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• W2048236220 abstract "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 vitroresults 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. 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 vitroresults 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. Many cellular processes are regulated coordinately with the progression of the cell division cycle (e.g. Refs. 1Koch C. Nasmyth K. Curr. Opin. Cell Biol. 1994; 6: 451-459Crossref PubMed Scopus (204) Google Scholar and 2Stillman B. Science. 1996; 274: 1659-1664Crossref PubMed Scopus (431) Google Scholar), 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 (3Gottesfeld J.M. Forbes D.J. Trends Biochem. Sci. 1997; 22: 197-202Abstract Full Text PDF PubMed Scopus (315) Google Scholar). Indeed, evidence indicates that the pre-mRNA splicing apparatus is disassembled during mitosis and has to be reassembled when cells exit mitosis (4Misteli T. Spector D.L. Trends Cell Biol. 1997; 7: 135-138Abstract Full Text PDF PubMed Scopus (99) Google Scholar). Because protein phosphorylation is involved in the regulation of virtually every aspect of cellular processes (5Hunter T. Cell. 1987; 50: 823-829Abstract Full Text PDF PubMed Scopus (774) Google Scholar, 6Edelman A.M. Blumenthal D.K. Krebs E.G. Annu. Rev. Biochem. 1987; 56: 567-613Crossref PubMed Scopus (1018) Google Scholar), 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 (7Takeuchi M. Yanagida M. Mol. Biol. Cell. 1993; 4: 247-260Crossref PubMed Scopus (64) Google Scholar), and it is homologous to a human SR protein kinase (8Gui J.F. Lane W.S. Fu X.-D. Nature. 1994; 369: 678-682Crossref PubMed Scopus (458) Google Scholar). 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 (9Kaufer N.F. Simanis V. Nurse P. Nature. 1985; 318: 78-80Crossref PubMed Scopus (121) Google Scholar, 10Prabhala G. Rosenberg G. Kaufer N.F. Yeast. 1992; 8: 171-182Crossref PubMed Scopus (90) Google Scholar, 11Urushiyama S. Tani T. Ohshima Y. Mol. Gen. Genet. 1996; 253: 118-127Crossref PubMed Scopus (53) Google Scholar). The dsk1 + gene was originally identified as a multicopy suppressor of cold-sensitive dis1 mutants (7Takeuchi M. Yanagida M. Mol. Biol. Cell. 1993; 4: 247-260Crossref PubMed Scopus (64) Google Scholar).dis1 mutants are defective in sister chromatid separation at the restrictive temperature, and mitosis never reaches completion in these mutants (12Ohkura H. Adachi Y. Kinoshita N. Niwa O. Toda T. Yanagida M. EMBO J. 1988; 7: 1465-1473Crossref PubMed Scopus (167) Google Scholar). The Dis1 protein is associated with microtubules and the spindle pole body and probably is phosphorylated by Cdc2 kinase (13Nabeshima K. Kurooka H. Takeuchi M. Kinoshita K. Nakaseko Y. Yanagida M. Genes Dev. 1995; 9: 1572-1585Crossref PubMed Scopus (127) Google Scholar). 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 (7Takeuchi M. Yanagida M. Mol. Biol. Cell. 1993; 4: 247-260Crossref PubMed Scopus (64) Google Scholar). 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 + (7Takeuchi M. Yanagida M. Mol. Biol. Cell. 1993; 4: 247-260Crossref PubMed Scopus (64) Google Scholar), the Dis1 protein is unlikely as a substrate for the Dsk1 kinase because dsk1 + also suppresses a null allele of dis1. 1M. Yanagida, unpublished observations. Dsk1 itself becomes highly phosphorylated at mitosis, and the Dsk1 protein isolated from mitotic cells is more active in phosphorylating myelin basic protein (MBP) 2The abbreviations used are: MBP, myelin basic protein; SRPK1, SR protein kinase 1; GST, glutathioneS-transferase; mAb, monoclonal antibody. in vitro (7Takeuchi M. Yanagida M. Mol. Biol. Cell. 1993; 4: 247-260Crossref PubMed Scopus (64) Google Scholar). 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 (8Gui J.F. Lane W.S. Fu X.-D. Nature. 1994; 369: 678-682Crossref PubMed Scopus (458) Google Scholar, 14Gui J.F. Tronchere H. Chandler S.D. Fu X.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10824-10828Crossref PubMed Scopus (185) Google Scholar). SR proteins are involved in constitutive splicing (15Zahler A.M. Lanes W.S. Stolk J.A. Roth M.B. Genes Dev. 1992; 6: 837-847Crossref PubMed Scopus (624) Google Scholar, 16Fu X.-D. Nature. 1993; 365: 82-85Crossref PubMed Scopus (232) Google Scholar) as well as being specific modulators in alternative splicing (17Kohtz J.D. Jamison S.F. Will C.L. Zuo P. Luhrmann R. Garcia-Blanco M.A. Manley J.L. Nature. 1994; 368: 119-124Crossref PubMed Scopus (529) Google Scholar). in vivo, SR proteins are phosphorylated, predominantly on serine residues in the RS domain (18Roth M.B. Zahler A.M. Stolk J.A. J. Cell Biol. 1991; 115: 587-596Crossref PubMed Scopus (267) Google Scholar, 19Colwill K. Pawson T. Andrews B. Prasad J. Manley J.L. Bell J.C. Duncan P.I. EMBO J. 1996; 15: 265-275Crossref PubMed Scopus (473) Google Scholar). 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 (20Mermoud J.E. Cohen P.T. Lamond A.I. EMBO J. 1994; 13: 5679-5688Crossref PubMed Scopus (274) Google Scholar, 21Tazi J. Kornstadt U. Rossi F. Jeanteur P. Cathala G. Brunel C. Luhrmann R. Nature. 1993; 363: 283-286Crossref PubMed Scopus (130) Google Scholar). 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 (22Xiao S.H. Manley J.L. Genes Dev. 1997; 11: 334-344Crossref PubMed Scopus (311) Google Scholar). The phosphorylation cycle of splicing factors is reminiscent of the mutually antagonistic kinase and phosphatase systems operating cell cycle control (23Hunt T. Kirschner M. Curr. Opin. Cell Biol. 1993; 5: 163-165Crossref PubMed Scopus (8) Google Scholar, 24Kirschner M. Trends Biochem. Sci. 1992; 17: 281-285Abstract Full Text PDF PubMed Scopus (52) Google Scholar). 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 (4Misteli T. Spector D.L. Trends Cell Biol. 1997; 7: 135-138Abstract Full Text PDF PubMed Scopus (99) Google Scholar). 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 (25Potashkin J. Naik K. Wentz-Hunter K. Science. 1993; 262: 573-575Crossref PubMed Scopus (89) Google Scholar), and human U2AF65 protein is a good substrate for SRPK1 in vitro (8Gui J.F. Lane W.S. Fu X.-D. Nature. 1994; 369: 678-682Crossref PubMed Scopus (458) Google Scholar). A prp2mutant was identified initially as a temperature-sensitive mutant defective in pre-mRNA splicing (26Potashkin J. Li R. Frendewey D. EMBO J. 1989; 8: 551-559Crossref PubMed Scopus (55) Google Scholar). Interestingly, anotherprp2 mutant allele, mis11-453, was isolated by a screen for mutants impaired in chromosome segregation with a high rate of minichromosome loss (27Takahashi K. Yamada H. Yanagida M. Mol. Biol. Cell. 1994; 5: 1145-1158Crossref PubMed Scopus (186) Google Scholar). 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. To construct pET-28adsk1 + encoding Dsk1 with a histidine tag at the NH2 terminus, a fragment of 340 base pairs from the 5′-portion of dsk1 + (7Takeuchi M. Yanagida M. Mol. Biol. Cell. 1993; 4: 247-260Crossref PubMed Scopus (64) Google Scholar) was synthesized using two primers (5′-GCCAGCCATGGATCCATGGGAAGTG-3′, including aBamHI site, and 5′-GGCAGCTCGATCATATGCAAGCCAAAC-3′, including the unique internal NdeI site) in a polymerase chain reaction (28Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). 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 (7Takeuchi M. Yanagida M. Mol. Biol. Cell. 1993; 4: 247-260Crossref PubMed Scopus (64) Google Scholar), including the remainder of dsk1 +, was added to regenerate the entiredsk1 + coding sequence. The BamHI/NotI fragment containing the constructeddsk1 + 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). AnNdeI-BamHI fragment containing the prp2 + sequence in pPrp2/RK171a (from Judith Potashkin) was then inserted to generate pET-28b GST-prp2 +. Escherichia coli strain BL21(DE3)pLysS (29Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6005) Google Scholar) carrying the plasmid of interest was grown to anA600 of 0.3–0.5 at 37 °C in LB containing 30 μg/ml chloramphenicol plus 30 μg/ml kanamycin for pET-28adsk1 + 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 β-d-thiogalactopyranoside at 30 °C for 3 h (30Kumagai A. Dunphy W.G. Cell. 1991; 64: 903-914Abstract Full Text PDF PubMed Scopus (358) Google Scholar). Cells were then centrifuged and washed with buffer A (10 mm Tris-HCl, pH 7.4, 100 mm NaCl, 1 mm MgCl2, 1 mmβ-mercaptoethanol for histidine-tagged proteins or 1 mmdithiothreitol 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 mmβ-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 mmphenylmethylsulfonyl 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. His6-Dsk1 was purified from the soluble fraction by nickel-IDA agarose chromatography (31Kumagai A. Dunphy W.G. Mol. Biol. Cell. 1995; 6: 199-213Crossref PubMed Scopus (110) Google Scholar), whereas GST fusion proteins were purified from both soluble and inclusion body fractions by glutathione-agarose chromatography (32Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar). IDA Sepharose Fast Flow (Pharmacia Biotech Inc.) was charged with 100 mm nickel chloride for 10 min at room temperature and washed extensively with water. The soluble fraction containing His6-Dsk1 was incubated with the nickel-IDA beads at 4 °C for 1 h with constant agitation in the presence of 5 mm EGTA and 10 mm imidazole. The protein-bound beads were packed in a column and washed with TBS (10 mmTris-HCl, pH 7.4, 150 mm NaCl) containing 5 mmEGTA, 20 mm imidazole, and 0.1–0.5% Nonidet P-40. Nonidet P-40 was omitted during the final wash, and His6-Dsk1 was eluted from the column in TBS containing 150–200 mm imidazole. Similarly, GST fusion proteins were bound to glutathione-agarose beads, washed with TBS, and eluted in TBS containing 5 mmglutathione. Purified proteins were aliquotted, frozen in liquid nitrogen, and stored at −80 °C. SRPK1 was a gift of Xiang-Dong Fu. SF2/ASF, SF2ΔRS, SRp30c, SRp40, SRp55, and Npl3 were gifts of Adrian Krainer. 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 (33Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). The anti-peptide antibodies were purified by affinity chromatography using the peptide coupled to Affi-Gel 15 resin (Bio-Rad) (34Kumagai A. Dunphy W.G. Cell. 1992; 70: 139-151Abstract Full Text PDF PubMed Scopus (336) Google Scholar). mAb104 was isolated from hybridoma cells (American Type Culture Collection CRL 2067) (33Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). Anti-GST polyclonal antibodies were from Santa Cruz Biotechnology. Anti-SF2/ASF monoclonal antibody was from Adrian Krainer. 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 mmMgCl2, 1 mm dithiothreitol) in the presence of 50 μm ATP and 2 μCi of [γ-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 (33Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar, 35Tang Z. Coleman T.R. Dunphy W.G. EMBO J. 1993; 12: 3427-3436Crossref PubMed Scopus (149) Google Scholar). 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 [γ-32P]ATP in a kinase buffer. As detected by gel electrophoresis and autoradiography, we observed that MBP was phosphorylated by the His6-Dsk1 (data not shown, but see Fig. 1, lane 10). Autophosphorylation of Dsk1 itself was detected after a longer exposure of the gel to x-ray film (data not shown; but see Fig. 2, lane 3), although the phosphorylation signal was much weaker than that of MBP. Both observations, which are consistent with our previous studies (7Takeuchi M. Yanagida M. Mol. Biol. Cell. 1993; 4: 247-260Crossref PubMed Scopus (64) Google Scholar), indicate that the purified His6-Dsk1 is active as a kinase.Figure 2Phosphorylation of various SR proteins by Dsk1 kinase. SRPK1 (lanes 4–7) or His6-Dsk1 (lanes 8–13) was incubated with various SR proteins, and samples were processed as described in Fig. 1. SF2/ASF protein was used as a positive control (lanes 4 and 8). SRp30c (lanes 6 and 10), SRp40 (lanes 7 and 11), SRp55 (lane 12), and SF2ΔRS (lanes 5 and 9) were tested at 0.5 μm. Budding yeast Npl3 (lanes 13 and 19) was tested at 1 μm. Each protein incubated without kinase served as a negative control (lanes 1–3 and 14–19).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The protein sequence of Dsk1 suggests that it is homologous to human SRPK1, which is an SR protein-specific kinase (8Gui J.F. Lane W.S. Fu X.-D. Nature. 1994; 369: 678-682Crossref PubMed Scopus (458) Google Scholar). To test the functional similarity between the two proteins, we compared the substrate specificity between Dsk1 and SRPK1 in a cell-free kinase assay. As a reference, Xenopus Cdc2-human cyclin B complex (Cdc2-cyclin B) was used in parallel for comparison because all three kinases belong to a superfamily of serine/threonine-specific kinases (7Takeuchi M. Yanagida M. Mol. Biol. Cell. 1993; 4: 247-260Crossref PubMed Scopus (64) Google Scholar, 8Gui J.F. Lane W.S. Fu X.-D. Nature. 1994; 369: 678-682Crossref PubMed Scopus (458) Google Scholar, 36Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Crossref PubMed Scopus (3812) Google Scholar). Cdc2-cyclin B complex is known as the maturation-promoting factor or the major M phase cyclin-dependent kinase for its function in inducing mitosis (37Dunphy W.G. Brizuela L. Beach D. Newport J. Cell. 1988; 54: 423-431Abstract Full Text PDF PubMed Scopus (555) Google Scholar, 38Nasmyth K. Science. 1996; 274: 1643-1645Crossref PubMed Scopus (324) Google Scholar). SR proteins are phosphoproteins in vivo and contain Cdc2 phosphorylation consensus sequences, (S/T)-P-X-(R/K); however, none of the SR proteins tested so far is an in vitro substrate for Cdc2 (8Gui J.F. Lane W.S. Fu X.-D. Nature. 1994; 369: 678-682Crossref PubMed Scopus (458) Google Scholar). Instead, SR proteins are phosphorylated by human SRPK1 and Clk/Sty in a highly specific and efficient manner (39Fu X.D. RNA. 1995; 1: 663-680PubMed Google Scholar, 40Manley J.L. Tacke R. Genes Dev. 1996; 10: 1569-1579Crossref PubMed Scopus (601) Google Scholar). Four proteins were tested as substrates in our assay: human SF2/ASF as an SR protein (39Fu X.D. RNA. 1995; 1: 663-680PubMed Google Scholar, 40Manley J.L. Tacke R. Genes Dev. 1996; 10: 1569-1579Crossref PubMed Scopus (601) Google Scholar, 41Valcarcel J. Green M.R. Trends Biochem. Sci. 1996; 21: 296-301Abstract Full Text PDF PubMed Google Scholar) in the form fused with GST (GST-SF2/ASF), bovine MBP, histone H1, and casein as serine/threonine-containing polypeptides. Histone H1 is typically used as a standard substrate for assaying the kinase activity of maturation-promoting factor (42Dunphy W.G. Newport J.W. Cell. 1989; 58: 181-191Abstract Full Text PDF PubMed Scopus (201) Google Scholar). As expected, MBP, histone H1, and casein served as good substrates for the Cdc2-cyclin B complex (Fig. 1, lanes 14–16), whereas GST-SF2/ASF was not phosphorylated by Cdc2-cyclin B (Fig. 1, lane 13). In contrast, SRPK1 and His6-Dsk1 did not act on histone H1 or casein (Fig. 1, lanes 7, 8, 11, and 12). Dsk1 displayed a relatively moderate kinase activity toward MBP (Fig. 1, lane 10) compared with SRPK1 (lane 6) or Cdc2-cyclin B (lane 14). Importantly, His6-Dsk1 phosphorylated GST-SF2/ASF protein very well (Fig. 1, lane 9), as did SRPK1 (lane 5). The specificity of SRPK1 and Dsk1 for GST-SF2/ASF is significant considering that GST-SF2/ASF (0.37 μm) was present at a level that was at least 20-fold less than the other three polypeptides (∼10 μm). Note that the concentrations of SF2/ASF and the other three substrates used in the assay are based on the standard assay conditions described previously (42Dunphy W.G. Newport J.W. Cell. 1989; 58: 181-191Abstract Full Text PDF PubMed Scopus (201) Google Scholar, 43Colwill K. Feng L.L. Yeakley J.M. Gish G.D. Caceres J.F. Pawson T. Fu X.-D. J. Biol. Chem. 1996; 271: 24569-24575Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). To examine further the specific activity of the Dsk1 kinase, we extended the list of substrates and included four additional human SR proteins in our assay (SF2/ASF, SRp30c, SRp40, and SRp55) (44Screaton G.R. Caceres J.F. Mayeda A. Bell M.V. Plebanski M. Jackson D.G. Bell J.I. Krainer A.R. EMBO J. 1995; 14: 4336-4349Crossref PubMed Scopus (246) Google Scholar). His6-Dsk1 phosphorylated SF2/ASF, SRp40, and SRp55 (Fig. 2, lanes 8, 11, and 12). In agreement, SRPK1 also phosphorylated SF2/ASF and SRp40 (Fig. 2, lanes 4 and 7). Interestingly, both kinases displayed very little activity toward SRp30c (Fig. 2, lanes 6 and 10). These results indicated the similarity between the two kinases in their substrate specificity. When SRp40 was used as substrate, an additional phosphorylated protein was detected which migrated in the gel considerably slower than SRp40 itself (Fig. 2, lanes 7 and 11). The nature of this high molecular weight protein is not clear. However, because SR proteins tend to aggregate without being phosphorylated (39Fu X.D. RNA. 1995; 1: 663-680PubMed Google Scholar), and the size of the slow migrating protein was about twice that of the recombinant SRp40, one likely explanation is that it might result from the association of two SRp40 proteins. The SRp55 used was a truncated version isolated from E. coli, which was missing a portion of its COOH-terminal domain (44Screaton G.R. Caceres J.F. Mayeda A. Bell M.V. Plebanski M. Jackson D.G. Bell J.I. Krainer A.R. EMBO J. 1995; 14: 4336-4349Crossref PubMed Scopus (246) Google Scholar). In addition to mammalian SR proteins, we also tested an SR protein from budding yeast Saccharomyces cerevisiae, Npl3 (39Fu X.D. RNA. 1995; 1: 663-680PubMed Google Scholar). As presented in Fig. 2, Npl3 is also a good substrate for His6-Dsk1 (lane 13). in vivo the majority of SR protein phosphorylation occurs on the serine residues in the RS domain (40Manley J.L. Tacke R. Genes Dev. 1996; 10: 1569-1579Crossref PubMed Scopus (601) Google Scholar). To test the importance of the RS domain for phosphorylation by Dsk1, a recombinant SF2/ASF with the RS region deleted (SF2ΔRS) was also used in the kinase assay (Fig. 2, lane 9). SF2ΔRS was not phosphorylated by SRPK1 (Fig. 2, lane 5; see Ref. 43Colwill K. Feng L.L. Yeakley J.M. Gish G.D. Caceres J.F. Pawson T. Fu X.-D. J. Biol. Chem. 1996; 271: 24569-24575Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The level of phosphorylation by Dsk1 was reduced drastically to a barely detectable level when the RS region was deleted from SF2/ASF (Fig. 2, compare lanes 8 and 9). These results show that phosphorylation by Dsk1 requires an arginine/serine-rich region on the protein substrate. 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 (19Colwill K. Pawson T. Andrews B. Prasad J. Manley J.L. Bell J.C. Duncan P.I. EMBO J. 1996; 15: 265-275Crossref PubMed Scopus (473) Google Scholar). 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 (45Roth M.B. Murphy C. Gall J.G. J. Cell Biol. 1990; 111: 2217-2223Crossref PubMed Scopus (158) Google Scholar). 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. 3 A, SF2/ASF or SF2ΔRS 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. 3 A, top panel), mAb104 antibody (middle panel), or anti-SF2/ASF antibody (bottom panel) (lanes 5–8). Control samples were also analyzed in parallel (Fig. 3 A, 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. 3 A, 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. 3 A, lanes 5 and 7). Neither the unphosphorylated SF2/ASF (Fig. 3 A, middle panel,lane 4) nor the SF2ΔRS 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. To show further that the recognition of SF2/ASF by mAb104 antibody is Dsk1-dependent, various amounts of Dsk1 protein (1, 5, or 50 units with 1 unit being 0.4 pmol) were incubated with SF2/ASF in a 20-μl kinase reaction; the samples were then analyzed by Western immunoblotting (Fig. 3 B, lanes 3–5). A concentration as low as 0.02 μm His6-Dsk1 was sufficient to generate the mAb104-reactive phosphoepitope (Fig. 3 B,lane 5, bottom panel), and the signal was increased substantially with the concentration of Dsk1 at 0.1 μm (lane 4). No further increase in intensity was observed when the concentration of His6-Dsk1 went up" @default.
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• W2048236220 title "Fission Yeast Mitotic Regulator Dsk1 Is an SR Protein-specific Kinase" @default.
• W2048236220 cites W1520514863 @default.
• W2048236220 cites W1531644439 @default.
• W2048236220 cites W1744830096 @default.
• W2048236220 cites W1873199501 @default.
• W2048236220 cites W1965619464 @default.
• W2048236220 cites W1968863900 @default.
• W2048236220 cites W1979341555 @default.
• W2048236220 cites W1980637163 @default.
• W2048236220 cites W1982036729 @default.
• W2048236220 cites W1991333381 @default.
• W2048236220 cites W1994762541 @default.
• W2048236220 cites W1999037332 @default.
• W2048236220 cites W1999521130 @default.
• W2048236220 cites W2002986711 @default.
• W2048236220 cites W2004313732 @default.
• W2048236220 cites W2004917770 @default.
• W2048236220 cites W2011314090 @default.
• W2048236220 cites W2013002081 @default.
• W2048236220 cites W2014427312 @default.
• W2048236220 cites W2017050402 @default.
• W2048236220 cites W2018775225 @default.
• W2048236220 cites W2020188860 @default.
• W2048236220 cites W2020257412 @default.
• W2048236220 cites W2023199595 @default.
• W2048236220 cites W2035289415 @default.
• W2048236220 cites W2047666280 @default.
• W2048236220 cites W2055157702 @default.
• W2048236220 cites W2068446924 @default.
• W2048236220 cites W2072736269 @default.
• W2048236220 cites W2075339456 @default.
• W2048236220 cites W2076787720 @default.
• W2048236220 cites W2080263723 @default.
• W2048236220 cites W2080568795 @default.
• W2048236220 cites W2081851185 @default.
• W2048236220 cites W2082491111 @default.
• W2048236220 cites W2086471743 @default.
• W2048236220 cites W2095296343 @default.
• W2048236220 cites W2099152453 @default.
• W2048236220 cites W2110449305 @default.
• W2048236220 cites W2116884417 @default.
• W2048236220 cites W2117939247 @default.
• W2048236220 cites W2134275666 @default.
• W2048236220 cites W2143889483 @default.
• W2048236220 cites W2397944365 @default.
• W2048236220 cites W4250083952 @default.
• W2048236220 cites W48376435 @default.
• W2048236220 cites W54260994 @default.
• W2048236220 doi "https://doi.org/10.1074/jbc.273.10.5963" @default.
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