clk/Sty and SRPK families of kinases are involved in the regulation of spliceosome assembly by mediating the localization and interaction of splicing factors in mammalian cells. It is, however, unlikely that they are completely redundant kinases, as each kinase may have a more defined spectrum of substrates. Alternatively, these kinases may be differentially expressed and/or regulated. To explore these possibilities, we examined the expression of these SR protein kinases by Northern blotting analysis in multiple human tissues as well as in many commonly used experimental cell lines. As shown in Fig. 8, SRPK1 and 2 are expressed as single 4.4- and 4.3-kb transcripts, respectively, in multiple human tissues. SRPK1 is highly expressed in pancreas whereas SRPK2 is highly expressed in brain. Both kinases are moderately expressed in other tissues including heart and skeletal muscle and the expression of both is low in lung, liver, and kidney. These results clearly show that SRPK1 and 2 are differentially expressed, which may contribute to the tissue specific regulation of both constitutive and alternative splicing.
In 11 experimental human cell lines examined, SRPK1
and 2 are ubiquitously expressed, although with notable
differences (Fig. 8, C and D). Compared to their expression in HeLa cells after normalizing against GAPDH,
SRPK1 is expressed at a higher level than SRPK2 in Weri-1
and Lan5, but much lower in Soas2 cells, and both kinases
exhibit a low level of expression in two leukemia cell lines,
THP1 and U937. Clk/Sty is expressed as two transcripts (3.2 and 1.8 kb) resulting from alternative splicing as characterized previously (Duncan et al., 1995). The 1.8-kb transcript corresponds to the primary cDNA for the active kinase, whereas the 3.2-kb mRNA results from a partially
spliced RNA, and therefore does not encode a functional
kinase (Duncan et al., 1995
). Interestingly, the 1.8-kb message is ubiquitously expressed, whereas the expression of
the 3.2-kb RNA is cell specific (Fig. 8 E, lane 3, for example), indicating that Clk/Sty may be regulated by alternative splicing in a cell specific fashion. These results will
guide us in choosing a cellular model to study the regulation of splicing by specific kinases in the future. Together,
these data demonstrate that SR protein kinases are differentially expressed. Thus, various SR protein kinases may
be regulated at multiple levels by differential expression,
alternative splicing, nuclear translocation, phosphorylation activation, or suppression, etc. These multiple regulation pathways in combination with their distinct substrate
specificity likely contribute to the complexity of regulation
of alternative splicing in mammalian cells and tissues, in
response to signaling, or during development.
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Discussion |
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Expression of Multiple SR Protein Kinases in Mammalian Cells
Two families of SR protein kinases (Clk/Sty and SRPK)
are now well characterized. The Clk/Sty family has three
members (Hanes et al., 1994) and the SRPK family has
two. Chromosome mapping suggests that the SRPK1 locus
is situated on mouse chromosome 17 and human chromosome 6, and SRPK2 on mouse chromosome 5 and human
chromosome 7, respectively (Wang, H.-Y., J.B. Bermingham, W. Lin, K.C. Arden, and X.-D. Fu, manuscript in
preparation). In addition, probes derived from the conserved kinase domains detected additional loci in mouse
chromosome X and human chromosome 8 and 18 (our unpublished results), indicating that these loci may encode
additional SRPK family members or pseudogenes. Consistent with this prediction, a cDNA fragment (named
WBP6) was recently isolated from a mouse library in a
two-hybrid screen, which resembles the human SRPK2
(Bedford et al., 1997
). The isolated partial cDNA has the
coding capacity for proline-rich and kinase domains nearly identifical to the corresponding domains in SRPK2. However, the partial clone lacks NH2 terminal sequence, and
the reported NH2 terminal part is completely different
from that of SRPK2. Thus, it remains unclear whether the
isolated cDNA represents the mouse homologue of human SRPK2 or another highly related kinase.
Kinases responsible for phosphorylation of RS domain-
containing splicing factors may not be confined to the two
families described above. Previous work showed that PKA
and protein kinase C can phosphorylate ASF/SF2 in vitro
(Colwill et al., 1996a), although the functional significance
of these phosphorylation events remains unclear. In addition, the NimA kinase selects arginines on both sides of
serine, suggesting that this kinase may also be involved in
phosphorylating SR proteins (Songyang et al., 1996
). Consistent with this possibility, a catalytically mutant form of
this kinase has been shown to colocalize with splicing factors in nuclear speckles (Lu et al., 1996
). However, unlike
SRPK2, NimA stringently requires a phenylalanine at the
3 position (Songyang et al., 1996
), indicating that the kinase may, at most, phosphorylate only a subset of SR proteins. NimA functions as a cell cycle regulator (Lu and
Hunter, 1995
), but whether or not it also has a role in splicing and is capable of phosphorylating some SR proteins
remain to be verified in future studies.
Structural Basis for the Activity and Specificity of the SRPK Family of Kinases
Recombinant SRPK1 and 2 are highly active and specific,
indicating that these kinases may have an open conformation for substrate recognition and catalysis. Although the
crystal structure of these kinases remains to be elucidated,
comparison with PKA revealed a basis for substrate specificity in the P+1 pocket as described in Results. In addition, the insertion in the linker loop within kinase domain
V (Fig. 1 B) may contribute to the separation between the
small and large lobes, and thus, to an open conformation
of the SRPK family of kinases for substrate recognition
and orientation. In PKA, Thr197 in the activation loop is
consititutively phosphorylated upon synthesis and the
phosphate on Thr197 is highly resistant to hydrolysis by
phosphatase (Steinberg et al., 1993). According to the
PKA crystal structure, this phosphothreonine forms ion pairs with His87 in the small lobe, and Arg165 and Lys189
in the large lobe, and these interactions are postulated to
be critical for the conformation of the active site in the enzyme (see discussion by Taylor and Radzio-Andzelm,
1994). The salt bridge between His87 and phosphothreonine197 is at least partially responsible for the closed
conformation of PKA (Knighton et al., 1991
), and the interaction is broken for the open conformation of the enzyme (Zheng et al., 1993
). As discussed earlier regarding
the structural features of Clk/Sty and SRPK1 (Colwill et
al., 1996a
), these two families of kinases have a completely
different set of amino acids in the corresponding positions.
As illustrated in Fig. 1 B, Thr197 in PKA is replaced by a
glutamic acid residue in both SRPK1 and 2; it is unclear
which amino acid corresponds to His87 as the region in kinase domain III contains a small, but critical deletion in
both SRPK1 and 2; and Arg165 and Lys189 are replaced
by threonine and cysteine, respectively. These differences
suggest that SRPK1 and 2 as well as Clk/Sty may have a
very different conformation than PKA at their active sites,
which may explain why these two families of kinases are
both highly active and selective in phosphorylating RS domain-containing splicing factors.
Proline-rich Domain in SRPK2
Characterization of the second SRPK family member not
only illustrates the complexity of regulation of splicing factors by multiple kinases, but also implies potential regulation of the kinase itself. One interesting structural feature
of SRPK2 is its NH2 terminal proline-rich domain, which
contains several consensus binding sites for an SH3 domain (Ren et al., 1993). To explore potential regulation of
splicing by signaling, we are currently testing whether
SRPK2 is associated with any SH3 domain-containing proteins. The proline-rich sequences are also frequent
binding targets for WW domain-containing proteins (Chen
and Sudol, 1995
; Chan et al., 1996
; Macias et al., 1996
; Sudol, 1996b
). Indeed, it was recently shown that a formin-binding protein interacts with a SRPK2-like kinase (WBP6)
from mouse in a two-hybrid screen (Bedford et al., 1997
).
Whether this association is of functional significance remains
to be investigated. The WW domain is present in a growing number of structural, regulatory, and signaling molecules (see an automatic update of WW domain-containing
proteins under the following electronic address: http://www.brok.embl-heidelberg.de/Modules/ww-gif.html; Sudol
et al., 1996a). Some splicing factors, such as Prp40 from yeast, also contain this domain (Kao and Siliciano, 1996
).
Therefore, it will be interesting to investigate whether the
NH2 terminal proline-rich domain in SRPK2 interacts
with WW domain proteins, especially WW domain-containing splicing factors. Such interactions may contribute
to a unique substrate specificity, regulation and/or cellular
targeting of the kinase in mammalian cells.
Implication of Nuclear and Cytoplasmic Localization of the SRPK Family of Kinases
The SRPK family of kinases is characterized by a large
spacer sequence dividing conserved catalytic kinase domains into two halves. Structural modeling of both kinases
suggests that the spacers constitute a unique domain,
which may function autonomously. This structural arrangement is rare among serine/threonine kinases, but
common in tyrosine kinases. For example, upon PDGF
binding on the cell surface, the PDGF receptor becomes
phosphorylated in its spacer, which then serves as a platform for signal relay by interacting with adaptor molecules
(for review see Ullrich and Schlessinger, 1990). By such an
analogy, the spacer sequences may also influence the biological function of SRPKs in mammalian cells.
A clue to the role of these spacer sequences comes from
the observation that deletion of the spacer from Dsk1, a
SRPK family member from fission yeast, resulted in exclusive nuclear localization of the mutant kinase in interphase
cells (Takeuchi and Yanagida, 1993). A similar phenomenon was also seen with both SRPK1 and 2 (our unpublished results). Thus, the spacer sequences may be involved in the regulation of nuclear translocation of these
SR protein kinases.
As shown above, SRPK1 and 2 are present in the nucleus and cytoplasm. The localization of these kinases in the nucleus is consistent with their putative function in splicing. The significance of their presence in the cytoplasm is unclear, but might reflect additional functions of these kinases in the cytoplasm. Alternatively, a fraction of these kinases may be restricted to the cytoplasm because oversupply of these kinases can completely alter the organization of the splicing machinery in the nucleus, and therefore, may be detrimental to normal cell physiology. We observed that both SRPK1 and 2 are localized in the cytoplasm in a nondiffused fashion, although the underlying cytoplasmic structure remains to be characterized. These findings, taken together with the observations that SRPK1 and 2 become exclusively localized in the nucleus after their spacers are removed (our unpublished observations), suggest that these kinases may be anchored in the cytoplasm through their spacer sequences.
Rules to Predict Preferred Phosphorylation Sites for the SRPK Family of Kinases
Our peptide selection studies have provided a structural
basis to explain the stringent substrate specificity observed
with the SRPK family of kinases. This information can
also be used to predict potential kinase substrates. As described in Results, these kinases appear to prefer phosphorylating an SR or RS dipeptide in a basic environment enriched with arginine or histidine, but not lysine. This rule
applies to all proven substrates tested so far in our lab. For
example, all standard SR proteins satisfy such a rule and
they can be efficiently phosphorylated by either SRPK1 or
2 in vitro. We also tested phosphorylation of other RS domain-containing splicing factors, such as human U2AF65
(Zamore et al., 1992) and U2AF35 (Zhang et al., 1992
),
and their homologues from Drosophila (dU2AF50 and
dU2AF38; Kanaar et al., 1993
; Rudner et al., 1996) and fission yeast (Prp2; Potashkin et al., 1993
). All these RS domain-containing splicing factors contain multiple SR or
RS dipeptides and are phosphorylated by SRPK1 (our unpublished observations).
It should be emphasized that not all RS domain-containing proteins implicated in splicing are substrates for
SRPK1 or 2. For example, Clk/Sty contains 10 SR or RS
repeats at the NH2 terminus (Ben-David et al., 1991).
However, it could not be phosphorylated in vitro by SRPK1
(our unpublished result), and inspection of its RS domain
indicates that none of those dipeptides satisfies the criteria
for recognition and phosphorylation by SRPK1 and 2 as
described above. In addition, Snp1p, a U1 70-K homologue from yeast, contains a number of dispersed SR or
RS dipeptides (Smith and Barrell, 1991
). Again, none of
those dipeptides is surrounded by arginine or histidine,
and Snp1p could not be phosphorylated by SRPK1. In
contrast, the B1C8 antigen, which was initially identified by an antinuclear matrix monoclonal antibody (Blencowe
et al., 1994
), was found to be a good substrate for SRPK1
during its biochemical purification, and recent cloning and
characterization show that it is an RS domain-containing
splicing factor (Blencowe et al., 1998
). Finally, we have recently cloned and characterized a novel splicing factor,
which is a U2AF35-related protein called Urp in a two-
hybrid screen using SRPK1 as bait (Tronchere et al., 1997
). Urp contains a typical RS domain at the COOH terminus and the protein was an excellent substrate for
SRPK1. Therefore, the stringent substrate specificity and
the sequence requirement defined by peptide selection
may be used to identify additional RS domain-containing proteins that may have a role in splicing.
How SR Protein Kinases May Affect Splicing
It was recently reported that Clk/Sty facilitates SR protein-protein interactions and prevents nonspecific protein-RNA interactions (Tacke and Manley, 1997; Xiao
and Manley, 1997). In this paper, we show that the SRPK
family of kinases has an analogous function in promoting
SR protein-protein interactions. Thus, it is conceivable that multiple kinases are involved in splicing in mammalian cells. However, the function of these SR protein kinases in splicing is just beginning to be understood. Previous studies using phosphatase inhibitors indicate that
phosphorylation of SR proteins is required for spliceosome assembly, and thus, SR protein kinases can be considered as activators for SR proteins (Mermoud et al.,
1994
). However, subsequent dephosphorylation is also important for splicing as blocking dephosphorylation by
phosphatase inhibitors prevented splicing from occurring
in the fully assembled spliceosome (Mermoud et al., 1994
).
In agreement with this finding, we observed that too much
SRPK1 is also inhibitory to splicing, likely by interfering with dephosphorylation in a later step of the splicing reaction (Gui et al., 1994a
). These observations are consistent
with the idea that the level of SR protein kinases in the nucleus must be regulated.
SR proteins are known to affect splice-site selection
both in vitro and in vivo, and, as such, it seems likely that
SR protein kinases might affect alternative splicing. Earlier studies demonstrated that SR proteins generally stimulate the selection of the proximal 5 and 3
splice sites if
two competing splice sites contain identical splicing signals
(Fu et al., 1992
). This situation, however, is unlikely to be
the case in most natural alternative splicing systems. In the
SV-40 T/t alternative splicing system, for example, different SR proteins were shown to prefer different splice sites
(Zahler et al., 1993
). Therefore, selection of a specific
splice site may be dictated by combinatory actions and
competition of mutiple SR proteins and other splicing factors. For this reason, it will be difficult to predict which direction a specific alternative splicing event will follow after
the activation of multiple SR proteins by a specific SR protein kinase. A specific alternative splicing event may be
regulated by the expression of a unique spectrum of SR
proteins, which may in turn be regulated by kinases with
distinct activity and substrate specificity. The identification of additional SR protein kinases will ultimately contribute to an understanding of regulatory events governing
alternative splicing.
![]() |
Footnotes |
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Received for publication Received for publication 3 October 1997 and in revised form 21 November 1997..
We thank Susan Taylor of University of California at San Diego (UCSD; La Jolla, CA) (UCSD) for many useful discussions on kinase structure and function, and Marius Sudol (Mount Sinai School of Medicine, New York) for many stimulating discussions. We are grateful to B. Blencowe and P. Sharp (Massachusetts Institute of Technology, Cambridge, MA) for B1C8 antibody, L. De Jong (University of Amsterdam) for the 5E10 anti-PML antibody, D. Black (University of California, Los Angeles, Los Angeles, CA) for Weri-1 and Lan5 cell lines, C. Glass (UCSD) for U937 and THP1 cell lines, M. Kamps (UCSD) for HBL100 and HT1080 cell lines, and J. Wang (UCSD) for Bosc23 and Soas2 cell lines. The authors thank members of the Fu lab for cooperation and suggestions during the course of this study, especially S. Chandler for help in SRPK2 expression and purification, and L. Feng for help in immunohistochemistry. ![]() |
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