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• W2011926249 abstract "Serine/arginine-rich proteins (SR proteins) are mainly involved in the splicing of precursor mRNA. RS domains are also found in proteins that have influence on other aspects of gene expression. Proteins that contain an RS domain are often located in the speckled domains of the nucleus. Here we show that the RS domain derived from a human papillomavirus E2 transcriptional activator can target a heterologous protein to the nucleus, as it does in many other SR proteins, but insufficient for localization in speckles. By using E2 as a bait in a yeast two-hybrid screen, we identified a human importin-β family protein that is homologous to yeast Mtr10p and almost identical to human transportin-SR. This transportin-SR2 (TRN-SR2) protein can interact with several cellular SR proteins. More importantly, we demonstrated that TRN-SR2 can directly interact with phosphorylated, but not unphosphorylated, RS domains. Finally, an indirect immunofluoresence study revealed that a transiently expressed TRN-SR2 mutant lacking the N-terminal region becomes localized to the nucleus in a speckled pattern that coincides with the distribution of the SR protein SC35. Thus, our results likely reflect a role of TRN-SR2 in the cellular trafficking of phosphorylated SR proteins. Serine/arginine-rich proteins (SR proteins) are mainly involved in the splicing of precursor mRNA. RS domains are also found in proteins that have influence on other aspects of gene expression. Proteins that contain an RS domain are often located in the speckled domains of the nucleus. Here we show that the RS domain derived from a human papillomavirus E2 transcriptional activator can target a heterologous protein to the nucleus, as it does in many other SR proteins, but insufficient for localization in speckles. By using E2 as a bait in a yeast two-hybrid screen, we identified a human importin-β family protein that is homologous to yeast Mtr10p and almost identical to human transportin-SR. This transportin-SR2 (TRN-SR2) protein can interact with several cellular SR proteins. More importantly, we demonstrated that TRN-SR2 can directly interact with phosphorylated, but not unphosphorylated, RS domains. Finally, an indirect immunofluoresence study revealed that a transiently expressed TRN-SR2 mutant lacking the N-terminal region becomes localized to the nucleus in a speckled pattern that coincides with the distribution of the SR protein SC35. Thus, our results likely reflect a role of TRN-SR2 in the cellular trafficking of phosphorylated SR proteins. serine/arginine-rich β-galactosidase hemagglutinin monoclonal antibody polymerase II nuclear localization signal adenosine 5′-O-(thiotriphosphate) transportin binding domain activation domain 5,6-dichloro-1-β-d-ribofuranosyl-benimidazole human papilloma virus arginine/serine SR1 proteins are a superfamily of eukaryotic proteins that contain repetitive serine-arginine dipeptides in a domain known as RS domains (1.Fu X.D. RNA. 1995; 1: 663-680PubMed Google Scholar, 2.Manley J.L. Tacke R. Genes Dev. 1996; 10: 1569-1579Crossref PubMed Scopus (599) Google Scholar, 3.Valcarcel J. Green M.R. Trends Biochem. Sci. 1996; 21: 296-301Abstract Full Text PDF PubMed Google Scholar). SR proteins are primarily involved in the splicing of precursor mRNA. Some SR splicing factors are essential for pre-mRNA splicing, and some can act as crucial players in alternative splicing by modulating splice site choice. A group of SR proteins that can be precipitated by magnesium and recognized by monoclonal antibody (mAb) 104 play both essential and regulatory roles in pre-mRNA splicing (4.Zahler A.M. Lane W.S. Stolk J.A. Roth M.B. Genes Dev. 1992; 6: 837-847Crossref PubMed Scopus (612) Google Scholar). Each of these SR proteins can complement splicing-deficient cytoplasmic S100 extracts as well as affect splice site selection at elevated concentrations (1.Fu X.D. RNA. 1995; 1: 663-680PubMed Google Scholar, 2.Manley J.L. Tacke R. Genes Dev. 1996; 10: 1569-1579Crossref PubMed Scopus (599) Google Scholar, 3.Valcarcel J. Green M.R. Trends Biochem. Sci. 1996; 21: 296-301Abstract Full Text PDF PubMed Google Scholar). In addition to splicing factors, RS domains are present in other proteins, such as a group of human papillomavirus E2 transcriptional activators (5.Birney E. Kumar S. Krainer A.R. Nucleic Acids Res. 1993; 21: 5803-5816Crossref PubMed Scopus (586) Google Scholar, 6.Lai M.-C. Teh B.H. Tarn W.-Y. J. Biol. Chem. 1999; 274: 11832-11841Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), RNA pol II-associated SR-like proteins (7.Yuryev A. Patturajan M. Litingtung Y. Joshi R.V. Gentile C. Gebara M. Corden J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6975-6980Crossref PubMed Scopus (288) Google Scholar), transcriptional coactivator PGC-1 (8.Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3000) Google Scholar), and pre-mRNA cleavage factor Im (9.Ruegsegger U. Blank D. Keller W. Mol. Cell. 1998; 1: 243-253Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Thus, RS domain-containing proteins can function in gene expression at different levels. Cytological studies have revealed that a variety of SR proteins are localized in nuclear speckled domains, which are thought to be the sites for storage/reassembly of splicing factors and/or supplying splicing factors to active genes (10.Spector D. Exp. Cell Res. 1996; 229: 189-197Crossref PubMed Scopus (104) Google Scholar, 11.Singer R.H. Green M.R. Cell. 1997; 91: 291-294Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). The RS domains of some, but not all, SR proteins have been shown to be necessary and sufficient for targeting to the nuclear speckles (12.Hedley M.L. Amrein H. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11524-11528Crossref PubMed Scopus (125) Google Scholar, 13.Caceres J.F. Misteli T. Screaton G.R. Spector D.L. Krainer A.R. J. Cell Biol. 1997; 138: 225-238Crossref PubMed Scopus (323) Google Scholar). Hyperphosphorylation of the RS domain by SR protein-specific kinases can relocate SR proteins from a speckled pattern into a more diffuse distribution (14.Gui J.F. Lane W.S. Fu X.D. Nature. 1994; 369: 678-682Crossref PubMed Scopus (451) Google Scholar, 15.Colwill K. Pawson T. Andrews B. Prasad J. Manley J.L. Bell J.C. Duncan P.I. EMBO J. 1996; 15: 265-275Crossref PubMed Scopus (464) Google Scholar, 16.Wang H.Y. Lin W. Dyck J.A. Yeakley J.M. Songyang Z. Cantley L.C. Fu X.D. J. Cell Biol. 1998; 140: 737-750Crossref PubMed Scopus (249) Google Scholar). Recent evidence indicates that a subset of SR proteins can continuously shuttle between the nucleus and the cytoplasm (17.Caceres J.F. Screaton G.R. Krainer A.R. Genes Dev. 1998; 12: 55-66Crossref PubMed Scopus (385) Google Scholar). The phosphorylation states of the RS domain appear to have influence on the shuttling properties of SR proteins (17.Caceres J.F. Screaton G.R. Krainer A.R. Genes Dev. 1998; 12: 55-66Crossref PubMed Scopus (385) Google Scholar). Thus, the nucleocytoplasmic transport and nuclear speckle localization of SR proteins are likely to be complex and regulated processes. Translocation of macromolecules across the nuclear envelope occurs through the nuclear pore complex (18.Nigg E.A. Nature. 1997; 386: 779-787Crossref PubMed Scopus (909) Google Scholar, 19.Gorlich D. EMBO J. 1998; 17: 2721-2727Crossref PubMed Scopus (286) Google Scholar, 20.Izaurralde E. Adam S. RNA. 1998; 4: 351-364PubMed Google Scholar, 21.Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1001) Google Scholar). Proteins targeted for the nucleus are initially complexed with corresponding soluble import receptors in the cytoplasm via specific signal-receptor recognition. Receptor-cargo complexes subsequently dock at saturable sites on the cytoplasmic face of the nuclear pore and then translocate through the pore to the nuclear interior. Nuclear translocation of cargo requires additional factors such as the small GTPase Ran and NTF2 (18.Nigg E.A. Nature. 1997; 386: 779-787Crossref PubMed Scopus (909) Google Scholar, 19.Gorlich D. EMBO J. 1998; 17: 2721-2727Crossref PubMed Scopus (286) Google Scholar, 20.Izaurralde E. Adam S. RNA. 1998; 4: 351-364PubMed Google Scholar, 21.Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1001) Google Scholar). Different import cargoes possess different nuclear localization signals (NLSs) (18.Nigg E.A. Nature. 1997; 386: 779-787Crossref PubMed Scopus (909) Google Scholar, 21.Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1001) Google Scholar). The prototypical NLS is composed of one or more clusters of basic amino acid residues and is recognized by importin-α, which functions as an adapter and in turn interacts with importin-β for nuclear pore complex docking. Nonclassic NLSs include the glycine-rich M9 sequence of heteronuclear ribonucleoprotein A1 (22.Michael W.M. Choi M. Dreyfuss G. Cell. 1995; 83: 415-422Abstract Full Text PDF PubMed Scopus (468) Google Scholar,23.Siomi H. Dreyfuss G. J. Cell Biol. 1995; 129: 551-560Crossref PubMed Scopus (433) Google Scholar), the arginine-rich sequence of human immunodeficiency virus regulatory proteins Rev and Tat (24.Palmeri D. Malim M.H. Mol. Cell. Biol. 1999; 19: 1218-1225Crossref PubMed Google Scholar, 25.Truant R. Cullen B.R. Mol. Cell. Biol. 1999; 19: 1210-1217Crossref PubMed Google Scholar), and the RGG box of the yeast RNA-binding protein Npl3p (26.Pemberton L.F. Rosenblum J.S. Blobel G. J. Cell Biol. 1997; 139: 1645-1653Crossref PubMed Scopus (92) Google Scholar, 27.Senger B. Simos G. Bischoff F.R. Podtelejnikov A. Mann M. Hurt E. EMBO J. 1998; 17: 2196-2207Crossref PubMed Scopus (157) Google Scholar). These NLSs, unlike the prototypical NLS, interact directly with their corresponding import receptors. All of the import receptors belong to the importin-β family. They are of similar size (90–130 kDa) and appear to consist of 18 or more helix-turn-helix HEAT repeats as revealed by the crystal structures of two importin-β proteins (28.Chook Y.M. Blobel G. Nature. 1999; 399: 230-237Crossref PubMed Scopus (287) Google Scholar, 29.Cingolani G. Petosa C. Weis K. Muller C.W. Nature. 1999; 399: 221-229Crossref PubMed Scopus (437) Google Scholar, 30.Mattaj I.W. Conti E. Nature. 1999; 399: 208-310Crossref PubMed Scopus (14) Google Scholar). It is well known that the N- and C-terminal halves of the importin-β proteins contribute to interaction with GTP-bound Ran and the NLS of cargo, respectively. However, importin-β interactions with Ran and NLS are mutually exclusive, implying the existence of a RanGTP-mediated cargo release mechanism. We previously showed that a RS domain-containing human papillomavirus (type 5) E2 transcriptional activator can function to facilitate the splicing of pre-mRNA made via transactivation by E2 itself (6.Lai M.-C. Teh B.H. Tarn W.-Y. J. Biol. Chem. 1999; 274: 11832-11841Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). This E2 transactivator colocalizes with cellular splicing factors in nuclear speckles, and its RS-rich hinge domain is required for colocalization (6.Lai M.-C. Teh B.H. Tarn W.-Y. J. Biol. Chem. 1999; 274: 11832-11841Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In the present study, we establish that the E2 hinge can target a heterologous protein to the nucleus but not to subnuclear speckle domains. To understand the mechanism of RS domain-mediated nuclear entry and its regulation, we searched for factors that could play a role in the nuclear trafficking of SR proteins. A human importin-β family protein was identified and its interactions with the phosphorylated SR proteins characterized. The β-gal-hinge and β-gal-RS expression vectors were constructed by insertion of a PCR product derived from the entire hinge region (amino acids 212–396) or the RS-rich subdomain (amino acids 212–346) of HPV-5 E2 into the uniqueKpnI site within pCH110 (Amersham Pharmacia Biotech), respectively. The resulting fusion proteins expressed in HeLa cells can be detected by monoclonal anti-β-gal antibody. The SRPK1 expression vector that produced FLAG-tagged SRPK1 in HeLa cells was kindly provided by X.-D. Fu (University of California, San Diego, CA) (16.Wang H.Y. Lin W. Dyck J.A. Yeakley J.M. Songyang Z. Cantley L.C. Fu X.D. J. Cell Biol. 1998; 140: 737-750Crossref PubMed Scopus (249) Google Scholar). Plasmid pGST-TRN-SR2C was obtained by subcloning the DNA fragment encoding the C-terminal 399 amino acids of the human TRN-SR2 protein into Escherichia coli expression vector pGEX-2T (Amersham Pharmacia Biotech). The resulting plasmid was used to produce the recombinant GST-TRN-SR2C protein in bacteria. ASF and SRPK1 open reading frames (gifts of X.-D. Fu) were also inserted into pGEX-2T to generate plasmids encoding GST-ASF and GST-SRPK1 fusion proteins, respectively. The pBluescript-derived plasmids encoding full-length and ΔN281 TRN-SR2 were constructed by appropriate restriction digestion of the parental pBluescript plasmid containing a 3.4-kilobase pair TRN-SR2 cDNA (see “Results”) to remove the 5′-untranslated region and 5′-untranslated region plus a region coding for the N-terminal 281 amino acid residues, respectively. The resulting plasmids generated in-frame fusion of TRN-SR2 to the first 34 amino acids of β-gal at the N terminus. Cell culture, transfection, and indirect immunofluorescence staining were performed essentially according to Lai et al. (6.Lai M.-C. Teh B.H. Tarn W.-Y. J. Biol. Chem. 1999; 274: 11832-11841Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). For treatment of cells with an RNA pol II inhibitor, transfected cells were incubated with 100 μm DRB for 4 h before fixation. The primary antibodies used included monoclonal anti-β-gal antibody (2 μg/ml; Promega), purified anti-TRN-SR2 antibodies (0.5 μg/ml), monoclonal anti-HA antibody (1:100 dilution from the supernatant of hybridoma culture medium; gift of S.-C. Cheng, Academia Sinica, Taipei, Taiwan), polyclonal anti-HA antibodies (1:50 dilution; Upstate Biotechnology Inc., Lake Placid, NY), and monoclonal anti-SC35 antibody (4.6 μg/ml; Sigma). The secondary antibodies used were fluorescein-conjugated anti-mouse IgG (7.5 μg/ml; Cappel Laboratories) for monoclonal primary antibodies and rhodamine-conjugated anti-rabbit IgG (12 μg/ml, Cappel Laboratories) for polyclonal primary antibodies. The specimens were observed using a laser confocal microscope (MRC 600 model; Bio-Rad) coupled with an image analysis system. HPV-5 E2 was cloned in frame into the GAL4 DNA binding domain (DB) plasmid pAS2–1 and then used as a bait to screen a HeLa cell cDNA library (CLONTECH) that was constructed in the GAL4 activation domain (AD)-containing pGAD GH vector. Yeast two-hybrid screening was performed as described in the protocol provided by the manufacturer. Initially, the bait plasmid was transformed intoSaccharomyces cerevisiae Y190 and maintained by selection in Trp− plates. The cDNA library was then transformed into the bait-containing yeast cells, and transformants were selected by the use of appropriate media. One of the positive clones, which encoded the C-terminal 399 amino acids of a human importin-β family protein, was named pGAD-TRN-SR2 (see “Results”). To obtain cDNAs encoding full-length TRN-SR2, the insert of pGAD-TRN-SR2 was used as a probe to screen a λZAP cDNA library made from HeLa cells (CLONTECH). The cDNA inserts of positive phage clones were excised into pBluescript phagemids as described in the manufacturer's instruction, and the sequences were then determined by autosequencing. The TRN-SR2 coding sequence matched perfectly to GenBank™ accession number AJ133769. To assay for pairwise interactions between TRN-SR2 and SR proteins, pGAD-TRN-SR2 and a pEG202-derived plasmid expressing individual SR proteins (gifts of J. Y. Wu, Washington University, St. Louis, MO) or domains as LexA fusion proteins were co-transformed into reporter (pSH18–34)-containing EGY48. The method of the liquid β-galactosidase assay was as described in theCLONTECH protocol. To examine whether phosphorylation of the RS domain is important for the TRN-SR2-SR protein interaction, pGAD-TRN-SR2 was co-transformed with pEG-ASF or pEG-SC35 into EGY48, corresponding sky1Δ strain (kind gift of X.-D. Fu; Ref. 31.Yeakley J.M. Tronchere H. Olesen J. Dyck J.A. Wang H.Y. Fu X.D. J. Cell Biol. 1999; 145: 447-455Crossref PubMed Scopus (118) Google Scholar), or sky1Δ expressing human SRPK1. Expression of SRPK1 was driven by the GPD promoter in the 2 μ plasmid pG-1. A pair of yeast plasmids encoding Gal4 AD-PRP19 and LexA DB-SNT309, respectively, were kindly provided by S.-C. Cheng (32.Chen H.-R. Jan S.-P. Tsao T.Y. Sheu Y.-J. Banroques J. Cheng S.-C. Mol. Cell. Biol. 1998; 18: 2196-2204Crossref PubMed Scopus (43) Google Scholar) and used as control. The recombinant GST-TRN-SR2C protein was overproduced in E. coli and purified by affinity chromatography via a glutathione-Sepharose column according to the manufacturer's instruction (Amersham Pharmacia Biotech). The purified fusion protein was subjected to cleavage by thrombin protease followed by preparative gel electrophoresis. Gel-purified TRN-SR2 C-terminal domain was then used to raise antiserum in rabbits. To purify anti-TRN-SR2 antibodies, antisera were incubated with nitrocellulose containing immobilized GST-TRN-SR2C protein at room temperature overnight. Antibodies were eluted from the filters with a solution containing 50 mm glycine (pH 2.3) and 150 mm NaCl, followed by neutralization with Tris base. His-tagged wild-type Ran and RanQ69L (gifts from I. W. Mattaj, European Molecular Biology Laboratory, Heidelberg, Germany) were expressed in E. coli strain BL21/pRep4 and purified on nickel-agarose (Novagen) essentially according to Gorlich et al. (33.Gorlich D. Pante N. Kutay U. Aebi U. Bischoff F.R. EMBO J. 1996; 15: 5584-5594Crossref PubMed Scopus (524) Google Scholar). Purified Ran and RanQ69L were initially dialyzed against 50 mm potassium phosphate (pH 7.0), 50 mm KCl, 5 mm MgCl2, 1 mmβ-mercaptoethanol, 8.7% glycerol, and 0.1 mm GDP (for Ran) or 0.1 mm GTP (for RanQ69L). Before use, they were loaded with 1 mm GDP and GTP, respectively, according to Floer and Blobel (34.Floer M. Blobel G. J. Biol. Chem. 1999; 274: 16279-16286Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). GST-ASF and GST-SRPK1 were overexpressed inE. coli strains BLR and XA90, respectively, upon isopropyl-1-thio-β-d-galactopyranoside induction. The two GST fusion proteins were purified using glutathione-Sepharose, and then dialyzed against buffer D containing 20 mm HEPES (pH 7.9), 50 mm KCl, 0.2 mm EDTA, 1 mmdithiothreitol, and 20% glycerol. Recombinant full-length and ΔN281 TRN-SR2 proteins were expressed in E. coli strain XA90 after induction with isopropyl-1-thio-β-d-galactopyranoside. The extracts were prepared by lysis of cells with modified transport buffer (35.Adam S.A. Sterne-Marr R. Gerace L. J. Cell Biol. 1991; 111: 807-816Crossref Scopus (763) Google Scholar) containing 20 mm HEPES (pH 7.3), 110 mm potassium acetate, 2 mm magnesium acetate, 5 mm sodium acetate, 2 mm dithiothreitol, 1 mm EGTA, 8.7% glycerol, and 1 mmphenylmethylsulfonyl fluoride. The lysates were stored in aliquots after removal of cell debris. HeLa cells (strain S3) were cultured in RPMI supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at a density of ∼5 × 105 cells/ml. The cytoplasmic extract was prepared from HeLa cells essentially according to Paschal (36.Paschal B.M. Celis J.E. Cell Biology: A Laboratory Handbook. 2. Academic Press, Orlando, FL1998: 305-313Google Scholar), and finally dialyzed against the transport buffer supplemented with 1 μg/ml each of aprotinin and leupeptin. The concentration of the cytoplasmic extract was ∼28 mg/ml. In vitrophosphorylation of GST-ASF was carried out in a 20-μl mixture containing 2 μg of GST-ASF, 2 mm MgCl2, 0.5 mm ATP (or 0.5 mm ATPγS), and 30 ng of GST-SRPK1 at 30 °C for 45 min. ATP was eliminated in the mock-phosphorylation reaction. Subsequently, phosphorylated or mock-phosphorylated GST-ASF was incubated with 35 μl of the HeLa cell cytoplasmic extract (equivalent to ∼1 mg of proteins) in a 60-μl mixture at 30 °C for 30 min. The reaction mix was then supplemented with an equal volume of NET-2 buffer (50 mm Tris-HCl (pH 7.4), 150 mm NaCl, and 0.05% Nonidet P-40) and subsequently incubated with 10 μl of glutathione-Sepharose at 4 °C for 2 h. The beads were washed extensively with NET-2 buffer. Bound proteins were extracted with SDS lysis buffer and analyzed by immunoblotting with purified anti-TRN-SR2 antibodies. The blot was stripped and then reprobed with mAb 104 to examine phosphorylated GST-ASF. To detect both phosphorylated and unphosphorylated GST-ASF, 1/20 volume each of the samples were fractionated in another SDS-polyacrylamide gel electrophoresis and subjected to Western blot analysis using anti-GST antibodies. Western blot analysis was performed by using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech). To test the interaction of GST-ASF with the recombinant TRN-SR2 protein, the pull-down experiments were carried out by using E. coli extract containing full-length or ΔN281 TRN-SR2 protein. The reactions were performed as described above. Bound proteins were analyzed as above, or, after blotting with anti-TRN-SR2, the filter was stained with Ponceau S. For Ran competition experiments, reaction mixtures containing phosphorylated GST-ASF, TRN-SR2-containing extract, and RanQ69L-GTP or Ran-GDP were incubated at 30 °C for 30 min. Pull-down experiments were performed as described above. Phosphorylation of the SR peptide [CGGG(RS)8R] was carried out in a 25-μl reaction mix containing 2.5 nmol of the peptide, 50 mm Tris-HCl (pH 7.4), 10 mmMgCl2, 1 mm dithiothreitol, 0.8 mmATP, and 0.24 μg of GST-SRPK1 at 30 °C for 45 min. Mock-phosphorylation was carried out in the reaction excluding ATP. Phosphorylated or mock-phosphorylated SR peptide was added to the reaction mix containing phosphorylated GST-ASF and recombinant TRN-SR2; the pull-down assay was performed as described above. The E2 protein encoded by epidermodysplasia verruciformis-associated papillomaviruses contains an RS-rich sequence in its hinge region. Previously, we showed that this hinge domain is essential for the colocalization of transiently expressed HPV-5 E2 protein with splicing factors in nuclear speckle domains (6.Lai M.-C. Teh B.H. Tarn W.-Y. J. Biol. Chem. 1999; 274: 11832-11841Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In an attempt to determine whether the RS-rich hinge is sufficient for nuclear speckle targeting, we inserted the entire hinge or the RS-rich subdomain into a heterologous protein, β-gal, and examined the cellular localization of the fusion protein. Fig. 1 shows that both fusion proteins localized predominantly in the nucleus (panels c and e), whereas β-gal itself was distributed throughout the whole cell (panel a). Although neither of the tested domains was sufficient for speckle targeting, this result suggests that the RS domain of E2 can serve as a functional NLS, like the RS domains or subdomains of some splicing factors (12.Hedley M.L. Amrein H. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11524-11528Crossref PubMed Scopus (125) Google Scholar, 13.Caceres J.F. Misteli T. Screaton G.R. Spector D.L. Krainer A.R. J. Cell Biol. 1997; 138: 225-238Crossref PubMed Scopus (323) Google Scholar, 17.Caceres J.F. Screaton G.R. Krainer A.R. Genes Dev. 1998; 12: 55-66Crossref PubMed Scopus (385) Google Scholar). We next asked whether the subcellular localization of the HPV-5 E2 protein could be altered or regulated upon phosphorylation. We first examined the ability of the E2 protein to serve as a substrate for cellular SR protein kinases in vivo. A human SR protein kinase, SRPK1, was transiently coexpressed with the full-length or hinge-deleted E2 protein in transfected HeLa cells. As shown in Fig.2 A, only full-length E2, but not hinge-deleted E2, changed its gel mobility in the presence of overexpressed SRPK1, suggesting phosphorylation of the hinge. Because the E2 protein purified from the baculovirus expression system is readily detectable by mAb 104, which recognizes phosphorylated epitopes in SR proteins (6.Lai M.-C. Teh B.H. Tarn W.-Y. J. Biol. Chem. 1999; 274: 11832-11841Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), E2 might be moderately phosphorylated by a cellular kinase even in the absence of exogenous SRPK1 in HeLa cells. Thus, our finding may indicate that excess SRPK1 extensively phosphorylates the E2 protein in the hinge. Furthermore, the E2 protein, similar to ASF/SF2, appeared to accumulate in the cytoplasm upon hyperphosphorylation of the hinge by excess SRPK1 (Fig. 2 B,panels b and f). In contrast, hinge-deleted E2 remained in the nucleus despite the presence of exogenous SRPK1 (panel d). Thus, the hinge of the HPV-5 E2 transactivator behaved similarly to the RS domains of several cellular SR proteins (12.Hedley M.L. Amrein H. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11524-11528Crossref PubMed Scopus (125) Google Scholar, 13.Caceres J.F. Misteli T. Screaton G.R. Spector D.L. Krainer A.R. J. Cell Biol. 1997; 138: 225-238Crossref PubMed Scopus (323) Google Scholar, 17.Caceres J.F. Screaton G.R. Krainer A.R. Genes Dev. 1998; 12: 55-66Crossref PubMed Scopus (385) Google Scholar) in both its nuclear targeting activity and cytoplasmic distribution upon hyperphosphorylation. We next performed a yeast two-hybrid screen to search for HPV-5 E2-interacting proteins from a HeLa cell cDNA library. We wished to identify candidates that could specifically recognize the RS domain of E2 and function as a nuclear transporter. A screen of 3.5 × 105 primary transformants yielded 37 positive clones. The majority of the isolated clones encoded known proteins including SF2p32, ASF/SF2, SC35, 9G8, Tra2β, and ribosomal proteins S4 and S14 (data not shown). One partial cDNA encoded a protein of about 400 amino acid residues homologous to the C terminus of yeast importin-β family protein Mtr10p and almost identical to human transportin-SR (Ref. 37.Kataoka N. Bachorik J.L. Dreyfuss G. J. Cell Biol. 1999; 145: 1145-1152Crossref PubMed Scopus (174) Google Scholar; hereafter termed TRN-SR1) and thus attracted our further attention. Such a clone had no detectable interaction with hinge-deleted E2, suggesting that its encoded protein interacts only with the RS-rich hinge domain of E2 (data not shown). Therefore, it is named transportin-SR2 (abbreviated as TRN-SR2). Northern blot analysis with the 3′ end of the coding region of human TRN-SR2 revealed a single transcript of ∼4.5 kilobase pairs that is ubiquitously expressed in all human tissues, with higher abundance in testis (data not shown). A ∼3.4-kilobase pair cDNA containing the possible entire open reading frame of human TRN-SR2 was obtained by screening a λZAP cDNA library made from HeLa cells. The human TRN-SR2 protein of 923 amino acid residues is ∼23% identical toS. cerevisiae Mtr10p and also shares similarities with the putative homologs in several other species (Fig.3, bottom panel). Intriguingly, TRN-SR2 lacks two regions of ∼30 amino acid residues from TRN-SR1 (37.Kataoka N. Bachorik J.L. Dreyfuss G. J. Cell Biol. 1999; 145: 1145-1152Crossref PubMed Scopus (174) Google Scholar) as shown in Fig. 3 (top panel). Further experiments are needed to clarify the relationship between these two proteins. In addition, human TRN-SR2 exhibited significant homology (∼22%) to another human open reading frame, namely the KIAA0724 protein (38.Nagase T. Ishikawa K. Suyama M. Kikuno R. Miyajima N. Tanaka A. Kotani H. Nomura N. Ohara O. DNA Res. 1998; 5: 277-286Crossref PubMed Scopus (125) Google Scholar), throughout the entire sequence. However, this putative importin-β family protein did not interact with SR proteins, as judged by the yeast two-hybrid interaction assay (data not shown). Since E2's RS-rich hinge behaved similarly to some splicing factors' RS domains in many different aspects (6 and see above), we next tested the interaction of TRN-SR2 with cellular SR splicing factors by using the yeast two-hybrid interaction assay. As shown in Fig. 4, human TRN-SR2 interacted with three tested SR proteins, i.e.ASF/SF2, SC35, and Tra2β, via its C-terminal 400 amino acid residues. TRN-SR2 interaction was also detected with truncated Tra2β, which possessed only the N-terminal RS domain, but not with the RNA binding domains of ASF and Tra2β (Fig. 4). This result suggests that the RS domain is sufficient to mediate the interaction of SR proteins with TRN-SR2. Thus, TRN-SR2 can probably interact with the whole family of SR protein splicing factors. Since cellular SR proteins represent key regulators in the expression of eukaryotic genes, and moreover, the hinge itself is dispensable for the nuclear entry of the E2 transactivators (6.Lai M.-C. Teh B.H. Tarn W.-Y. J. Biol. Chem. 1999; 274: 11832-11841Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 39.Skiadopoulos A.H. McBride A.A. J. Viol. 1996; 70: 1117-1124Crossref PubMed Google Scholar), we focused further experiments on SR splicing factors instead of HPV E2. To test whether phosphorylation plays any role in TRN-SR2-SR protein interactions, we performed a protein-protein interaction assay in a SR protein kinase-deficient (sky1Δ) yeast strain (31.Yeakley J.M. Tronchere H. Olesen J. Dyck J.A. Wang H.Y. Fu X.D. J. Cell Biol. 1999; 145: 447-455Crossref PubMed Scopus (118) Google Scholar). The interaction of TRN-SR2 with either ASF/SF2 or SC35 was severely affected" @default.
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