NF-κB Is Transported into the Nucleus by Importin α3 and Importin α4
2005; Elsevier BV; Volume: 280; Issue: 16 Linguagem: Inglês
10.1074/jbc.m500814200
ISSN1083-351X
AutoresRiku Fagerlund, Leena Kinnunen, Matthias Köhler, Ilkka Julkunen, Krister Melén,
Tópico(s)RNA Research and Splicing
ResumoNF-κB transcription factors are retained in the cytoplasm in an inactive form until they are activated and rapidly imported into the nucleus. We identified importin α3 and importin α4 as the main importin α isoforms mediating TNF-α-stimulated NF-κB p50/p65 heterodimer translocation into the nucleus. Importin α3 and α4 are close relatives in the human importin α family. We show that importin α3 isoform also mediates nuclear import of NF-κB p50 homodimer in nonstimulated cells. Importin α3 is shown to directly bind to previously characterized nuclear localization signals (NLSs) of NF-κB p50 and p65 proteins. Importin α molecules are known to have armadillo repeats that constitute the N-terminal and C-terminal NLS binding sites. We demonstrate by site-directed mutagenesis that NF-κB p50 binds to the N-terminal and p65 to the C-terminal NLS binding site of importin α3. In vitro competition experiments and analysis of cellular NF-κB suggest that NF-κB binds to importin α only when it is free of IκBα. The present study demonstrates that the nuclear import of NF-κB is a highly regulated process mediated by a subset of importin α molecules. NF-κB transcription factors are retained in the cytoplasm in an inactive form until they are activated and rapidly imported into the nucleus. We identified importin α3 and importin α4 as the main importin α isoforms mediating TNF-α-stimulated NF-κB p50/p65 heterodimer translocation into the nucleus. Importin α3 and α4 are close relatives in the human importin α family. We show that importin α3 isoform also mediates nuclear import of NF-κB p50 homodimer in nonstimulated cells. Importin α3 is shown to directly bind to previously characterized nuclear localization signals (NLSs) of NF-κB p50 and p65 proteins. Importin α molecules are known to have armadillo repeats that constitute the N-terminal and C-terminal NLS binding sites. We demonstrate by site-directed mutagenesis that NF-κB p50 binds to the N-terminal and p65 to the C-terminal NLS binding site of importin α3. In vitro competition experiments and analysis of cellular NF-κB suggest that NF-κB binds to importin α only when it is free of IκBα. The present study demonstrates that the nuclear import of NF-κB is a highly regulated process mediated by a subset of importin α molecules. NF-κB 1The abbreviations used are: NF-κB, nuclear factor κB; NLS, nuclear localization signal; arm, armadillo motif; TNF-α, tumor necrosis factor-α; LMB, leptomycin B; NES, nuclear export signal; NPC, nuclear pore complex; GST, glutathione S-transferase; FITC, fluorescein isothiocyanate; STAT, signal transducers and activators of transcription. 1The abbreviations used are: NF-κB, nuclear factor κB; NLS, nuclear localization signal; arm, armadillo motif; TNF-α, tumor necrosis factor-α; LMB, leptomycin B; NES, nuclear export signal; NPC, nuclear pore complex; GST, glutathione S-transferase; FITC, fluorescein isothiocyanate; STAT, signal transducers and activators of transcription. p50/p65 transcription factor has a central role in controlling host cell immune and inflammatory responses, cell differentiation, and apoptosis (1Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2919) Google Scholar, 2Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4585) Google Scholar). Dysregulation of NF-κB has been associated with several common diseases such as cancer and diabetes (3Baldwin Jr., A.S. J. Clin. Investig. 2001; 107: 3-6Crossref PubMed Google Scholar). Cytoplasmic NF-κB can be rapidly activated by various physiological and nonphysiological stimuli such as cytokines, growth factors, bacterial or viral infection and UV irradiation. Activation of NF-κB is followed by its rapid translocation into the nucleus where it activates the transcription of numerous genes including those encoding for cytokines and cell adhesion molecules. Some genes can be transcriptionally up-regulated within minutes after NF-κB activation (2Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4585) Google Scholar, 4Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5552) Google Scholar). NF-κB transcription factors are dimers belonging to the Rel family (5Sen R. Baltimore D. Cell. 1986; 46: 705-716Abstract Full Text PDF PubMed Scopus (1924) Google Scholar). All five mammalian NF-κB subunits, p65 (RelA), RelB, c-Rel, p50 (and its precursor p105), and p52 (and its precursor p100) contain an N-terminal Rel homology domain responsible for their dimerization, nuclear localization, and DNA binding (6Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2011) Google Scholar, 7Verma I.M. Stevenson J.K. Schwarz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1655) Google Scholar). NF-κB subunits can form various dimers, but the classical, best characterized form is composed of p50 and p65 (1Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2919) Google Scholar, 7Verma I.M. Stevenson J.K. Schwarz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1655) Google Scholar, 8Thanos D. Maniatis T. Mol. Cell. Biol. 1995; 15: 152-164Crossref PubMed Scopus (127) Google Scholar). p65, RelB, and c-Rel contain a C-terminal transcription activation domain, and they can therefore form transcription-activating dimers with each other and with p50 or p52. p50 and p52 proteins lack the transcription activation domain, and the homodimers they form are mostly suppressors of gene expression (9Zhong H. May M.J. Jimi E. Ghosh S. Mol. Cell. 2002; 9: 625-636Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar). NF-κB dimer is held in an inactive state in the cytoplasm by an inhibitor protein (IκB) that masks the NLSs of the subunits (10Beg A.A. Ruben S.M. Scheinman R.I. Haskill S. Rosen C.A. Baldwin Jr., A.S. Genes Dev. 1992; 6: 1899-1913Crossref PubMed Scopus (610) Google Scholar, 11Ganchi P.A. Sun S.C. Greene W.C. Ballard D.W. Mol. Biol. Cell. 1992; 3: 1339-1352Crossref PubMed Scopus (204) Google Scholar, 12Henkel T. Zabel U. van Zee K. Müller J.M. Fanning E. Baeuerle P.A. Cell. 1992; 68: 1121-1133Abstract Full Text PDF PubMed Scopus (304) Google Scholar, 13Zabel U. Henkel T. Silva M.S. Baeuerle P.A. EMBO J. 1993; 12: 201-211Crossref PubMed Scopus (266) Google Scholar). IκBα preferentially inhibits the nuclear translocation of the p50/p65 heterodimer. Other IκB molecules found in higher vertebrates include IκBβ, IκBϵ, and Bcl3. All IκB molecules contain ankyrin repeats, which mediate specific interactions with the Rel-homology domains of NF-κB molecules. The C-terminal regions of p100 and p105 proteins also contain ankyrin repeats and they can function as an IκB (2Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4585) Google Scholar, 4Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5552) Google Scholar, 14Rice N.R. MacKichan M.L. Israel A. Cell. 1992; 71: 243-253Abstract Full Text PDF PubMed Scopus (343) Google Scholar). Crystal structures of most NF-κB Rel domains bound to DNA or IκB have been determined (15Cramer P. Larson C.J. Verdine G.L. Müller C.W. EMBO J. 1997; 16: 7078-7090Crossref PubMed Google Scholar, 16Jacobs M.D. Harrison S.C. Cell. 1998; 95: 749-758Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar, 17Huxford T. Huang D.B. Malek S. Ghosh G. Cell. 1998; 95: 759-770Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar, 18Chen F.E. Huang D.B. Chen Y.Q. Ghosh G. Nature. 1998; 391: 410-413Crossref PubMed Scopus (331) Google Scholar, 19Ghosh G. van Duyne G. Ghosh S. Sigler P.B. Nature. 1995; 373: 303-310Crossref PubMed Scopus (505) Google Scholar, 20Huang D.B. Chen Y.Q. Ruetsche M. Phelps C.B. Ghosh G. Structure (Camb). 2001; 9: 669-678Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 21Berkowitz B. Huang D.B. Chen-Park F.E. Sigler P.B. Ghosh G. J. Biol. Chem. 2002; 277: 24694-24700Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 22Malek S. Huang D.B. Huxford T. Ghosh S. Ghosh G. J. Biol. Chem. 2003; 278: 23094-23100Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). p50/p65 heterodimers are activated by the IκB kinase (IKK) complex that phosphorylates IκBα on two N-terminal serine residues (23Verma I.M. Stevenson J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11758-11760Crossref PubMed Scopus (179) Google Scholar, 24Stancovski I. Baltimore D. Cell. 1997; 91: 299-302Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 25DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1900) Google Scholar, 26Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1841) Google Scholar, 27Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (845) Google Scholar, 28Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (945) Google Scholar, 29Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4058) Google Scholar). Phosphorylation triggers polyubiquitination of IκBα, which is then rapidly degraded by the proteasome (29Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4058) Google Scholar, 30Henkel T. Machleidt T. Alkalay I. Kronke M. Ben-Neriah Y. Baeuerle P.A. Nature. 1993; 365: 182-185Crossref PubMed Scopus (1034) Google Scholar). As a consequence, the NLSs of p50 and p65 proteins are unmasked, and the dimers are translocated into the nucleus where they activate NF-κB responsive genes. p50/p65 heterodimers and p50 homodimers are considered the most abundant NF-κB types in most cells. p50 homodimer formation has been suggested to take place cotranslationally. During this process p50/p105 intermediates are formed, where the C-terminal ankyrin repeat containing domain of p105 functions as an IκB (IκBγ). Additional post-translational steps regulate p50 homodimer formation (14Rice N.R. MacKichan M.L. Israel A. Cell. 1992; 71: 243-253Abstract Full Text PDF PubMed Scopus (343) Google Scholar, 29Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4058) Google Scholar, 31Lin L. DeMartino G.N. Greene W.C. EMBO J. 2000; 19: 4712-4722Crossref PubMed Google Scholar). Under nonstimulated conditions 10–20% of p105 proteins are processed to form p50 homodimers (32Ciechanover A. Gonen H. Bercovich B. Cohen S. Fajerman I. Israel A. Mercurio F. Kahana C. Schwartz A.L. Iwai K. Orian A. Biochimie (Paris). 2001; 83: 341-349Crossref PubMed Scopus (57) Google Scholar). The affinity between p50 and p65 proteins is stronger than between two p50 proteins, but the actual mechanism of p50/p65 heterodimer formation in the cytoplasm is poorly understood (18Chen F.E. Huang D.B. Chen Y.Q. Ghosh G. Nature. 1998; 391: 410-413Crossref PubMed Scopus (331) Google Scholar, 31Lin L. DeMartino G.N. Greene W.C. EMBO J. 2000; 19: 4712-4722Crossref PubMed Google Scholar). Eukaryotic cells are compartmentalized by the nuclear envelope into the cytoplasm and the nucleus. The nuclear envelope contains nuclear pore complexes (NPCs), which mediate the molecular traffic between the two compartments. The nucleocytoplasmic traffic of large molecules (>25 nm in diameter) is regulated by specific nuclear import and export systems. Proteins that contain classical NLSs are imported into the nucleus by importin α/β heterodimers. Importin α binds to NLS containing proteins, and importin β is responsible for the docking of the importin-cargo complex to the cytoplasmic side of the NPC followed by translocation of the complex through the NPC (33Görlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-660Crossref PubMed Scopus (1663) Google Scholar, 34Macara I.G. Microbiol. Mol. Biol. Rev. 2001; 65: 570-594Crossref PubMed Scopus (737) Google Scholar). A classical monopartite NLS consists of a stretch of basic amino acids, arginines and lysines (35Morin N. Delsert C. Klessig D.F. Mol. Cell. Biol. 1989; 9: 4372-4380Crossref PubMed Scopus (40) Google Scholar, 36Dingwall C. Laskey R.A. Trends Biochem. Sci. 1991; 16: 478-481Abstract Full Text PDF PubMed Scopus (1708) Google Scholar). Classical NLSs are found in p50 and p65 (37Gilmore T.D. Temin H.M. J. Virol. 1988; 62: 703-714Crossref PubMed Google Scholar, 38Blank V. Kourilsky P. Israel A. EMBO J. 1991; 10: 4159-4167Crossref PubMed Scopus (127) Google Scholar). Recent studies have shown that some signaling molecules are transported into the nucleus by NLS- and importin-independent processes by associating directly with proteins of the NPC (39Xu L. Massague J. Nat. Rev. Mol. Cell. Biol. 2004; 5: 209-219Crossref PubMed Scopus (217) Google Scholar). Six importin α family members have been identified in humans; importin α1 (Rch1, hSRP1α), importin α3 (Qip1), importin α4 (hSRP1γ), importin α5 (hSRP1, NPI1), importin α6, and importin α7 (40Cuomo 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 (163) Google Scholar, 41Cortes P. Ye Z.S. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7633-7637Crossref PubMed Scopus (168) Google Scholar, 42Köhler M. Ansieau S. Prehn S. Leutz A. Haller H. Hartmann E. FEBS Lett. 1997; 417: 104-108Crossref PubMed Scopus (204) Google Scholar, 43Seki T. Tada S. Katada T. Enomoto T. Biochem. Biophys. Res. Commun. 1997; 234: 48-53Crossref PubMed Scopus (81) Google Scholar, 44Nachury 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, 45Köhler 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). The crystal structures of two importin α molecules, yeast karyopherin α and mouse importin α2, have been determined (46Conti E. Uy M. Leighton L. Blobel G. Kuriyan J. Cell. 1998; 94: 193-204Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, 47Fontes M.R. Teh T. Kobe B. J. Mol. Biol. 2000; 297: 1183-1194Crossref PubMed Scopus (313) Google Scholar). Importin α molecules contain a large central domain that consists of 10 tandemly repeated armadillo (arm) motifs, which mediate the interactions with the NLS-containing cargo protein. Each importin α molecule has two potential NLS binding sites that directly interact with the NLS of the cargo. The arm repeats 2–4 comprise the N-terminal NLS binding site and the arm repeats 7–9 the C-terminal NLS binding site (46Conti E. Uy M. Leighton L. Blobel G. Kuriyan J. Cell. 1998; 94: 193-204Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, 47Fontes M.R. Teh T. Kobe B. J. Mol. Biol. 2000; 297: 1183-1194Crossref PubMed Scopus (313) Google Scholar, 48Melén K. Fagerlund R. Franke J. Köhler M. Kinnunen L. Julkunen I. J. Biol. Chem. 2003; 278: 28193-28200Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). We now report that TNF-α-induced nuclear import of NF-κB p50/p65 heterodimers is mediated by importin α3 and importin α4. Importin α3 is also involved in uninduced import of p50 homodimers. Importin α molecules bind to the previously identified NLSs of p50 and p65 proteins. Moreover, by site-directed mutagenesis we show that p50 is bound by the N-terminal and p65 by the C-terminal NLS binding site of importin α3. Antibodies—In Western blot analysis rabbit anti-p65 (sc-109x; 1:5000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), anti-p50 (H-119x; 1:2500; Santa Cruz Biotechnology), anti-IκBα (9242; 1:1000; Cell Signaling Technology, Inc., Beverly, MA) and anti-phospho(serine)-IκBα (9241; 1:1000; Cell Signaling Technology) antibodies were used as suggested by the manufacturer. Anti-importin α1 and α3 antibodies have been described previously (45Köhler 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). In Western blotting secondary horseradish peroxidase-conjugated goat anti-rabbit (1:2000; Dako, Glostrup, Denmark) immunoglobulins were used. For immunoprecipitation anti-p50 (sc-1191; 5 μg/reaction; goat polyclonal; Santa Cruz Biotechnology) and anti-p65 (sc-372x; 5 μg/reaction; goat polyclonal; Santa Cruz Biotechnology) immunoglobulins were used. For confocal laser microscopy rabbit anti-p50 (H-119x; 1:50; Santa Cruz Biotechnology) and anti-c-Myc (sc-789; 1:100; Santa Cruz Biotechnology) or mouse anti-FLAG M5 (1:400; Sigma) and anti-Penta-His (1:50; Qiagen Inc., Valencia, CA) antibodies were used. Secondary antibodies used were FITC-labeled sheep anti-rabbit and anti-mouse IgG F(ab′)2 fragment (1:100; Roche Applied Science, Mannheim, Germany) and Rhodamine Red-X-labeled goat anti-mouse and anti-rabbit immunoglobulins (1:100; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Cells, Media, and Other Reagents—Human A549 lung carcinoma cell line (ATCC, CCL 185) was maintained in continuous culture in minimum Eagle's medium-α (Invitrogen) supplemented with 0.6 μg/ml penicillin, 60 μg/ml streptomycin, and 10% fetal calf serum (Integro, Zaandam, the Netherlands). Human hepatocellular carcinoma HuH7 (49Nakabayashi H. Taketa K. Miyano K. Yamane T. Sato J. Cancer Res. 1982; 42: 3858-3863PubMed Google Scholar) cells were maintained in minimum Eagle's medium-α with supplements as above. In transfection experiments the cells were cultured in the growth medium supplemented with 2% fetal calf serum. Human tumor necrosis factor-α (TNF-α) was purchased from R&D systems (Abingdon, UK). For cell stimulation 5 ng/ml of TNF-α was used. Leptomycin B (LMB) (10 μg/ml) was kindly provided by Dr. Minoru Yoshida from The University of Tokyo, Japan. Monolayers and suspension cultures of Spodoptera frugiperda Sf9 cells that were used for baculovirus expression were maintained in TNM-FH medium as described previously (50Summers M.D. Smith G.E. Tex. Agric. Exp. Stn. Bull. 1986; 1555: 1-57Google Scholar). For in vitro translation 35S-labeled PRO.MIX (>100 Ci/mmol) was used, and it was obtained from Amersham Biosciences (Buchinghamshire, UK). Plasmids and DNA Manipulations—Escherichia coli-produced GST-importins α1, α3, α5, α7, and β as well as the mutants in the arm repeats 3 and 8 of importin α3 have been described previously (48Melén K. Fagerlund R. Franke J. Köhler M. Kinnunen L. Julkunen I. J. Biol. Chem. 2003; 278: 28193-28200Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). To create arm repeat 7 mutations to GST-importin α3 we used Quick-Change™ site-directed mutagenesis kit (Stratagene). The primer used was 5′-GAG AAA ATT AAT AAA GAA GCA GTG GCC TTC CTC TCC GCC ATC ACT GCA GGA AAT CAG CAG CAG. Human p50 and p65 cDNAs in plasmids RcCMV and pCMV were kindly provided by Dr. Jorma Palvimo (University of Helsinki, Helsinki, Finland) and Dr. John Hiscott (McGill University, Montreal, Canada), respectively. To create NLS mutations to p50 and p65 the primers were 5′-CAA AGA TAA AGA AGA AGT GCA GAG GGC AGC TCA GAA GCT CAT GCC CAA TTT TTC G (p50 NLS: K362A, R363A) and 5′-GAT CGT CAC CGG ATT GAG GAG GCA GCT GCA AGG ACA TAT GAG ACC TTC AAG AGC (p65 NLS: K301A, R302A, K303A). To create FLAG-tagged p50 and c-Myc-His-tagged p65 transient transfection constructs for indirect immunofluorescence and confocal laser microscopy, wild type and mutated cDNAs were modified by PCR to create N- and C-terminal BamHI and BglII sites, respectively, for further cloning into the BamHI site of FLAG-pcDNA3.1(+) (51Melén K. Julkunen I. J. Biol. Chem. 1997; 272: 32353-32359Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) and pcDNA3.1/Myc-His(–) (Invitrogen) expression vectors. The primers used were 5′-ATA TAT AGA TCT ACC ATG GCA GAA GAT GAT CCA TAT TTG (5′-oligonucleotide, BglII codons in bold face and initiation codon underlined) and 5′-ATA TAT AGA TCT TTA CAT GGT TCC ATG CTT CAT CCC AGC ATT AGA TTT AG (3′-oligonucleotide) for p50 and 5′-ATA TAT GGA TCC ACC ATG GAC GAA CTG TTC CCC CTC ATC (5′-oligonucleotide, BamHI codons in bold face) and 5′-ATA TAT GGA TCC GGA GCT GAT CTG ACT CAG CAG GGC (3′-oligonucleotide) for p65. All DNA manipulations were performed according to standard protocols. To create a GST fusion vector for the baculovirus expression system, GST-encoding cDNA was first modified by PCR using the pGEX-2T fusion vector (Amersham Biosciences) as a template. To modify an N-terminal GST with a new BamHI cloning site the primers were 5′-A GAA AAC (AGA TCT) ACC ATG TCC CCT ATA CTA GGT TAT TG (5′-oligonucleotide) and 5′-G TGG TGC (AGA TCT) TTA GGA TCC ACG CGG AAC CAG ATC CGA TTT TG (3′-oligonucleotide, BglII sites in parentheses, ATG and STOP codons underlined, and the newly created BamHI cloning site in bold face). The PCR product was first digested with BglII and then subcloned into the BamHI cloning site of the pAcYM1 baculovirus expression vector (50Summers M.D. Smith G.E. Tex. Agric. Exp. Stn. Bull. 1986; 1555: 1-57Google Scholar). Human importin α1, α3, α4, α7, p50, and p65 cDNAs were modified by PCR to create N- and C-terminal BglII (for importin α4 and p50) or BamHI (for importin α1, α3, α7, and p65) sites for further cloning into the BamHI site of GST-pAcYM1 vector. Primers used were: 5′-GCA CAA GGA TCC ACC ATG TCC ACC AAC GAG AAT GCT AAT AC (5′-oligonucleotide, BamHI codons in boldface and initiation codon underlined) and 5′-TGC TTG GGA TCC CTA AAA GTT AAA GGT CCC AGG AGC CCC (3′-oligonucleotide) for importin α1, 5′-GCA CAA GGA TCC ACC ATG GCG GAC AAC GAG AAA CTG GAC (5′-oligonucleotide) and 5′-TGC TTG GGA TCC TTA CTA AAA CTG GAA CCC TTC TGT TGG TAC (3′-oligonucleotide) for importin α3, 5′-GG AGC AGA TCT ACC ATG GCC GAG AAC CCC AGC TTG (5′-oligonucleotide) and 5′-ATA TAT AGA TCT TCA TTA AAA ATT AAA TTC TTT TGT TTG (3′-oligonucleotide) for importin α4, 5′-G AGC GGA TCC ACC ATG GAG ACC ATG GCG AGC CC (5′-oligonucleotide) and 5′-TTC TTA GGA TCC CTA TTA TAG CTG GAA GCC CTC (3′-oligonucleotide) for importin α7, 5′-GCA CAA AGA TCT ACC ATG GCA GAA GAT GAT CCA TAT TTG (5′-oligonucleotide) and 5′-GCA CAA AGA TCT TTA CAT GGT TCC ATG CTT CAT CCC AGC ATT AGA TTT AG (3′-oligonucleotide) for p50 and 5′-ATA TAT GGA TCC ACC ATG GAC GAA CTG TTC CCC CTC ATC (5′-oligonucleotide) and 5′-ATA TAT GGA TCC TTA GGA GCT GAT CTG ACT CAG CAG GGC (3′-oligonucleotide) for p65. Recombinant viruses were obtained as described previously (50Summers M.D. Smith G.E. Tex. Agric. Exp. Stn. Bull. 1986; 1555: 1-57Google Scholar). Production of GST Fusion Proteins in E. coli and Sf9 Cells, and Preparation of Cell Lysate from A549 Cells—Human importins α1, α3, α5, α7, and β as well as mutants in the arm repeats 3, 3+8, and 7+8of importin α3 were expressed in E. coli BL21 cells as GST fusion proteins at +21 °C for 4 h under 0.2 mm isopropyl-1-thio-β-d-galactopyranoside induction. Bacteria were lysed in 50 mm Tris-HCl buffer, pH 7.4, 150 mm NaCl, 5 mm EDTA and 1% Triton X-100 (lysis (L) buffer) with 5 mg/ml lysozyme (Sigma) and protease inhibitors (Complete; Roche Applied Science, Basel, Switzerland) for 30 min at room temperature, briefly sonicated, and clarified by Eppendorf centrifugation (13,000 rpm, 5 min). For protein production of GST-tagged human importin α1, α3, α4, α7, p50, and p65 in Sf9 cells, the cells were first infected with importin, p50 or p65-expressing baculoviruses for 42 h and then collected, and whole cell extracts were prepared by disrupting the cells in l-buffer on ice for 10 min. The cells were disrupted by passing them through a syringe. Cell extracts were clarified by Eppendorf centrifugation (13,000 rpm, 10 min). For preparation of cell lysate, A549 cells were stimulated with TNF-α (5 ng/ml) or left nonstimulated. The cells were washed, harvested, and lysed in l-buffer containing 1 mm NaVO4 and protease inhibitors on ice for 10 min. The cells were disrupted by passing them through a syringe. Cell debris was removed by centrifugation at 13,000 rpm at +4 °C for 10 min. Importin Binding Assay, Immunoprecipitation, SDS-PAGE, and Western Blotting—For GST pull-down experiments, GST fusion proteins were first allowed to bind to 25 μl of glutathione-Sepharose 4 Fast Flow beads (Amersham Biosciences) at +4 °C for 60 min in L-buffer followed by washing twice with the buffer. 25 μl of glutathione-Sepharose-immobilized GST fusion proteins was mixed with 200 μl of cell lysate and rotated at +4 °C for 2 h followed by washing three times with L-buffer. Sepharose beads were dissolved in 30 μl of 2× Laemmli sample buffer, and the proteins were separated on 10% SDS-PAGE (52Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206620) 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 (ECL) (Amersham Biosciences) as recommended by the manufacturer. To detect the binding of in vitro translated proteins in GST pull-down experiments, 25 to 50 μlof in vitro translated p50, p65, or IκBα proteins (TnT-coupled reticulocyte lysate systems, Promega) were allowed to bind to 25 μl of Sepharose-immobilized GST-importin on ice for 60 min followed by washing three times with l-buffer. GST importin-bound 35S-labeled proteins were dissolved in 30 μl of 2× Laemmli sample buffer and separated on 10% SDS-PAGE. The gels were fixed and treated with Amplify reagent (Amersham Biosciences) as specified by the manufacturer and autoradiographed. For immunoprecipitation experiments, 25 μl of protein A-Sepharose (Amersham Biosciences) was incubated with 5 μg of goat immunoglobulins against NF-κB p50 or NF-κB p65 proteins in L-buffer for 1 h, followed by washing three times with the buffer. Protein A-Sepharose beds were then mixed with 1 ml of A549 cell lysate (stimulated with TNF-α (5 ng/ml) for 30 min or left nonstimulated), rotated at +4 °C for 6 h, followed by washing twice with l-buffer and once with washing buffer (10 mm Tris-HCl, pH 6.8, 1 mm EDTA). Proteins were separated on 12% SDS-PAGE and Western blots were stained with rabbit immunoglobulins against p50, p65, importin α3, and IκBα. Oligonucleotide Precipitation—A549 cells were stimulated with 5 ng/ml of TNF-α for 0, 15, 30, or 60 min. The cells were collected, and samples were treated as described by Rosen et al. (53Rosen R.L. Winestock K.D. Chen G. Liu X. Hennighausen L. Finbloom D.S. Blood. 1996; 88: 1206-1214Crossref PubMed Google Scholar). Upper strands of CCL5 (RANTES) (5′-gga tcc CTC CCC TTA GGG GAT GCC CCT CAA CT) and CXCL10 (IP-10) (5′-gga tcc GCA GAG GGA AAT TCC GTA ACT TGG) promoter NF-κB elements were synthesized with BamHI overhangs as spacers, and they were 5′-biotinylated (DNA Technologies Inc., Gaithersburg, MD). Lower strands were nonbiotinylated. Oligonucleotides were annealed in 0.5 m NaCl and incubated with streptavidin-agarose beads (Neutravidin; Pierce) at +4 °C for 2 h in a ratio to yield maximum saturation of the beads with the biotinylated oligonucleotide. The samples were incubated with the agarose beads saturated with the oligonucleotide at +4 °C for 2 h in binding buffer containing 10 mm HEPES, 133 mm KCl, 10% glycerol, 2 mm EDTA, 1 mm EGTA, 0.01% Triton X-100, 0.5 mm dithiothreitol, 1 mm NaVO4, and protease inhibitors. After washing oligonucleotide-bound proteins were dissolved in 75 μl of 2× Laemmli sample buffer and separated on 10% SDS-PAGE. Proteins were transferred onto Immobilon-P membranes and visualized with antibodies against p50 and p65 proteins. Indirect Immunofluorescence and Confocal Laser Microscopy—For indirect immunofluorescence and confocal laser microscopy transiently transfected HuH7 cells were grown on glass coverslips. Cells were left untreated or pretreated with LMB (2 ng/ml) (54Kudo N. Khochbin S. Nishi K. Kitano K. Yanagida M. Yoshida M. Horinouchi S. J. Biol. Chem. 1997; 272: 29742-29751Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) for 20 min followed by stimulation with TNF-α (100 ng/ml) in the presence of LMB (2 ng/ml) for 30 min. The cells were fixed with methanol at –20 °C for 10 min and processed for immunofluorescence as previously described (55Melén K. Keskinen P. Ronni T. Sareneva T. Lounatmaa K. Julkunen I. J. Biol. Chem. 1996; 271: 23478-23486Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The cells positive for FLAG, c-Myc, and His tags or expressing native p50 protein, as indicated in the figures, were visualized and photographed on a Leica TCS NT confocal microscope. NF-κB Binds to Importin α3 and Importin α4—Upon activation NF-κB is translocated from the cytoplasm into the nucleus. The nucleocytoplasmic shuttling of NF-κB has been thought to be mediated via importin α/β- and exportin 1-dependent pathways. However, the molecular mechanisms of the nuclear import of NF-κB have remained unknown. To investigate the possible interactions of NF-κB with different importin isoforms, we stimulated human A549 lung carcinoma cells with TNF-α. Different expression systems were used for importin α3, since all isoforms could not be expressed in E. coli or by baculovirus expression. Cell extracts were prepared, and the cellular proteins were allowed to bind to Sepharose-immobilized bacterially expressed GST-importins α1, α3, α5, α7, or β (Fig. 1A) or baculovirus-expressed GST-importins α1, α3, α4, or α7 (Fig. 1B). Proteins bound to GST-importins were analyzed
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