1 Program in Genes and Disease, Centre de Regulació Genòmica-CRG,
Passeig Marítim 37-49, 08003-Barcelona, Spain
2 Institut per la Recerca i Estudis Avançats-ICREA, Barcelona,
Spain
* Author for correspondence (e-mail: susana.luna{at}crg.es)
Accepted 15 April 2003
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Summary |
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Key words: DYRK1A, kinase, speckle disassembly, splicing, histidine repeat, cycling T1
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Introduction |
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DYRK1A (Guimera et al.,
1996) is a protein kinase, the exact cellular functions of which
are still unknown, although may participate in different cellular processes
and signal transduction pathways (Yang et
al., 2001
; Hammerle et al.,
2002
; Mao et al.,
2002
), including central nervous system development
(Fotaki et al., 2002
). DYRK1A
belongs to a highly conserved family of protein kinases called DYRKs
(dual-specificity tyrosine-regulated
kinases or dual-specificity
yak-related kinases), with members from yeast
to humans (Becker et al.,
1998
). The lower eukaryotic members of this family, such as Yak1p
in Saccharomyces cerevisiae
(Garrett et al., 1991
), Pom1p
in Schizosaccharomyces pombe
(Bahler and Pringle, 1998
) and
YakA in Dictyostelium discoideum
(Souza et al., 1998
), have
been associated with growth control and development. The DYRK1A homologue in
Drosophila, minibrain, seems to be involved in postembryonic
neurogenesis (Tejedor et al.,
1995
). The human DYRK1A gene maps to chromosome 21, and
it is ubiquitously expressed in adult and fetal tissues, with a high level of
expression in the brain (Guimera et al.,
1996
; Guimera et al.,
1999
). Its expression pattern in the adult central nervous system
(Marti et al., 2003
) and in
neurogenesis (Hammerle et al.,
2002
), its overexpression in Down's syndrome fetal brains
(Guimera et al., 1999
), and
the phenotype of DYRK1A transgenic and knock-out mice
(Altafaj et al., 2001
;
Fotaki et al., 2002
) make it
likely that DYRK1A contributes to some of the neuropathological traits
observed in Down's syndrome.
Five members of the DYRK kinase subfamily exist in mammals (DYRK1A, DYRK1B,
DYRK2, DYRK3 and DYRK4) that share a high degree of conservation in the
catalytic domain, but are very divergent in their N- and C-terminal domains
(Becker et al., 1998). Features
of DYRK1A are a bipartite nuclear localization signal (NLS) at the N-terminus
and a PEST domain, a histidine tail and a region rich in serines and
threonines at the C-terminus. The last two domains, both of unknown function,
are exclusively present in DYRK1A. It has been defined as a dual specificity
kinase because of its ability to phosphorylate serine/threonine and tyrosine
residues (Kentrup et al.,
1996
; Himpel et al.,
2001
). Substrate specificity studies have identified a consensus
phosphorylation sequence (RPX(S/T)P), which defines DYRK1A as a
proline-directed kinase (Himpel et al.,
2000
). A few DYRK1A substrates have been reported over recent
years, including cytosolic proteins, such as the
subunit of eukaryotic
initiation factor 2B (eIF2B
), the microtubule-associated protein tau
(Woods et al., 2001a
) and
dynamin (Chen-Hwang et al.,
2002
), and several transcription factors, such as FKHR
(Woods et al., 2001b
), CREB
(Yang et al., 2001
) and Gli1
(Mao et al., 2002
).
In this study we have analyzed the subcellular localization of DYRK1A, characterizing a second NLS at the C-terminal end of the catalytic domain. We show that DYRK1A accumulates in nuclear speckles or SFCs targeted by the histidine-rich segment present in its C-terminal region, and thus we identify a specific role for this domain of the kinase and define it as a novel speckle-targeting signal that is different from those previously described. Supporting this, we have also identified a signal with similar characteristics in cyclin T1. Furthermore, we show that the overexpression of DYRK1A, but not that of a kinase inactive mutant or its close relative DYRK1B, induces the redistribution of SC35 splicing factor from speckles to the nucleoplasm. Together, these results suggest a new potential role for DYRK1A in RNA synthesis or processing.
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Materials and Methods |
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Plasmids
The cloning of DYRK1A full-length open reading frame (the 754 amino acids
alternative spliced form) as an N-terminal HA-tagged fusion protein has
already been described (Marti et al.,
2003). HA-DYRK1A deletion mutants were generated either by
removing different fragments with restriction enzymes and subsequent ligation
or by PCR amplification using specific primers with appropriate restriction
sites for subcloning. DYRK1A cDNA was also cloned into a pEGFP-C1 vector (BD
Clontech, Palo Alto, CA) to generate an N-terminal GFP fusion protein. All the
GFP constructs containing different fragments of DYRK1A were prepared by
subcloning PCR products into pEGFP-C1 or by creating stop codons using the
Quick-Change kit of Stratagene (La Jolla, CA). The DYRK1A inactive mutant
K179R was generated by site-directed mutagenesis.
The pEGFP-DYRK1A (1-176) (Becker et al.,
1998) and pEGFP-DYRK1B (Leder
et al., 1999
) plasmids were kindly provided by W. Becker (Aachen,
Germany). To generate the DYRK1B/A chimeric protein, a PstI fragment
(amino acids 386-589) in pEGFP-DYRK1B was replaced by a PstI fragment
containing the C-terminal region of DYRK1A (amino acids 465-754).
The SUMO-1 open reading frame was amplified from a human brain cDNA library with specific primers and inserted in-frame into the BamHI and XhoI sites of pcDNA-HA1 to generate an N-terminal HA-tagged fusion protein. UBC-9 cDNA was amplified from mouse brain RNA with specific primers and cloned into pRcCMV (Invitrogen, Carlsbad, CA). Human cyclin T1 cDNA fragments (nucleotides 1621-1920 and 1836-1920 in GenBank Acc. No. NM_001240) were amplified from a human brain cDNA library with specific primers and cloned in-frame into the BglII and SalI sites of pEGFP-C1.
All constructs made by PCR, as well as all the in-frame fusions, were verified by DNA sequencing.
Cell culture and transfection
COS-7 and HeLa cells were purchased from the European Collection of Cell
Cultures (ECACC) and maintained at 37°C in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum (FCS) and antibiotics. Treatment
of COS-7 cells with -amanitin (50 µg/ml; Sigma, St Louis, MI) was
carried out for 5 hours at 37°C. Transient transfections were performed
using the calcium phosphate precipitation method.
Western blotting
COS-7 cells were plated in six-well dishes (1x105
cells/well), transfected with 3 µg of plasmid DNA and harvested in ice-cold
phosphate-buffered saline (PBS) 48 hours post transfection. Cells were lysed
in Laemmli's SDS buffer (250 mM Tris-HCl pH 6.8, 4.6% SDS, 10%
2-mercaptoethanol, 20% glycerol and 20 µM bromophenol blue). Samples were
subjected to 10% SDS-PAGE, transferred onto a nitrocellulose membrane (Hybond
C, Amersham Biosciences) and blocked with 10% skimmed milk in PBS. The
membrane was incubated for 1 hour with the anti-HA antibody (1:2000 in PBS
with 0.1% Tween-20 and 5% skimmed milk) and then with an HRP-conjugated rabbit
anti-mouse antibody (1:2000 in PBS with 0.1% Tween-20 and 5% skimmed milk) for
45 minutes. Washes were done in PBS-0.1% Tween-20. Detection was performed by
enhanced chemiluminiscence (SuperSignal West Pico, Pierce, Rockford, IL).
Immunofluorescence
COS-7 cells growing on coverslips in six-well dishes were transfected with
different expression constructs. 48 hours after transfection the cells were
washed in PBS, fixed in 4% paraformaldehyde in PBS for 15 minutes and
permeabilized in 0.1% Triton X-100 in PBS for 10 minutes. In general the cells
were blocked in 10% FCS in PBS, except for the detection of Sm proteins, in
which case a blocking solution containing 50% FBS, 6% skimmed milk, 3% BSA and
0.2% Tween-20 was used. Cells were incubated with primary antibodies for 1 to
2 hours and washed extensively with PBS before and after incubation with
secondary antibodies for 45 minutes. All incubations were done at room
temperature. Anti-HA, anti-PML, anti-SC35 and Y12 antibodies were used at
1:800, 1:100, 1:100 and 1:100 dilutions in PBS-1% FCS, respectively.
FITC-conjugated goat anti-mouse, Texas-Red-conjugated sheep anti-mouse and
Cy3-conjugated goat anti-rabbit antibodies were used at 1:400, 1:50 and 1:1000
dilutions in PBS-10% FCS, respectively. Coverslips were mounted onto slides
using Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA) with
0.2 µg/ml 4,6-diamidino-2-phenylindole (DAPI) and analyzed with an Olympus
BX60 microscope with the appropriate filters. Images were captured using a
digital camera (Spot RT Colour, Diagnostic Instruments) with SPOT Advanced
version 3.2.4. (Diagnostic Instruments) software and processed for
presentation with Adobe Photoshop. When indicated in the figure legend, images
were acquired in an inverted Leica SP2 Confocal Microscope, using an HCX PL
APO 63x 1,32 Oil Ph3 CS objective, and the double images were all taken
sequentially. GFP was excited with the 488 nm line of the Argon laser and IgG
Texas Red was excited with a 543 nm HeNe laser.
Kinase assay
COS-7 cells were plated in 10-cm dishes at 7x105
cells/plate, transfected with 14 µg of the different HA-fusion constructs
and harvested in ice-cold PBS 48 hours after transfection.
Cells were lysed in a buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM EDTA, 25 mM sodium fluoride, 30 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1% Nonidet P-40 (NP-40) and a protease inhibitor cocktail (Roche, Manheim, Germany). Cell extracts were clarified by centrifugation (10,000 g, 15 minutes) and incubated overnight at 4°C with protein-G Sepharose beads (Amersham Biosciences) pre-bound with anti-HA antibody. The beads were washed four times with a buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl and 2 mM EDTA, adding 0.1% NP-40 for the initial washes.
The immunocomplexes were subjected to an in vitro kinase assay using the
peptide DYRKtide as exogenous substrate, as described previously
(Himpel et al., 2000).
Briefly, the immunocomplexes were incubated for 20 minutes at 30°C in 20
µl of phosphorylation buffer containing 200 µM DYRKtide (a kind gift of
W. Becker), in the presence of 10 µM [32P]-ATP
(5x10-3 µCi/pmol). Phosphorylation of DYRKtide was assayed
with the phosphocellulose method and samples were resolved in 10% SDS-PAGE and
visualized by autoradiography of the dried gels to detect DYRK1A
autophosphorylation. The presence of HA-DYRK1A proteins in the immunocomplexes
was detected by western blot.
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Results |
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|
Identification of a nuclear localization signal in the catalytic
domain of DYRK1A
PSORT II
(http://psort.ims.u-tokyo.ac.jp/)
identified, apart from the N-terminal NLS (NLS1), a consensus monopartite type
NLS, PKRAKFF, at amino acid residues 380-386 (NLS2). As previously described
(Becker et al., 1998), a GFP
fusion protein comprising the N-terminal region of DYRK1A (NLS1) was found
exclusively in the nucleus (Fig.
2, 1-176). When a similar approach was taken for the predicted
additional NLS2, the fusion failed to completely accumulate GFP in the nucleus
(Fig. 2, 378-386). A more
detailed analysis of the primary sequence around the region revealed the
presence of another stretch of basic residues between amino acids 397-407
(KKTKDGKREYK), suggesting the possibility that this sequence element was also
necessary for nuclear targeting, as a bipartite-like NLS. A GFP fusion protein
including both polybasic sequences was also unable to fully accumulate GFP in
the nucleus (Fig. 2, 378-407).
However, a fusion protein with an extended fragment of the region, defined
above, localized exclusively in the nucleus
(Fig. 2, 378-466), indicating
that the flanking residues of the predicted NLS2 are also determinant for the
nuclear enrichment. To confirm that the predicted NLS2 was nevertheless
necessary for nuclear targeting, amino acids 380-386 were removed from the
last construct, which induced the accumulation of the fusion protein both in
the nucleus and the cytosol (Fig.
2, 390-466). These results, together with the deletion analysis,
indicate that DYRK1A contains two independent NLSs, each of which individually
is sufficient for targeting DYRK1A to the nucleus.
|
DYRK1A has a novel nuclear speckle-targeting signal
Having defined a region between amino acids 588-616
(Fig. 1) that seems to be
required for the targeting of DYRK1A to the nuclear speckles, we wanted to
investigate whether this particular domain of DYRK1A was sufficient for
localizing a heterologous protein to the speckles. We first generated a GFP
fusion protein with an extended fragment of the minimal region defined above
(378-616) to include the second NLS identified in case nuclear import was
necessary for targeting to the subnuclear compartment. This fusion protein
showed the same staining pattern as that seen for the full-length DYRK1A fused
to GFP (Fig. 3, compared
wildtype and 378-616 images). When the amino acid sequence containing the
histidine tail was deleted, accumulation in speckles was no longer detected,
although nuclear localization was maintained
(Fig. 3, 378-588). A fusion
protein, consisting of the histidine repeat alone, clearly accumulated in the
speckle compartment (Fig. 3, 590-616), indicating that the presence of this sequence is sufficient for
nuclear speckle localization. Furthermore, since this GFP fusion protein does
not harbour an NLS, and accordingly is not restricted to the nucleus, the
results allow us to suggest that the speckle targeting promoted by this
sequence does not depend on receptor-mediated nuclear import. Therefore, a
novel signal, both necessary and sufficient for targeting to nuclear speckles,
is present in DYRK1A.
|
Colocalization of DYRK1A with splicing factors
Next, we attempted to determine whether DYRK1A associates with any of the
known subnuclear structures by means of colocalization studies of GFP-DYRK1A
using reported markers for different subnuclear compartments. On the basis of
the type and number of DYRK1A nuclear dots, we concentrated on the splicing
factor compartment, PODs, and SUMO nuclear bodies, the last two based on the
fact that the protein kinase HIPK2, a close relative of the DYRK family of
proteins, localizes to nuclear dots when modified by SUMO
(Kim et al., 1999) and
accumulates within PML nuclear bodies
(Trost et al., 2000
).
Immunostaining with an antibody against the promyelocytic leukemia protein (PML), a major component of PML bodies, did not show colocalization (Fig. 4A). Nor were we able to detect any colocalization of DYRK1A with SUMO when exogenously expressed, neither in the absence or presence of its E2 enzyme UBC-9 (Fig. 4B and data not shown). However, a marked colocalization was observed when we used an antibody to the SC35 protein, a non-snRNP splicing factor of the SR family of proteins that is commonly used to define splicing nuclear speckles (Fig. 4C, upper panel). To determine whether DYRK1A associates with components of the spliceosome in addition to SC35, we tested an antibody to Sm proteins (Y12 antibody) that recognizes an epitope found on a number of snRNPs, and colocalization was also detected (Fig. 4C, lower panel). Results with the fusion GFP-DYRK1A 590-616 confirmed that the discrete foci for the novel targeting signal were also SC35 positive (Fig. 4D).
|
Cyclin T1 has been shown to localize to SFCs, and a region (amino acids
433-533) containing a histidine-rich segment has been defined by deletion
analysis as important for its localization in this compartment
(Herrmann and Mancini, 2001).
Not only the fusion GFP-cyclin T1 433-533 but also a shorter version including
only the histidine segment (HKEKHKTHPSN(H)5NHHSHKHSHSQ) clearly
colocalized with SC35 staining in the transfected cells
(Fig. 4E).
Although the results from above strongly suggest that kinase activity is
not required for DYRK1A nuclear speckle localization, we wanted to confirm it
by studying the behaviour of a kinase-negative mutant. The mutant (K179R),
generated by replacing the conserved lysine in the ATP-binding site with an
arginine, was unable to autophosphorylate and failed to phosphorylate a
peptide substrate (DYRKtide) (Fig.
5A), a result similar to that obtained with the equivalent mutant
(DYRK1A K188R) in the 763 amino acids rat DYRK1A form
(Kentrup et al., 1996). This
inactive kinase mutant accumulated in nuclear speckles and colocalized with
the SC35 splicing factor (Fig.
5B). These results, all together, indicate that DYRK1A
concentrates in nuclear speckles with splicing factors and that it does not
require its kinase activity for such localization.
|
Effect of transcriptional inhibition on DYRK1A localization
When cells are treated with transcription inhibitors, splicing activity is
reduced and foci associated with RNA processing undergo dynamic changes, in
particular, speckles labelled with SC35 become fewer, increase in size and
round up (O'Keefe et al.,
1994). To determine whether the localization of DYRK1A is
dependent on the transcriptional activity of the cell, transfected cells were
treated with
-amanitin, an inhibitor of transcription mediated by RNA
polymerase II. As shown in Fig.
6A, DYRK1A localized to larger speckles with a complete overlap
with SC35. The same effect was observed for the kinase-negative mutant (DYRK1A
K179R) and for DYRK1A 590-616 (Fig.
6B,C), although in this case a few GFP-positive foci were not
co-stained with SC35. Therefore, DYRK1A accumulation is sensitive to the
transcriptional state of the cell, responding as other members of the splicing
machinery with regard to such behaviour.
|
DYRK1A disassembles nuclear speckles
In DYRK1A-expressing cells we repeatedly detected a marked population of
cells in which DYRK1A staining appeared to be diffuse. In these cells the
endogenous SC35 staining was not concentrated in speckles, but nucleoplasmic
staining was still detected (Fig.
7A). Quantification of this phenotype
(Fig. 7B) indicates that, in
around 40% of the transfected cells, overexpression of DYRK1A wildtype, but
not of the kinase-negative mutant, was able to induce the disassembly of
nuclear speckles. DYRK1A 590-616 did not affect disassembly, supporting the
release of splicing factors from speckles is dependent on DYRK1A kinase
activity. Surprisingly, overexpression of a truncated form, lacking the
C-terminal region of DYRK1A (DYRK1A 1-522, C) and unable to localize to
speckles, also induced the redistribution of endogenous splicing factors at a
similar level to DYRK1A wildtype (Fig.
7B). Treatment with
-amanitin had no effect on the
percentages of speckle disassembly (Fig.
7B). Therefore, DYRK1A, similarly to CLKs or SRPKs
(Gui et al., 1994
;
Colwill et al., 1996
;
Duncan et al., 1997
;
Wang et al., 1998
), is able to
induce the redistribution of SR proteins from speckles when the active kinase
is overexpressed.
|
DYRK1A has a paralogous gene in humans named DYRK1B. The two
proteins share over 80% amino-acid identity in the catalytic region, with the
similarity extending towards the N-terminus but not to the C-terminus. As
previously reported (Leder et al.,
1999), a fusion protein GFP-DYRK1B localized primarily in the
nucleus, with no detectable accumulation in nuclear speckles, as was expected
because of a lack of the histidine repeat, which is not conserved. Moreover,
all cells overexpressing the fusion protein presented a normal SC35 staining
pattern (Fig. 7C). In contrast,
a chimeric protein (DYRK1B/A) in which the C-terminal regions of both proteins
have been exchanged, regained the ability to localize to speckles, but not to
cause the release of SR proteins (Fig.
7C). These results suggest that, if speckle disassembly is
mediated by the phosphorylation of certain factors present in the nuclear
speckles, some substrate specificity, which may be different in DYRK1A and
DYRK1B, should be involved in the process.
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Discussion |
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We have shown that both NLSs may work independently, because they are both
capable of targeting a heterologous protein to the nucleus and because their
deletion in DYRK1A protein still leaves a protein competent in nuclear
localization. The existence of multiple NLSs in a single protein has been
associated with a faster nuclear uptake of the protein
(Boulikas, 1993). It is also
possible that if each NLS is subject to different regulatory pathways, they
may contribute to the fine control of DYRK1A concentration within the
nucleus.
The nuclear speckles in which DYRK1A accumulates have been identified as
the splicing factor compartment or SFC, as indicated by DYRK1A colocalization
with SC35 and Sm proteins. We did not detect colocalization with PML or
SUMO-1, as described for the DYRK family relative HIPK2
(Kim et al., 1999;
Trost et al., 2000
), although
we cannot exclude the possibility that DYRK1A may be modified by other SUMO
family members not tested here. Speckle localization is not cell type specific
because it can be readily observed in several mammalian cell lines, including
NIH 3T3, HeLa and SH-SY5Y (data not shown). DYRK1A protein levels are very low
in all these cell lines, precluding the possibility of studying the endogenous
pattern. However, in in vitro cultured mouse cerebellar neurons, endogenous
DYRK1A immunostaining, as shown by confocal microscopy, resembles the pattern
found when exogenously expressed (Marti et
al., 2003
), excluding an overexpression-mediated effect.
Very little is known about the protein signals that direct accumulation in
specific subnuclear sites. In the case of SFCs a few protein domains have been
described: the RS-domain and the RNA-recognition motif for SR proteins and
CrkRS (Caceres et al., 1997;
Gama-Carvalho et al., 1997
;
Ko et al., 2001
); the
`forkhead-associated' domain in the case of the protein phosphatase-1
regulator NIPP1 (Jagiello et al.,
2000
); the ankyrin repeats in IkappaBL
(Semple et al., 2002
), the
TP-domain in SF3b155/SAP155
(Eilbracht and Schmidt-Zachmann,
2001
), and a newly described region in SRm160
(Wagner et al., 2003
).
According to this, the speckle-targeting signal described here is completely
novel. We consider it a bona fide signal, given that it meets the criteria of
both being necessary for DYRK1A localization and being sufficient to mediate
the speckle localization of a heterologous protein. The fact that a similar
type of signal is responsible for cyclin T1 localization to SC35 foci
highlights the importance of this novel domain and leads us to suggest that
the histidine-rich region represents a novel class of targeting signals to
SFCs. At present we do not know whether the novel histidine-rich targeting
signal works as an RNA-binding domain or as a protein interacting surface. Our
attempts to detect interactions from SR-protein-enriched fractions using the
DYRK1A targeting signal as a bait in GST pull-downs have been unsuccessful
(data not shown), suggesting that the target may not be any of the major SR
proteins. Taking into account that the signal is shared by cyclin T1, a
regulator of transcription, it would be possible that the target is not a
member of the splicing machinery but of RNA synthesis complexes.
Spliceosome formation is a dynamic process, and there is continuous
switching of binding partners, both protein and RNA components, throughout the
splicing reaction. Protein phosphorylation seems to significantly contribute
to the modulation of both the structural organization of the spliceosomal
complex and the catalytic steps of the splicing reaction. In spite of this,
not many kinases have been shown to modulate mRNA splicing
(Gui et al., 1994;
Duncan et al., 1997
;
Xiao and Manley, 1998
;
Prasad et al., 1999
;
Wang et al., 1999
). Although
some kinases accumulate in SFCs: PITSLREp110
(Loyer et al., 1998
),
phosphatidylinositol 3-kinase C2
(Didichenko and Thelen, 2001
),
phosphatidylinositol phosphate kinases
(Boronenkov et al., 1998
),
casein kinase I (Gross et al.,
1999
), cyclin T-cdk9 (Herrmann
and Mancini, 2001
), CrkRS (Ko
et al., 2001
) and hPRP4
(Kojima et al., 2001
), only
members of the CLK and SRPK families have been described as able to disturb
the normal appearance of SFCs when overexpressed
(Gui et al., 1994
;
Colwill et al., 1996
;
Kuroyanagi et al., 1998
;
Wang et al., 1998
). The
results shown here indicate that DYRK1A belongs to the very short list of
kinases capable of disassembling nuclear speckles. Disassembly occurs in about
40% of cells overexpressing the protein, and it does not result from the
displacement of a speckle component by competition because the GFP fusion
containing only the targeting signal does not cause speckle redistribution.
Moreover, on the basis of the results of the kinase-defective mutant, it seems
that the effect is definitely dependent on having an active kinase that may
either phosphorylate spliceosome components or act as a regulatory kinase for
another speckle-disassembly kinase. The pattern shown by the DYRK1B protein
targeted to the speckles through the C-terminal region of DRYK1A points to a
target of phosphorylation being DYRK1A specific. An intriguing finding is that
disassembly is not an `all or nothing' effect, because it is detectable only
in a population of transfected cells. The percentages found cannot be related
to differences in the level of protein expression because we have not been
able to correlate high expression level and speckle redistribution. One
possibility is that the kinase is active in a defined cell population, for
example, during a specific phase of the cell cycle. Preliminary data
indicating that DYRK1A-overexpressing cells are arrested in G1 would suggest
that speckle reorganization is not related to cells being in the S, G2 or M
phases of the cell cycle (S. Aranda and S.L., unpublished). The effect can
also be explained if the phosphorylation target is not present in speckles in
all cells. Future experiments will be directed towards addressing these
questions, identifying DYRK1A partners in the speckles and discovering whether
DYRK1A kinase activity is able to regulate splicing by modulating the
biogenesis of nuclear speckles.
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Acknowledgments |
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