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Vol. 17, Issue 2, 876-885, February 2006
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* Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093-0651;
Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0651; and
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0651
Submitted October 18, 2005;
Accepted November 21, 2005
Monitoring Editor: Joseph Gall
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Manley and Tacke, 1996; Graveley, 2000). RRMs are important for sequence-specific binding to commit pre-mRNA to the splicing pathway (Fu, 1993), whereas the RS domain in SR proteins is thought to promote protein-protein interactions during spliceosome assembly (Wu and Maniatis, 1993). Recently, the RS domain in SR proteins was found to directly bind RNA in assembled spliceosomes (Shen et al., 2004; Shen and Green, 2004).
SR proteins are regulated by reversible phosphorylation. Phosphorylated SR proteins seem to be required for initiating spliceosome assembly at the earliest detectable stage (Mermoud et al., 1994; Kohtz et al., 1994; Cao et al., 1997). Once the spliceosome is fully assembled, dephosphorylation seems to be essential for splicing to take place in the spliceosome (Mermoud et al., 1992; Cao et al., 1997). Interestingly, a later study indicates that a given SR protein may not be obligated to go through a complete phosphorylation-dephosphorylation cycle in each splicing reaction, implying that a dephosphorylated SR protein is able to substitute for dephosphorylation-resistant SR proteins to fulfill the late requirement in splicing (Xiao and Manley, 1998). Consistent with these biochemical observations, ASF/SF2 mutants containing negatively charged aspartic acids or glutamic acids in place of serines in the RS domain are fully functional in complementing the essential function of ASF/SF2 in cell viability (Lin et al., 2005).
Reversible phosphorylation of SR proteins has also been shown to play a critical role in coupling splicing with both upstream and downstream events during gene expression. It has been well established that splicing takes place cotranscriptionally in the nucleus; thus, splicing factors have to be recruited to nascent transcripts. This recruitment process seems to require proper phosphorylation of SR proteins (Misteli et al., 1997, 1998). A series of in vitro and in vivo experiments show that phosphorylation is important for SR proteins to interact with the C-terminal domain of polymerase II (Pol II) to coordinate transcription with splicing (Misteli and Spector, 1999; Hirose and Manley, 2000). In linking splicing to RNA export, SR proteins have been found to function as adaptors for nuclear export of spliced mRNA (Huang and Steitz, 2001; Huang et al., 2003, 2004; Lai and Tarn, 2004). The adaptor function seems to be confined within a subset of SR proteins capable of shuttling between the nucleus and the cytoplasm. Interestingly, several shuttling SR proteins can directly interact with the nuclear export factor TAP via sequences in the N-terminal RRMs, but the interaction requires dephosphorylation of the C-terminal RS domain. Although prevention of a single SR protein from shuttling is not sufficient to impair mRNA export in vivo, it remains to be determined whether shuttling SR proteins as a class are important for mRNA export (Lin et al., 2005). Once exported to the cytoplasm, SR proteins are reimported to the nucleus by interacting with the import receptor hMtr10/Transportin-SR in a phosphorylation-dependent manner (Yun and Fu, 2000; Lai et al., 2000, 2001; Gilbert et al., 2001; Yun et al., 2003). It has been proposed that phosphorylation of shuttling SR proteins in the cytoplasm may act as a switch between mRNA unloading and SR protein reimport (Gilbert and Guthrie, 2004).
Mammalian cells express several kinases key to phosphorylation regulation of SR proteins and RNA metabolism in general. The first SR protein-specific kinase SRPK1 was cloned during the characterization of an activity in regulating SR protein redistribution during the cell cycle (Gui et al., 1994). We now know that SRPK1 belongs to a family of kinases that are evolutionarily conserved from budding yeast to humans (Takeuchi and Yanagida, 1993; Wang et al., 1998; Kuroyanagi et al., 1998; Siebel et al., 1999; Koizumi et al., 1999; Tang et al., 1998, 2000). All SRPK family members share highly conserved kinase domains, which are separated by a unique spacer sequence in individual family members, indicating that specific SRPKs may be uniquely regulated by the spacer sequence. The second family of SR protein kinases is called Clk/Sty (for cyclin-like kinase or serine/threonine/tyrosine kinase), which has three family members in mammalian cells. Clk/Sty or Clk-1 was initially cloned as a cyclin-like kinase by degenerate PCR (Ben-David et al., 1991; Howell et al., 1991). Its connection to SR proteins was made when Clk/Sty was shown to interact with SR proteins in a two-hybrid screen (Colwill et al., 1996). The best evidence for its involvement in splicing regulation came from studies in Drosophila where a Clk/Sty orthologue could phosphorylate endogenous SR proteins and mutations in the kinase altered specific alternative splicing events in the sex determination pathway (Du et al., 1998).
Here, we report the mechanism for the regulation of SRPKs by a unique spacer sequence in each kinase. The cytoplasmic localization of SRPK1 is controlled by multiple elements in the spacer, deletion of which had little effect on kinase activity, but caused a quantitative translocation of the kinase to the nucleus. Contrary to an early work indicating the presence of active nuclear export signal (NES) in the spacer of the SRPK family member in fission yeast, we found that cellular partitioning of SRPK1 is likely achieved by a spacer-mediated anchoring mechanism in mammalian cells. The importance of partitioning the kinase during interphase is demonstrated by induced aggregation of splicing factors when an excessive amount of a spacer-deleted kinase is present in the nucleus. Interestingly, the kinase translocates to the nucleus in response to a cell cycling signal before the initiation of the M phase, indicating that the kinase may play a role in cell cycle progression. Together, these findings raise the possibility that the SRPK family of kinases may function as an important class of signal molecules for splicing regulation in higher eukaryotic cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) or pcDNA3 (Invitrogen, Carlsbad, CA). SRPK1-K109M contains one point mutation at position 109 from Lys to Met, which impairs the ATP binding site in the kinase (Wang et al., 1998). The nuclear localization signal (NLS) from the SV40 large T antigen was engineered into the N terminus of the kinase coding sequence by using an extended PCR primer. Spacer deletion series were constructed using proper restriction sites in the spacer in combination with PCR. Details in plasmid construction were available upon request. All mutant constructs were confirmed by sequencing.
Kinetic Analysis
The phosphorylation of glutathione S-transferase (GST)-ASF/SF2 was carried out in a buffer containing 50 mM 2-(N-morpholino)ethanesulfonic acid, pH 7.0, 10 mM Mg2+, 1 mg/ml bovine serum albumin (BSA), and [
-32P]ATP (600-1000 cpm pmol-1) as described previously (Aubol et al., 2003). Typically, enzyme and substrate were preincubated for 3 min before reaction initiation with 0.2 mM ATP in a total volume of 20 µl. Upon reaction quenching with 10 µl of SDS-PAGE loading buffer after designated time periods, phosphorylated GST-ASF/SF2 was separated from unreacted [32P]ATP and enzyme in a 12% SDS-PAGE gel. The appropriate bands were cut out of the dried gel and counted in liquid scintillant. The amount of phosphorylated GST-ASF/SF2 was calculated using the specific activity of the [32P]ATP. As described previously (Aubol et al., 2003), steady-state kinetics was used to derive the Km, and single turnover experiments over a range of kinase concentrations were performed to calculate the Kd for both wild-type (wt) and spacer-deleted SRPK1.
Cell Culture and Synchronization
HeLa and HeLa tTA cells were cultured in DME supplemented with 10% fetal bovine serum. For immunofluorescence staining, cells were first seeded on coverslips coated with 0.1% polylysine. Transfection was carried out using Lipofectamine 2000 reagent (Invitrogen). Cells were synchronized to S phase by adding 2.5 mM thymidine to the growth medium for 24 h as described previously (Gui et al., 1994). G2/M phase cells were prepared by releasing S-phase cells after the thymidine block to the culture media for 7-8 h. Transcription and translation were blocked by using 50 µg/ml actinomycin D and 100 µg/ml cycloheximide for 3 h, respectively. NES-mediated nuclear export was blocked with 10 ng/ml leptomycin B (LMB) for 1 h.
Immunofluorescence Microscopy
Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at room temperature followed by permeabilization for 10 min in 0.1% Triton X-100/phosphate-buffered saline. We used 0.1% BSA in PBS to block nonspecific binding during antibody staining. Endogenous SRPK1 was detected using monoclonal anti-SRPK1 (catalog no. 611072; BD Transduction Laboratories, Lexington, KY), and SC35 was stained with either polyclonal anti-SC35 (Himmelspach et al., 1995) or monoclonal anti-SC35 (Fu and Maniatis, 1990). Transfected wild-type and mutant SRPK1 were localized by using polyclonal rabbit anti-myc (catalog no. 2272; Cell Signaling Technology, Beverly, MA). Transfected wild-type and spacer-deleted SRPK2 were localized by using monoclonal anti-FLAG (M2 from Sigma-Aldrich, St. Louis, MO). Fluorescent-conjugated secondary antibodies were Alexa 594-conjugated donkey anti-mouse IgG and Alexa 488-conjugated goat anti-rabbit IgG (catalog nos. A-21203 and A-11070, Molecular Probes, Eugene, OR). Coverslips were mounted in a mounting solution containing 4,6-diamidino-2-phenylindole (DAPI) (Vectashield; Vector Laboratories, Burlingame, CA) and analyzed under a Zeiss Axiophot microscope and images acquired with a Hamamatsu ORCA-ER digital camera with Improvision OpenLab 3.1.5 software. Deconvolution images were taken in the image core of the University of California San Diego Cancer Center with a DeltaVision restoration microscope system (Applied Precision, Issaquah, WA) using a Photometrics Sony CoolSNAP HQ charged-coupled device camera system (10 MHz; 12 bit; 1392 x 1040) attached to an inverted, wide-field fluorescent microscope (Nikon TE-200).
Heterokaryon Assay
HeLa tTA cells were transfected with various SRPK constructs. Transfected cells were trypsinized 2 h later and mixed with mouse embryonic fibroblasts at the ratio of 2:3. After overnight coculturing, cycloheximide was added to block protein synthesis at 10 µg/ml for 1 h and then increased to 100 µg/ml for 30 min before cell fusion. Cell fusion was induced with 50% polyethylene glycol (PEG)3350 (wt/vol) in PBS for 2 min at 37°C. PEG was washed away with PBS (5 times), and treated cells were incubated in the growth medium containing 100 µg/ml cycloheximide for additional 2 h. Actin was labeled with Alexa 594-phaloidin to identify fused cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Yeakley et al., 1999). To determine the localization of endogenous kinase, we stained HeLa cells with a specific monoclonal antibody against SRPK1 (see Materials and Methods). The endogenous kinase was mainly localized in the cytoplasm, consistent with our previous observations. In addition, a fraction of the kinase was visible in the nucleus under a standard inverted microscope (Figure 1, A-C). This result was further confirmed by confocal microscopic analysis of center sections of the nucleus (Figure 1, D-F). No signal was detected if primary antibodies were omitted (Figure 1, G-I). Colocalization of SRPK1 with endogenous SC35 revealed that nuclear SRPK1 seems generally associated with SC35 in nuclear speckles under a standard inverted microscope (Figure 1C, note that staining with the polyclonal anti-SC35 peptide antibodies used here is more diffused than the pattern detected with the monoclonal, phosphoepitope-specific, anti-SC35 antibody, see images in Figure 3). However, both the kinase and the SR protein clearly have a large population diffusely distributed in the nucleus as seen under a confocal microscope (Figure 1F).
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; Prasad et al., 1999). Therefore, kinases for SR proteins must be regulated, which, in SRPKs, seems to be achieved by partitioning the kinase in the cytoplasm, analogous to many protein kinases involved in signaling (Cyert, 2001). Although SRPKs are serine-specific kinases, they resemble tyrosine kinases by having a unique spacer that separates the small and large catalytic lobes. Spacers in tyrosine kinases are frequently serving as docking sites for signaling molecules and thus are important regulatory modules in those kinases (Hunter, 2000). Deletion of the spacer in both SRPK1 and SRPK2 resulted in translocation of the kinases to the nucleus (Figure 2, A-H). Similar nuclear translocation was also reported for the SRPK family members in fission and budding yeast (Takeuchi and Yanagida, 1993; Siebel et al., 1999). Interestingly, the spacers in SRPK1 and SRPK2 seem to function as an autonomous unit because insertion of the spacer of SRPK2 into SRPK1 restored the cytoplasmic localization of the kinase (Figure 2, I and J). These findings, coupled with previous studies of SRPK orthologues in low eukaryotic cells, demonstrate that the spacers are regulatory domains critical for the cellular distribution of the SRPK family of kinases.
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Kinase Activity Required for Nuclear Targeting
Both wt and mutant SRPK1 lacking the kinase activity (because of a mutation in the ATP binding site) were localized in the cytoplasm (Figure 3, A-C and D-F). When the spacer was deleted, the kinase translocated to the nucleus where it colocalized with splicing factors in nuclear speckles (Figure 3, G-I). Accumulation of the spacer-deleted SRPK1 in the nucleus then induced aggregation of splicing factors (Figure 3, J-L), which is similar to the cellular response to inhibition of splicing by injected oligos or by the transcription inhibitor actinomycin D (O'Keefe et al., 1994). These observations are consistent with the finding that expression of the spacerdeleted Sky1p in budding yeast resulted in a lethal phenotype, together indicating that cellular partitioning of SRPKs is essential for cell physiology (Siebel et al., 1999).
To determine whether the kinase activity is critical for induced aggregation of splicing factors in the nucleus, we tested the spacer-deleted SRPK1 in which the ATP binding site was mutated. Surprisingly, the mutation efficiently blocked the accumulation of the spacer-deleted kinase in the nucleus (Figure 3, M-O). Thus, the kinase activity seems to be essential for nuclear entrance of the spacer-deleted SRPK1.
Impact of the Spacer Sequence on Kinase Activity
The requirement for an enzymatically active kinase to enter the nucleus is consistent with our previous biochemical studies that the spacer-deleted SRPKs are active in phosphorylating SR proteins in vitro (Siebel et al., 1999; Nolen et al., 2001). It has been unclear, however, whether deletion of the spacer further activates the kinase (as recombinant full-length SRPKs are already active) or modulates other biochemical aspects of the kinase because many kinases translocate to the nucleus upon activation (Cyert, 2001). We therefore set out to conduct detailed kinetic characterization of wt and the spacer-deleted SRPK1 using ASF/SF2 as a model substrate.
In steady-state kinetic assays (Figure 4A), the spacer increased the turnover number (k) by a factor of 2 (5 versus 10 min-1), but it had no impactcaton the K for ASF/SF2 (Km of 
200 nM). To ensure that the observed changes in kcat were not because of inactive enzyme in our purification, single turnover experiments were performed under conditions of excess enzyme concentration. Under these constraints, the observed rate constant for substrate phosphorylation should be independent of the total enzyme concentration and thus report directly on the overall phosphorylation rate in the active site. As shown in Figure 4B, the single exponential rate constants for ASF/SF2 phosphorylation using excess SRPK1 and SRPK1
S (i.e., [E] >> Kd) differed by approximately threefold, indicating that the spacer enhanced substrate turnover by a small but real amount.
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Signals in the Spacer and Potential Action Mechanism
Because the effect of spacer removal from SRPK1 on kinase activity does not explain the quantitative shift of the kinase to the nucleus, we next focused on potential signals in the spacer sequence that may dictate the cellular distribution of SRPK1. A previous study showed that a motif in the spacer of the fission yeast kinase Dsk1 could act as a NES in an LMB-sensitive manner (Fukuda et al., 1997). However, the motif does not seem to be conserved among SRPK family members. To pursue the possibility that the cytoplasmic localization of SRPK1 may be directed by a divergent NES, we treated the cell with LMB. Although nuclear export of c-Abl was effectively blocked by LMB as documented previously (Taagepera et al., 1998), we observed no effect of a similar treatment on SRPK1 localization (Figure 5A). To substantiate this observation, we determined the potential NES signal in SRPK1 by examining its ability to shuttle between the nucleus and the cytoplasm. For this purpose, a classic NLS from the SV40 large T antigen was fused to the kinase, and the nucleus-targeted kinase was tested in a heterokaryon assay. The NLS was sufficient to shift the kinase to the nucleus, indicating that the NLS acted in a dominant manner over potential NES. However, the nuclear-targeted kinase was unable to shuttle (Figure 5B, a-c). Similarly, we fused the spacer from SRPK1 to an NLS-green fluorescent protein (GFP) reporter and found that the fusion protein was also unable to shuttle (Figure 5B, d-f). These data indicate that the spacer in SRPK1 does not carry a detectable NES.
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Dissection of the Localization Signal in the Spacer
The dramatic effect of spacer deletion on the localization of SRPK1 does not seem to be explained by a large change in enzymatic kinetics or the presence of a measurable NES. We therefore considered the possibility that the spacer may carry a cytoplasmic anchoring signal(s), which may enable the tight regulation of the kinase in the cell. To pursue this possibility and pinpoint potential regulatory motifs in the spacer, we conducted deletion analysis to characterize the potential cytoplasmic anchoring signal in SRPK1. Systematic deletion from the N or C terminus of the spacer indicated that none of the smaller deletions had a major effect on shifting the kinase to the nucleus (our unpublished data), which again argued against the existence of a single functional NES in the regulation of the cellular distribution of the kinase.
Inspection of the impact on kinase distribution by a large number of spacer deletion mutants suggests that the spacer may be roughly divided into three segments. Any combination of two segments (deletion of any single segment) was sufficient to retain the kinase in the cytoplasm, whereas any single segment (deletion of any two segments) had a partial effect, resulting in both cytoplasmic and nuclear localization of the kinase in transfected cells (Figure 6). These observations suggest that a simple conformational change may not be sufficient to explain the dramatic effect of spacer deletion on nuclear translocation of the kinase (because at least some single deletions tested were expected to cause a major conformational change in the spacer). Instead, the spacer may contain multiple redundant elements involved in interaction with cytoplasmic proteins, which together promote the localisation of the kinase in the cytoplasm. Interestingly, whereas the SRPK family of kinases is evolutionarily conserved from yeast to human, the spacer sequences in them are highly divergent (Siebel et al., 1999), suggesting that spacer sequences in SRPKs may have coevolved with multiple interaction partners in different species and that SRPK family members may be uniquely regulated via their spacer sequences.
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) and the localization of the RNA binding proteins in stress granules in the cytoplasm (van der Houven van Oordt et al., 2000; Allemand et al., 2005). However, none of these treatment conditions were found to alter the localization of SRPK1 in HeLa cells (our unpublished data). It was reported earlier that Dsk1 entered to the nucleus in the M phase during the cell cycle in fission yeast, indicating that a cell cycle signal may trigger the translocation of the kinase to the nucleus to facilitate cell cycle progression at the M phase (Takeuchi and Yanagida, 1993). To determine whether SRPKs in mammalian cells were able to respond to cell cycle signaling, we examined SRPK1 localization in synchronized HeLa cells (Figure 7). On release of thymidine-blocked HeLa cells at different time points, we observed that a significant fraction of SRPK1 became accumulated in the nucleus before the beginning of the M phase (compare SRPK1 localization in cells released from the thymidine block by 7 and 7.5 h in Figure 7, A-D).
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Because nuclear envelop breaks down during the cell cycle in mammalian cells (which does not happen in fission yeast), the localization experiment during the G2 to M transition requires careful monitoring of the integrity of the nuclear envelop to determine whether SRPK1 entered the nucleus before nuclear envelop breakdown in response to cell cycle signaling. We accomplished this by monitoring the structure of nuclear lamins. As shown in Figure 7, E-H, we detected nuclear translocation of SRPK1 at the late G2 phase (7.5-7.7 h after releasing from the thymidine block)when nuclear envelope was still intact. To make sure that the kinase was not merely piled up on top of the nuclear envelop, we conducted deconvolution microscopy, and the data clearly showed the presence of the kinase within the nucleus (Figure 7, I-L). These data demonstrate that SRPK1 is able to respond to cell cycle signaling to enter the nucleus during the cell cycle. It will be interesting to determine in the future how SRPK1 and other SRPK family members may respond to specific signaling in interphase cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Yun et al., 2000, 2003). Second, a fraction of SRPKs is also detected in the nucleus where they may join other SR protein kinases such as the Clk/Sty family members to regulate the function of SR proteins in many different steps from splicing to export. Because both hypo- and hyperphosphorylation of SR proteins are harmful to splicing, the kinase activity has to be regulated. Here, we show that the presence of a large amount of active SRPK1 in the nucleus after deleting the regulatory spacer caused aggregation of splicing factors, which likely leads to general inhibition of gene expression. One of the key aspects affected may be at the transition from splicing to export because dephosphorylation of shuttling SR proteins is required for interaction with the key export factor TAP (Huang et al., 2003, 2004; Lai and Tarn, 2004). It is thus conceivable that inhibition of SR protein dephosphorylation in the presence of an excessive amount of an SR protein kinase would be detrimental to the orderly transition from splicing to export. Finally, localization of SRPKs in the cytoplasm may also play a key role in releasing shuttling SR proteins from exported mRNA and for proper recycling of shuttling SR proteins back to the nucleus (Gilbert and Guthrie, 2004). Together, these works clearly demonstrate a strategic role for the localization of SRPKs in the cytoplasm. We now provide key evidence that such cellular partitioning is mainly achieved by the action of the unique spacer sequences within the kinases in mammalian cells.
Potential Import Mechanism for SRPKs
By immunocytochemistry, bulk of the endogenous SRPK1 is found in the cytoplasm with a fraction of the kinase in the nucleus, indicating that SRPKs are able to enter the nucleus. Nuclear import of SRPK1 is accelerated when the "anchoring signal" in the spacer is removed. Interestingly, nuclear accumulation of the spacer-deleted SRPK1 depends on its kinase activity, which suggests a potential mechanism for how the kinase might be imported. Proteins may be imported by diffusion (if the size is small enough), by receptor-mediated nuclear transport, or by a so-called piggy-back mechanism. The diffusion mechanism clearly does not apply to SRPK1 as the kinase is a large protein of 655 amino acids, migrating as an 90-kDa protein in SDS-PAGE (Gui et al., 1994). We considered the possibility for receptor-mediated import of SRPK1. Our current results show that the kinase core without the spacer is sufficient to enter the nucleus, but its import depends on the kinase activity. This observation indicates that the kinase core, but not the spacer, may carry an NLS. If this is the case, such a putative NLS has to be fully exposed in the active kinase and become tightly masked in the inactive kinase containing a point mutation in the ATP binding site. We considered this scenario less likely because the mutation in the ATP binding site is not expected to cause a major conformation change in the kinase. Therefore, the piggy-back mechanism becomes a viable choice and is most consistent with the observations we have made so far.
SRPK1 is known to bind tightly to SR protein substrates via a mitogen-activated protein kinase kinase insert in the kinase (Ngo et al., 2005). In addition, RS domain phosphorylation is catalyzed by SRPK1 in a processive manner, which seems to be mediated by a docking motif upstream the RS domain (Aubol et al., 2003; Ngo et al., 2005). As a result, the substrate remains docked on the kinase without being released during or even after phosphorylation. Because only phosphorylated RS domain in typical SR proteins is able to interact with hMtr10 (Lai et al., 2000; Yun et al., 2003), the interaction may result in the formation of a tripartite complex, which is competent for nuclear import. By this mechanism, the kinase may be piggy-backed to the nucleus in a manner that is dependent on the phosphorylation state of its substrates. The spacer may carry a dominant signal to prevent efficient nuclear translocation of SRPK1, thus keeping a balanced distribution of the kinase in the cell. When the spacer is removed, increased binding and/or decreased anchoring may result in efficient import of the kinase to the nucleus.
In contrast to nuclear import, nuclear export of the spacer-deleted kinase may be because of the association of the kinase with shuttling SR proteins. Shuttling SR proteins are dephosphorylated after splicing and remain associated with spliced mRNA during mRNA export (Huang et al., 2003, 2004; Lai and Tarn, 2004; Lin et al., 2005). SRPK1 may interact with a fraction of dephosphorylated shuttling SR proteins during the transition from splicing to export. Because export of shuttling SR proteins actually requires them in a hypophosphorylation state (Lin et al., 2005), the associated SRPK1 may be exported as part of the ribonucleoprotein complex to the cytoplasm.
Signals in the Spacer for Cytoplasmic Localization of SRPK1
It is clear that the spacer in the kinase plays a key regulatory role. Our kinetic analysis indicates that removal of the spacer modestly increased the stability of the kinase-substrate complex, which may contribute to nuclear import of the spacer-deleted kinase via a potential piggy-back mechanism. This model is consistent with the observation that overexpression of ASF/SF2 increased nuclear localization of the full-length kinase in transfected cells (Ngo et al., 2005). However, the ASF/SF2 induced shift was not as quantitative as we detected with the spacer-deleted kinase. Thus, although a modest enhancement of the kinase-substrate stability may have helped nuclear translocation of the kinase upon the removal of the spacer, it is unlikely to count for the quantitative shift observed. Furthermore, it is unlikely that SR protein synthesis and/or shuttling are significantly elevated at the end of the G2-phase to massively induce the nuclear translocation of the endogenous kinase.
A previous study in fission yeast indicated an active NES in the spacer of kinase Dsk1 (Fukuda et al., 1997). By a number of criteria, we could not detect any NES activity in the spacer of SRPK1. We therefore considered some sort of anchoring mechanism for the localization of SRPK1 in the cytoplasm of mammalian cells. Consistent with this possibility, we found multiple elements in the spacer that seem to act in a combinatory manner to cause the localization of the kinase in the cytoplasm. Strikingly, although the spacers of SRPK1 and SRPK2 share little homologue in their sequences, they seem to function interchangeably in the regulation of kinase cellular partitioning. Interestingly, we also found that the full-length SRPK1 did not efficiently export out of the nucleus in comparison with the spacer-deleted SRPK1. One explanation might be that the spacer may also exert some anchoring effect in the nucleus by engaging in interactions with some nonshuttling nucleus proteins (Nikolakaki et al., 2001). Future studies are required to identify specific spacer binding proteins in order to formally test these potential mechanisms.
Function and Regulation of SRPK1 beyond Splicing?
The finding that SRPKs can translocate to the nucleus in response to a cell cycle signal indicates that the kinase system has the potential to be regulated by signaling in interphase cells. How SRPK1 may respond to signals remains unknown and represents an interesting subject for future studies. Although SRPKs seem to be constitutive kinases, a recent study indicates that SRPK1 may be further activated by CK2 (Mylonis and Giannakouros, 2003). However, it remains to be determined whether CK2 has any effect on nuclear translocation of the kinase.
The functional aspects of nuclear translocation of SRPK1 in the end of the G2 phase are also interesting, which may or may not relate to phosphorylation regulation of pre-mRNA processing. In keeping with this possibility, SRPK1 has been shown to phosphorylate substrates that are not related to pre-mRNA splicing. For example, SRPK1 has been found to phosphorylate a RS repeat region in the lamin B receptor, and SRPK1-mediated phosphorylation seems to play a role in the attachment of the lamin B receptor to chromatin (Takano et al., 2004). This function may require the translocation SRPK1 to the nucleus before initiation of the M phase.
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ACKNOWLEDGMENTS |
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Footnotes |
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These authors contributed equally to this work. 
Address correspondence to: Xiang-Dong Fu (xdfu{at}ucsd.edu).
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REFERENCES |
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) and K1-
). For SRPK1, kcat and Km are 9.5 ± 0.5 min-1 and 190 ± 40 nM, respectively, and for K1-
), and 360 nM (
) K1-
