ABSTRACT
The
prp4
gene of
Schizosaccharomyces pombe
encodes a protein kinase. A physiological substrate is not yet known. A mutational
analysis of
prp4
revealed that the protein consists of a short N-terminal domain, containing several essential motifs, which is followed by the kinase catalytic domain comprising the C- terminus of the protein. Overexpression of N-terminal mutations disturbs mitosis and produces elongated
cells. Using a PCR approach, we isolated a putative homologue of Prp4 from
human and mouse cells. The mammalian kinase domain is 53% identical to the
kinase domain of Prp4. The short N-terminal domains share <20% identical amino acids, but contain conserved motifs. A fusion protein consisting of the N-terminal region from
S.pombe
followed by the mammalian kinase domain complements a temperature- sensitive
prp4
mutation of
S.pombe.
Prp4 and the recombinant yeast/mouse protein kinase phosphorylate the human SR
splicing factor ASF/SF2
in vitro
in its RS domain.
The
prp4
gene of
Schizosaccharomyces pombe
was identified in a pool of temperature-sensitive (ts)
prp
(pre-mRNA processing) mutants whose molecular phenotype is the accumulation of
pre-mRNA at the restrictive temperature (
1
). The gene is located on chromosome III and is essential for growth. The amino
acid sequence of
prp4
predicts a serine/threonine protein kinase catalytic domain at the C-terminus of the protein. In addition to the kinase domain the protein consists of an N-terminus comprising 157 amino acids. The predicted
M
r
of Prp4 is 55 000 (
2
).
According to the protein kinase classification system of Hanks and Hunter (
3
), which is based on similarity in the amino acid sequence of the kinase
domains, Prp4 belongs to the Clk (Cdc-like kinase) family. This family includes the mammalian SRPK1 and Clk/Sty protein kinases (
4
,
5
). The mammalian protein kinase Clk/Sty and Prp4 of fission yeast show the same domain arrangement: a
short N-terminal region is followed by the catalytic kinase domain.
Both mammalian kinases have been shown to phosphorylate the RS (arginine/serine-rich) domains of pre-mRNA splicing factors, called SR proteins,
in vitro
. The SR proteins are involved in constitutive and alternative splicing (
4
,
6
-
9
). It has been suggested that these two kinases do not act directly at the spliceosome, but co-localize with the SR splicing factors in subnuclear structures, called
speckles (
9
,
10
). Although the specific function of these kinases is still unknown, there is
some evidence that they play an important role in regulating the traffic of SR
splicing factors between speckles and the location of spliceosome assembly (
4
,
9
,
11
).
Five snRNPs (U1, U2, U4/U6 and U5) are required for pre-mRNA splicing (
12
,
13
). In mammalian cells a protein kinase activity co-purifying with the U1 snRNP has been detected. This kinase activity
specifically phosphorylates the U1 70K protein in its RS domain. When the human
SR splicing factor ASF/SF2 is added to an
in vitro
assay, the U1 snRNP-associated kinase activity also phosphorylates the RS domain of this
protein (
14
). The gene for this kinase activity has not been identified.
As yet we do not know a physiological substrate of Prp4. The notion that Prp4 is
involved in pre-mRNA splicing is based on the observation that intron-containing genes accumulate pre-mRNA at the restrictive temperature (36oC) when the
prp4-73
allele is in the genetic background. When the culture is shifted back to the
permissive temperature (26oC) mature message appears again after 30 min. This observation gives no
hint of whether Prp4 is directly or indirectly involved in the splicing
process. In many reports it has been demonstrated that phosphorylation and
dephosphorylation of spliceosomal components play a crucial role in spliceosome assembly and disassembly. The specific functions of the protein kinase(s) and phosphatase(s) involved are, however, still
elusive (
15
-
19
).
In this report we describe a mutational analysis of the N-terminal and kinase domains of Prp4 protein kinase of
S.pombe
. This analysis revealed short motifs in the N-terminus which are essential for function
in vivo
. Overproduction of Prp4 containing the mutated motifs revealed phenotypes which
indicate that the cells are impaired in mitosis.
We also describe the isolation of a mammalian kinase which is composed of an N-terminal region of ~170 amino acids followed by a catalytic protein kinase domain. The
mammalian kinase domain shares 53% identical amino acids with Prp4sp of fission
yeast. The N-terminal domain of the mammalian kinase, however, appears to be different,
sharing <20% identical amino acids with the yeast N-terminus. The essential motifs found in the N-terminus of Prp4sp are highly conserved in the mouse N-terminus. When the complete mouse cDNA is expressed in
S.pombe
it does not complement the ts allele
prp4-73
, however a fusion of the yeast N-terminus with the mammalian kinase domain was complementary.
We show that Prp4sp of
S.pombe
and the recombinant yeast/mouse kinase phosphorylate predominantly the RS
domain of the human splicing factor ASF/SF2
in vitro
(
20
-
22
).
The strains used in this study were L972, L975,
h
-s
ura4-294 prp4-73
,
h
-s
ura4-D18 leu1-32 prp4-73
. The prp4 ts mutant strain has been described by Rosenberg
et al.
(
1
). Standard classical and molecular genetic procedures and media for growth of
the
S.pombe
strains used have been described by Gutz
et al.
(
23
) and Moreno
et al.
(
24
) Transformation of
S.pombe
with shuttle plasmids and linearized fragments for integration was performed as
previously described by Gatermann
et al.
(
25
).
The system used for site-specific mutagenesis was based on the method developed by Kunkel (
26
). The procedure was performed as described previously (
25
). The following synthetic oligonucleotides were phosphorylated and annealed to the appropriate single- stranded uracil-containing phage DNA:
[Delta]NLS, 5'-GATGAAATTATACAGCAATTGTACCAACAGACTGGCAAT-3';
[Delta][Delta]NLS, 5'-GAAATTATACAGCAATTCGAAGATGTCGACCAAGTCTCT-3';
SX1, 5'-AGTACTACTGGTGATTTGCCCGCTATCAAATCTTCTGT-3';
SX2, 5'-ATGTTTGCAGATATCCCTTTGCCTGCTGTTAAGCGGCA-3';
EGY1, 5'-TGCAGGTTAACTCGGTCGTTTTTGCAGATAATAATACGGAAGTTCTTATGG-3';
EGY2, 5'-TAATTGGGACGTTAACGCAGATAATAATACGGAAGTTCTTATGGAGG-3';
mut1, 5'-CTTACTGATTTCTGCAGCACGATAAAATCG-3';
mut2, 5'-CTCACGAGGTACCGTGTTATTTCATTTTCAG-3';
mut3, 5'-CTCACGAGGTACCGTGTTATTTCATTTTCAG-3';
mut4, 5'-CTCACGAGGTAAGCTGTTATT TCATTTTCAG-3';
mut5, 5'-CGAGGTACGGTGCTATTTCATTTTCAGAGGC-3';
mut6, 5'-CTCACGAGGAACGGTGTTATTTCATTTTCAG-3';
mut7, 5'-CTCACGAGGAACGGTGCTATTTCATTTTCAG-3'.
All mutation constructs were sequenced and subsequently subcloned into the
apropriate vectors for integration into the
ura4
locus.
The human cDNA was isolated using a UNI-Zap XR HeLa S3 cDNA library (Stratagene), the sense primer comprising part
of the T-loop of the human sequence HsPK 27 (5'-CTGCTAGGATCCTCGGCTTCACATGTTGCGGA-3'; EMBL accession no. Z25435) and the T7 primer
(5'-GTAATACGACTCACTATAGGGC-3'). A 1.7 kb PCR fragment produced in this reaction was
isolated from the agarose gel and radiolabelled with [[alpha]-
32
P]dCTP as described previously (
2
). This labelled fragment was used to screen the HeLa S3 cDNA. The filter
hybridization conditions used were according to the protocol from Stratagene.
The inserts of the hybridizing plaques were subcloned into pBluescript SK(-) by
in vivo
excision following the protocol from Stratagene. The mouse
prp4
cDNA was isolated using a [lambda]gt10 library from embryonic stem cells (Clontech). A 600 bp
Mun
I-
Eco
RV fragment comprising part of the ORF of the human
PRP4
cDNA was radiolabelled and hybridized to the filters. Recombinant phage DNA was
isolated from positive plaques. Since the inserts contained an
Eco
RI site, the DNA was digested partially with this enzyme and subsequently cloned
into pUC18.
The subcloned inserts from the human and mouse libraries were sequenced with [
35
S]ATP using the universal and the reverse primer. Based on the first sequences
we synthesized oligonucleotides which were used as primers in further sequence
reactions. Databank accession numbers for the human and
mouse sequences are U488736 and U488737 respectively.
cDNA of
S.pombe
prp4
was isolated using the primers 5'-GAGCTCGGATCCGACGATAGATTTGCAGAAGAT-3' and 5'-ATATGGATCCATGAACCCGCAGTTTATT-3' and a
S.pombe
cDNA library. The 1.4 kb PCR product was inserted into the
Bam
HI site of a pREP1 vector in which the
Sal
I site in the multiple cloning site was destroyed by filling in the protruding
ends (
27
). For the deletion construct
prp4
[Delta]
XI
the second primer was 5'-TCTAGAGGATCCTCAGGCGGTACGTTTCTCTG-3'. The same approach was used to insert a complete mouse cDNA into pREP1.
The primers used were 5'-ATATGGATCCATGAAAGTTGAGCAAGAGTCT-3' and 5'-CCGGGGATCCTTAAATTTTTTCCTGGATGAATGC-3'. The PCR reaction was
performed with one of the isolated mouse clones and revealed a 1.4 kb product. This fragment was cloned into the
Bam
HI site of pREP1. Both constructs were sequenced to confirm the sequence and the proper ORF. The first swap
leading to the construct Sp/Mm1 (Fig.
2
) was a simple exchange of fragments from the pREP1 constructs using the
Eco
RV restriction site which both cDNAs share at the same position in the ORF. For
the swap construct Sp/Mm2 (Fig.
4
) we introduced an
Eco
RI restriction site 614 bp downstream of the start codon ATG of the
S.pombe
cDNA, performing site-specific mutagenesis as described previously (
25
). This manipulation allowed us then to exchange fragments from the pREP1
constructs using this
Eco
RI site, since the mouse cDNA contains an
Eco
RI site at the same position in the ORF. Sequence analysis of the constructs
confirmed the proper ORF. The swap constructs Sp/Mm3 and Mm/Sp (Fig.
4
) were constructed in a two step PCR reaction, called the `Megaprimer' method,
developed by Sarkar and Sommer (
28
). In the first reaction each chosen N- and C-terminal fragment was independently amplified using primers which
create an overlap between the N- and C-terminal fragments of yeast and mouse. We used primers which had a
Mun
I site in this overlap. The
Mun
I site is only present in the mouse cDNA, not in the
S.pombe
cDNA. In the second PCR reaction we combined the N- and C-terminal PCR products and used a primer sequence of the N-terminal fragment containing a
Bam
HI site and a primer of the C-terminal fragment also equipped with a
Bam
HI site. This PCR reaction revealed products of 1.4 kb which were isolated and
cloned into the
Bam
HI site of pREP1. The primers used for the
S.pombe
N-terminal fragment were 5'-GAGCTCGGATCCGACGATAGATTTGCAGAAGATG-3' and 5'-TGCATCTGTCCAATTGTCCTGCATATCTG-3'; for the C-terminal
fragment 5'-ACCTC- AGAGACAATTGGGACGATATTGAAG-3' and 5'-ATATGGATCCATGAACCCGCAGTTTATT-3'. The primers used for
the mouse N-terminal fragment were 5'-ATATGGATCCATGAAAGTTGAGCAAGAGTCT-3' and 5'-AATATCGTCCCAATTGTCTCTGAGGTTGG-3'; for the C-terminal
fragment 5'-CCGGGGATC- CTTAAATTTTTTCCTGGATGAATGC-3'. The constructs were sequenced to confirm the
proper ORF.
A 1 kb
Bam
HI fragment of the
S.pombe
cDNA was ligated into plasmid pGEX2T (Pharmacia KB). The recombinant protein
contains at the N-terminus gluthatione S-transferase (GST) followed by the N-terminal sequence of Prp4 and two thirds of the kinase domain.
After transformation in
Escherichia coli
protein production was induced with 1 mM IPTG. Bacterial extract was prepared
as described by Krämer
et al.
(
29
). Most of the recombinant protein appeared in the inclusion bodies. This material was separated by SDS-PAGE and the 69 kDa recombinant GST-Prp4 protein was electroeluted. The electroeluted protein was used
as antigen. Antibodies were raised in rabbits by several injections with 150-250 [mu]g recombinant GST-Prp4 (Pogonos Rabbit Farm, PA). We purified the antibodies
using protein A-Sepharose.
For Western blot analysis proteins were separated on 7.5% SDS-polyacrylamide gels and transferred to nitrocellulose. For antibody
detection we used the ECL Western blotting analysis system from Amersham following the manufacturer's instructions.
To prepare native and denatured protein extract we exactly followed the
procedure described by Moreno
et al
. (
24
). For the preparation of native extract exponentially growing cells were used.
Samples of 2 * 10
8
cells were resuspended in 200 ml extraction buffer HB (
24
) and broken with glass beads by vortexing. For immunoprecipitation 400 [mu]l protein extract were used and incubated with anti-GST-Prp4 overnight at 4oC, then 25 [mu]l protein A-Sepharose were added and incubated for a
further 2 h at the same temperature. The precipitate was washed three times in
HE buffer (50 mM Tris, pH 8, 150 mM NaCl, 50 mM [beta]-glycerolphosphate, 50 mM NaF, 10 mM EDTA, 5 mM EGTA, 0.1 mM sodium vanadate, 1 mM DTT, 20 [mu]g/ml leupeptin, 40 [mu]g/ml aprotinin, 30 [mu]g/ml pepstatin, 50 [mu]g/ml Pefubloc SC) followed by three washes with
kinase buffer I (20 mM HEPES, pH 7.9, 50 mM KCl, 3 mM MgCl
2
, 5% glycerol).
The immunocomplex of Prp4 bound to anti-Prp4-protein A- Sepharose beads was used in the kinase assay. The kinase assay was
routinely performed in a 20 [mu]l volume containing kinase buffer II (20 mM HEPES, pH 9, 50 mM KCl, 1.5 mM MgCl
2
, 0.5 mM DDT), 5 [mu]Ci [[gamma]-
32
P]ATP, 10 mM ATP and 5 [mu]l immunoprecipitate. As possible substrates we added, in general, 36 pmol
bacterially produced ASF/SF2, ASF/SF2[Delta]RS (provided by R.Lührmann, Marburg;
21
,
22
), histone H1 (Boehringer Mannheim), myelin basic protein (MBP; Sigma) or [beta]-casein (Sigma). This is 1 [mu]g for protein ASF/SF2. The samples were incubated at 37oC for 30 min. The reaction was stopped by adding 20 [mu]l 2* SDS sample buffer and boiling for 2 min.
Samples were run on a 12.5% SDS-PAGE gel, transferred to nitrocellulose and exposed to X-ray film.
To learn about the function of the two domains of Prp4 we undertook a mutational analysis and tested the effect of the mutations
in vivo
by measuring the capability to complement the ts
prp4-73
allele. The mutation causing the temperature sensitivity of Prp4 is in kinase subdomain IV, changing the cysteine residue at position 235
to a tyrosine (Fig.
3
). Haploid cells containing
prp4-73
grow normally at 26oC, but do not grow at all at 36oC (
1
). The mutation constructs were integrated into the genome via homologous
recombination using the
ura4
+
gene to target the
ura4-294
allele in a strain containing the
prp4-73
allele. This manipulation leads to two
prp4
alleles on chromosom III. As controls we constructed strains containing the
prp4-73
allele and in the
ura4
locus either the wild-type or the
prp4
-
73
allele. The wild-type gene complements
prp4-73
at 36oC, whereas the strain containing two
prp4-73
alleles shows no growth at 36oC (Fig.
1
, WT and TS). The effects of the mutations were checked by spotting cells, which
were grown to mid log phase at 26oC, on plates and monitoring growth at 36oC.
We made mutations in the N-terminus using a cDNA of
prp4
. The constructs were inserted into a vector which places them under the control
of the
nmt1
promoter. The
nmt1
promoter allows down-regulation of expression in medium containing thiamine and leads to strong
expression in thiamine-free medium (
27
).
The N-terminus of Prp4 of
S.pombe
consists of 157 amino acids. At position 15 in Prp4 we detected a putative nuclear localization signal (NLS)
of the SV40 type, consisting of the five basic amino acid residues RRKRR (Fig.
1
A). Five basic amino acids in the N-terminus of polymerase [alpha] of
S.pombe
have been shown to be solely responsible for moving the protein into the
nucleus (
30
). We deleted 11 amino acids including the putative NLS and also made a more
extensive deletion of 30 amino acids (Fig.
1
, [Delta]NLS and [Delta][Delta]NLS). Neither the deletion of 11 amino acids nor the second
deletion had an effect on growth at 36oC. Both deletion constructs complemented the
prp4-73
allele under repressed (Fig.
1
A) and derepressed conditions (results not shown).
In positions 90-95 and 112-117 we find two elements, SDSPSI and SPSPSV, which we call serine
elements (Fig.
1
A, SX1 and SX2). We replaced the serines with other amino acids as shown in
Figure
1
A. Changing the serines in one of the elements had no effect. These mutations
still complemented the
prp4-73
allele. However, the mutation construct in which the serines of both elements
had been replaced by other residues did not complement the
prp4-73
allele (Fig.
1
A, SX1+SX2), indicating that these two motifs together play a crucial role in the proper functioning of Prp4.
The amino acid sequence DNWDDIEGYYKV starting at position 138 is highly
conserved in sequence and position in Prp4sp of
S.pombe
and a mammalian protein kinase which we have isolated (see below, Fig.
3
). Therefore, we changed this sequence in Prp4sp of
S.pombe
to VNSVVFADNNTE (Fig.
1
, EGY1). In a second construct DIEGYYKV was changed to VNADNTE (Fig.
1
, EGY2). Neither mutation construct complemented the
prp4-73
allele (Fig.
1
A). These results suggest that this motif including EGYYK is essential for
function.
When we overexpress the mutation constructs at 26oC by growing the cells in medium without thiamine, the strains containing the mutations
which do not complement the
prp4-73
allele (SX1+SX2, EGY1 and EGY2) exhibit significantly elongated cells after 20
h (Fig.
1
B). The DAPI staining pattern of these cells suggests that overexpression of the
mutated Prp4 proteins impairs mitosis. Unequal and puffy staining patterns of
DNA in one cell can be observed, particularly in panels (c) and (d) of Figure
1
B.
The kinase catalytic domain of Prp4 consists of 321 amino acids and shows the 12
subdomains defined by the signature sequences indicative of the
serine/threonine protein kinase family (Fig.
3
;
3
). We searched the databases using the sequence between the signatures DFG and
APE of Prp4 as query sequence (Fig.
2
A). This region of a protein kinase is called the T-loop and serves in some kinases as a switch for up- and down-regulating activity (
31
). The search revealed a partial human sequence that was isolated in a screen
for Cdc2-like kinases (
32
). Between the signature sequence DFG and APE the human sequence HsPK 27 and
Prp4sp share 79% identical amino acid residues (Fig.
2
A). We also found in this search the T-loops of the MAP kinases Erk1 and Erk2 and of the stress-activated kinases (SAPKs). The T-loops of these kinases contain the sequences TEY and TPY (Fig.
2
A). Activation of these kinases requires both threonine and tyrosine
phosphorylation in these sites (
31
,
33
). Interestingly, these phosphorylation sites resemble the sequence found in the
Prp4 and HsPK T-loops (Fig.
2
A).
To test whether the T-loop sequence plays a regulatory role in the function of Prp4, we replaced
threonine with alanine and tyrosine with phenylalanine. In one construct we
made a double mutation replacing threonine and tyrosine. We also changed other
amino acid residues in the T-loop region (Fig.
2
B). As a control we mutated the signature sequence APE to AAE (Fig.
2
B, mut1). The constructs were integrated into the
ura4
locus as described before, however, in this series of experiments the
prp4
gene was under the control of its own promoter. As expected, the mutation mut1
in the signature sequence APE does not complement the
prp4-73
allele (Fig.
2
B). However, all the other mutations rescued the ts phenotype (Fig.
2
B) and none, including the double mutation mut7, had any influence on growth.
Thus, under these growth conditions the T-loop appears not to play a regulatory role for Prp4.
We produced PCR fragments using a HeLa cDNA library and a sequence of the human
T-loop HsPK 27 and the T7 sequence in the [lambda] phage arm as primers (Fig.
2
A). The PCR products were used as a probe to screen the HeLa library. Out of
this screen we isolated and sequenced several cDNAs. A defined fragment of the
ORF of the human cDNA was then used to screen the mouse cDNA library. With this
approach we isolated cDNA from human and mouse encoding a protein kinase which
shares 98% identical amino acids. Overall the mammalian sequence shares 44%
identical amino acids with the yeast sequence. Throughout the kinase domains,
however, we find 53% identical amino acids. This high conservation changes apruptly in the N-terminal domains. The N-termini share <20% identical amino acids. However, the EGY motif mentioned above
appears to be conserved in the mammalian and yeast N-termini (Fig.
3
). We also detected in the mammalian N-terminus a serine element, SRSPSP, which resembles the serine elements
found in Prp4sp (Fig.
3
).
To test whether the mammalian gene complements the ts mutation we inserted the
mouse cDNA behind the
nmt1
promoter into the expression vector pREP1 and transformed it into a strain
containing the
prp4-73
allele. The pREP1 vector containing the complete
S.pombe prp4
cDNA complements the ts mutation under thiamine repressing conditions as well as under derepressed conditions (Fig.
4
, Prp4sp). The vector containing the complete mouse cDNA was not complementary (Fig.
4
, Prp4m). Therefore, we designed swap constructs switching mouse with yeast sequences as shown in
Figure
4
. The construct Sp/Mm1, containing 55% of the mammalian kinase domain,
complements
prp4-73
under repressed and derepressed conditions, whereas the construct Sp/Mm2,
spanning 75% of the mammalian kinase domain, complements only under derepressed
conditions (Fig.
4
). Neither construct Sp/Mm3 nor Mm/Sp complemented the
prp4-73
allele. As a negative control we used a
prp4
cDNA of
S.pombe
which has a 13 amino acid deletion in kinase subdomain XI. This construct does
not rescue the ts mutation (Fig.
4
, Prp4sp[Delta]XI). In addition, a
prp4
cDNA of
S.pombe
in which the N-terminal region was deleted did not rescue the ts mutation (results not
shown).
The swap construct Sp/Mm2 complemented only when the recombinant protein was
highly expressed (Fig.
4
). This indicates some differences in kinase subdomains I-II. The simplest explanation for this result would be that the fusion
products of Sp/Mm2 and Sp/Mm3 induce some structural changes in that region
which causes the decrease and loss of activity
in vivo
respectively. The results also indicate that differences in the primary
sequence of the N-termini of the proteins might account for the failure of the mouse N-terminus to complement.
As yet we have no
in vitro
data on the kinase activity of Prp4sp, since we do not know a physiological
substrate. All kinase activities in mammalian cells which have been implicated
as involved directly or indirectly in pre-mRNA splicing phosphorylate SR proteins
in vitro
. It has been shown that these kinases and kinase activities preferably
phosphorylate the RS domains of SR proteins (
4
,
9
,
14
,
34
). Therefore, we decided to test the mammalian SR protein ASF/SF2 and a mutated version of ASF/SF2 as
in vitro
substrates. The mutated protein ASF/SF2[Delta]RS lacks the RS dipeptides in the RS domain at the C-terminus (
21
,
22
).
A polyclonal antiserum raised against a glutathione S-transferase- Prp4sp fusion (GST-Prp4, see Materials and Methods) was used to examine the
proteins expressed with pREP1. Protein extracts of
S.pombe
cells containing the Prp4sp cDNA, the mouse cDNA and the chimeric constructs
(shown in Fig.
4
) were isolated and probed in a Western analysis with polyclonal antibodies. The
antibodies recognized the product of the Prp4sp cDNA, but failed to detect a
product of the mouse cDNA (Fig.
5
A, lanes 1 and 2); recombinant proteins, however, that contained the N-terminus of the
S.pombe
protein were recognized (Fig.
5
A, lane 4). This suggests that the antibody population consists mostly of antibodies against epitopes in the N-terminus of the
S.pombe
protein. Based on this result and the fact that
prp4sp
cDNA and the chimeric yeast/mouse gene (Sp/Mm2, Fig.
4
) complemented the
prp4-73
allele, we immunoprecipitated Prp4sp and the Sp/Mm2 proteins with GST-Prp4 antibodies and performed
in vitro
kinase assays using [[gamma]-
32
P]ATP. Prp4 and the recombinant yeast/mouse protein phosphorylated human ASF/SF2 protein
in vitro
(Fig.
5
B, lanes 2 and 6, arrows). ASF/SF2[Delta]RS protein was hardly phosphorylated in this assay (Fig.
5
B, lanes 5 and 7). These results suggest that
in vitro
the kinase activity phosphorylates ASF/SF2 protein at the RS domain. We
conclude that this
in vitro
kinase activity is due to Prp4 and the recombinant yeast/mouse protein, since
the immunoprecipitate containing the Prp4[Delta]XI deletion protein did not phosphorylate ASF/SF2 (Fig.
5
B, lane 1). This is consistent with the observation that
in vivo
the deletion construct Prp4sp[Delta]XI does not complement the ts mutation (Fig.
4
). Furthermore, in the autoradiographs we see additional distinct bands in the
range 30-45 kDa (Fig.
5
B circles, all lanes except lane 1). This suggests that proteins which were
phosphorylated by Prp4 co-precipitated.
This is the first report of a protein kinase of the fission yeast
S.pombe
involved in pre-mRNA splicing showing
in vitro
kinase activity. Prp4sp and the recombinant yeast/mouse kinase are capable of
phosphorylating the the RS domain of the mammalian splicing factor ASF/SF2.
Splicing factor ASF/SF2 is a member of the SR family of phosphoproteins which
appears to be highly conserved throughout metazoan organisms (
11
,
35
). SR proteins, including ASF/SF2, which have been studied in a mammalian
in vitro
pre-mRNA splicing system play a role in constitutive and alternative splicing
(
6
,
8
,
11
,
36
,
37
). In particular, ASF/SF2 has been shown to be an important component in
determining 5'-splice sites of alternatively spliced genes (
21
,
22
). Typical SR proteins involved in pre-mRNA splicing have not been reported from the yeasts
Saccharomyces cerevisiae
and
S.pombe
. However, very recently we isolated a gene
srp1
of
S.pombe
which encodes a 30 kDa protein containing a RNA recognition motif (RRM) and a
domain which resembles metazoan RS domains (T.Groß, C.Mierke and N.F.Käufer, unpublished results, EMBL accession no. U66833). Whether Srp1
is a potential substrate of Prp4 is currently under investigation.
The complementation studies with the swap constructs indicate a difference in
kinase subdomains I and II of the yeast and mammalian proteins. It is
conceivable that for optimal
in vivo
activity of the protein kinase other factors, including the substrate(s), might
need to interact with the protein kinase through the N-terminus and the early kinase domain. Taking the differences in the N-terminus of these proteins into consideration, it is, therefore,
possible that
in vivo
the fission yeast factors cannot interact properly with the recombinant product of
Sp/Mm3
(Fig.
4
).
We know from experiments with fusions of Prp4 and green fluorescent protein
(GFP) that Prp4 accumulates in the nucleus (results not shown). Transport into
the nucleus appears not to be solely dependent on commonly known NLS signals
(Fig.
1
;
38
). However, the information for targetting the nucleus appears to be in the N-terminus, since the chimeric yeast/mouse construct Sp/Mm2, which contains
the
S.pombe
N-terminus and the mouse kinase domain, is detected in the nucleus, whereas
a fusion protein of GFP with the kinase domain of Prp4sp is detected in the
cytoplasm (results not shown). The mouse Prp4-GFP fusion protein does not reach the nucleus when expressed in
S.pombe
(results not shown). The N-terminus of the mouse protein contains a putative NLS which resembles a
bipartite NLS of the nucleoplasmin type, but it does not fit the consensus well (Fig.
3
;
38
).
It has been proposed that Clk/Sty and SRPKI regulate ASF/SF2 function
in vivo
by phosphorylating the protein in the RS domain to induce release from the
speckles (
9
). The repeats of RS/SR dipeptides in RS domains have been implicated in protein-protein interaction (
39
,
40
,
41
). The N-terminus of the mammalian protein kinase Clk/Sty does not contain a
typical RS domain, but scattered throughout it contains 10 SR/RS dipeptides and
one RSRS motif. The N-terminus of Clk/Sty has been shown to interact with SR proteins (
9
). With this in mind we have substituted the serines in the serine elements of
Prp4sp (Fig.
1
). When the serines are replaced in both elements, the mutated protein does not
compelement the
prp4-73
allele. This indicates that the two serine elements are part of the protein
architecture which might be involved in interaction with other components.
The results of the mutational analysis of the N-terminus of Prp4sp are consistent with the idea that the N-terminus is involved in interaction with other components. These
components might be substrates, but interaction partners with other functions,
such as inhibitor or docking functions, are also conceivable. We still do not
know whether Prp4 is associated with spliceosomes or, perhaps, as demonstrated
for the mammalian kinases Clk/Sty and SRPKI, located in subnuclear structures such as speckles (
4
,
9
).
The highly elongated phenotype of cells caused by overexpression of mutations in the N-terminus of Prp4sp warrants some comment. Overproduced mutated proteins do
not impair cell growth. The cells appear to be disrupted in mitosis. It has
been shown that overexpression of the kinases Clk/Sty and SRPKI in mammalian
cells causes disruption and rearrangement of speckles containing splicing
components (
4
,
9
). Speckles are subnuclear structures embedded in the nuclear scaffold (
10
,
42
). It is conceivable that overproduced mutated Prp4 protein effects nuclear
segregation by disrupting orderly rearrangement of the nuclear scaffold. This notion, however, needs further investigation. In any case, a functional connection of pre-mRNA splicing and cell cycle events has been observed in
S.cerevisiae
and
S.pombe
(
4
3
,
4
4
). The fission yeast gene
cdc28
encodes an RNA-dependent ATPase/helicase and is allelic with
prp8.
The temperature-sensitive alleles
cdc28-P8
and
prp8-1
accumulate pre-mRNA at the restrictive temperature and show a cell cycle phenotype (
4
4
).
Interestingly, a homologue of the mammalian SRPKI kinase in
S.pombe
appears to be Dsk1 (
4
). The
dsk1
gene has been detected as a multicopy suppressor of
dis1
mutants. These mutants are blocked in mitosis due to non-disjunction of sister chromatids (
34
,
4
2
). Whether Dsk1 kinase in
S.pombe
is involved in splicing is not known. We first detected the role of Prp4
protein kinase in pre-mRNA splicing in fission yeast with a genetic approach, isolating
prp
mutants which accumulate pre-mRNA at the restrictive temperature (
1
,
2
). The data presented in this paper indicate that the function of Prp4 might be
pleiotropic. Further studies in the fission yeast and mammalian systems will
help to elucidate the biological role of Prp4 protein kinase.
We are grateful to Dr R.Lührmann (Marburg) for providing the proteins ASF/SF2 and ASF/SF2[Delta]RS. Dr Silke Fetzer (Marburg) we thank for her helpful advice with the kinase assays, Antje Nickel for excellent technical assistance, Beate Schnell for sequencing
the mouse cDNA, Christoph Peter for preparation of the antibody and Claudia
Mierke for her help with the microscopy. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to N.F.K.
*To whom correspondence should be addressed. Tel: +49 531 391 5774; Fax: +49 531
391 5765; Email: n.kaeufer@tu-bs.de
+
Present address: CuraGen Corporation, 322 East Main Street, Branford, CT 06405,
USA
[sect]
U488736, U488737, U66833 and L10739
REFERENCES
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