Arginine/Lysine-rich Structural Element Is Involved in Interferon-induced Nuclear Import of STATs
2001; Elsevier BV; Volume: 276; Issue: 19 Linguagem: Inglês
10.1074/jbc.m008821200
ISSN1083-351X
AutoresKrister Melén, Leena Kinnunen, Ilkka Julkunen,
Tópico(s)interferon and immune responses
ResumoSignal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors, which mediate interferon (IFN), interleukin, and some growth factor and peptide hormone signaling in cells. IFN stimulation results in tyrosine phosphorylation, dimerization, and nuclear import of STATs. In response to IFN-γ stimulation, STAT1 forms homodimers, whereas IFN-α induction results in the formation of STAT1·STAT2 heterodimers, which assemble with p48 protein in the nucleus. Phosphorylation as such is not sufficient to target STATs into the nucleus; rather, the dimerization triggered by phosphorylation is essential. Although IFN-induced nuclear import of STATs is mediated by the importin/Ran transport system, no classic nuclear localization signal (NLS) has been found in STATs. In the three-dimensional structure of STAT1, we observed a structural arginine/lysine-rich element within the DNA-binding domain of the molecule. We created a series of point mutations in these elements of STAT1 and STAT2 and showed by transient transfection/IFN stimulation assay that this site is essential for the nuclear import of both STAT1 and STAT2. The results suggest that two arginine/lysine-rich elements, one in each STAT monomer, are required for IFN-induced nuclear import of STAT dimers. Import-defective STAT1 and STAT2 proteins were readily phosphorylated and dimerized, but they functioned as dominant negative molecules inhibiting the nuclear import of heterologous STAT protein. Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors, which mediate interferon (IFN), interleukin, and some growth factor and peptide hormone signaling in cells. IFN stimulation results in tyrosine phosphorylation, dimerization, and nuclear import of STATs. In response to IFN-γ stimulation, STAT1 forms homodimers, whereas IFN-α induction results in the formation of STAT1·STAT2 heterodimers, which assemble with p48 protein in the nucleus. Phosphorylation as such is not sufficient to target STATs into the nucleus; rather, the dimerization triggered by phosphorylation is essential. Although IFN-induced nuclear import of STATs is mediated by the importin/Ran transport system, no classic nuclear localization signal (NLS) has been found in STATs. In the three-dimensional structure of STAT1, we observed a structural arginine/lysine-rich element within the DNA-binding domain of the molecule. We created a series of point mutations in these elements of STAT1 and STAT2 and showed by transient transfection/IFN stimulation assay that this site is essential for the nuclear import of both STAT1 and STAT2. The results suggest that two arginine/lysine-rich elements, one in each STAT monomer, are required for IFN-induced nuclear import of STAT dimers. Import-defective STAT1 and STAT2 proteins were readily phosphorylated and dimerized, but they functioned as dominant negative molecules inhibiting the nuclear import of heterologous STAT protein. signal transducers and activators of transcription Janus tyrosine kinase interferon nuclear localization signal antibody fluorescein isothiocyanate hemagglutinin polyacrylamide gel electrophoresis wild-type interleukin-6 IFN-α/β receptor IFN-γ receptor nuclear export signal nucleoprotein interactor 1 tetramethylrhodamine isothiocyanate electrophoretic mobility shift assay Signal transducers and activators of transcription (STATs)1 are latent transcription factors, which function as signal transducers in the cytoplasm and activators of transcription in the nucleus. Presently, seven mammalian STAT proteins, STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6 have been described. Binding of cytokines to their specific cell surface receptors leads to the activation of Janus tyrosine kinase (JAK)/STAT pathway (1Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3399) Google Scholar). In response to type I IFNs (IFN-α, -β, and -ω), IFN-α/β receptor-associated JAK1 and TYK2 are phosphorylated and activated (2Velazquez L. Fellous M. Stark G.R. Pellegrini S. Cell. 1992; 75: 313-322Abstract Full Text PDF Scopus (712) Google Scholar, 3Müller M. Briscoe J. Laxton C. Guschin D. Ziemiecki A. Silvennoinen O. Harpur A.G. Barbieri G. Witthuhn B.A. Schindler C. Pellegrini S. Wilks A. Ihle J.N. Stark G.R. Kerr I.A. Nature. 1993; 366: 129-135Crossref PubMed Scopus (644) Google Scholar). JAKs in turn tyrosine phosphorylate STAT1 and STAT2 at Tyr-701 and Tyr-690, respectively. Phosphorylation triggers STAT1 and STAT2 to dimerize, translocate into the nucleus, and, together with p48 protein, bind to well-conserved DNA sequences in the promoter regions of IFN-α/β-responsive target genes and activate gene transcription (4Fu X.-Y. Kessler D.S. Veals S.A. Levy D.E. Darnell J.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8555-8559Crossref PubMed Scopus (343) Google Scholar, 5Kessler D.S. Veals S.A. Fu X.-Y. Levy D.E. Darnell Jr., J.E. Genes Dev. 1990; 4: 1753-1765Crossref PubMed Scopus (256) Google Scholar, 6Fu X.-Y. Schindler C. Improta T. Aebersold R. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7840-7843Crossref PubMed Scopus (453) Google Scholar, 7Veals S.A. Schindler C. Leonard D. Fu X.-Y. Aebersold R. Darnell Jr., J.E. Levy D.E. Mol. Cell. Biol. 1992; 12: 3315-3324Crossref PubMed Scopus (348) Google Scholar, 8Schindler C. Fu X.-Y. Improta T. Aebersold R. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7836-7839Crossref PubMed Scopus (544) Google Scholar). Binding of type II IFN (IFN-γ) to its receptor leads to activation of JAK1 and JAK2 and tyrosine phosphorylation of STAT1 (also at Tyr-701). STAT1 homodimerizes, translocates into the nucleus, and activates IFN-γ-inducible gene expression (1Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3399) Google Scholar, 9Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1267) Google Scholar). Both in cytokine-induced and uninduced cells the majority of STAT protein seem to be associated with high molecular mass complexes, and the amount of free STAT monomers is very small (10Lackmann M. Harpur A.G. Oates A.C. Mann R.J. Gabriel A. Meutermans W. Alewood P.F. Kerr I.M. Stark G.R. Wilks A.F. Growth Factors. 1998; 16: 39-51Crossref PubMed Scopus (47) Google Scholar, 11Ndubuisi M.I. Guo G.G. Fried V.A. Etlinger J.D. Sehgal P.B. J. Biol. Chem. 1999; 274: 25499-25509Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). STAT1 has the ability to form heterocomplexes with several other proteins and is capable of being imported into the nucleus also in its unphosphorylated form (12Chatterjee-Kishore M. Wright K.L. Ting J.P.-Y. Stark G.R. EMBO J. 2000; 19: 4111-4122Crossref PubMed Scopus (276) Google Scholar). Tyrosine phosphorylation triggered dimerization is crucial in IFN-induced nuclear import of STATs (13Mowen K. David M. J. Biol. Chem. 1998; 273: 30073-30076Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 14Milocco L. Haslam J. Rosen J. Seidel M. Mol. Cell. Biol. 1999; 19: 2913-2920Crossref PubMed Scopus (43) Google Scholar). Active nuclear transport of large macromolecules occurs via the nuclear pore complex (15Görlich D. Mattaj I.W. Science. 1996; 271: 1513-1518Crossref PubMed Scopus (1067) Google Scholar). Proteins to be imported into the nucleus contain a nuclear localization signal (NLS), which interacts with a specific NLS receptor, usually importin-α (16Adam E.J.H. Adam S.A. J. Cell Biol. 1994; 125: 547-555Crossref PubMed Scopus (257) Google Scholar). Importin α binds to importin-β, which docks the NLS-containing cargo·importin-α/β complex at the cytoplasmic side of the nuclear pore (17Görlich D. Henklein P. Laskey R.A. Hartmann E. EMBO J. 1996; 15: 1810-1817Crossref PubMed Scopus (363) Google Scholar, 18Weiss K. Ryder U. Lamond A.I. EMBO J. 1996; 15: 1818-1825Crossref PubMed Scopus (224) Google Scholar). Importin β interacts with Ran GTPase and p10/NTF2, and the complex is translocated into the nucleus in an energy-dependent manner (15Görlich D. Mattaj I.W. Science. 1996; 271: 1513-1518Crossref PubMed Scopus (1067) Google Scholar, 19Melchior F. Paschal B. Evans E. Gerace L. J. Cell Biol. 1993; 123: 1649-1659Crossref PubMed Scopus (472) Google Scholar, 20Moore M.S. Blobel G. Nature. 1993; 365: 661-663Crossref PubMed Scopus (641) Google Scholar). Importin-α recognizes the classic mono- and bipartite basic-type NLSs (16Adam E.J.H. Adam S.A. J. Cell Biol. 1994; 125: 547-555Crossref PubMed Scopus (257) Google Scholar, 21Görlich D. Prehn S. Laskey R.A. Hartmann E. Cell. 1994; 79: 767-778Abstract Full Text PDF PubMed Scopus (601) Google Scholar). The classic monopartite NLS consists of one and the bipartite NLS of two arginine/lysine-rich clusters of basic amino acids separated by a spacer region ranging from the usual 10 amino acids up to 37 residues (22Dingwall C. Laskey R.A. Trends Biochem. Sci. 1991; 16: 478-481Abstract Full Text PDF PubMed Scopus (1713) Google Scholar, 23Morin N. Delsert C. Klessig D.F. Mol. Cell. Biol. 1989; 9: 4372-4380Crossref PubMed Scopus (40) Google Scholar). Although IFN-γ-induced nuclear import of STAT1 has been shown to be mediated by at least one importin-α molecule, NPI-1 (24Sekimoto T. Imamoto N. Nakajima K. Hirano T. Yoneda Y. EMBO J. 1997; 16: 7067-7077Crossref PubMed Scopus (306) Google Scholar), and to be dependent on RanGTPase (25Sekimoto T. Nakajima K. Tachibana T. Hirano T. Yoneda Y. J. Biol. Chem. 1996; 272: 31017-31020Abstract Full Text Full Text PDF Scopus (99) Google Scholar), the molecular mechanisms or elements in STATs responsible for the nuclear import have remained unresolved. In the present work we show that STAT1 and STAT2 have a structural arginine/lysine-rich element involved in IFN-induced nuclear import. The structural element situates in the DNA-binding domain of the molecule, and two of these elements, one in each monomer, are required for nuclear import, because nuclear import-defective mutant STAT1 proteins inhibit the nuclear accumulation of STAT2 and vice versa. Human hepatocellular carcinoma HuH7 (26Nakabayashi H. Taketa K. Miyano K. Yamane T. Sato J. Cancer Res. 1982; 42: 3858-3863PubMed Google Scholar) cells were maintained in minimal essential medium, supplemented with penicillin (0.6 mg/ml), streptomycin (60 mg/ml), glutamine (2 mm), HEPES buffer, pH 7.4 (20 mm), and 10% fetal calf serum (Integro, Zaandam, Netherlands). Before stimulation with IFNs, the cells were cultured in the growth medium supplemented with 2% fetal calf serum for 24 h. Monolayers and suspension cultures ofSpodoptera frugiperda Sf9 cells were maintained in TNM-FH medium as described previously (27Summers M.D. Smith G.E. Tex. Agric. Exp. Stn. Bull. 1986; 1555: 1-57Google Scholar). Human leukocyte IFN-α (6 × 106 IU/ml) was kindly provided by Dr. Kari Cantell at our Institute (28Cantell K. Hirvonen S. Kauppinen H.-L. Myllylä G. Methods Enzymol. 1981; 78: 29-38Crossref PubMed Scopus (158) Google Scholar). Human IFN-γ (1 × 106 IU/ml) was obtained from the Finnish Red Cross Blood Transfusion Service and was prepared and purified as described (29Cantell K. Hirvonen S. Kauppinen H.-L. Methods Enzymol. 1986; 119: 54-63Crossref PubMed Scopus (36) Google Scholar). ANTI-FLAG M5 Abs (Sigma Chemical Co., St. Louis, MO) were used in indirect immunofluorescence microscopy and in immunoprecipitation (1:1000 dilution). Mouse anti-human STAT1 (1:50 dilution, Transduction Laboratories, Lexington, KY) and rabbit anti-human STAT1 (STAT1 p91 c-24), STAT2 (STAT2 p113 c-20), and p48 (ISGF-3γ p48 c-20) were obtained commercially (1:50–1:200 dilutions in immunofluorescence microscopy, Santa Cruz Biotechnology, Santa Cruz, CA). FITC- and TRITC-labeled goat anti-mouse and anti-rabbit immunoglobulins were used as secondary Abs (1:200 dilution, Cappel, Organon Teknika Co., West Chester, PA). In Western blotting secondary horseradish peroxidase-conjugated goat anti-mouse immunoglobulins (1:2000 dilution; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) were used. For the detection of tyrosine phosphorylation of STAT proteins, immunoprecipitated or gel-filtrated samples were stained with rabbit or mouse anti-phosphotyrosine Abs (1:500 dilution, Transduction Laboratories), followed by biotin-SP-conjugated goat anti-rabbit or anti-mouse (1:10,000 dilution; Jackson ImmunoResearch Laboratories) and horseradish peroxidase-conjugated streptavidin (1:2000 dilution; Jackson ImmunoResearch Laboratories). Polymerase chain reaction was used to modify STAT1, STAT2, andp48 genes. The noncoding sequences were removed by inserting unique BamHI or BclI restriction sites immediately upstream of the first ATG codon and downstream of the STOP codon of STAT1, STAT2, and p48 genes, respectively. The primers used for STAT1 were GCA CAA (GGA TCC) GCC ATG TCT CAG TGG TAC GAA CTT CAG-(sense) and AAA AAT T(GG ATC C) CT ATA CTG TGT TCA TCA TAC TGT C-(antisense); for STAT2 the primers were CTA ATC (TGA TCA) GCC ATG GCG CAG TGG GAA ATG CTG CAG-(sense) and GAA ATG (TGA TCA) CTA GAA GTC AGA AGG CAT CAA GGG-(antisense); and for p48 the primers were GGA CAG GAT CCC GCC ATGGCA TCA GGC AGG GCA CGC-(sense) and TGG GTC GGA TCC TCA TTACAC CAG GGA CAG AAT GGC TGC-(antisense) (BamHI sites forSTAT1 and p48 and BclI sites forSTAT2 in parentheses, initiation and STOP codonsunderlined). The polymerase chain reaction products were digested with BamHI or BclI, isolated from agarose gel, and subcloned into the BamHI site of a modified transient FLAG-tagged pCDNA 3.1(+) expression vector (Invitrogen, Carlsbad, CA) or HA-tagged pBC12/CMV expression vector (30Melén K. Julkunen I. J. Biol. Chem. 1997; 272: 32353-32359Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). To construct a FLAG-tagged transient expression vector, oligonucleotides with a new BamHI cloning site withBclI-compatible ends (sense oligonucleotide, 5′-CTA GCA CCA TGG ACT ACA AGG ACG ACG ATG ACA AGG GAT CCC; and antisense, 5′-TCG AGG GAT CCC TTG TCA TCG TCG TCC TTG TAG TCC ATG GTG; the initiation codon is underlined) were synthesized. The oligonucleotides were annealed, and the resulting double-stranded DNA fragment was subcloned into the BamHI site of the pCDNA 3.1(+), to create a vector pCDNA 3.1(+)-FLAG-tag. All DNA manipulations were performed according to standard protocols, and the newly created gene constructs were partially sequenced. Point mutations to the arginine/lysine-rich elements of STAT1 and STAT2 (FLAG-tagged STAT1 and STAT2) in the modified pCDNA 3.1(+)-FLAG-tag expression vector were constructed using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Primers used were 5′-CAA CTC AGT CCT GAT AGC TCC AGT TCC TTT AGG (Y701A), 5′-GCC CAA AAT GTT GAA GGC CGC AAA TCC TTT TAC TG (R378A,K379A), 5′-GTG CCA GCA TTT GCC TGT TCT GCC AAT TGC AGG TGC CG (K410A,K413A), and 5′-CCT CAT TCG TTG CGG TGC CAG CAT TGG CCT GTT CTT TC (K413A,R418A) for STAT1 and 5′-GTC AGA ATG TTG AAC GCC GCG AAG CCT TGT AAT TG (R374A,K375A) and 5′-CTT ATT GCT GCC CGC TCC TGA ACC ACC TGA AGC TTG CTC CAC C (R409A,K415A) for STAT2. The newly created mutations were verified by sequence analysis. wtSTAT1, STAT1 Y701A, STAT1 K410A,K413A, wtSTAT2, and wtp48 were also subcloned into the BamHI site of the baculovirus expression vector pAcYM1 (27Summers M.D. Smith G.E. Tex. Agric. Exp. Stn. Bull. 1986; 1555: 1-57Google Scholar). Baculovirus expression vectors of wt murine JAK2 (31Silvennoinen O. Witthuhn B. Quelle F.W. Cleveland J.L. Yi T. Ihle J.N. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8429-8433Crossref PubMed Scopus (437) Google Scholar) and kinase-negative JAK2 L882G (knJAK2) (32Quelle F.W. Thierfelder W. Witthun B.A. Tang B. Cohen S. Ihle J.N. J. Biol. Chem. 1995; 270: 20775-20780Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) were kindly provided by Dr. Olli Silvennoinen (University of Tampere, Finland). HuH7 cells were transfected with HA-STAT1- and HA-STAT2-pBC12/CMV or FLAG-STAT1- and FLAG-STAT2-pCDNA 3.1(+) gene constructs, using FuGENE6 transfection reagent (Roche Molecular Biochemicals, Mannheim, Germany). Transfected HuH7 cells were left untreated or were treated with IFN-α (1000 IU/ml, 30 min). The cells were collected, washed with phosphate-buffered saline, and lysed in immunoprecipitation binding buffer (IP buffer) (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, and 1% Triton X-100) on ice for 30 min. The cell lysates were cleared by centrifugation. The samples of cleared cell lysates were immunoprecipitated with monoclonal anti-FLAG Abs bound to protein A-Sepharose CL-4B (Amersham Pharmacia Biotech, Uppsala, Sweden) at 4 °C for 2 h. Immunoprecipitates were washed three times, and Laemmli sample buffer was added (33Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207438) Google Scholar). SDS-PAGE was performed on 6–12% polyacrylamide gels. Proteins separated on gels were transferred onto Immobilon-P membranes (polyvinylidene difluoride, Millipore, Bedford, MA) and visualized with the enhanced chemiluminescence system (ECL) (Amersham Pharmacia Biotech, Buckinghamshire, UK) as recommended. Monolayers of Sf9 cells were infected with STAT1, STAT1 Y701A, STAT1 K410A,K413A, STAT2, p48, wtJAK2, or knJAK2 recombinant baculoviruses. 28 or 38 h after infection, cells were disrupted in lysis buffer (10 mm HEPES-KOH, pH 7.9, 100 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 10% glycerol, and 100 μm Na3VO4). Lysates were cleared of insoluble material by centrifugation and used for electrophoretic mobility shift assay using ISRE15 and GAS consensus sequence oligonucleotides as described (34Lehtonen A. Matikainen S. Julkunen I. J. Immunol. 1997; 159: 794-803PubMed Google Scholar). Lysates (above) of baculovirus-infected Sf9 cells were gel-filtrated in the above lysis buffer using a 24-ml Superose 12 fast protein liquid chromatography (Amersham Pharmacia Biotech) gel filtration column. Molecular weight standards were obtained from Sigma. For indirect immunofluorescence and confocal laser microscopy, transiently transfected HuH7 cells were grown on glass coverslips, either treated with IFN-α (1000 IU/ml) for 45 min or left untreated and performed as described previously (35Melén 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 tag were visualized and photographed on a Zeiss Axiophot photomicroscope or a Leica TCS NT confocal microscope. In some experiments, to better visualize STAT and p48 proteins in immunofluorescence, the cells were primed with IFN-γ (10 IU/ml for 24 h), which is known to up-regulate their expression (34Lehtonen A. Matikainen S. Julkunen I. J. Immunol. 1997; 159: 794-803PubMed Google Scholar,36Koster M. Hauser H. Eur. J. Biochem. 1999; 260: 137-144Crossref PubMed Scopus (54) Google Scholar). The transport kinetics of endogenous STAT1 and STAT2 was analyzed by treating HuH7 cells with 1000 IU/ml of IFN-α. As detected by confocal laser microscopy (Fig. 1), nuclear import of both STAT1 and STAT2 could be seen starting at 10 min after IFN-α treatment. The nuclear accumulation was at its maximum within 30–60 min, and the cytoplasmic recycling was completed within 3 h. The kinetics of nuclear transport of STAT1 and STAT2 was dependent on IFN dose, because low doses of IFN-α (1–10 IU/ml) resulted to reduced transport kinetics (not shown). As visualized by confocal laser microscopy (Fig. 2, a andb), in untreated HuH7 cells STAT1 was located evenly in the cytoplasm and nucleus, whereas STAT2 was predominantly cytoplasmic and no colocalization with STAT1 could be seen in the cell nucleus. 30 min after IFN-α treatment both STAT1 and STAT2 translocated effectively to the cell nucleus and showed marked nuclear colocalization (Fig. 2, c and d). To study the intracellular location of p48 protein, we carried out double-staining experiments in HuH7 cells (Fig. 3). p48 protein was predominantly nuclear already in untreated HuH7 cells, and no detectable change in intracellular distribution was seen after IFN-α treatment. STAT1, instead, was effectively imported into the nucleus and showed marked colocalization with p48 protein 30 min after IFN-α treatment (Fig. 3).Figure 2Confocal images of indirect immunofluorescence staining for STAT1 and STAT2 proteins in untreated and IFN -α-treated HuH7 cells. a, HuH7 cells were left untreated or treated (c) with 1000 IU/ml IFN-α for 30 min and double stained with monoclonal anti-STAT1 Abs, followed with TRITC-labeled anti-mouse Abs (red) and polyclonal anti-STAT2 Abs, followed with FITC-labeled anti-rabbit Abs (green). a andc, focus was adjusted through the center of the nucleus.b, staining profiles of STAT1 and STAT2 in untreated HuH7 cells (white line in a). d, staining profiles of STAT1 and STAT2 in IFN-α-treated HuH7 cells (white line in c). Nuclear areas are indicated.Bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Colocalization of STAT1 and p48 proteins in HuH7 cells detected by confocal microscopy using indirect immunofluorescence staining. The cells were first primed with 10 IU/ml of IFN-γ for 24 h, then treated with 1000 IU/ml of IFN-α for 45 min, or left untreated and double-stained with anti-STAT1 and anti-p48 Abs. Bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because IFN-induced nuclear import of STAT1 has been suggested to be mediated by NPI-1 (24Sekimoto T. Imamoto N. Nakajima K. Hirano T. Yoneda Y. EMBO J. 1997; 16: 7067-7077Crossref PubMed Scopus (306) Google Scholar), we reasoned that STATs are likely to contain an arginine/lysine-rich element that would be involved in their nuclear import. In most DNA-binding proteins the NLS overlaps or is immediately adjacent to the DNA-binding domain (37LaCasse E.C. Lefebvre Y.A. Nucleic Acids Res. 1995; 23: 1647-1656Crossref PubMed Scopus (191) Google Scholar). Although STATs do not contain a classic NLS, computer analysis of the STAT1 structure (38Chen X. Vinkemeier U. Zhao Y. Jeruzalmi D. Darnell J. Kuriyan J. Cell. 1998; 93: 827-839Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar) revealed a cluster of basic amino acids in the DNA-binding domain of the molecule within the DNA-binding domain (Fig. 4, aand c). This element includes amino acids Arg-378, Lys-379, Lys-410, Lys-413, and Arg-418. All of these amino acids are conserved in STAT1, STAT3, and STAT4 proteins and are mostly conserved in STAT2 (human and pig), which is lacking the lysine corresponding to K410 of STAT1. STAT5a, STAT5b, and STAT6 are more distantly related to the other STAT molecules (Fig. 4 b), and they lack the basic residues corresponding to Arg-378 and Lys-379 of STAT1. Instead, they have two additional arginine or lysine residues further downstream (Fig. 4 a). To analyze whether this structural site rich in basic amino acids is involved in nuclear import of human STAT1 and STAT2, we made a series of double point mutations and studied the nuclear import of the FLAG-tagged, mutated STAT proteins in transiently transfected HuH7 cells by indirect immunofluorescence microscopy (Fig.5). HuH7 cells were selected, because they were effectively transfected and possessed excellent cellular morphology in microscopic studies. Although HuH7 cells had their own functional STAT1 and STAT2 proteins, using FLAG-tagged gene constructs, we were able to follow the transport of transfected mutant proteins. To rule out the possibility that the FLAG-tag affected the transport, we also carried out the experiments with influenza virus hemagglutinin (HA)-tagged STAT gene constructs with identical results (not shown). Weak basal nuclear accumulation of intrinsic (Fig. 1) as well as of transiently expressed (Fig. 5) STAT1 and to a lesser extent of STAT2 was observed in HuH7 as well as in several other cell lines studied. Although FLAG-tagged transiently expressed wild-type STAT1 protein was readily transported into the nucleus after IFN-α or IFN-γ stimulation, all arginine/lysine mutant STAT1 proteins showed impaired IFN-induced nuclear import (Fig. 5). Fig.6 shows the nuclear import of the corresponding STAT forms in a quantitative manner. In STAT1 R378A,K379A and K413A,R418A mutant proteins nuclear import was partially inhibited, whereas STAT1 K410A,K413A protein appeared to be fully nuclear transport incompetent after stimulation with either IFN type (Fig. 5,j, k, l). A revertant, where residues 413 and 418 where mutated back to those of the wild-type STAT1, showed normal IFN-induced nuclear translocation (Fig. 5,p, q, r and Fig. 3). Mutation in tyrosine 701 of STAT1 (STAT1 Y701A in Fig. 5), which has previously been shown to result in lack of tyrosine phosphorylation and subsequent dimerization (39Shuai K. Schindler C. Prezioso V.R. Darnell Jr., J.E. Science. 1992; 259: 1808-1812Crossref Scopus (659) Google Scholar), rendered the protein cytoplasmic. The corresponding mutations in STAT2 molecule showed similar effects to those seen in STAT1 (Figs. 5 and 6). IFN-α-induced nuclear import of STAT2 R374A,K375A and STAT2 R409A,K415A proteins were partially and completely blocked, respectively (Fig. 5, u, v,w, x). It is unlikely that p48 protein, which together with STAT1 and STAT2 proteins forms the ISGF3 complex (4Fu X.-Y. Kessler D.S. Veals S.A. Levy D.E. Darnell J.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8555-8559Crossref PubMed Scopus (343) Google Scholar), would be involved in STAT1·STAT2 heterodimer transport, because both transfected as well as intrinsic p48 protein was always found in the nucleus and its cellular distribution was not altered by IFN treatment (Fig. 3). Although the tyrosine phosphorylation site is far from the arginine/lysine-rich element of STAT proteins (Fig. 4 c), we considered the possibility that mutations in the arginine/lysine-rich element of STAT1 or STAT2 proteins would affect their tyrosine phosphorylation properties. Therefore, we transfected HuH7 cells with wt or mutantSTAT genes for 48 h, followed by stimulation with IFN-α for 1 h. Cells were collected, and transgene STAT protein expression was analyzed by anti-FLAG Abs. All STAT1constructs were equally well expressed, and in response to IFN-α stimulation there was a clear increase in the molecular weight of all tested STAT1 proteins except Y701A mutant construct (Fig.7 a). Immunoprecipitation, followed by Western blotting with anti-P-Tyr Abs, revealed that all STAT forms except STAT1 Y701A were readily tyrosine-phosphorylated (Fig. 7 b). This result suggests that point mutations in the arginine/lysine-rich element do not affect IFN-induced phosphorylation status of STAT proteins. Because STAT1 and STAT2 heterodimerize after stimulation with IFN-α, we studied whether the transport-defective STATs inhibit the nuclear import of heterologous STAT molecules. wt and transport-defective STAT proteins were transiently expressed in HuH7 cells, and colocalization of STAT1, STAT2, and p48 proteins was analyzed. IFN-α-induced nuclear import of STAT2 in wt STAT1 transfected cells was readily seen (Fig.8 a), and it was comparable to that seen in Figs. 1 and 5. Instead, the cytoplasmic FLAG-STAT1 K410A,K413A mutant effectively blocked the nuclear import of endogenous STAT2, functioning as a dominant negative molecule in IFN-α-treated cells (Fig. 8 a). To analyze the possible effect of STAT2 expression on IFN-γ induction in HuH7 cells both wt and nuclear transport-defective STAT2 were transiently expressed, and colocalization of STAT1 and STAT2 was detected. Overexpression of both wt and nuclear transport-defective FLAG-STAT2 blocked IFN-γ induction in transiently transfected HuH7 cells (Fig. 8 b). Transiently transfected FLAG-STAT1 Y701A did not inhibit IFN-α-induced nuclear import of endogenous STAT2, suggesting that mutant STAT1 Y701A is incapable of forming dimers with endogenous STAT2 and thus does not block its nuclear import. It is likely that the weak nuclear accumulation of STAT2 was due to the endogenous STAT1 in HuH7 cells (Fig. 8 a). Transiently transfected transport-defective FLAG-STAT2 R409A,K415A, instead, completely blocked IFN-α-induced nuclear import of endogenous STAT1. This suggests that FLAG-STAT2 R409A,K415A mutant protein and endogenous STAT1 formed complexes in the cytoplasm but they were not translocated into the cell nucleus after IFN-α induction (Fig. 8 a). The results suggest that in a heterodimer both arginine/lysine-rich elements, one in each STAT, have to be intact before IFN-induced nuclear import of STAT dimers can take place. To study STAT dimerization and DNA binding in a system lacking intrinsic human STAT proteins we reconstituted STAT activation and DNA binding analysis using a baculovirus expression system. We infected Sf9 cells with STAT1, STAT2, p48, and JAK2 protein-expressing recombinant baculoviruses. Sf9 cells and a baculovirus expression system is selected, because JAK2 kinase has earlier been shown to tyrosine-phosphorylate and activate STAT1 protein (32Quelle F.W. Thierfelder W. Witthun B.A. Tang B. Cohen S. Ihle J.N. J. Biol. Chem. 1995; 270: 20775-20780Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). As shown in Fig. 9 (a and b), wtSTAT1 was tyrosine-phosphorylated, dimerized, and exhibited DNA binding to the GAS oligonucleotide probe in the presence of wtJAK2 kinase, but not with that of knJAK2, as analyzed by gel filtration, anti-phosphotyrosine blotting, and EMSA, respectively. The nuclear import-defectiv
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