Importin α Nuclear Localization Signal Binding Sites for STAT1, STAT2, and Influenza A Virus Nucleoprotein
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m303571200
ISSN1083-351X
AutoresKrister Melén, Riku Fagerlund, Jacqueline Franke, Matthias Köhler, Leena Kinnunen, Ilkka Julkunen,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoProteins actively transported into the nucleus via the classical nuclear import pathway contain nuclear localization signals (NLSs), which are recognized by the family of importin α molecules. Importin α contains 10 armadillo (arm) repeats, of which the N-terminal arm repeats 2–4 have been considered as the "major" NLS binding site. Interferon-activated, dimerized signal transducers and activators of transcription (STAT1 and STAT2) directly bind to importin α5 via a dimeric nonclassical NLS. Here we show by site-directed mutagenesis that the very C-terminal arm repeats 8 and 9 of importin α5 form a unique binding site for STAT1 homodimers and STAT1-STAT2 heterodimers. Influenza A virus nucleoprotein also contains a nonclassical NLS that is recognized by the C-terminal NLS binding site of importin α5, comprising arm repeats 7–9. Binding of influenza A virus nucleoprotein to importin α3 also occurs via the C-terminal arm repeats. Simian virus 40 large T antigen instead binds to the major N-terminal arm repeats of importin α3, indicating that one importin α molecule is able to use either its N- or C-terminal arm repeats for binding various NLS containing proteins. Proteins actively transported into the nucleus via the classical nuclear import pathway contain nuclear localization signals (NLSs), which are recognized by the family of importin α molecules. Importin α contains 10 armadillo (arm) repeats, of which the N-terminal arm repeats 2–4 have been considered as the "major" NLS binding site. Interferon-activated, dimerized signal transducers and activators of transcription (STAT1 and STAT2) directly bind to importin α5 via a dimeric nonclassical NLS. Here we show by site-directed mutagenesis that the very C-terminal arm repeats 8 and 9 of importin α5 form a unique binding site for STAT1 homodimers and STAT1-STAT2 heterodimers. Influenza A virus nucleoprotein also contains a nonclassical NLS that is recognized by the C-terminal NLS binding site of importin α5, comprising arm repeats 7–9. Binding of influenza A virus nucleoprotein to importin α3 also occurs via the C-terminal arm repeats. Simian virus 40 large T antigen instead binds to the major N-terminal arm repeats of importin α3, indicating that one importin α molecule is able to use either its N- or C-terminal arm repeats for binding various NLS containing proteins. Regulated import of molecules into the nucleus through the nuclear pores is a vital event in eukaryotic cells. Importins (also known as karyopherins) are the major cargo carriers from the cytoplasm into the nucleus. Large molecules (>40 kDa) that cannot passively diffuse through the nuclear pores use a signal-mediated transport system. The importin α/importin β-mediated import pathway was the first one to be discovered, and it is also referred to as the classical pathway. Proteins transported into the nucleus contain nuclear location signals (NLSs) 1The abbreviations used are: NLSs, nuclear localization signals; NP, nucleoprotein; STAT, signal transducers and activators of transcription; IFN, interferon; GST, glutathione S-transferase; BSA, bovine serum albumin. that are recognized by importin α/importin β heterodimers. Importin α recognizes and binds the NLS, and importin β docks the complex to the nuclear pore and translocates it into the nucleus (1Görlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-660Crossref PubMed Scopus (1676) Google Scholar, 2Macara I.G. Microbiol. Mol. Biol. 2001; 65: 570-594Crossref PubMed Scopus (746) Google Scholar, 3Weis K. Cell. 2003; 112: 441-451Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar). Six different human importin α molecules have been identified: importin α1 (Rch1, hSRP1α), importin α3 (Qip1), importin α4 (hSRP1γ), importin α5 (hSRP1, NPI-1), importin α6, and importin α7 (4Cuomo C.A. Kirch S.A. Gyuris J. Brent R. Oettinger M.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6156-6160Crossref PubMed Scopus (164) Google Scholar, 5Köhler M. Ansieau S. Prehn S. Leutz A. Haller H. Hartmann E. FEBS Lett. 1997; 417: 104-108Crossref PubMed Scopus (206) Google Scholar, 6Cortes P. Ye Z.S. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7633-7637Crossref PubMed Scopus (170) Google Scholar, 7Nachury M.V. Ryder U.W. Lamond A.I. Weis K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 582-587Crossref PubMed Scopus (96) Google Scholar, 8Seki T. Tada S. Katada T. Enomoto T. Biochem. Biophys. Res. Commun. 1997; 234: 48-53Crossref PubMed Scopus (82) Google Scholar, 9Köhler M. Speck C. Christiansen M. Bischoff F.R. Prehn S. Haller H. Görlich D. Hartmann E. Mol. Cell. Biol. 1999; 19: 7782-7791Crossref PubMed Google Scholar). Importin α proteins have remained structurally and functionally conserved throughout evolution. The six human importin α proteins show over 60% sequence similarity. The crystal structures of two importin α molecules, yeast importin α (10Conti E. Uy M. Leighton L. Blobel G. Kuriyan J. Cell. 1998; 94: 193-204Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar) and mouse importin α2 (11Fontes M.R.M. Teh T. Kobe B. J. Mol. Biol. 2000; 297: 1183-1194Crossref PubMed Scopus (315) Google Scholar), have been determined. Importin α is composed of a large central domain that consist of 10 tandemly repeated armadillo (arm) motifs, which are organized in a superhelix flanked by small N- and C-terminal domains. The 10 arm repeats generate an array of binding pockets that are situated within a long helical surface groove. One binding pocket typically includes a tryptophan residue, followed by an asparagine residue 4 residues downstream. The arm repeats are variable within one protein, but they are remarkably conserved in sequence and order when homologous proteins from yeast to humans are compared. The N-terminal importin β binding domain of importin α mediates binding to importin β (12Görlich D. Henklein P. Laskey R.A. Hartmann E. EMBO J. 1996; 15: 1810-1817Crossref PubMed Scopus (363) Google Scholar, 13Weis K. Ryder U. Lamond A.I. EMBO J. 1996; 15: 7120-7128Crossref PubMed Scopus (120) Google Scholar). In the absence of cargo and importin β, the importin β binding domain blocks the NLS binding site of importin α (14Kobe B. Nat. Struct. Biol. 1999; 6: 388-397Crossref PubMed Scopus (323) Google Scholar, 15Harreman M.T. Hodel M.R. Fanara P. Hodel A.E. Corbett A.H. J. Biol. Chem. 2003; 278: 5854-5863Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The C-terminal domain of importin α mediates interactions with the export receptor CAS (16Herold A. Truant R. Wiegand H. Cullen B.R. J. Cell Biol. 1998; 143: 309-318Crossref PubMed Scopus (107) Google Scholar, 17Moroianu J. Blobel G. Radu A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7059-7062Crossref PubMed Scopus (90) Google Scholar). The NLS used by the classical nuclear import pathway is a short stretch of positively charged amino acids, arginines and lysines, that lack strict consensus sequence (18Dingwall C. Laskey R.A. Trends Biochem. Sci. 1991; 16: 478-481Abstract Full Text PDF PubMed Scopus (1713) Google Scholar). A monopartite NLS, like that of simian virus 40 (SV40) large T antigen, is composed of a cluster of five to seven basic amino acids (19Kalderon D. Roberts B.L. Richardson W.D. Smith A.E. Cell. 1984; 39: 499-509Abstract Full Text PDF PubMed Scopus (1874) Google Scholar, 20Lanford R.E. Butel J.S. Cell. 1984; 37: 801-813Abstract Full Text PDF PubMed Scopus (367) Google Scholar). A typical bipartite NLS contains two clusters of basic amino acids separated by a linker of 10–11 amino acids (21Dingwall C. Sharnick S.V. Laskey R.A. Cell. 1982; 30: 449-458Abstract Full Text PDF PubMed Scopus (337) Google Scholar). Monopartite NLSs have been shown to bind to the "major" binding site of importin α, which is formed by arm repeats 2–4 (10Conti E. Uy M. Leighton L. Blobel G. Kuriyan J. Cell. 1998; 94: 193-204Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar). The downstream cluster of the bipartite NLS is also recognized by these arm repeats, whereas the upstream cluster is recognized by arm repeats 7 and 8, also called the "minor" binding site (11Fontes M.R.M. Teh T. Kobe B. J. Mol. Biol. 2000; 297: 1183-1194Crossref PubMed Scopus (315) Google Scholar). Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors that regulate the expression of a number of genes involved in host defense and growth (22Levy D.E. Darnell Jr., J.E. Nat. Rev. Mol. Cell. Biol. 2002; 3: 651-662Crossref PubMed Scopus (2526) Google Scholar). Binding of interferon (IFN) to its specific cell-surface receptor leads to the activation of receptor-associated Janus (Jak) tyrosine kinases, which phosphorylate STATs. This leads to dimerization and translocation of STATs into the nucleus. Dimerization is an essential and sufficient step for nuclear import (23Milocco L.H. Haslam J.A. Rosen J. Seidel H.M. Mol. Cell. Biol. 1999; 19: 2913-2920Crossref PubMed Scopus (43) Google Scholar). Although bound by importin α5, STATs do not contain a classical NLS (24Sekimoto T. Imamoto N. Nakajima K. Hirano T. Yoneda Y. EMBO J. 1997; 16: 7067-7077Crossref PubMed Scopus (306) Google Scholar). Instead, it is supposed that both STAT1 and STAT2 contain in the DNA binding domain a nonclassical NLS that seems to become operative in STAT dimers (25Melén K. Kinnunen L. Julkunen I. J. Biol. Chem. 2001; 276: 16447-16455Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 26Fagerlund R. Melén K. Kinnunen L. Julkunen I. J. Biol. Chem. 2002; 277: 30072-30078Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 27Meyer T. Begitt A. Lödige I. van Rossum M. Vinkemeier U. EMBO J. 2002; 21: 344-354Crossref PubMed Scopus (145) Google Scholar). In response to IFN-γ stimulation, STAT1 homodimers are formed, whereas IFN-α stimulation results in the formation of STAT1-STAT2 heterodimers. Both STAT1 homodimers and STAT1-STAT2 heterodimers are specifically bound to importin α5 (24Sekimoto T. Imamoto N. Nakajima K. Hirano T. Yoneda Y. EMBO J. 1997; 16: 7067-7077Crossref PubMed Scopus (306) Google Scholar, 26Fagerlund R. Melén K. Kinnunen L. Julkunen I. J. Biol. Chem. 2002; 277: 30072-30078Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 28McBride K.M. Banninger G. McDonald C. Reich N.C. EMBO J. 2002; 21: 1754-1763Crossref PubMed Scopus (195) Google Scholar). The STAT1-binding site of importin α5 has been suggested to be located in the very C-terminal end of STAT1 apart from the arm repeat domain (24Sekimoto T. Imamoto N. Nakajima K. Hirano T. Yoneda Y. EMBO J. 1997; 16: 7067-7077Crossref PubMed Scopus (306) Google Scholar). Also other regions or completely different mechanisms have been suspected to affect the nuclear translocation of STATs. Strehlow and Schindler (29Strehlow I. Schindler C. J. Biol. Chem. 1998; 273: 28049-28056Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) suggested that the most N-terminal amino acids (the first 129 residues) regulate the nuclear import of STAT molecules. Subramaniam et al. (30Subramaniam P.S. Larkin III, J. Mujtaba M.G. Walter M.R. Johnson H.M. J. Cell Sci. 2000; 113: 2771-2781PubMed Google Scholar) suggested that the C-terminal nuclear localization sequence of IFN-γ regulates STAT1α nuclear import, and Bild et al. (31Bild A.H. Turkson J. Jove R. EMBO J. 2002; 21: 3255-3263Crossref PubMed Scopus (127) Google Scholar) showed that receptor-mediated endocytosis is necessary for STAT3 nuclear import. Influenza A virus nucleoprotein (NP) and SV40 large T antigen have functioned as important model molecules for analyzing the nuclear import machinery. Influenza A virus NP encapsidates the viral genome and is essential for viral transcription, replication, and packaging. NP interacts with viral RNA, NP, and other viral proteins as well as cellular proteins including importins α1 and α5 (32O'Neill R.E. Palese P. Virology. 1995; 206: 116-125Crossref PubMed Scopus (133) Google Scholar, 33Görlich D. Prehn S. Laskey R.A. Hartmann E. Cell. 1994; 79: 767-778Abstract Full Text PDF PubMed Scopus (601) Google Scholar). Two NLSs have been identified in this protein, an unusual NLS at the N terminus (34Wang P. Palese P. O'Neill R.E. J. Virol. 1997; 71: 1850-1856Crossref PubMed Google Scholar) and a classical bipartite NLS in the middle of the molecule (35Weber F. Kochs G. Gruber S. Haller O. Virology. 1998; 250: 9-18Crossref PubMed Scopus (91) Google Scholar). Apparently, the N-terminal NLS mediates NP interaction with importins (34Wang P. Palese P. O'Neill R.E. J. Virol. 1997; 71: 1850-1856Crossref PubMed Google Scholar, 36Neumann G. Castrucci M.R. Kawaoka Y. J. Virol. 1997; 71: 9690-9700Crossref PubMed Google Scholar). SV40 large T antigen has a monopartite NLS that interacts with importin α (19Kalderon D. Roberts B.L. Richardson W.D. Smith A.E. Cell. 1984; 39: 499-509Abstract Full Text PDF PubMed Scopus (1874) Google Scholar, 20Lanford R.E. Butel J.S. Cell. 1984; 37: 801-813Abstract Full Text PDF PubMed Scopus (367) Google Scholar, 37Conti E. Kuriyan J. Struct. Fold. Des. 2000; 8: 329-338Abstract Full Text Full Text PDF Scopus (255) Google Scholar). Here we show by site-directed mutagenesis that importin α5 recognizes the nonclassical STAT1, STAT2, and influenza A virus NP NLSs by its arm repeat domain. The binding site for STAT NLS is composed of arm repeats 8 and 9 and for influenza A virus NP of arm repeats 7–9. Both binding sites differ from the previously described major and minor classical NLS binding sites of importin α. We also show that influenza A virus NP binds to the C terminus and SV40 large T antigen to the N-terminal NLS binding site of importin α3, indicating that one importin α molecule is able to use different binding sites for various NLSs. Cells—Monolayers and suspension cultures of Spodoptera frugiperda Sf 9 cells were maintained in TNM-FH medium and used for baculovirus expression as described (38Summers M.D. Smith G.E. Tex. Agric. Exp. Stn. Bull. 1986; 1555: 1-57Google Scholar). Human NK-92 cell line was maintained in continuous culture in minimum Eagle's medium-α (Invitrogen) supplemented with 12% horse serum (Invitrogen), 12% fetal calf serum, 0.2 mm inositol, 20 mm folic acid, 40 mm 2-mercaptoethanol, 2 mm l-glutamine, antibiotics, and 100 IU/ml of human recombinant interleukin-2 (Chiron, Emeryville, CA). Interferons and Other Reagents—Human leukocyte IFN-α (6 × 106 IU/ml) was kindly provided by Dr. Kari Cantell at our Institute (39Cantell K. Hirvonen S. Kauppinen H.-L. Methods Enzymol. 1986; 119: 54-63Crossref PubMed Scopus (36) Google Scholar). 35S-Labeled PRO-MIX (>1000 Ci/mmol) was obtained from Amersham Biosciences. Antibodies—In Western blot analysis rabbit anti-STAT1 (c-24; 1:10,000; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-STAT2 (c-20; 1:2000; Santa Cruz Biotechnology), and mouse monoclonal anti-SV40 large T antigen (sc-147; 1:1000; Santa Cruz Biotechnology) immunoglobulins were used as suggested by the manufacturer. Influenza A NP antibodies (40Ronni T. Sareneva T. Pirhonen J. Julkunen I. J. Immunol. 1995; 154: 2764-2774PubMed Google Scholar) were used at a 1:500 dilution. In Western blotting secondary horseradish peroxidase-conjugated goat anti-rabbit (1:2000; Dako, Glostrup, Denmark) or anti-mouse immunoglobulins (1:5000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) were used. Plasmids and DNA Manipulations—The importin α1 and α3 gene constructs encoding C-terminal His-tagged proteins have been described previously (9Köhler M. Speck C. Christiansen M. Bischoff F.R. Prehn S. Haller H. Görlich D. Hartmann E. Mol. Cell. Biol. 1999; 19: 7782-7791Crossref PubMed Google Scholar). His tags were replaced by GST tags from pFA6aGST-HIS3Mx6 via BamHI/HindIII using PCR mutagenesis. Importin β and α5 cDNAs in GST expression vectors were kindly provided by Drs. D. Görlich and M. Malim, respectively. To create N- and C-terminal point mutations to GST-importin α5, we used QuikChange™ Site-directed mutagenesis kit (Stratagene, La Jolla, CA). The primers used are 5′-CAC TGC AGT TTG AAT CAG CTG CGG TAC TGG CAG CTA TTG CTT CAG GAA ATT CTC (arm 2), 5′-GAT GTC CAG GAA CAG GCA GTC GCG GCT CTT GGC GCC ATT GCT GGA GAT AGT ACC ATG (arm 3), 5′-CGG AAT GCA GTA GCG GCT TTG TCT AAT C (arm 4), 5′-GAA TCT ATC AAA AAG GAA GCA TGT GCG ACG ATA GCT GCT ATT ACA GCT GGA AAT AGG GC (arm 7), 5′-CGG ACA AGA AAA GAA GCA GCT GCG GCC ATC ACG GCT GCA ACT TCT GGA GGA TCA GCT G (arm 8), and 5′-GAT TGT ACA GGT TGC CCT AGC CGG CTT GGA AGC TAT CCT GAG GCT TGG AGA AC (arm 9). To create arm repeat mutations to GST-importin α3, we used QuikChange™ Site-directed mutagenesis kit (Stratagene). The primers used are 5′-GTC TGT GAG CAA GCA GTG GCG GCA TTG GGA GCT ATC ATA GGT GAT GGG CCC CAG (arm 3) and 5′-GGC ACT CAA AAA GAA GCT GCT GCC GCC ATA AGT GCC TTA ACA ATT AGT GGA AGG (arm 8). Human importin α7 gene (9Köhler M. Speck C. Christiansen M. Bischoff F.R. Prehn S. Haller H. Görlich D. Hartmann E. Mol. Cell. Biol. 1999; 19: 7782-7791Crossref PubMed Google Scholar) (GenBank™ accession number AF060543) was PCR-modified with oligonucleotides GAG CGG ATC CAC CAT GGA GAC CAT GGC GAG CCC (5′ oligonucleotide, initiation codon underlined) and TTC TTA GGA TCC CTA TTA TAG CTG GAA GCC CTC (3′ oligonucleotide) to create BamHI cloning sites (in boldface) on both sides of the gene coding region. After BamHI digestion the insert was cloned into the pGEX-2T GST fusion vector (Amersham Biosciences). Baculovirus expression constructs of SV40 large T antigen and influenza A virus NP have been described previously (26Fagerlund R. Melén K. Kinnunen L. Julkunen I. J. Biol. Chem. 2002; 277: 30072-30078Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 41Cantalupo P. Saenz-Robles M.T. Pipas J.M. Methods Enzymol. 1999; 306: 297-307Crossref PubMed Scopus (12) Google Scholar). All DNA manipulations were performed according to standard protocols, and the newly created gene constructs were partially sequenced. Baculovirus Expression—Human Tyk2 cDNA in the baculovirus expression plasmid pVL1392 and SV40 large T antigen cDNA in the baculovirus expression plasmid were kindly provided by Dr. Sandra Pellegrini (Institute Pasteur, Paris) and Dr. J. M. Pipas (University of Pittsburgh, Pittsburgh (41Cantalupo P. Saenz-Robles M.T. Pipas J.M. Methods Enzymol. 1999; 306: 297-307Crossref PubMed Scopus (12) Google Scholar)), respectively. Wt STAT1 and STAT2 baculovirus constructs were as described (25Melén K. Kinnunen L. Julkunen I. J. Biol. Chem. 2001; 276: 16447-16455Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Influenza A (PR8) virus NP gene was inserted into the BamHI site of the pAcYM1 expression plasmid, and recombinant viruses were obtained by plaque purification as described (38Summers M.D. Smith G.E. Tex. Agric. Exp. Stn. Bull. 1986; 1555: 1-57Google Scholar). For protein production Sf 9 cells were coinfected with Tyk2 and STAT protein-expressing baculoviruses for 42 h. Influenza A NP and SV40 large T antigen were produced without coinfection with Tyk2. Virus-infected Sf 9 cells were collected, and the whole cell extracts were prepared by disrupting the cells in 50 mm Tris-HCl buffer, pH 7.4, 150 mm NaCl, 5 mm EDTA, and 1% Triton X-100 (immunoprecipitation (IP) buffer) on ice for 30 min. The cells were disrupted with a syringe. Cell extracts were clarified by Eppendorf centrifugation (13,000 rpm, 10 min). Importin Binding Assay, SDS-PAGE, and Western Blotting—Human importins β, α1, α3, α5, and α7 as well as mutants in the arm repeats 2, 3, 2 + 3, 2 + 4, 3 + 4, 2 + 3 + 4, 2 + 3 + 7, 2 + 3 + 8, 7, 8, and 9 of importin α5 and mutants in the arm repeats 3 and 8 of importin α3 were expressed in Escherichia coli BL21 cells as GST fusion proteins under isopropyl-1-thio-β-d-galactopyranoside induction. Bacteria were lysed in IP buffer with 5 mg/ml lysozyme (Sigma) at room temperature for 30 min, briefly sonicated, and clarified by Eppendorf centrifugation (13,000 rpm, 5 min). From 0.1 to 1.0 ml of bacterial cell extracts containing GST-importin proteins were allowed to bind to 25 μl of glutathione-Sepharose 4 Fast Flow beads (Amersham Biosciences) in IP buffer with agitation at +4 °C for 60 min, followed by washing twice with the buffer. From 0.1 to 1.0 ml of baculovirus cell extracts containing STATs, influenza A NP, or SV40 large T antigen as well as 50 μl of in vitro translated influenza A virus NP protein (TnT Coupled Reticulocyte Lysate Systems, Promega, Madison, WI) were allowed to bind in IP buffer to Sepharose-immobilized GST-importins α3 and α5 or GST-importin β at +4 °C for 2 h, followed by washing 3 times with the buffer. Sepharose beads were dissolved in 50 μl of 2× Laemmli sample buffer, and the proteins were separated on 8% SDS-PAGE (42Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). The gels were stained with Coomassie Brilliant Blue or transferred onto Immobilon-P membranes (polyvinylidine difluoride; Millipore, Bedford, MA), followed by staining with primary and secondary antibodies and visualization of the proteins with the enhanced chemiluminescence system (Amersham Biosciences) as recommended by the manufacturer. 35S-Labeled protein samples were separated on 8% SDS-PAGE. The gels were stained and treated with Amplify reagent (Amersham Biosciences) as recommended and autoradiographed. Structural Features of the Conserved Importin α Molecules— Importin α molecules are evolutionarily conserved and can be found in eukaryotes from yeast to humans. In humans, six importin α isoforms have been identified (Fig. 1A). Yeast cells contain only one importin α isoform that shows over 55% sequence similarity with the human importins. The similarity of the six human importins is between 56 and 89% (Fig. 1A). The central domain of importin α is composed of 10 arm repeats (Fig. 1B). The well conserved tryptophan and asparagine residues in arm repeats 2–4 and 7–9 in the helical surface groove of the importin α molecule are thought to be crucial for NLS binding (10Conti E. Uy M. Leighton L. Blobel G. Kuriyan J. Cell. 1998; 94: 193-204Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar, 11Fontes M.R.M. Teh T. Kobe B. J. Mol. Biol. 2000; 297: 1183-1194Crossref PubMed Scopus (315) Google Scholar) (Fig. 1, B and C). An alignment of the central domain of human importin α molecules is shown in Fig. 1C. Typical arm repeats contain a tryptophan-asparagine pair at analogous positions. A tryptophan-asparagine pair was found in arm repeats 2–4 and 7–8. Arm repeats 5 and 6, which do not contain this pair, have not been shown to participate directly in NLS binding. Arm repeat 9 contains the conserved asparagine residue, but the tryptophan residue is replaced either by an aspartic acid (importins α1, α3, and α4) or an asparagine (importins α5, α6, and α7). Based on published crystal structures (10Conti E. Uy M. Leighton L. Blobel G. Kuriyan J. Cell. 1998; 94: 193-204Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar, 11Fontes M.R.M. Teh T. Kobe B. J. Mol. Biol. 2000; 297: 1183-1194Crossref PubMed Scopus (315) Google Scholar) and the alignment in Fig. 1C, series of mutations were created to analyze the interactions between importin α and various NLSs. Arm repeat 9 was included in our analysis, because the two asparagine residues appeared as part of a possible NLS binding pocket (Fig. 1C). IFN-stimulated STAT Dimers Bind Specifically to Importin α5—At present, STAT1 homodimers and STAT1-STAT2 heterodimers are the only STAT dimers that have been shown to interact with importin α molecules (24Sekimoto T. Imamoto N. Nakajima K. Hirano T. Yoneda Y. EMBO J. 1997; 16: 7067-7077Crossref PubMed Scopus (306) Google Scholar, 26Fagerlund R. Melén K. Kinnunen L. Julkunen I. J. Biol. Chem. 2002; 277: 30072-30078Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 28McBride K.M. Banninger G. McDonald C. Reich N.C. EMBO J. 2002; 21: 1754-1763Crossref PubMed Scopus (195) Google Scholar). Recent mutational analyses suppose that leucine 407 and lysines 410 and 413 within the DNA binding domain of STAT1 function as an NLS and mediate binding to importin α5 (25Melén K. Kinnunen L. Julkunen I. J. Biol. Chem. 2001; 276: 16447-16455Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 26Fagerlund R. Melén K. Kinnunen L. Julkunen I. J. Biol. Chem. 2002; 277: 30072-30078Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 28McBride K.M. Banninger G. McDonald C. Reich N.C. EMBO J. 2002; 21: 1754-1763Crossref PubMed Scopus (195) Google Scholar). To determine the possible interactions of STAT1-STAT2 dimers with other importin isoforms than α5, we stimulated the human NK-92 cell line with 1000 IU/ml of IFN-α for 30 min. Cell extracts were prepared, and the cellular proteins were allowed to bind to Sepharose-immobilized GST-importins α1, α3, α5, or α7 at +4 °C for 2 h, followed by an analysis of the bound STAT proteins by Western blotting. As shown in Fig. 2, STAT1-STAT2 heterodimers were readily bound to importin α5, whereas no binding to importins α1, α3, or α7 in IFN-α-stimulated cells was detected. Clearly, binding of STAT1-STAT2 dimers to importin α5 is highly specific. Tyrosine-phosphorylated STAT Dimers, Influenza A NP, and SV40 Large T Antigen Bind to Different Importin α Isoforms—To characterize further STAT binding to different importin α isoforms, we used a baculovirus expression system to reconstitute STAT activation (26Fagerlund R. Melén K. Kinnunen L. Julkunen I. J. Biol. Chem. 2002; 277: 30072-30078Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Coinfection of Sf 9 cells with recombinant Tyk2 and STAT protein expressing baculoviruses resulted in an efficient expression and tyrosine phosphorylation of STAT1 and STAT2 proteins. Tyrosine-phosphorylated STAT1 and STAT2 formed dimers, which enabled us to analyze the potential interactions of dimeric STAT complexes with importins (25Melén K. Kinnunen L. Julkunen I. J. Biol. Chem. 2001; 276: 16447-16455Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 26Fagerlund R. Melén K. Kinnunen L. Julkunen I. J. Biol. Chem. 2002; 277: 30072-30078Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). As shown in Fig. 3, baculovirus-expressed STAT1 homodimers or STAT1-STAT2 heterodimers bound specifically to Sepharose-immobilized GST-importin α5 but not to importin α1, α3, or α7 isoforms. These results, together with those shown in Fig. 2 using natural IFN-α-stimulated cells, show that the reconstituted STAT activation in the baculovirus expression system is a reliable method to study STAT-importin α interactions. We also analyzed interactions of Sepharose-immobilized GST-importins α1, α3, α5, or α7 with baculovirus-expressed influenza A NP and SV40 large T antigen. Two NLSs have been identified in influenza A virus NP, a nonclassical NLS at the N terminus (34Wang P. Palese P. O'Neill R.E. J. Virol. 1997; 71: 1850-1856Crossref PubMed Google Scholar) and a classical bipartite NLS in the middle of the molecule (35Weber F. Kochs G. Gruber S. Haller O. Virology. 1998; 250: 9-18Crossref PubMed Scopus (91) Google Scholar). It has been shown that the N-terminal NLS of NP is involved in the interaction with importins (34Wang P. Palese P. O'Neill R.E. J. Virol. 1997; 71: 1850-1856Crossref PubMed Google Scholar, 36Neumann G. Castrucci M.R. Kawaoka Y. J. Virol. 1997; 71: 9690-9700Crossref PubMed Google Scholar). Here we show that influenza A virus NP bound strongly to importins α1 and α5 and to a lesser extent to importin α3, whereas no marked binding to importin α7 isoform was detected (Fig. 3C). Importins α1 and α5 have been shown to bind efficiently SV40 large T antigen NLS peptide or T antigen NLS conjugated to BSA (24Sekimoto T. Imamoto N. Nakajima K. Hirano T. Yoneda Y. EMBO J. 1997; 16: 7067-7077Crossref PubMed Scopus (306) Google Scholar, 43Miyamoto Y. Imamoto N. Sekimoto T. Tachibana T. Seki T. Tada S. Enomoto T. Yoneda Y. J. Biol. Chem. 1997; 272: 26375-26381Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 44Nadler S.G. Tritschler D. Haffar O.K. Blake J. Bruce A.G. Cleaveland J.S. J. Biol. Chem. 1997; 272: 4310-4315Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Instead, our binding experiments of SV40 large T antigen with Sepharose-immobilized GST-importins revealed that full-length T antigen bound strongly to importin α3 and to a lesser extent to importin α1 but not to importin α5 or α7 isoforms (Fig. 3D), indicating that the natural context of an NLS can greatly affect its accessibility to importins. A C-terminal Unique NLS Binding Site, Including Arm Repeats 8 and 9 of Importin α5, Binds STATs—Because STAT NLS is formed by a cluster of basic amino acids within the DNA binding domain of STAT1 and STAT2 (25Melén K. Kinnunen L. Julkunen I. J. Biol. Chem. 2001; 276: 16447-16455Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 27Meyer T. Begitt A. Lödige I. van Rossum M. Vinkemeier U. EMBO J. 2002; 21: 344-354Crossref PubMed Scopus (145) Google Scholar) and recognized by importin α5 (26Fagerlund R. Melén K. Kinnunen L. Julkunen I. J. Biol. Chem. 2002; 277: 30072-30078Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 28McBride K.M. Banninger G. McDonald C. Reich N.C. EMBO J. 2002; 21: 1754-1763Crossref PubMed Scopus (195) Google Scholar), we reasoned that importin α5 arm repeats might be involved in STAT NLS binding. To analyze the role of arm repeats in STAT-importin α5 interaction, we created several mutations in the binding pockets of arm repeats 2–4 and 7–9 of importin α5 (Figs. 1 and 4). Mutations in arm repeat 2 (W149A, T152A, and N153A), arm repeat 3 (W191A and N195A), arm repeat 4 (W234A), arm repeat 7 (W360A, S363A, and N364A), o
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