From the Department of Molecular Biology, Beckman Research
Institute of the City of Hope, Duarte, California 91010 and the
Department of Biophysics, Faculty of Science, Kyoto
University, Kyoto 606, Japan
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ABSTRACT |
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Intricate interplay may exist between pre-mRNA splicing and the cell division cycle, and fission yeast Dsk1 appears to play a role in such a connection. Previous genetic analyses have implicated Dsk1 in the regulation of chromosome segregation at the metaphase/anaphase transition. Yet, its protein sequence suggests that Dsk1 may function as a kinase specific for SR proteins, a family of pre-mRNA splicing factors containing arginine-serine repeats. Using an in vitro system with purified components, we showed that Dsk1 phosphorylated human and yeast SR proteins with high specificity. The Dsk1-phosphorylated SF2/ASF protein was recognized strongly by a monoclonal antibody (mAb104) known to bind the in vivo phosphoepitope shared by SR proteins, indicating that the phosphorylation sites resided in the RS domain. Moreover, the fission yeast U2AF65 homolog, Prp2/Mis11 protein, was phosphorylated more efficiently by Dsk1 than by a human SR protein-specific kinase, SRPK1. Thus, these in vitro results suggest that Dsk1 is a fission yeast SR protein-specific kinase, and Prp2/Mis11 is likely an in vivo target for Dsk1. Together with previous genetic data, the studies support the notion that Dsk1 may play a role in coordinating pre-mRNA splicing and the cell division cycle.
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INTRODUCTION |
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Many cellular processes are regulated coordinately with the progression of the cell division cycle (e.g. Refs. 1 and 2), and RNA splicing is likely to be included in this regulation. For example, RNA splicing may be down-regulated in the cell when transcription is repressed during mitosis and up-regulated when RNA precursors are produced actively during cell growth (3). Indeed, evidence indicates that the pre-mRNA splicing apparatus is disassembled during mitosis and has to be reassembled when cells exit mitosis (4). Because protein phosphorylation is involved in the regulation of virtually every aspect of cellular processes (5, 6), we decided to look for protein kinases in fission yeast which are involved in regulating splicing during the cell cycle. The Dsk1 kinase in Schizosaccharomyces pombe is an excellent candidate because it has a role in cell cycle progression (7), and it is homologous to a human SR protein kinase (8). Compared with budding yeast, fission yeast (S. pombe) has the advantage of being more similar to higher eukaryotes especially with regard to the appearance of introns in protein-encoding genes (9-11).
The dsk1+ gene was originally identified as a multicopy suppressor of cold-sensitive dis1 mutants (7). dis1 mutants are defective in sister chromatid separation at the restrictive temperature, and mitosis never reaches completion in these mutants (12). The Dis1 protein is associated with microtubules and the spindle pole body and probably is phosphorylated by Cdc2 kinase (13). dsk1+ gene is not essential for viability, probably because of a redundancy in its function in fission yeast, but overexpression of dsk1+ results in a delay at the G2/M phase transition (7). dsk1+ encodes a 61-kDa protein kinase, but its in vivo substrate has yet to be identified. Although dis1 mutants are suppressed by increasing the expression level of dsk1+ (7), the Dis1 protein is unlikely as a substrate for the Dsk1 kinase because dsk1+ also suppresses a null allele of dis1.1 Dsk1 itself becomes highly phosphorylated at mitosis, and the Dsk1 protein isolated from mitotic cells is more active in phosphorylating myelin basic protein (MBP)2 in vitro (7). Interestingly, the localization of Dsk1 is also cell cycle-dependent; Dsk1 is localized in the cytoplasm during interphase, but it is found mostly in the nucleus at mitosis. Hence the Dsk1 protein may play a role in mitotic control by altering its cellular location and its target proteins.
The sequence similarity between Dsk1 and human SRPK1 (SR protein kinase 1) suggests that the in vivo substrates for Dsk1 may be proteins containing serine/arginine repeats. SRPK1 specifically phosphorylates a family of pre-mRNA splicing factors called SR proteins, at their arginine/serine-rich domain, the RS domain (8, 14). SR proteins are involved in constitutive splicing (15, 16) as well as being specific modulators in alternative splicing (17). In vivo, SR proteins are phosphorylated, predominantly on serine residues in the RS domain (18, 19). The phosphorylation of SR proteins apparently is not only important for the splicing reaction itself but also affects the location of the SR proteins within the nucleus. A cycle of phosphorylation-dephosphorylation of splicing factors is necessary for splicing to take place (20, 21). The stage-dependent sensitivity of pre-mRNA splicing to phosphatases and phosphatase inhibitors observed in mammalian nuclear extracts may simply reflect the dynamics in differential phosphorylation of SR proteins in one round of the splicing reaction. Recent evidence demonstrates that phosphorylation of SR proteins enhances protein-protein interactions while also inhibiting nonspecific interactions with RNA (22). The phosphorylation cycle of splicing factors is reminiscent of the mutually antagonistic kinase and phosphatase systems operating cell cycle control (23, 24). Thus, the cellular localization and possibly the activity of SR proteins are regulated during the cell cycle by their phosphorylation status in a stage-specific manner (4).
Prp2 protein is the only SR-like protein identified so far which is required for pre-mRNA splicing in S. pombe. The prp2+ gene encodes a homolog of the 65-kDa subunit of human splicing factor U2AF (25), and human U2AF65 protein is a good substrate for SRPK1 in vitro (8). A prp2 mutant was identified initially as a temperature-sensitive mutant defective in pre-mRNA splicing (26). Interestingly, another prp2 mutant allele, mis11-453, was isolated by a screen for mutants impaired in chromosome segregation with a high rate of minichromosome loss (27). Progression through G1 and G2 phases is blocked in mis11 mutant cells which leads to reduced cell size. Thus, the Prp2/Mis11 protein is a very attractive candidate as a substrate for Dsk1.
The fission yeast Dsk1 protein is of particular interest because of its possible function in coordinating pre-mRNA splicing with the progression of the cell division cycle. It may provide a model system to unravel, at the molecular level, the mechanism for synchronous regulation between pre-mRNA splicing and the cell division cycle. To determine whether Dsk1 is a functional homolog of human SRPK1 in fission yeast, we purified recombinant Dsk1 and various SR proteins to characterize the kinase properties of Dsk1. Using a cell-free assay we established biochemically that Dsk1 is an SR protein-specific kinase in fission yeast. The results also provided the first evidence that the essential splicing factor in S. pombe, the Prp2/Mis11 protein, is likely an in vivo target for Dsk1 function. Our studies, combined with the previous genetic data, suggest that Dsk1 may be a dual functional protein involved in both pre-mRNA splicing and the cell division cycle.
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EXPERIMENTAL PROCEDURES |
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Plasmid Construction-- To construct pET-28a dsk1+ encoding Dsk1 with a histidine tag at the NH2 terminus, a fragment of 340 base pairs from the 5'-portion of dsk1+ (7) was synthesized using two primers (5'-GCCAGCCATGGATCCATGGGAAGTG-3', including a BamHI site, and 5'-GGCAGCTCGATCATATGCAAGCCAAAC-3', including the unique internal NdeI site) in a polymerase chain reaction (28). The polymerase chain reaction fragment was inserted into the PCR II vector (Invitrogen). The NdeI-EcoRV fragment of 1,400 base pairs from pDS113-6 (7), including the remainder of dsk1+, was added to regenerate the entire dsk1+ coding sequence. The BamHI/NotI fragment containing the constructed dsk1+ was then inserted into pET-28a (Novagen) to generate pET-28a dsk1+. To construct pET-28b GST-prp2+ encoding Prp2 fused at the COOH terminus of glutathione S-transferase (GST), the NcoI-NdeI fragment in pET-28b (Novagen) containing the histidine tag was replaced by the GST sequence from pET-14b GST-SF2/ASF (from Xiang-Dong Fu). An NdeI-BamHI fragment containing the prp2+ sequence in pPrp2/RK171a (from Judith Potashkin) was then inserted to generate pET-28b GST-prp2+.
Production and Purification of Recombinant
Proteins--
Escherichia coli strain BL21(DE3)pLysS (29)
carrying the plasmid of interest was grown to an
A600 of 0.3-0.5 at 37 °C in LB containing 30 µg/ml chloramphenicol plus 30 µg/ml kanamycin for pET-28a
dsk1+ and pET-28b
GST-prp2+ or plus 50 µg/ml carbenicillin for
pET-14b GST-SF2/ASF. The culture was induced in the presence of 0.4 mM isopropyl -D-thiogalactopyranoside at
30 °C for 3 h (30). Cells were then centrifuged and washed with
buffer A (10 mM Tris-HCl, pH 7.4, 100 mM NaCl,
1 mM MgCl2, 1 mM
-mercaptoethanol for histidine-tagged proteins or 1 mM
dithiothreitol for GST fusion proteins). The cell pellet was
resuspended at ~0.1 g of cells/ml or <1010 cells/ml in
buffer B (50 mM Tris-HCl, pH 7.4, 100 mM NaCl,
5% glycerol, 5 mM EGTA for histidine-tagged proteins or 2 mM EDTA for GST fusion proteins, 1 mM
-mercaptoethanol for histidine-tagged proteins or dithiothreitol for
GST fusion proteins) plus protease inhibitors (5 µg/ml of pepstatin,
5 µg/ml of chymostatin, 5 µg/ml of leupeptin, 1 mM
phenylmethylsulfonyl fluoride, and 25 µg/ml aprotinin). The cells
were lysed by repetitive freezing and thawing followed by sonication
using a sonicator (Polytron, Kinematica AG, Switzerland) for 30 s
at setting 5. The lysate was centrifuged at 15,000 rpm for 15 min in an
SA600 rotor (Sorvall) to separate the soluble fraction (supernatant)
and inclusion bodies (pellet). The inclusion bodies were washed in
buffer A, suspended in buffer B containing 1% Nonidet P-40 and the
protease inhibitors (at 20-30 mg of the starting cell mass/ml of
buffer), and sonicated.
Antibodies-- A peptide corresponding to the COOH-terminal end of Dsk1, ATGEDVPGWATEIR, was conjugated to keyhole limpet hemocyanin, and the conjugate was used for immunization of rabbits (33). The anti-peptide antibodies were purified by affinity chromatography using the peptide coupled to Affi-Gel 15 resin (Bio-Rad) (34). mAb104 was isolated from hybridoma cells (American Type Culture Collection CRL 2067) (33). Anti-GST polyclonal antibodies were from Santa Cruz Biotechnology. Anti-SF2/ASF monoclonal antibody was from Adrian Krainer.
Kinase Assay--
Purified kinase was incubated at 23 °C for
30 min with the substrate in a total volume of 20 µl containing a
kinase buffer (50 mM Tris-HCl, pH 7.4, 10 mM
MgCl2, 1 mM dithiothreitol) in the presence of
50 µM ATP and 2 µCi of [-32P]ATP. The
kinase reaction was terminated by boiling in SDS sample buffer, and the
sample was resolved on a 10% or 12% SDS-polyacrylamide gel. Protein
phosphorylation was detected by autoradiography. For Western blot
analysis, the kinase reaction was performed by employing an
ATP-regenerating system (10 mM creatine phosphate, 1 mM ATP, and 0.1 mg/ml creatine phosphokinase) without
radioisotopes. Immunoblotting was performed as described (33, 35).
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RESULTS |
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The Dsk1 Kinase Has Distinct Substrate Specificity for SR Proteins-- Full-length Dsk1 protein was produced as a fusion construct with an NH2-terminal tag of 6 histidine residues (designated His6-Dsk1) in E. coli. The histidine-tagged Dsk1 protein in the soluble fraction of the bacterial lysate was bound to nickel-IDA agarose and eluted with buffers containing 150-200 mM imidazole. The His6-Dsk1 protein was recognized by polyclonal antibodies against its COOH-terminal peptide (ATGEDVPGWATEIR) as a 65-kDa protein (data not shown).
To assess whether the purified fusion His6-Dsk1 protein was catalytically active, we examined whether it was capable of phosphorylating MBP. Dsk1 was incubated with MBP in the presence of [
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Phosphorylation by Dsk1 Generates a Phosphoepitope Shared by Native
SR Proteins--
We showed above that Dsk1 phosphorylated various SR
proteins having budding yeast to human origins with a specificity and efficiency similar to human SRPK1. SRPK1, as well as Clk/Sty, not only
phosphorylates serine in the RS domain in vitro, but also
the pattern of phosphorylation closely resembles that occurring in vivo (19). A unique feature of the phosphorylation by
SRPK1 or Clk/Sty is the formation of a phosphoepitope within the RS domain, which can be recognized by mAb104 monoclonal antibody, which
was found originally to recognize a specific phosphoepitope present in
native SR proteins isolated from mammalian cells (45). To provide
further direct biochemical evidence that Dsk1 is an SR protein-specific
kinase, we asked whether Dsk1 produces the mAb104-reactive
phosphoepitope upon phosphorylating SR proteins. In the experiment
depicted in Fig. 3A, SF2/ASF
or SF2RS was incubated with Dsk1 or SRPK1 in the presence of an
ATP-regenerating system. The samples were then analyzed by Western
blotting with anti-Dsk1 antibody (Fig. 3A, top
panel), mAb104 antibody (middle panel), or anti-SF2/ASF
antibody (bottom panel) (lanes 5-8). Control
samples were also analyzed in parallel (Fig. 3A, lanes
1-4). Note that probing the samples with anti-SF2/ASF antibody
revealed that phosphorylation of SF2/ASF by both kinases caused a
slight up-shift in gel mobility (Fig. 3A, bottom
panel, lanes 5 and 7). Only two reactions in which full-length SF2/ASF was incubated with either SRPK1 or His6-Dsk1 resulted in the formation of an mAb104-reactive signal (Fig.
3A, lanes 5 and 7). Neither the
unphosphorylated SF2/ASF (Fig. 3A, middle panel,
lane 4) nor the SF2
RS incubated with either kinase (lanes 6 and 8) was recognized by mAb104. Thus,
the phosphoepitope generated by Dsk1 resided in the RS domain and was
the same as that formed by SRPK1. These results allow us to argue
strongly that Dsk1 is an SR protein-specific kinase in fission
yeast.
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Dsk1 Phosphorylates the Fission Yeast Prp2 Protein in
Vitro--
What is the in vivo target for Dsk1 in fission
yeast? So far, Prp2 is the only protein identified in fission yeast
which contains an RS domain and functions in pre-mRNA splicing
(39). The protein sequence of Prp2 bears extensive similarity to
mammalian splicing factor U2AF65 (25). The human U2AF65 has been shown
to be a good substrate for both SRPK1 and Clk/Sty in vitro
(8, 19). Therefore, we decided to test the phosphorylation of Prp2
protein by Dsk1 using the cell-free kinase assay system. Unlike the
recombinant His6-Dsk1 and GST-SF2/ASF proteins, the expression level of
recombinant Prp2 protein in E. coli was quite low (Fig.
4B, lanes 3 and
4). Thus, we examined whether the unpurified fission yeast
Prp2 protein in the bacterial lysate could be phosphorylated by Dsk1.
Lysates (soluble and inclusion body fractions) from bacteria
transformed with a plasmid encoding the prp2+
gene or the vector alone were incubated separately with SRPK1 (Fig.
4A, lanes 4-7) or His6-Dsk1 (lanes
10-13) in the presence of [-32P]ATP. A
phosphorylated protein with the apparent molecular mass expected for
Prp2 protein (~60 kDa) was observed specifically in the lysates from
the bacteria containing the prp2+ gene (Fig.
4A, lanes 4, 5, 10, and
11). Two bacterial proteins (one of which is indicated by
the lower arrow in Fig. 4, A and B)
were phosphorylated independent of SRPK1 or Dsk1. Interestingly, another bacterial protein (indicated by the top arrow in
Fig. 4, A and B) was specifically labeled with
32P upon incubation with SRPK1 (Fig. 4A,
lanes 4-7) or His6-Dsk1 (lanes 10-13). The
identity of this bacterial protein was not investigated, but it
provided an internal control for comparing the specific activity of the
two kinases: the phosphorylation intensity of the bacterial protein was
higher than that of Prp2 in the case of SRPK1 (Fig. 4A,
lanes 4 and 5), but the intensity of the two was
quite similar in the case of Dsk1 (lanes 11 and 12). These observations suggested that Dsk1 has a higher
specific activity toward Prp2 than does SRPK1 and that Prp2 may be an
in vivo substrate for Dsk1 in fission yeast.
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DISCUSSION |
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In this study, we have purified recombinant forms of both Dsk1 and Prp2/Mis11 proteins of fission yeast S. pombe and characterized the potential biochemical pathway linking these two proteins. Using a cell-free assay, we have demonstrated that Dsk1 phosphorylates SR proteins with high specificity, and Prp2 is the best substrate among various SR proteins tested. This study has provided the first molecular evidence that Dsk1 is an SR protein kinase in fission yeast. These data also reveal the possible role of Dsk1 in vivo and suggest that Dsk1 may act directly on Prp2 protein by phosphorylation, thus affecting pre-mRNA splicing. In addition to its mitotic role in cell cycle regulation implicated by previous genetic studies, our results argue that Dsk1 may have dual functions in both pre-mRNA splicing and the cell division cycle.
Interestingly, many observations indicating the possible interplay between pre-mRNA splicing and the cell division cycle are obtained in fission yeast, more so than in budding yeast (for discussion, see Refs. 11 and 46). Out of 14 pre-mRNA processing (prp) mutants so far identified in fission yeast, 12 of them exhibit a cell division cycle (cdc) phenotype (11). One remarkable example is the two allelic genes, cdc28 and prp8, which were independently discovered as a cdc and a prp mutant, respectively; yet each mutant displays both cdc and prp phenotypes (46). This may be attributed partly to the much higher intron content in the genome of S. pombe than that of S. cerevisiae (11). It is not surprising therefore that any abnormality in pre-mRNA splicing or in the regulation of pre-mRNA splicing may be manifested in fission yeast more readily than in budding yeast. Additionally, progression through the cell division cycle in fission yeast can be monitored easily by the size of the cell; therefore, perturbations in the cell cycle control points, particularly the G2/M phase transition, can be conveniently identified.
Although the present investigation has established that the fission yeast Dsk1 protein functions as an SR protein-specific kinase in vitro, several important questions remain to be addressed. What are the physiological consequences of the phosphorylation by Dsk1 in pre-mRNA splicing and cell cycle control? Is the role of Dsk1 protein in cell cycle regulation executed through the splicing pathway or by acting directly on the cell cycle control system at metaphase?
To answer these questions, it is necessary to determine in vivo targets of Dsk1 protein in fission yeast. Although dis1 mutants are suppressed by increasing the expression level of dsk1+ (7), the Dis1 protein is unlikely to be a substrate for the Dsk1 kinase because dsk1+ also suppresses a null allele of dis1.1 It is interesting to note that an intron is present in the dis1 gene (13). On the other hand, the Prp2/Mis11 protein is a very attractive candidate as an in vivo substrate for Dsk1. Besides being phosphorylated in vitro by Dsk1 with high specificity based on our biochemical data, Prp2/Mis11 protein also seems to possess a dual functional feature with respect to its role in both pre-mRNA splicing and the cell division cycle (see the Introduction). Although prp2+ and mis11+ are in the same gene, a gene homologous to the human splicing factor U2AF65, the effect of the mis11-453 mutation on pre-mRNA splicing has not been examined. To assess whether pre-mRNA splicing is defective, the mis11 mutant was shifted to the restrictive temperature, and the total RNA was isolated for Northern blot analysis. A block of pre-mRNA splicing in the mis11 mutant was detected3 which is similar to that observed in prp2 mutants (26). Thus, the mis11-453 mutation not only causes minichromosome loss at mitosis and cell growth block in interphase (27) but also abolishes pre-mRNA splicing at the restrictive temperature. The splicing defect observed in mis11 mutant cells, together with previous genetic and cellular studies, again is consistent with our hypothesis that Dsk1 and Prp2/Mis11 protein may be interrelated in pathways connecting pre-mRNA splicing to the cell division cycle in fission yeast. It remains to be investigated whether there is a cause-consequence relationship between splicing and chromosome loss or whether Prp2/Mis11 uniquely has a dual role in these two cellular processes.
Could dsk1 regulate pre-mRNA splicing and cell cycle progression through a pathway other than or in addition to affecting splicing activity per se? It is formally possible that an RS domain could also exist in proteins involved in other cellular processes in fission yeast, which are substrates for Dsk1. Interestingly, a large cyclophilin protein homologous to a natural killer tumor recognition protein has been found to contain an RS domain (47). Nonetheless, it is also conceivable that the splicing apparatus, as an indispensable part of proper nuclear architecture, may undergo stage-specific changes during the cell cycle; that is, the splicing machinery may be structurally and functionally distinct at mitosis and in the interphase. The Dsk1 protein may facilitate these changes in a cell cycle-dependent fashion. Specifically, at the metaphase/anaphase transition, Dsk1 may prepare the nuclear structure for the exit from mitosis by interacting with factors involved in chromosome segregation and components of the splicing machinery. This hypothesis would predict that if the concentration of Dsk1 protein in the cell is too high, the nuclear architecture may be retained in the interphase configuration and consequently prevent the cells from entering mitosis. Consistent with this theory, overexpression of the dsk1+ gene leads to a delay at G2/M phase transition (7).
Cell viability is not affected by disruption of the dsk1+ gene (7). It may simply reflect the fact that additional protein kinases exist in fission yeast with an overlapping function to Dsk1. Four kinases involved in SR protein phosphorylation have been identified, and two of them have been cloned (for review, see Refs. 39 and 40). In the mammalian system, it has been demonstrated that in addition to SRPK1, the Clk/Sty protein (Cdc28/Cdc2-like kinase) also phosphorylates SR proteins and controls the distribution of SR proteins within the nucleus (19, 43). The Clk/Sty family of protein kinases is conserved through evolution from yeast to human (19). The genome project of fission yeast reveals that the ka23+ gene may be one of the members of this family based on the sequence similarity (48). Another candidate is the fission yeast Prp4 protein (49) which has been shown recently to phosphorylate human SF2/ASF protein in vitro (50). Because Dsk1 protein appears to be distributed in the cytoplasm during interphase (7), a Dsk1-like kinase may localize in the nucleus to act on SR proteins for efficient assembly of spliceosome and splicing of pre-mRNA in interphase. On the other hand, we cannot rule out the possibility that in interphase a small amount of Dsk1 is present in the nucleus. In addition, Dsk1 protein is phosphorylated differentially during the cell cycle (7), which may exert another level of regulation.
To investigate further the function of Dsk1, we propose as a model that there may exist a regulatory network of protein kinases and phosphatases to couple pre-mRNA splicing to cell cycle control in fission yeast (Fig. 6). The network would consist of a family of Dsk1-like kinases (Dlks) specific for SR proteins, factors to regulate the kinases during the cell cycle, and their substrates in pre-mRNA splicing or in the cell cycle control pathway. As delineated briefly in the model (Fig. 6), the activity, substrate specificity, and intracellular distribution of Dsk1 may be altered by phosphorylation in a cell cycle-dependent manner. More specifically, upon phosphorylation, Dsk1 may favor one substrate over the other and thus, gain different or additional functions. Dsk1 may act by phosphorylating regulatory and structural proteins, such as splicing factors or cell cycle control elements, to closely coordinate pre-mRNA splicing with the cell division cycle. This hypothesis accommodates the possibilities that Dsk1 may have multiple substrates in vivo, and other Dsk1-like protein kinases may exist to overlap with Dsk1 function. New information revealing the mode of Dsk1 action will undoubtedly advance our understanding of the synchronous regulation between pre-mRNA splicing and the cell division cycle.
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ACKNOWLEDGEMENTS |
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We thank Xiang-Dong Fu (University of
California at San Diego) for providing SRPK1 and pET-14b
GST-SF2/ASF; Adrian Krainer (Cold Spring Harbor Laboratory) for
SF2/ASF, SF2RS, SRp30c, SRp40, SRp55, Npl3, and the anti-SF2/ASF
antibody; Akiko Kumagai (California Institute of Technology) for
Cdc2-cyclin B; and Judith Potashkin (Chicago Medical School) for
pPrp2/RK171a. We also thank Melissa Holtz for proofreading the
manuscript and the reviewers for constructive comments.
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FOOTNOTES |
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* This work was supported by the Beckman Research Institute of the City of Hope.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Molecular Biology, Beckman Research Institute of the City of Hope, 1450 E. Duarte Rd., Duarte, CA 91010-3011. Tel.: 626-301-8286; Fax: 626-301-8280; E-mail: rlin{at}smtplink.coh.org.
1 M. Yanagida, unpublished observations.
2 The abbreviations used are: MBP, myelin basic protein; SRPK1, SR protein kinase 1; GST, glutathione S-transferase; mAb, monoclonal antibody.
3 Z. Tang, and R.-J. Lin, unpublished data.
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REFERENCES |
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