Wellcome/CRC Institute, Tennis Court Road, Cambridge, CB2 1QR, UK
*
Author for correspondence (e-mail:
j.pines{at}welc.cam.ac.uk
)
Accepted April 23, 2001
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SUMMARY |
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Key words: Phosphorylation, RS domain, Nuclear speckles, MPM-2, RNA polymerase II
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INTRODUCTION |
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The Crk family comprises a diverse set of proteins that vary from 42 to 55%
identity to cdc2 in the kinase domain (Meyerson et al.,
1992). Individual Crk family
members are often referred to by the one letter code of the amino acid
sequence in the region corresponding to the `PSTAIRE'
-helix of cdc2
that interacts with the cyclin.
The PCTAIRE proteins are most closely related to cdc2 (51-55% identity)
(Meyerson et al., 1992; Okuda
et al., 1992
). There are three
human PCTAIRE genes, and the proteins are abundant in nerve cells (Gao et al.,
1996
; Sladeczek et al.,
1997
). There are at least two
PCTAIRE genes in the mouse, of which PCTAIRE-1 is abundant in differentiated
spermatids and in adult neurones (Besset et al.,
1999
). A closely related
protein kinase, PFTAIRE, is found in postnatal and adult nerve cells (Lazzaro
et al., 1997
), and has a
Drosophila homologue (Sauer et al.,
1996
). However, the functions
of both PCTAIRE and PFTAIRE kinases are unknown.
The PITSLRE kinase subfamily have been implicated in apoptosis (Beyaert et
al., 1997; Lahti et al.,
1995
). There are three
tandemly linked human PITSLRE genes (A, B and C) (Lahti et al.,
1994
) that give rise to eight
isoforms (40-130 kDa) through alternative splicing (Xiang et al.,
1994
), and internal initiation
in G2 phase (Cornelis et al.,
2000
). The 110 kDa isoform
localises to nuclear speckles and binds to the RNPS1 RNA binding protein
(Loyer et al., 1998
). The
PISSLRE kinase is a 39 kDa protein kinase that is most closely related to
PITSLRE (50% identity). It is subject to a complicated pattern of splicing,
but has not been correlated with apoptosis (Brambilla and Draetta,
1994
; Crawford et al.,
1999
; Grana et al.,
1994
).
Lastly, CHED (cholinesterase-related cell division controller) is a 418
amino acid serine/threonine kinase with 42% identity to human cdc2
(Lapidot-Lifson et al., 1992).
CHED has the sequence PITAIRE in the `PSTAIRE'
-helix and has been
suggested to be involved in haematopoesis. Mouse bone marrow cells treated
with antisense oligonucleotides directed against the CHED mRNA showed a
reduction in the number of mature, polynuclear megakaryocytes, and an increase
in the number of early, mononuclear cells (Lapidot-Lifson et al.,
1992
). CHED is also the
closest relative to a protein kinase identified in mosquitos that have been
subjected to stress. This kinase is not present in normal Aedes
aegypti mosquitos, but is induced after trauma or bacterial innoculation
(Chiou et al., 1998
).
In this paper we report the cloning and characterisation of a Cdc2-related kinase, CrkRS, which may represent a novel kinase involved in the regulation of transcription and splicing.
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MATERIALS AND METHODS |
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Northern blotting
Northern blots were performed on multiple tissue mRNA blots (Clontech).
Blots were probed using two different CrkRS cDNA [-32P]dCTP
random-prime labelled probes. One probe was from subdomains I-V of the kinase
domain of CrkRS and the other from the unique 3' untranslated region
(UTR) of CrkRS.
Southern blotting and genomic mapping
Filters were hybridised in Church buffer with [-32P]dCTP
labelled probes. For the human genomic DNA blot, DNA was extracted from MRC5
diploid lung fibroblasts and 10 µg of DNA was digested with 100 units of
BamHI, EcoRI, HindIII or PstI for a total
of 19 hours at 37°C. DNA fragments were transferred to nylon membrane
under alkaline conditions. Probes were generated from either the kinase domain
(subdomains I to V) or the 3' UTR. Filters were probed at 42°C for
20 hours, washed and exposed to preflashed film with an intensifying screen
for 6-10 days at -70°C. The 3' UTR probe recognised one band in
BamHI-, HindIII- and PstI-digested DNAs, and one
strong band and one faint band in EcoRI-digested DNA. The kinase
domain probe recognised one band in the BamHI and PstI
digests, and two bands in the other digests. CrkRS was mapped onto the mouse
genome by restriction fragment length polymorphism analysis between
interspecific crosses of Mus spretus and C57BL/6J mice through the
Jackson Laboratory DNA mapping resource (Bar Harbor, ME) (Rowe et al.,
1994
). Filters were hybridised
to the 3' coding region of CrkRS (nucleotides 3662-4260), which is
unique to CrkRS. CrkRS co-segregated with the mouse HoxB cluster, and
therefore mapped to the distal end of mouse chromosome 11 (56.0 cM) (Mouse
Genome Database (MGD), Mouse Genome Informatics, The Jackson Laboratory, Bar
Harbor, ME.)
Expression and purification of GST-CrkRS C-terminal fusion
protein
A HindIII fragment of the CrkRS C-terminal coding region (amino
acids 1218-1490) was subcloned into the HindIII and NotI
sites of pGEX-5X-1 (Pharmacia, Sweden) and expressed in Escherichia
coli C41(DE3). Overnight cultures (grown in LB, 100 µg/ml ampicillin)
were diluted 1:10 in prewarmed media plus 100 µg/ml ampicillin and 2%
glucose, and grown with shaking at 37°C until mid-log phase
(OD600nm 0.3-0.4). IPTG (final concentration 0.1-0.5 mM) and
ethanol (final concentration, 3%) were added and cells were induced for 4
hours at either 30°C or 37°C. Cells were lysed and the fusion protein
was purified on glutathione sepharose 4B according to the manufacturer's
instructions (Pharmacia, Sweden).
Affinity purification of anti-CrkRS polyclonal antibodies
Rabbit polyclonal antisera were raised against a GST-CrkRS C-terminal
fusion protein (GST-CCT). GST and a GST-CCT protein columns were constructed
by crosslinking proteins to glutathione agarose beads with dimethyl
pimelimidate-HCL (DMP; Sigma). Raw serum from inoculated rabbits was passed
through the GST column to remove anti-GST antibodies and passed down a GST-CCT
protein column. Anti-CrkRS antibodies were eluted from the column with glycine
pH 2.5 and immediately neutralised with Tris pH 7.4.
Immunoblotting
Cell lysates were resolved by SDS-PAGE and transferred to Immobilon-P
membrane (Millipore) and incubated with primary antibodies in TBS with 0.2%
Tween-20. The working dilutions for the MPM-2 monoclonal antibodies (Upstate
Biotechnology), affinity-purified anti-CrkRS antibodies, goat horseradish
peroxidase anti-rabbit and anti-mouse secondary antibodies (ICN/Cappel) were
1:1000, 1:3000, 1:5000 and 1:3000, respectively. Immunoreactive proteins were
visualised using enhanced chemiluminescence (Amersham).
In vitro coupled transcription/translation
CrkRS cDNA was subcloned into the NheI and EcoRI sites of
pCI-neo (Promega) and transcribed and translated in vitro with the TNT® T7
Coupled Transcription/Translation System (Promega).
Cell culture and synchronisation
HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 5%
newborn calf serum, 5% fetal calf serum plus fungizone, penicillin,
streptomycin and glutamine at 37°C in 10% CO2. Cells were
synchronised by sequential thymidine (Sigma) and aphidicolin (Sigma)
treatment. Mitotic cells were obtained by using aphidicolin/nocodazole
(Aldrich) treatment. The extent of synchronisation was determined by DNA flow
cytometry analysis on a FACSort (Becton Dickinson).
Nuclear preparation and immunoprecipitation
HeLa cells were grown to 70-80% confluency, harvested and washed in PBS,
and lysed in hypotonic buffer (20 mM Hepes (pH 7.4), 20 mM NaCl, 5 mM
ß-mercaptoethanol, 5 mM EDTA, 50 mM NaF, 1 mM
Na3VO4, protease inhibitor cocktail (BCL)) with a loose
fitting dounce homogeniser. The lysate was centrifuged at 1000 rpm for 7
minutes, and the pellet extracted with nuclear extraction buffer (NEB) (50 mM
Tris-HCL (pH7.4), 150 mM NaCl, 50 mM NaF, 2 mM Na3VO4,
40 mM ß-glycerophosphate, 1 µM Microcystin LR (Calbiochem), 1-10 mM
ATP--S (Calbiochem), 5 mM EDTA (or EGTA), 0.1-0.5% sodium deoxycholate,
0.25-1%Tween-20, 5 mM ß-mercaptoethanol and protease inhibitor cocktail
(BCL, UK)] for 30-60 minutes at 4°C. The nuclear extract was centrifuged
at 32,000 g (20 minutes, 4°C) to separate the insoluble
fraction, and the supernatant centrifuged at 285,000 g (1
hour, 4°C). The soluble nuclear supernatant was pre-cleared with protein G
or protein A beads (Pharmacia) for 1 hour. 0.1-0.5% Triton X-100, 8-10 µl
of affinity purified anti-CrkRS antibodies and 25 µl of protein G or
protein A were used per immunoprecipitation. After immunoprecipitating for a
total of 2 hours at 4°C, the lysate was removed and the beads were washed
four times with HSNEB (0.3-1 M NaCl+NEB) for 15 minutes. Anti-CrkRS
immunocomplexes were resolved on either 7.5% or 12.5% SDS-PAGE and
immunoblotted as above. Control rabbit IgG (Jackson ImmunoResearch Labs) and
pre-cleared affinity purified anti-CrkRS were used as controls.
In vitro kinase assay
Anti-CrkRS IPs were washed once in kinase buffer (50 mM Tris-HCL (pH 7.4),
100 mM NaCl, 20 mM ß-glycerophosphate, 10 mM MgCl2, 0.25%
Tween-20, 0.1% Triton X-100, 50 µM cold ATP, 1 mM DTT and protease
inhibitor cocktail (BCL)) and resuspended in 15 µl of kinase buffer with
3.0 µCi of [-33P]ATP for 30 minutes at 30°C. Samples
were resolved on SDS-PAGE and quantified on a phosphoimager (Fuji) using
MacBAS V2.5 and Image Reader V1.4E software. Exogenous substrates (0.4-1 µg
per reaction) used were ASF (a kind gift of Angus Lamond, Dundee University),
casein (Sigma), histone HI (BCL), myelin basic protein (MBP; Upstate
Biotechnology) and yeast GST C-terminal domain of RNA polymerase II (GST-CTD;
a kind gift of Jeff Corden, Johns Hopkins Medical School).
protein phosphatase reactions
200 U of protein phosphatase (Biolabs) was used per 50 µl
reaction in (50 mM Tris-HCl (pH 7.5), 0.1 mM Na2EDTA, 5 mM DTT,
0.01% Brij 35, 0.5% Tween-20, 0.1% Triton X-100 and 2 mM MnCl2). To
inhibit the phosphatase, 50 mM NaF, 10 mM Na3 VO4, and
50 mm EDTA was used as recommended by the manufacturer. The reaction mix was
incubated for 30 minutes at 30°C and stopped with SDS- sample buffer.
Immunofluorescence microscopy
Cells were fixed and permeabilised with 3% paraformaldehyde/0.5% Triton
X-100. Affinity purified anti-CRKRS, MPM-2 and anti-SC35 antibodies were used
respectively at 1:400, 1:400 and 1:2. FITC-conjugated goat anti-rabbit IgG,
and Texas red-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Labs)
were used at 1:200. Images were collected on a Nikon Optiphot microscope
(Nikon) equipped with the MRC 1024 lasersharp confocal imaging system (Bio-Rad
Laboratories). In the -amanitin experiment, cells were incubated with
50 µg ml-1 of
-amanitin (Sigma) for 8 hours (Huang et
al., 1994) prior to fixation.
Microinjection and YFP imaging
Cells were grown to 70-80% confluence on metasilicate-coated coverslips,
transferred to a 0.15 mm T dish (Bioptechs) and incubated in
CO2-independent medium without phenol red at 37°C. Cells were
microinjected with yellow fluorescent protein (YFP)-tagged DNA constructs (0.1
mg/ml) using a semi-automatic microinjector (Eppendorf) on a Leica DMIRBE
inverted microscope (Leica). Cells were fixed for 4-6 hours after
microinjection. YFP was visualised by confocal laser scanning microscopy with
a broad band FITC-filter set.
Expression and partial purification of CrkRS from insect cells
CrkRS tagged at the N-terminus with (His)6 was subcloned into
the transfer vector pVL1393 at the XbaI and NotI sites.
Recombinant baculoviruses were generated using BaculogoldTM (Pharmingen).
800 ml of exponential growing Sf9 (Spodoptera frugiperda) cells (cell density:
1-1.5x106) were infected with CRKRS recombinant baculovirus
at a multiplicity of infection of five. Infected cells were harvested 40
hours post infection. Unfortunately, most CRKRS was associated with the
insoluble fraction.
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RESULTS |
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A prominent feature of CrkRS is an arginine/serine rich (RS) domain (Fig.
1A,C,
originally found in pre-mRNA splicing factors that are important in
spliceosome assembly and in the regulation of alternative splicing (reviewed
by Valcarcel and Green, 1996).
In the first 400 amino acids of CrkRS there are 21 RS motifs, and only one
other RS motif in the remaining 1000 amino acids.
After the RS domain there is an acidic patch between residues 74 and 106
(38% glutamic acid or aspartic acid). Overall the protein has a pI of 7.4.
Between the charged N-terminus and the catalytic domain are a number of
proline/leucine rich repeats. The C-terminus is also rich in proline residues,
including a run of 9 consecutive prolines. These N- and C-terminal regions
contain the consensus binding sites for 15 class I and 3 class II SH3 binding
sites (Alexandropoulos et al.,
1995; Lim et al.,
1994
; Pawson and Schlessinger,
1993
; Weng et al.,
1995
)
(Fig. 1C). Of the class I sites
there are 14 potential c-Abl SH3 binding sites, and one c-Src SH3 binding
site. Furthermore, six of these have the sequence PPLP, which also acts as a
ligand for the WW domain (Bedford et al.,
1997
).
The high proportion of proline and acidic residues in CrkRS indicated that
the protein may contain `PEST' regions that are correlated with protein
degradation (Rogers et al.,
1986). The PESTFIND program
identified seven potential PEST sequences in CrkRS (triangular symbols in
Fig. 1C).
CrkRS is conserved through evolution
A BLAST search of the NCBI database revealed that the closest relative to
CrkRS is the CHED protein kinase (Lapidot-Lifson et al.,
1992). The two proteins are
89% identical over 421 amino acids, including the kinase domain, but are
completely unrelated at both the amino acid and nucleotide level outside this
sequence (Fig. 1B). A BLAST
search of the human genome revealed that the two proteins are encoded by
separate genes; CHED is encoded by a gene on chromosome 7, whereas the CrkRS
locus is on chromosome 17 (see below).
The next closest relative to CrkRS is the 1157 amino acid gene product of the CG7597 gene in Drosophila. This gene was sequenced as part of the Drosophila genome sequencing project, and maps to 78D7-E1 but there are no recorded alleles at this locus. This protein kinase is very likely to be the homologue of CrkRS because the protein kinase domains are 77% identical (Fig. 1B). In addition, the sequences C-terminal to the protein kinase domain are 48% identical (whereas CHED and CG7597 are only 27% identical in this region). The Drosophila protein also resembles CrkRS in that it has an RS domain at the N-terminus followed by a proline-rich region (Fig. 1C). Overall, CrkRS and the Drosophila protein are 41% identical.
There is also a 77 kDa protein kinase in the C. elegans database, hypothetical protein B0285, that is closely related to both CrkRS and CG7597. B0285 has been annotated as a putative CDK9 homolog, to which its protein kinase domain is 37% identical, but the sequence is much more similar to the CrkRS proteins. B0285 is 53% identical to human CrkRS and 54% identical to Drosophila CG7597; the `PSTAIRE' helices are identical, and the region just C-terminal to the protein kinase domain is highly conserved (Fig. 1B). Moreover, in the 285 amino acids that are N-terminal to the B0285 protein kinase domain, there is an RS domain followed by a segment that is rich in proline residues, including two putative class I SH3 ligands. Thus, the overall structure of B0285 is very like that of human CrkRS and Drosophila CG7597 (Fig. 1C).
CrkRS is ubiquitously expressed in tissues and maps to a single
genetic locuss
To determine the tissue distribution of CrkRS we screened a panel of RNAs
from specific human tissues with two different probes. One was to the 3'
untranslated region (UTR) of CrkRS, which should be specific to the CrkRS
mRNA. The second was to kinase domains I-V. Two primary transcripts were
recognised, 6.8 and 8.5 kb in length, and these were present in all
tissues (Fig. 2A). An
additional transcript of
5.5 kb was detected specifically in the testis.
The same pattern of transcripts was recognised by both probes under low or
high stringency conditions. These findings show that CrkRS expression is
ubiquitous, and suggest that the two primary transcripts arise by alternative
splicing. Moreover, it is likely that the CrkRS mRNAs contain long 5'
UTRs that may indicate regulation at the translational level.
|
To determine whether CrkRS was encoded by a single gene we probed human genomic DNA with either the kinase domain (subdomains I-V) or the 3' UTR probe. The 3' UTR probe recognised one band in BamHI-, HindIII- and PstI- digested DNAs, and one strong band and one faint band in EcoRI digested DNA (Fig. 2B). The kinase domain probe recognised one band in the BamHI and PstI digests, and two bands in the other digests, showing that CrkRS is encoded by a single gene. Analysing the draft human genome sequence revealed that CrkRS is encoded by a gene containing seven exons on chromosome 17q21 (contig number NT010838). This agrees well with the position of the mouse CrkRS homologue that we mapped to the distal end of mouse chromosome 11 (56.0 cM, data not shown).
Affinity-purified anti-CrkRS antibodies recognise a 180 kDa nuclear
protein that has associated protein kinase activity
To characterise the endogenous CrkRS product in human cells, we raised
polyclonal antibodies against a GST-CrkRS fusion protein expressed in E.
coli, and affinity-purified the antibodies as described in Materials and
Methods. We used the unique C-terminal fragment of CrkRS (amino acids
1198-1490) as the antigen. These antibodies recognised GST-CrkRS expressed in
bacteria, but not GST alone (Fig.
3A). Pre-incubating the antibodies with GST-CrkRS abolished the
signal on the immunoblot (Fig.
3A). The anti-CrkRS antibodies specifically recognised a protein
of apparent Mr 180,000 on immunoblots of cell lysates from
Sf9 cells infected with a baculovirus expressing CrkRS, but not those infected
with a control baculovirus (Fig.
3B). This was the same apparent Mr on SDS-PAGE
as the in vitro translation product of the CrkRS cDNA
(Fig. 3C). These results
demonstrate that the antibodies are specific for CrkRS.
|
We used the anti-CrkRS antibodies to characterise CrkRS. On immunoblots of fractionated HeLa cell lysates, anti-CrkRS antibodies recognised a single protein band of 180 kDa in the nuclear fraction (Fig. 4A). No proteins were recognised by anti-CrkRS antibodies that had been pre-incubated with bacterially expressed GST-CrkRS (Fig. 4A). Both the CrkRS expressed using the baculovirus system, and the protein recognised by anti-CrkRS antibodies in HeLa cell lysates, had an apparent Mr on SDS-PAGE of 180,000, showing that we had isolated the full-length open reading frame of CrkRS (Fig. 3B; Fig. 4A). This was approximately 30 kDa larger than the Mr predicted by the CrkRS ORF. It was likely that the aberrant migration on SDS-PAGE was caused by the large number of proline rich regions and phosphorylation (see below).
|
We then assayed whether we were able to detect kinase activity in anti-CrkRS immunoprecipitates. We found that anti-CrkRS immunoprecipitates, but not immunoprecipitates using control rabbit IgG, were able to phosphorylate CrkRS itself and an, as yet unidentified, 85 kDa protein. When exogenous substrates were assayed, anti-CrkRS immunoprecipitates were able to phosphorylate the SR-type splicing factor ASF, myelin basic protein and a GST-fusion protein of the RNA polymerase II CTD (Fig. 4B). Neither casein nor histone HI were phosphorylated to a significant extent (Fig. 4B). Unfortunately, CrkRS expressed in baculovirus-infected cells proved to be highly insoluble and could not be purified to homogeneity, therefore we were unable to measure specifically its associated kinase activity. This also meant that we were unable to exclude the possibility that other kinases present in the anti-CrkRS immunoprecipitates are responsible for some of the phosphorylation activity.
CrkRS is phosphorylated in a cell cycle-dependent manner
Given the number of potential PEST regions in CrkRS, we considered it
possible that the levels of CrkRS vary through the cell cycle. Therefore, we
immunoblotted whole cell lysates of HeLa cells synchronised at defined cell
cycle stages. This analysis revealed that CrkRS was present at approximately
constant levels throughout the cell cycle
(Fig. 4C), but we noticed that
CrkRS in the mitotic samples migrated more slowly on SDS-PAGE, which is often
the hallmark of phosphorylation. To show whether this modification was due to
phosphorylation, we treated anti-CrkRS immunoprecipitates with
protein phosphatase in the presence or absence of phosphatase inhibitors.
protein phosphatase was able to increase the mobility of mitotic
CrkRS on SDS-PAGE, but only in the absence of phosphatase inhibitors,
providing good evidence that CrkRS is phosphorylated in mitosis
(Fig. 4D). Moreover, CrkRS from
interphase cells migrated slightly faster on SDS-PAGE after phosphatase
treatment, suggesting that Crk is also phosphorylated in interphase
(Fig. 4D).
CrkRS is localised to SC35 speckles
To characterize CrkRS further, we performed an immunofluorescence analysis
with anti-CrkRS antibodies. We found that anti-CrkRS antibodies exclusively
stained the nucleus. Furthermore, CrkRS appeared to be localized in a discrete
pattern of nuclear speckles and excluded from nucleoli
(Fig. 5A). We observed a
similar pattern whether the cells were prepared by fixing with
paraformaldehyde or with methanol (data not shown). The staining was abolished
when the antibodies were pre-incubated with purified GST-CrkRS, and was not
observed with the control rabbit IgG (Fig.
5A).
|
The pattern of anti-CrkRS staining resembled the `nuclear speckles' that
correspond to perichromatin fibrils (PFs) and interchromatin granule clusters
(IGCs) observed by electron microscopy when cells are stained with antibodies
against components of the splicing machinery (Mintz et al.,
1999). Current evidence
indicates that IGCs may be storage sites for spliceosome proteins and that
sites of active transcription surround the IGCs, suggesting that spliceosomal
proteins may be mobilised from the IGCs to active transcription sites
(reviewed by Huang et al.,
1997
). To demonstrate whether
CrkRS colocalised with nuclear speckles we co-stained cells with antibodies
against CrkRS and with antibodies against a component of the spliceosome, the
SC35 protein (Fu and Maniatis,
1992a
; Fu and Maniatis,
1992b
) (kind gift of A.
Lamond, Dundee University). We found that the staining pattern of CrkRS
significantly overlapped with that of SC35, suggesting that CrkRS might be
associated with nuclear speckles (Fig.
5A). The speckled pattern of CrkRS staining was not altered by
treatment with either RNase, or DNase, or both
(Fig. 6A), in contrast to the
disruption of snRNP staining after RNase treatment as visualised by the Y12
antibody (Fig. 6A). Thus, like
SC35, CrkRS was not primarily localised to speckles through its association
with either chromatin or RNA. To explore further the possibility that CrkRS
was associated with nuclear speckles we treated cells with the RNA polymerase
II inhibitor
-amanitin. This was shown to cause a decrease in the
number and an increase in the size of nuclear speckles. We found that
-amanitin caused similar changes in the immunofluorescence pattern with
both SC35 and anti-CrkRS antibodies (Fig.
6B). CrkRS colocalized with nuclear speckles throughout
interphase. In mitosis, CrkRS dispersed throughout the cell and was excluded
from condensed chromosomes (Fig.
6C).
|
CrkRS is a constitutive MPM-2 antigen
The change in the pattern of anti-CrkRS staining in mitotic cells could
have been due to the change in its phosphorylation state. A number of proteins
phosphorylated in mitosis have been recognised by the MPM-2 monoclonal
antibody, a phospho-epitope specific antibody. Several MPM-2 antigens migrated
at a similar size to CrkRS, therefore we immunoblotted anti-CrkRS
immunoprecipitates with the MPM-2 antibody. This demonstrated that CrkRS was
recognised by the MPM-2 antibody in both mitosis and interphase
(Fig. 7A). The recognition of
CrkRS by MPM-2 was dependent on phosphorylation because staining was abolished
by treating samples with phosphatase
(Fig. 7A). On
immunofluorescence, MPM-2 was shown to stain interphase cells in a speckled
pattern and we found that this pattern of staining overlapped with anti-CrkRS
staining (Fig. 7B). Thus CrkRS
is a novel MPM-2 antigen in both interphase and mitotic cells.
|
The RS domain of CrkRS plays the major role in targeting CrkRS to the
nuclear speckles
To determine which region of CrkRS is required to target CrkRS to nuclear
speckles we generated a series of different deletion mutants of CrkRS. These
were tagged in-frame at the C-terminus with yellow fluorescent protein (YFP).
The full-length CrkRS-YFP protein colocalised with nuclear speckles.
N-terminal deletions showed that the first 414 amino acids of CrkRS, which
included the RS domain, were required for the protein to localise to nuclear
speckles (Fig. 8, compare i
with ii-v). All but one of the other deletion mutants were localised uniformly
within the nucleoplasm but were excluded from the nucleoli
(Fig. 8ii-iv). A mutant that
contained only the last 284 amino acids of CrkRS and lacked any consensus
bipartite nuclear localisation signals, remained in the cytoplasm
(Fig. 8v). To refine this
analysis we generated a fusion protein between YFP and the first 414 amino
acids of CrkRS. This fusion protein was predominantly localised to nuclear
speckles (Fig. 8vi). However,
all further deletions did not localise properly to nuclear speckles
(Fig. 8B, compare vi with
vii-ix). Furthermore, these deletion mutants accumulated in the nucleoli. Thus
the first 414 amino acids, including the RS domain, were absolutely required
for the proper targeting of CrkRS to nuclear speckles.
|
![]() |
DISCUSSION |
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The presence of Drosophila and C. elegans homologues with very similar protein kinase domains, and with N-terminal RS domains separated from the protein kinase domain by a proline rich region, indicates that this protein kinase has been conserved through animal evolution. (There does not appear to be a homologue in Saccharomyces cerevisiae.) CrkRS and the Drosophila CG7597 protein are 77% identical in the protein kinase domain and 41% identical overall. The C. elegans protein B0285 is 53% identical in the protein kinase domain and 45% identical overall to CrkRS. In the proline-rich region between the RS and the protein kinase domains, B0285 has two class I SH3 ligands (PxxxPxxP) that are also the most abundant type of SH3 ligand in human CrkRS.
The nearest neighbour to CrkRS in humans is the 46 kDa CHED protein kinase.
The kinase domains of these two proteins are very similar (89% identical) but
they are encoded by two separate genes. CHED was implicated as a protein
kinase involved in haematopoesis by antisense experiments on mouse bone marrow
cells. After antisense treatment there was a shift in the proportion of cells
from mature, polynuclear megakaryocytes to early, mononuclear cells
(Lapidot-Lifson et al., 1992).
The antisense oligonucleotide used in these experiments,
5'-ATTGACTGGGGAAAA has 3 mismatches to the analogous sequence in Crk
7,5'-AGCGACTGGGGGAAA. Therefore, it is unlikely that CrkRS would also
have been eliminated in these experiments, although this remains a formal
possibility.
The ATP binding site of CrkRS contains an adjacent threonine and tyrosine in analagous positions to those used to regulate cdc2 and CDK2. Therefore, CrkRS could be regulated by post-translational modification by dualspecificity threonine/tyrosine kinases and phosphatases in an analagous fashion to cdc2. However, CrkRS-associated kinase activity does not appear to vary through the cell cycle when assayed using myelin basic protein and GST-CTD (data not shown).
At present we do not know whether CrkRS needs to bind to a cyclin partner
to be activated (if it does, this would make it a cyclin-dependent kinase), or
whether, like other members of the Crk family such as PITSLRE, it does not
need an activatory cyclin. Thus far we have not found an association between
CrkRS and a number of candidate cyclins including cyclins K, T and M (data not
shown). However, CrkRS might interact with an as yet uncharacterised cyclin,
such as cyclins L, O or P identified in the draft human genome sequence
(Murray and Marks, 2001). The
tightly associated
85 kDa protein that coimmunoprecipitates with CrkRS is
an attractive candidate for an activatory partner.
The CrkRS sequence provides an almost embarrassing number of candidate
protein-protein interaction domains. There are three class II SH3 binding
sites, corresponding to the consensus PxxPxR/K (Alexandropoulos et al.,
1995), which are potential
interaction sites for proteins such as Crk. There are 14 potential ligands for
the c-Abl SH3 domain corresponding to the consensus PxxxxPxxP, and one to the
c-Src SH3 domain, RxxPxxP (Alexandropoulos et al.,
1995
). Because CrkRS is a
nuclear protein, an interaction with the nuclear c-Abl kinase (Van Etten et
al., 1989
) is more likely than
one with c-Src, which is cytoplasmic. The conservation of proline-rich regions
in the CrkRS proteins in all species suggests that this feature of the protein
kinase is important for its function.
CrkRS is a novel nuclear speckle kinase
Subcellular fractionation and immunofluorescence studies show that CrkRS is
a nuclear protein. The sequence of CrkRS has four regions that match the
consensus for a bipartite nuclear localisation signal (NLS) (Dingwall and
Laskey, 1991; Makkerh et al.,
1996
); three are in the
N-terminus and the fourth at the C-terminus. By deletion analysis we have
found that the first three NLS motifs are sufficient to localise CrkRS to the
nucleus. More specifically, a large proportion of CrkRS appears to be
localised to discrete structures in the nucleus that appear as speckles, and
this localization depends on the RS domain in CrkRS. A proportion of these
structures stain with antibodies against components of the splicing machinery
and against RNA polymerase II (reviewed by Spector,
1993
). CrkRS is very strongly
associated with the nuclear matrix. In subcellular fractionation studies the
bulk of CrkRS remains in the insoluble fraction of the nucleus, even after
nuclease and detergent treatment (data not shown). Thus CrkRS might act as
both an enzyme in modulating the activity of RNA polymerase II and/or the
splicing machinery, and as a scaffold between these components and the nuclear
matrix.
A phosphorylated form of the major subunit of RNA polymerase II is
recognised in interphase by the MPM-2 monoclonal antibody (Albert et al.,
1999). MPM-2 primarily
recognises mitotic phosphoproteins (Davis et al.,
1983
) but in interphase it
stains nuclei in a speckled pattern. This pattern overlaps with that of CrkRS,
and CrkRS from both interphase and mitotic cells is recognised on immunoblots
by MPM-2. These results, allied with our finding that anti-CrkRS
immunoprecipitates can phosphorylate the CTD of RNA polymerase II in vitro,
raises the possibility that CrkRS regulates RNA polymerase II activity.
However, CrkRS does not co-purify with the RNA polymerase II holoenzyme (J.
Parvin, personal communication), indicating that it is probably not part of
the core transcriptional apparaus. In addition, we are unable to exclude the
possibility that a co-immunoprecipitating kinase, and not CrkRS itself, is
responsible for phosphorylating RNA polymerase II CTD, although the balance of
probability remains that CrkRS does phosphorylate the CTD because this is the
physiological substrate of a number of the other Cdc2-family members in the
cell. Thus, CrkRS may be involved in, for example, transcription or
alternative splicing in response to cues such as differentiation. CrkRS
co-immunprecipitates with p150, an as yet unidentified protein recognised by
the CC-3 monoclonal antibody raised against phosphoepitopes of the RNA
polymerase CTD (Albert et al.,
1999
) (data not shown).
The splicing machinery has been shown to be regulated by phosphorylation.
For example, the phosphorylation of RS proteins alters their sequence specific
binding to RNA, and, therefore, can regulate alternative splicing (Fetzer et
al., 1997; Tacke et al.,
1997
). The SRPK and Clk/Sty
protein kinase families regulate the localisation of RS proteins. The SRPK1
protein kinase is specific for RS proteins (Gui et al.,
1994b
) and can cause the
disassembly of nuclear speckles (Gui et al.,
1994a
). Furthermore, high
levels of SRPK1 inhibit an in vitro splicing reaction (Gui et al.,
1994a
). The Clk/Sty protein
kinase has a less extensive RS domain than the one in CrkRS, but this domain
is important for interactions between Clk/Sty and splicing factors (Colwill et
al., 1996
). When Clk/Sty is
overexpressed, it causes a redistribution of splicing factors from nuclear
speckles to the nucleoplasm (Colwill et al.,
1996
). Overexpressing CrkRS to
a high level also causes RS proteins and RNA polymerase II to become more
evenly distributed through the nucleoplasm (data not shown), indicating an
increase in transcriptional activity. However, it is an exciting possibility
that CrkRS differs from Clk/Sty and SPRK in directly phosphorylating the RNA
polymerase II CTD. Thus, CrkRS could represent a novel, evolutionarily
conserved RNA polymerase II CTD kinase that might directly link transcription
with the splicing machinery.
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ACKNOWLEDGMENTS |
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